AU2016228053B2 - Uses of insecticidal protein - Google Patents

Abstract

Provided is a method for controlling prodenia litura. The method comprises: allowing prodenia litura to be in contact with a Cry2Ab protein. The method specifically comprises: growing genetically modified plants by using polynucleotide coded with a Cry2Ab protein; and allowing the prodenia litura to be in contact with the Cry2Ab protein by allowing the prodenia litura to eat genetically modified plants, wherein after the contact, the prodenia litura is restrained in growth and/or finally dies, so as to prevent the prodenia litura doing harm to plants.

Description

USES OF INSECTICIDAL PROTEIN

Cross-reference to related applications

The present application claims the priority of Chinese patent application No. 201510096598.3 filed on March 04, 2015, the full text of which is incorporated here by reference.

Field of the invention

The present invention relates to uses of insecticidal protein, in particular, involved in a Cry2Ab protein used in protecting plant form the harm of pest Spodoptera litura by expressing the Cry2Ab protein in the plant.

Background of the invention

Spodoptera litura belongs to Lepidoptera, Noctuidae, which is a polyphagous and gluttonous pest, is harmful to many kinds of hosts, besides corn and soybean, it also attacks melons, eggplants, beans, shallots, leeks, spinach as well as cruciferae plant, grain and economic crops etc. in total of nearly 100 families and more than 300 species of plants. Spodoptera litura distributes around the world and occurs in various parts of China, mainly in Yangtze 20 River Basin and Yellow River Basin. Pest Spodoptera litura is mainly harmful to whole plant when they are larvae and live on leaves’ back in group and gnaw at leaves when they are low instar; after the third instar, they will scatter and be harmful to leaves and tender stems, the aging larvae may eat into fruit.

Com and soybean are important food crops in China, Spodoptera litura cause tremendous grain 25 loss every years, it even affects the living conditions of the local populations. At present, agricultural control, chemical control and physical control are usually applied to control Spodoptera litura.

Agricultural control is a method to comprehensively manage multiple factors of the whole farmland ecological system, by means of the regulation of crops, pests and the environmental 30 factors, a farmland ecological environment is created, which is conducive to the crop growth and nonadvantagous to the outbreaking of Spodoptera litura. Such as the removal of weeds,

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2016228053 16 Oct 2018 harvesting after drying or irrigation, to destroy or worsen their pupa sites, help to reduce the insect source; or combined with the management of the removal of eggs and cluster hazards of newly hatched larvae to reduce the insect source. Because most of the agricultural controls are preventive measures, the application of agriculture control is limited and cannot serve as an 5 emergency measures. It doesn’t work when Spodoptera litura outbreaks.

Chemical control, i.e. pesticides control, is a method to kill pests by using chemical pesticide, chemical control is an important part of the comprehensive treatment of Spodoptera litura, it is rapid, convenient, simple and economically, chemical control is an indispensable measure for emergency when Spodoptera litura outbreaks, Spodoptera litura 10 can be eliminated before it causes harm and losses by using chemical control. Current chemical control method mainly refers to spraying of medical solution. But chemical control also has its limitations, for example, the improper operation can usually cause crop phytotoxicity, and pest resistance to drugs, in addition, natural enemies can also be killed, chemical pesticides may cause the environmental pollution and destruct the farmland 15 ecosystem as well, furthermore, pesticide residues may pose a threat to the safety of people and animals and leads to other serious results.

Physical control is to control pests by using physical factors, such as: light, electricity, color, humidity, temperature and etc as well as mechanical equipment to trap and kill the pests and sterilize the pests by irradiation based on the response of pests to physical factors 20 in the environmental conditions. The methods widely applied at present mainly include:

attracting moths with lamp, syrupacetiacid bait trap, and trapping and killing moths by dipping and sprinkling 500 dipterex using willow branch; although the above methods show a control effect to some extent, they have certain difficulty in operation.

In order to solve the limitations of the agricultural control, chemical control and physical 25 control in practical application, the scientists found that, by means of transfecting genes encoding pesticidal protein into plants, some insect-resistant transgenic plants were obtained to control plant pests. Cry2Ab pesticidal protein is one of the numerous pesticidal proteins, which is an insoluble meridian crystal protein produced by Bacillus thuringiensis.

Cry2Ab protein is ingested into midgut by insects, the protoxin of toxic protein is dissolved 30 in an alkaline environment of insect midgut. N-end and C-end of the protein are digested by alkaline protease and the protoxin is transformed into active fragments; the active fragments are bound with the receptors on the upper surface of epithelial membrane of insect midgut and inserted into intestinal membrane, resulting in perforation lesions of

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2016228053 16 Oct 2018 cytomembrane, destroying osmotic pressure change and pH balance between inside and outside of the cytomembrane, disturbing the digestion process of insects and eventually resulting in death.

It has been proved that Cry2Ab protein can resist the encroachment of Spodoptera litura, so far there is no report about the application of transgenic plants expressing Cry2Ab protein to control Spodoptera litura.

Summary of the invention

The present invention is to provide uses of insecticidal protein, particularly provide a 10 method for controlling harm of Spodoptera litura to plants by producing transgenic plants expressing Cry2Ab protein, which effectively overcomes the technical limitations of the prior art such as agricultural control, chemical control and physical control.

In order to achieve the purpose mentioned above, the present invention provides following technical solutions, specifically:

The first aspect of the present application relates to a method for controlling Spodoptera litura, wherein the method includes contacting Spodoptera litura at least with Cry2Ab protein which is preferably used as a individual insecticidal active ingredient;

preferably, Cry2Ab protein at least exists in a host cell expressing Cry2Ab protein, and Spodoptera litura at least contacts with Cry2Ab protein by ingestion of the host cell;

more preferably, Cry2Ab protein at least exists in bacteria or a transgenic plant expressing Cry2Ab protein, and Spodoptera litura at least contacts with Cry2Ab protein by ingestion of the bacteria or a tissue of the transgenic plant; thereafter, the growth of Spodoptera litura is inhibited and/or Spodoptera litura dies, so as to achieve controlling the damage of Spodoptera litura to the plant.

In further embodiments, as the method for controlling Spodoptera litura, wherein the transgenic plant is in any growth period; and/or the tissues of the transgenic plant are leaves, stems, fruits, tassels, ears, buds, anthers or filaments; and/or the control of the damage of Spodoptera litura to the plant does not depend on planting location and/or planting time; and/or

Further, as said method for controlling pest Spodoptera litura, the plant is selected from the

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2016228053 16 Oct 2018 group consisting of corn, soybean, cotton, sweet potato, taro, lotus, tianjing, tobacco, beet, cabbage or eggplant.

Further, as said method for controlling Spodoptera litura, prior to contacting, the method includes a step of planting a seedling containing a polynucleotide encoding Cry2Ab 5 protein.

Further, as said method for controlling Spodoptera litura, the amino acid sequence of Cry2Ab protein comprises an amino acid sequence shown by SEQ ID NO:1;

preferably, the nucleotide sequence encoding Cry2Ab protein comprises a nucleotide sequence shown by SEQ ID NO:2.

Further, as said method for controlling Spodoptera litura, the plant also contains at least a second nucleotide sequence which is different from the nucleotide sequence encoding Cry2Ab protein, but the second nucleotide sequence does not encode a Cry 1 A. 105 protein;

preferably, the second nucleotide sequence encodes a Cry-type insecticidal protein, a Vip-type insecticidal protein, a protease inhibitor, lectin, α-amylase, peroxidase or a 15 dsRNA inhibiting an important gene of target insect or pest;

more preferably, the second nucleotide sequence encodes Cry 1 Fa protein or Vip3Aa protein;

still preferably, the amino acid sequence of CrylFa protein comprises an amino acid sequence shown by SEQ ID NO:3;

most preferably, the second nucleotide comprises a nucleotide sequence shown by SEQ ID NO:4.

Further, as the method for controlling Spodoptera litura, Spodoptera litura is in the crop field.

On the other hand, the present invention provides use of Cry2Ab protein to control 25 Spodoptera litura, preferably, wherein the Cry2Ab protein is used as ai ndividual insecticidal active ingredient.

And on another hand, the present invention provides use of a plant cells, a plant tissues, a plant, a bacteria which is transformed with Cry2Ab gene to control Spodoptera litura, preferably, Cry2Ab gene is used as a coding gene for a individual insecticidal active 30 ingredient.

Again on the other hand, the present invention provides a method for producing a plant that

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2016228053 16 Oct 2018 controls Spodoptera litura, the method includes a step of introducing a polynucleotide sequence encoding Cry2Ab protein into the genome of a plant.

Also on the one hand, the present invention provides a method for producing a plant seed that controls Spodoptera litura, the method includes a step of hybridizing a first plant 5 produced by the method according to said methods with a second plant, thus produces a seed containing a polynucleotide sequence encoding Cry2Ab protein.

On the other hand, the present invention provides a seed that controls Spodoptera litura produced by said methods.

And on another hand, the present invention provides a method for cultivating a plant that 10 controls Spodoptera litura, the method includes the following steps:

planting at least one seed whose genome contains a polynucleotide sequence encoding Cry2Ab protein;

making the seed grow into a plant;

making the plant grow under the condition of artificial inoculation and/or natural 15 occurrence of Spodoptera litura, and harvesting the plant with weakened plant damage and/or increased plant yield compared to other plant without the polynucleotide sequence encoding Cry2Ab protein.

Again on the other hand, the present invention provides a plant that controls Spodoptera litura produced by said methods.

Also on the one hand, the present invention provides a plant cell, a plant tissue, a plant or a bacteria that controls Spodoptera litura, wherein the plant cell, the plant tissue, the plant or the bacteria contains the polynucleotide encoding Cry2Ab protein;

preferably, the amino acid sequence of Cry2Ab protein comprises an amino acid sequence shown by SEQ ID NO:1;

more preferably, the nucleotide sequence encoding Cry2Ab protein comprises nucleotide sequence shown by SEQ ID NO:2.

Further, as said plant cell, the plant tissue, the plant or the bacteria that controls Spodoptera litura, wherein the plant cell, the plant tissue, the plant or the bacteria at least contains a second nucleotide sequence which is different from the nucleotide sequence encoding 30 Cry2Ab protein, but the second nucleotide sequence does not encode a Cry 1 A. 105 protein;

preferably, the second nucleotide sequence encodes a Cry-type insecticidal protein, a Vip-type insecticidal protein, a protease inhibitor, lectin, α-amylase, peroxidase or a dsRNA inhibiting an important gene of target insect or pest;

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2016228053 16 Oct 2018 more preferably, the second polynucleotide sequence encodes CrylFa protein or Vip3Aa protein;

still preferably, the amino acid sequence of CrylFa protein comprises an amino acid sequence shown by SEQ ID NO:3;

most preferably, the second nucleotide sequence comprises a nucleotide sequence shown by SEQ ID NO:4.

Further, as said plant cell, the plant tissue, the plant or the bacteria that controls Spodoptera litura, the plant is selected from the group consisting of corn, soybean, cotton, sweet potatoe, taro, lotus, tianjing, tobacco, sugar beet, cabbage or eggplant, and the tissues of the 10 transgenic plant are leaves, stems, fruits, tassels, ears, buds, anthers or filaments.

The “contact” in the present application refers to that insects and/or pests touch, stay and/or intake plant, plant organs, plant tissues or plant cells. The plant, plant organs, plant tissues or plant cells refer to they can expresse insecticidal proteins in vivo or the surface of the plant, plant organs, plant tissues or plant cells has insecticidal proteins or microorganisms 15 generating insecticidal proteins.

Terms “control” and/or “prevent” in the present application refers to that pest Spodoptera litura comes into contact with Cry2Ab protein, and after the contact, the growth of pest Spodoptera litura is inhibited and/or the pest Spodoptera litura dies. Further more, the pest Spodoptera litura contacts with the Cry2Ab protein through intaking plant tissue, after the 20 contact, the growth of all or some of the pest Spodoptera litura is inhibited and/or all or some of them die. Inhibition refers to sub-lethality, i.e.: it does not refer to lethal, but it may arouse certain effects in such aspects such as growth and development, behavior, physiology, biochemistry and tissues, for example: the growth and development is slow and/or stops. Meanwhile, the plant should be normal in morphology and can be cultured 25 under conventional methods in order to use them for consumption and/or generation of products. Besides, compared with non-transgenic wild plant, the plant and/or plant seeds controlling pest Spodoptera litura which contain polynucleotide sequence encoding Cry2Ab protein have weakened plant damage under the condition that the pest Spodoptera litura does harm through artificial inoculation and/or natural occurrence. The concrete 30 manifestation includes but without limitation: improved stalk resistance, and/or increased grain weight and/or yield. The “control” and/or “prevent” function of Cry2Ab protein over the pest Spodoptera litura may exist independently and will not abate and/or disappear due to the existence of other substances which can “control” and/or “prevent” pest Spodoptera litura. Specifically, if the tissues of transgenic plant (containing polynucleotide sequence

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2016228053 16 Oct 2018 encoding Cry2Ab protein) simultaneously and/or asynchronously contain and/or generate Cry2Ab protein and/or another substance which can control pest Spodoptera litura, then the existence of another substance will neither affect the “control” and/or “prevent” function of Cry2Ab protein over Spodoptera litura, nor can result in that the “control” and/or “prevent” 5 function is realized completely by another substance, while irrelevance with Cry2Ab protein. Under normal conditions, on farmland, the ingestion process of plant tissues by pest Spodoptera litura is short and can hardly be observed by naked eyes, therefore, under the condition that pest Spodoptera litura does harm through artificial inoculation and/or natural occurrence, if any tissue of transgenic plant (containing polynucleotide sequence encoding Cry2Ab protein) has dead pest Spodoptera litura, and/or pest Spodoptera litura whose growth is inhibited stays on them, and/or plant damage is weakened compared with non-transgenic wild plant, it means the method and use of the present application are realized, i.e.: the method and/or use for controlling the pest Spodoptera litura is realised through the contact between pest Spodoptera litura and Cry2Ab protein.

In present invention, the Cry2Ab protein is expressed in a transgenic plant accompanied by the expressions of one or more Cry-class insecticidal proteins and/or Vip-class insecticidal proteins. This co-expression of more than one kind of insecticidal toxins in a same transgenic plant can be achieved by transfecting and expressing the genes of interest in plants by genetic engineering. In addition, Cry2Ab protein can be expressed in one plant 20 (Parent 1) through genetic engineering operations and Cry-class insecticidal protein and/or Vip-class insecticidal proteins can be expressed in the second plant (Parent 2) through genetic engineering operation. The progeny expressing all genes of Parent 1 and Parent 2 can be obtained by crossing Parent 1 and Parent 2.

RNA interference (RNAi) refers to a highly conserved and effective degradation of specific homologous mRNA induced by double-stranded RNA (dsRNA) during evolution. Therefore RNAi technology is applied to specifically knock out or shut down the expression of a specific gene of the target insect pest in present invention.

In the classification system, generally, Lepidoptera is divided into suborder, superfamily, family etc. according to morphological characteristics such as the neuration of adult wings, 30 chain mode and antenna type, and the Noctuidae is the most abundant species of Lepidoptera, more than 20,000 species of insects have been found around the world, and most of them are agricultural pests, 2110 species of Noctuidae insects have been reported in China which belong to 18 subfamilies, 514 genera, among them, the most common pests in China are Helicoverpa armigera, Agrotis ypsilon, Sesamia inferen and Spodoptera

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2016228053 16 Oct 2018 frugiperda, etc. Although, Spodoptera litura belongs to Noctuidae as well as Helicoverpa armigera, Agrotis ypsilon and Sesamia inferen, in addition to the similarity in the classification criteria, there are significant differences in other morphological structures, it is akin to strawberries and apples in plants (both belong to Rosaceae Rosaceae), they have 5 characteristics of flowers bisexual, radiation symmetry, 5 petals and so on, but they have tremendous differences in fruits and plant morphology. Spodoptera litura has its unique characteristics form morphological point of view when it’s larval or adult. For example, Spodoptera litura larvae have dark brown head, changeable ch est, the corlor of the chest may be khaki, black or green; the adult body are dark brown, the back of the chest have white fur, grayish brown forehead with many patterns. Spodoptera frugiperda, which also belongs to the species Noctuidae, has green body when newly-hatched; the adult of Spodoptera frugiperda are grayish brown, the wings of female adult are gray or grayish brown, the wings of male adult are black and covered with dark spots and light gray lines.

Insects belong to the family Noctuidae are not only different in morphological characteristics, but also differ in feeding habits. For example, Helicoverpa armiger, which also belongs to Noctuidae, damages cotton bolls or corn ears by drilling, Agrotis ypsilon damages the base of corn and other stems, Spodoptera frugiperda damages corn by eating leaves, cutting root, or drilling into corncob, while Spodoptera litura prefers to bite leaves 20 and only left the main veins; the aging larvae can eat fruits. Differences in feeding habits also suggest that the enzyme and the receptor protein produced by the digestive system are different. The enzymes produced by the digestive tract are the key point of the B.t. gene effection, the only way to make one certain B.t. gene resist insect, is that the insect has the enzyme or receptor protein which can bind with the certain B.t. protein. More and more 25 reports show that, insects in the same species of different order, and even the different family of the same species, have different sensitivity to the same B.t. protein. For example, Cry2Ab protein shows high toxicity to Helicoverpa armigera Hubner which belongs to Noctuidae, but do not have effect to Spodoptera exigua Hiibner which also belongs to Noctuidae, Cry2Ac protein shows high toxicity to Helicoverpa armigera Hubner and 30 cabbage looper, but only shows minor inhibition on Spodoptera exigua Hiibner. The cases above fully demonstrate that the interaction between B.t. protein and the insect’s enzyme and receptor is complex and unpredictable; it also shows that the response of the same insect (Spodoptera exigua Hiibner) to different B.t. proteins (Cry2Ab and Cry2Ac) is also different. This is because the naming of endotoxin from Bacillus thuringiensis is based on

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2016228053 16 Oct 2018 the similarity of the amino acid sequence, and has nothing to do with its insecticidal activity, that a B. t. protein (such as Cry2Ac) has insecticidal activity against an insect (such as Spodoptera exigua Hiibner) does not necessarily indicates that the other protein (such as Cry2Ab) in the same category have insecticidal activity on the same insect.

Meanwhile, there is a general understanding in the research community of Bacillus thuringiensis, the Cry2 toxin has unique function on insect, it do not has significant homology with CrylA endotoxin, not only in amino acid sequence, but also exhibit different binding and pore formation properties, it is not possible to determine whether Cry2 toxin has control/prevent effect on the target pests when it is known that Cry2 toxin is 10 used in combination with CrylA endotoxin to control/prevent CrylA endotoxin target pests.

The genome of the plants, the plant tissues or the plant cells described in the present invention, refers to any genetic material in the plants, the plant tissues, or the plant cells, including the nucleus, plastids and the genome of mitochondrial.

As described in the present invention, polynucleotides and/or nucleotides form a complete “gene”, encoding proteins or polypeptides in the host cells of interest. It is easy for one skilled in the art to realize that polynucleotides and/or nucleotides in the present invention can be introduced under the control of the regulatory sequences of the target host.

As well known by one skilled in the art, DNA exists typically as double strands, which are complementary with each other. When DNA is replicated in plants, other complementary strands of DNA are also generated. Therefore, the polynucleotides exemplified in the sequence listing and complementary strands thereof are comprised in this invention. The “encoding strand” generally used in the art refers to a strand binding with an antisense strand. For protein expression in vivo, one of the DNA strands is typically transcribed into a complementary strand 25 of mRNA, which serves as the template of protein expression. Actually, mRNA is transcribed from the “antisense” strand of DNA. “Sense strand” or “encoding strand” contains a series of codons (codon is a triplet of nucleotides that codes for a specific amino acid), which might be read as open reading frames (ORF) corresponding to genes that encode target proteins or peptides. RNA which is functionally equivalent with the exemplified DNA was also 30 contemplated in this invention.

Nucleic acid molecule or fragments thereof were hybridized with the present application Cry2Ab pesticidal gene under stringency condition in this invention. Any regular methods of nucleic acid hybridization or amplification can be used to identify the existence of the Cry2Ab gene in

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2016228053 16 Oct 2018 present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing with other nucleic acid molecules under certain conditions. In present invention, if two nucleic acid molecules can form an antiparallel nucleic acid structure with double strands, it can be determined that these two molecules can hybridize with each other specifically. If two 5 nucleic acid molecules are completely complementary, one of two molecules is called as the “complement” of another one. In this invention, when every nucleotide of a nucleic acid molecule is complementary with the corresponding nucleotide of another nucleic acid molecule, it is identified the two molecules are “completely complementary”. If two nucleic acid molecules can hybridize with each other so that they can anneal to and bind to each other with enough 10 stability under at least normal “low-stringency” conditions, these two nucleic acids are identified as “minimum complementary”. Similarly, if two nucleic acid molecules can hybridize with each other so that they can anneal to and bind to each other with enough stability under normal “high-stringency” conditions, it is identified that these two nucleic acids are “complementary”. Deviation from “completely complementary” can be allowed, as long as the deviation does not 15 completely prevent the two molecules to form a double-strand structure. A nucleic acid molecule which can be taken as a primer or a probe must have sufficiently complementary sequences to form a stable double-strand structure in the specific solvent at a specific salt concentration.

In this invention, basically homologous sequence refers to a nucleic acid molecule, which can specifically hybridize with the complementary strand of another matched nucleic acid molecule 20 under “high-stringency” condition. The stringency conditions for DNA hybridization are well-known to one skilled in the art, such as treatment with 6.0*sodium chloride/sodium citrate (SSC) solution at about 45 °C and washing with 2.0*SSC at 50 °C. For example, the salt concentration in the washing step is selected from 2.0*SSC and 50 °C for the “low-stringency” conditions and 0.2*SSC and 50 °C for the “high-stringency” conditions. In addition, the 25 temperature in the washing step ranges from 22 °C for the “low-stringency” conditions to 65 °C for the “high-stringency” conditions. Both temperature and the salt concentration can vary together or only one of these two variables varies. Preferably, the stringency condition used in this invention might be as below. SEQ ID NO:2 is specifically hybridized in 6.0*SSC and 0.5% SDS solution at 65 °C. Then the membrane was washed one time in 2*SSC and 0.1% SDS 30 solution and 1*SSC and 0.1% SDS solution, respectively.

Therefore, the insect-resistant sequences which can hybridize with SEQ ID NO:2 under stringency conditions were comprised in this invention. These sequences were at least about 40%-50% homologous or about 60%, 65% or 70% homologous, even at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homologous to the sequences io

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2016228053 16 Oct 2018 of present invention.

Genes and proteins described in the present invention not only include the specifically exemplified sequences, but also include parts and/or fragments (including deletion(s) in and/or at the end of the full-length protein), variants, mutants, substitutes (proteins containing substituted 5 amino acid(s)), chimeras and fusion proteins retaining the pesticidal activity thereof. The “variants” or “variation” refers to the nucleotide sequences encoding the same one protein or encoding an equivalent protein having pesticidal activity. The “equivalent protein” refers to the claimed proteins or that have the same or the substantially same bioactivity of anh-Spodoptera litura.

The “fragment” or “truncation” of the DNA or protein sequences as described in this invention refers to a part or an artificially modified form thereof (e.g., sequences suitable for plant expression) of the original DNA or protein sequences (nucleotides or amino acids) involved in present invention. The sequence length of said sequence is variable, but it is long enough to ensure that the (encoded) protein is an insect toxin.

It is easy to modify genes and to construct genetic mutants by using standard techniques, such as the well-known point mutation technique. Another example method is that described in the U. S. patent 5605793 of randomly splitting DNA and then reassembling them to create other diverse molecules. Commercially available endonucleases can be used to make gene fragments of full-length gene, and exonuclease can also be operated following the standard procedures. For 20 example, enzymes such as Βαΐ31 or site-directed mutagenesis can be used to remove nucleotides systematically from the ends of these genes. Various restriction enzymes can also be applied to obtain genes encoding active fragments. In addition, active fragments of these toxins can be obtained directly using the proteases.

In the present invention, the equivalent proteins and/or genes encoding these proteins could be derived from B.t. isolates and/or DNA libraries. There are many ways to obtain the pesticidal proteins of the invention. For example, the antibodies raised specifically against the pesticidal protein disclosed and protected in present invention can be used to identify and isolate other proteins from protein mixtures. In particular, the antibody may be raised against the most constant part of the protein and the most different part from other B. t. proteins. These antibodies then can be used to specifically identify equivalent proteins with the characteristic activity using methods of immunoprecipitation, enzyme linked immunosorbent assay (ELISA) or Western blotting assay. It is easy to prepare the antibodies against the proteins, equivalent proteins or the protein fragments disclosed in the present invention using standard procedures in this art. The

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2016228053 16 Oct 2018 genes encoding these proteins then can be obtained from microorganisms.

Due to redundancy of the genetic codons, a variety of different DNA sequences can encode one same amino acid sequence. It is available for one skilled in the art to achieve substitutive DNA sequences encoding one same or substantially same protein. These different DNA sequences are 5 comprised in this invention. The “substantially same protein refers to a sequence in which certain amino acids are substituted, deleted, added or inserted but pesticidal activity thereof is not substantially affected, and also includes the fragments remaining the pesticidal activity.

Substitution, deletion or addition of some amino acids in amino acid sequences in this invention is conventional technique in the art. Preferably, such an amino acid change includes: minor 10 characteristics change, i.e. substitution of reserved amino acids which does not significantly influence the folding and/or activity of the protein; short deletion, usually a deletion of about 1-30 amino acids; short elongation of amino or carboxyl terminal, such as a methionine residue elongation at amino terminal; short connecting peptide, such as about 20-25 residues in length.

The examples of conservative substitution are the substitutions happening in the following amino acids groups: basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (e.g., glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (e.g., phenylalanine, tryptophan and tyrosine), and small molecular amino acids (such as glycine, alanine, serine and threonine and methionine). Amino acid substitutions generally not changing 20 specific activity are well known in the art and have been already described in, for example, “Protein” edited by N. Neurath and R. L. Hill, published by Academic Press, New York in 1979. The most common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/ Glu and Asp/Gly, and reverse substitutions thereof.

Obviously, for one skilled in the art, such a substitution may happen outside of the regions which are important to the molecular function and still cause the production of active polypeptides. For the polypeptide of the present invention, the amino acid residues which are required for their activity and chosen as the unsubstituted residues can be identified according to the known methods of the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.

Cunningham and Wells, 1989, Science 244:1081-1085). The latter technique is carried out by introducing mutations in every positively charged residue in the molecule and detecting the insect-resistant activity of the obtained mutation molecules so as to identify the amino acid residues which are important to the activity of the molecules. Enzyme-substrates interaction sites

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2016228053 16 Oct 2018 can also be determined by analyzing its three-dimensional structure, which can be determined through some techniques such as nuclear magnetic resonance (NMR) analysis, crystallography, or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255:306-312, ; Smith, et al., 1992, J. Mol. Biol 224:899-904; Wlodaver et al„ 1992, FEBS Letters 309:59-64).

In the invention, Cry2Ab protein includes but is not limited to SEQ ID NO:1, amino acid sequences which have certain homology with the amino acid sequences set forth in SEQ ID NO: 1 are also comprised in this invention. The sequence similarity/homology between these sequences and the sequences described in the present invention are typically more than 78%, preferably more than 85%, more preferably more than 90%, even more preferably more than 10 95% and more preferably more than 99%. The preferred polynucleotides and proteins in the present invention can also be defined according to more specific ranges of the homology and/or similarity. For example, they have a homology and/or similarity of 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the sequences described in this invention.

In this invention, transgenic plants that produce the Cry2Ab protein include but are not limited to the Mon89034 transgenic maize event and/or plant material comprising the Mon89034 transgenic maize event (as described in CN101495635A), MON87751 transgenic soybean event and/or plant material containing the MON87751 transgenic soybean event (as described in USDA APHIS Non-Regulated State Application 13-337-01p), or Monl5985 transgenic cotton 20 event and/or plant materials containing Monl5985 transgenic cotton event (as described in CN 101413028B), which can achieve the method and/or the use of the present invention, i.e. by contacting the Cry2Ab protein with at least the pest Spodoptera litura to achieve the method and/or the use of controlling pest Spodoptera litura. It is understood by those skilled in the art that the method and/or the use of the present invention can also be achieved by expressing the 25 Cry2Ab protein in the above-described transgenic event in different plants. More specifically, the Cry2Ab protein at least exists in a transgenic plant producing the Cry2Ab protein, the pest Spodoptera litura at least contacted with the Cry2Ab protein by ingestion of the tissue of the transgenic plant, after the contact step, pest Spodoptera litura is inhibited and/or leads to death in order to control the pest Spodoptera litura from damaging the plants.

Regulatory sequences described in this invention include but are not limited to a promoter, transit peptide, terminator, enhancer, leading sequence, introns and other regulatory sequences that can be operably linked to the pesticidal gene.

The promoter is a promoter expressible in plants, wherein said “a promoter expressible in plants”

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2016228053 16 Oct 2018 refers to a promoter which ensures that the encoding sequences bound with the promoter can be expressed in plant cells. The promoter expressible in plants can be a constitutive promoter. The examples of promoters capable of directing the constitutive expression in plants include but are not limited to 35S promoter derived from Cauliflower mosaic virus, Ubi promoter, promoter of 5 GOS2 gene derived from rice and the like. Alternatively, the promoter expressible in plants can be a tissue-specific promoter, which means that the expression level directed by this promoter in some plant tissues such as in chlorenchyma, is higher than that in other tissues of the plant (can be measured through the conventional RNA test), such as the PEP carboxylase promoter. Alternatively, the promoter expressible in plants can be wound-inducible promoters as well.

Wound-inducible promoters or promoters that direct wound-inducible expression manners refer to the promoters by which the expression level of the encoding sequences can be increased remarkably compared with those under the normal growth conditions when the plants are subjected to mechanical wound or wound caused by the gnaw of an insect. The examples of wound-inducible promoters include but are not limited to the promoters of genes of protease inhibitor of potato and tomato (pin I and pin II) and the promoter of maize proteinase inhibitor gene (MPI).

The transit peptide (also called secretary signal sequence or leader sequence) directs the gene products into specific organelles or cellular compartment. For the receptor protein, the transit peptide can be heterogeneous. For example, sequences encoding chloroplast transit peptide are 20 used to lead to chloroplast; or ‘KDEL’ reserved sequence is used to lead to the endoplasmic reticulum or CTPP of the barley lectin gene is used to lead to the vacuole.

The leader sequences include but are not limited to small RNA virus leader sequences, such as EMCV leader sequence (encephalomyocarditis virus 5’ non encoding region); Potato virus Y leader sequences, such as MDMV (maize dwarf Mosaic virus) leader sequence; human 25 immunoglobulin heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of Alfalfa Mosaic virus (AMV RNA4); Tobacco Mosaic virus (TMV) leader sequence.

The enhancer includes but is not limited to Cauliflower Mosaic virus (CaMV) enhancer, Figwort mosaic virus (FMV) enhancer, carnations etched ring virus (CERV) enhancer, cassava vein 30 Mosaic virus (CsVMV) enhancer, mirabilis mosaic virus (MMV) enhancer, Cestrum yellow leaf curling virus (CmYLCV) enhancer, Cotton leaf curl Multan virus (CLCuMV), Commelina yellow mottle virus (CoYMV) and peanut chlorotic streak caulimovirus (PCLSV) enhancer.

For the application of monocotyledon, the introns include but are limited to maize hsp70 introns,

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2016228053 16 Oct 2018 maize ubiquitin introns, Adh intron 1, sucrose synthase introns or rice Actl introns. For the application of dicotyledonous plants, the introns include but are not limited to CAT-1 introns, pKANNIBAL introns, PIV2 introns and “super ubiquitin” introns.

The terminators can be the proper polyadenylation signal sequences playing a role in plants.

They include but are not limited to polyadenylation signal sequence derived from Agrobacterium tumefaciens nopaline synthetase (NOS) gene, polyadenylation signal sequence derived from protease inhibitor II (pin II) gene, polyadenylation signal sequence derived from peas ssRUBISCO E9 gene and polyadenylation signal sequence derived from α-tubulin gene.

The term “operably linked” described in this invention refers to the linking of nucleic acid 10 sequences, which provides the sequences the required function of the linked sequences. The term “operably linked” described in this invention can be to link a promoter with the sequences of interest, which makes the transcription of these sequences under the control and regulation of the promoter. When the sequence of interest encodes a protein and the expression of this protein is required, the term “operably linked” indicates that the linking of the promoter and said sequence 15 makes the obtained transcript to be effectively translated. If the linking of the promoter and the encoding sequence results in transcription fusion and the expression of the encoding protein are required, such a linking is generated to make sure that the first translation initiation codon of the obtained transcript is the initiation codon of the encoding sequence. Alternatively, if the linking of the promoter and the encoding sequence results in translation fusion and the expression of the 20 encoding protein is required, such a linking is generated to make sure that the first translation initiation codon of the 5’ untranslated sequence is linked with the promoter, and such a linking way makes the relationship between the obtained translation products and the open reading frame encoding the protein of interest meet the reading frame. Nucleic acid sequences that can be operably linked include but are not limited to sequences providing the function of gene 25 expression (i.e. gene expression elements , such as a promoter, 5’untranslated region, introns, protein-encoding region, 3’ untranslated region, polyadenylation sites and/or transcription terminators); sequences providing the function of DNA transfer and/or integration (i.e., T-DNA boundary sequences, recognition sites of site-specific recombinant enzyme, integrase recognition sites); sequences providing selectable function (i.e., antibiotic resistance markers, biosynthetic 30 genes); sequences providing the function of scoring markers; sequences assistant with the operation of sequences in vitro or in vivo (polylinker sequences, site-specific recombinant sequences) and sequences providing replication function (i.e. origins of replication of bacteria, autonomously replicating sequences, centromeric sequences).

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The term “pesticidal” or “insect resistance” described in this invention means it is poisonous to crop pests, in order to “control” and/or “prophylaxis” crop pests. Preferably, said “pesticidal” or “insect resistance” means killing crop pests. More specifically, the target insect is pest Spodoptera litura.

Cry2Ab protein of this invention is poisonous to most pests of Spodoptera litura. The plants mentioned in the invention, especially the cron and soybean, contain exogenous DNA in their genome. The exogenous DNA contains Cry2Ab gene sequenc, pest Spodoptera litura is inhibited and/or leaded to death when it contacts with the protein through intaking plant tissues. Inhibition refers to lethal or sub-lethality. Meanwhile, the plant should be normal in 10 morphology and can be cultured under conventional methods in order to use them for consumption and/or generation of products. In addition, the requirement of chemical or biological pesticides of the plant can be essentially eliminated (the chemical or biological pesticides are the ones against pest Spodoptera litura targeted by the protein encoded by Cry2Ab gene).

The expression level of pesticidal crystal proteins (ICP) in the plant materials can be determined using various methods described in this field, such as the method of quantifying mRNA encoding the pesticidal protein in the tissue through using specific primers, or the method of quantifying the pesticidal protein directly and specifically.

The pesticidal effect of ICP in the plants can be detected by using different tests. The target 20 insects of the present invention are mainly pests of Spodoptera litura.

In the present application, the Cry2Ab protein may have the amino acid sequence shown by SEQ ID NO:1 in the sequence listing. In addition to including the encoding region of Cry2Ab protein, other elements may also be included, such as: the elements encoding the selective labeled protein.

Further, the expression cassette containing the nucleotide sequence encoding Cry2Ab protein of the present application may also be expressed together with at least one gene encoding herbicide resistance protein. The herbicide resistance gene includes without limitation: phosphinothricin resistance gene (such as: bar gene and pat gene), phenmedipham resistance gene (such as: pmph gene), glyphosate resistance gene (such as: EPSPS gene), bromoxynil resistance gene, 30 sulfonylurea resistance gene, herbicide dalapon resistance gene, cyanamide resistance gene or glutamine synthetase inhibitor (such as: PPT) resistance gene, thereby obtaining transgenic plant with both high insecticidal activity and herbicide resistance.

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In the present application, exogenous DNA is introduced into plant. For example, the gene or expression cassette or recombinant vector encoding the Cry2Ab protein is introduced into plant cells. The conventional transformation methods include without limitation: agrobacterium tumefaciens-mediated transformation, trace emission bombardment, direct ingestion DNA into 5 protoplast, electroporation or silica whisker mediated DNA introduction.

The present invention provides a pesticidal protein and use thereof with the following advantages:

1. Control through internal cause. The prior art controls the harm of pest Spodoptera litura mainly through external action, i.e.: external cause, for example, agricultural control, chemical control and physical control; While the present application controls pest

Spodoptera litura through generating Cry2Ab protein which can kill Spodoptera litura inside plant, i.e.: controls pest Spodoptera litura through internal cause.

2. No pollution and no residue. The chemical control method used in prior art plays certain role in controlling the harm of pest Spodoptera litura, but in the same time, it also causes pollution, destruction and residue to human, livestock and farmland ecosystem. Using the method for controlling pest Spodoptera litura provided in the present application may eliminate the foregoing bad consequences.

3. Control throughout all growth period. All the methods for controlling pest Spodoptera litura used in prior art are staged, while the present application protects the plant throughout all growth period so that transgenic plant (Cry2Ab protein) can be free from the encroachment of Spodoptera litura from sprouting, growth and till blooming and fruit.

4. Control over whole plant. The method for controlling pest Spodoptera litura used in the prior art mostly are localised, for example foliage spray; while the present application protects whole plant, for example, the leaves, stalks, fruits, tassels, pistils, buds, anthers or filaments and so on of transgenic plant (Cry2Ab protein) all may resist the harm of

Spodoptera litura.

5. Effect stability. Both agricultural control method and physical control method used in the prior art need to utilize environmental conditions to control pests and have many variable factors. The present application makes the Cry2Ab protein be expressed inside the plant and effectively avoids the defect of unstable environmental conditions. Further, the control effect of transgenic plant (Cry2Ab protein) provided by the present application is stable and consistent in different places, different time and different genetic backgrounds.

6. Simple, convenient and economical. The physical control method in the prior art have

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2016228053 16 Oct 2018 certain difficulties on agricultural production and operation, while the present application only needs to plant the transgenic plant which can express Cry2Ab protein and does not need to take other measures, thereby saving a large amount of human, material and financial resources.

7. Thorough effect. The method for controlling pest Spodoptera litura used in the prior art does not have a thorough effect and only plays a role of mitigation, while transgenic plant (Cry2Ab protein) provided by the present application may cause mass death of newly hatched larvae of Spodoptera litura and significantly inhibit the growth of the small portion survived larvae. 3 days later, larvae are basically in a newly hatching state or a state 10 between new hatching and negative control, all suffer maldevelopment and have stopped development. The transgenic plant largely suffers mild damage.

The technical solutions of this invention will be further described through the appended figures and examples as following.

Brief description of the drawings

Figure 1 shows the scheme to construct the recombinant cloning vector DBN01-T containing Cry2Ab nucleotide sequence for uses of insecticidal protein in this invention;

Figure 2 shows the scheme to construct the recombinant expression vector DBN100033 containing Cry2Ab nucleotide sequence for uses of insecticidal protein in this invention;

Figure 3 shows the leaf damage of transgenic com plants for uses of insecticidal protein against Spodoptera litura in this invention;

Figure 4 shows the leaf damage of transgenic soybean plants for uses of insecticidal protein against Spodoptera litura in this invention.

Detailed description of the invention

The technical solutions of this invention for uses of insecticidal protein will be further illustrated through the following examples.

Example 1: The obtaining and synthesis of gene

1. Obtaining of nucleotide sequence

Amino acid sequence of Cry2Ab pesticidal protein (634 amino acids) was shown as SEQ ID

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NO:1 in the sequence listing; Nucleotide sequence of Cry2Ab gene (1905 nucleotides) encoding the corresponding amino acid sequence of Cry2Ab pesticidal protein was shown as SEQ ID NO:2 in the sequence listing.

Amino acid sequence of CrylFa pesticidal protein (605 amino acids) was shown as SEQ ID

NO:3 in the sequence listing; the nucleotide sequence of CrylFa gene (1818 nucleotides) encoding the corresponding amino acid sequence of CrylFa pesticidal protein was shown as SEQ ID NO:4 in the sequence listing.

2. Synthesis of the nucleotide sequence as described above

The Cry2Ab nucleotide sequence (shown as SEQ ID NO:2 in the sequence listing) and the

CrylFa nucleotide sequence (shown as SEQ ID NO:4 in the sequence listing) were synthesized by GenScript CO., LTD, Nanjing, P.R. China. The synthesized Cry2Ab nucleotide sequence (SEQ ID NO:2) was linked with a Ncol restriction site at the 5’ end and a Spel restriction site at the 3’ end. The synthesized CrylFa nucleotide sequence (SEQ ID NO:4) was linked with an Asci restriction site at the 5’ end and a BamHI restriction site at the 3’ end.

Example 2: Construction of recombinant expression vectors and the transfection of Agrobacterium with the recombinant expression vectors

1. Construction of the recombinant cloning vectors containing Cry2Ab gene

The synthesized Cry2Ab nucleotide sequence was sub-cloned into cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), to get cloning vector DBN01-T following the 20 instructions of Promega pGEM-T vector, and the construction process was shown in Figure 1 (wherein the Amp is ampicillin resistance gene; fl is the replication origin of phage fl; LacZ is initiation codon of LacZ; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; Cry2Ab is Cry2Ab nucleotide sequence (SEQ ID NO:2); MCS is multiple cloning sites).

The recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cell (Transgen, Beijing, China, the CAT: CD501) through heat shock method. The heat shock conditions were as follows: 50μ1 of E. coli T1 competent cell and 10μ1 of plasmid DNA (recombinant cloning vector DBN01-T) were incubated in water bath at 42 °C for 30 seconds. Then the E. coli cells were incubated in water bath at 37 °C for 1 h (100 rpm in a shaking incubator) and then were grown on a LB plate (lOg/L Tryptone, 5g/L yeast extract, lOg/L NaCl, 15g/L Agar and pH was adjusted to 7.5 with NaOH) coated on the surface with IPTG (Isopropyl thio-beta-D-galactoseglucoside), X-gal (5-bromine-4-chlorine-3-indole-beta-D-galactose

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2016228053 16 Oct 2018 glucoside) and ampicillin (lOOmg/L) overnight. The white colonies were picked out and cultivated in LB broth (lOg/L Tryptone, 5g/L yeast extract, lOg/L NaCl, 100 mg/L ampicillin and pH was adjusted to 7.5 with NaOH) at 37 °C overnight. The plasmids thereof were extracted using alkaline lysis method as follows: the cell broth was centrifuged for 1 min at 12000 rpm, the 5 supernatant was discarded and the pellet was resuspended in 100 μΐ of ice-chilled solution I (25 mM Tris- HC1, 10 mM EDTA (ethylenediaminetetraacetic acid) and 50 mM glucose, pH 8.0); then 200μ1 of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)) was added and the tube was reversed 4 times, mixed and then put on ice for 3-5 min; 150μ1 of cold solution III (3 M potassium acetate and 5 M acetic acid) was added, thoroughly mixed 10 immediately and incubated on ice for 5-10 min; the mixture was centrifuged at 12000 rpm at 4 °C for 5 min, two volumes of anhydrous ethanol were added into the supernatant, mixed and then placed at room temperature for 5 min; the mixture was centrifuged at 12000 rpm at 4 °C for 5 min, the supernatant was discarded and the pellet was dried after washed with 70% ethanol (V/V); 30 μΐ TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing RNase (20pg /ml) was 15 added to dissolve the precipitate; the mixture was incubated at 37 °C in a water bath for 30 min to digest RNA and stored at - 20 °C for the future use.

After the extracted plasmids were confirmed with restriction enzymes EcoRI and Xhol, the positive clones were verified through sequencing. The results showed that the Cry2Ab nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the sequence set forth in 20 SEQ ID NO:2 in the sequence listing, indicating that Cry2Ab nucleotide sequence was correctly inserted.

The synthesized nucleotide sequence Cry 1 Fa was inserted into cloning vector pGEM-T to get recombinant cloning vector DBN02-T following the process for constructing cloning vector DBN01-T as described above, wherein CrylFa was CrylFa nucleotide sequence (SEQ ID NO:4).

The CrylFa nucleotide sequence in the recombinant cloning vector DBN02-T was verified to be correctly inserted with restriction enzyme digestion and sequencing.

2. Construction of the recombinant expression vectors containing Cry2Ab gene

The recombinant cloning vector DBN01-T and expression vector DBNBC-01 (Vector backbone: pCAMBIA2301, available from CAMBIA institution) were digested with restriction enzymes 30 Ncol and Spel. The cleaved Cry2Ab nucleotide sequence fragment was ligated between the restriction sites Ncol and Spel of the expression vector DBNBC-01 to construct the recombinant expression vector DBN100033. It is a well-known conventional method to construct expression vector through restriction enzyme digestion. The construction scheme was shown in Figure 2

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2016228053 16 Oct 2018 (Kan: kanamycin gene; RB: right border; CaMV35S: cauliflower mosaic virus 35S gene promoter (SEQ ID NO:5); Cry2Ab: Cry2Ab nucleotide sequence (SEQ ID NO:2); Nos, terminator of nopaline synthetase gene (SEQ ID NO:6); Hpt: hygromycin phosphotransferase gene (SEQ ID NO:7); LB: left border).

The recombinant expression vector DBN100033 was transformed into E. coli T1 competent cells with heat shock method as follows: 50μ1 of E. coli T1 competent cell and 10μ1 of plasmid DNA (recombinant expression vector DBN 100033) were incubated in water bath at 42 °C for 30 seconds. Then the E. coli cells were incubated in water bath at 37 °C for 1 h (100 rpm in a shaking incubator) and then were grown on a LB solid plate (10 g/L Tryptone, 5 g/L yeast 10 extract, 10 g/L NaCl, 15 g/L Agar and pH was adjusted to 7.5 with NaOH) containing 50 mg/L kanamycin at 37 °C for 12 hrs. The white colonies were picked out and cultivated in LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 mg/L kanamycin and pH was adjusted to 7.5 with NaOH) at 37 °C overnight. The plasmids thereof were extracted using alkaline lysis method. After the extracted plasmids were confirmed with restriction enzymes Ncol and Spel, 15 the positive clones were verified through sequencing. The results showed that the nucleotide sequence between restriction sites Ncol and Spel in the recombinant expression vector DBN100033 was the nucleotide sequence set forth in SEQ ID NO:2 in the sequence listing, i.e. Cry2Ab nucleotide sequence.

Eollowing the process for constructing recombinant expression vector DBN100033 as described 20 above, recombinant cloning vectors DBN01-T and DBN02-T were digested with restriction enzymes Ncol/Spel and AscI/BamHI respectively to cleave the Cry2Ab nucleotide sequence and CrylEa nucleotide sequence which then were inserted into the expression vector DBNBC-01 to get the recombinant expression vector DBN 100076. Restriction enzyme digestion and sequencing verified that recombinant expression vector DBN100076 contained the nucleotide 25 sequences set forth in SEQ ID NO:2 and SEQ ID NO:4 in the sequence listing, i.e. the nucleotide sequences of Cry2Ab and CrylEa, the nucleotide sequences of Cry2Ab and CrylEa may linked with CaMV35S promoter and Nos terminator.

3. Transfection of Agrobacterium tumefaciens with the recombinant expression vectors

The correctly constructed recombinant expression vectors DBN100033 and DBN100076 were 30 transfected into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, Cat. No: 18313-015) following liquid nitrogen rapid-freezing method as follows: 100 pL Agrobacterium LBA4404 and 3 pL plasmid DNA (recombinant expression vector) were put into liquid nitrogen for 10 min and then incubated in water bath at 37 °C for 10 min. Then the transfected Agrobacterium

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LBA4404 cells were inoculated in LB broth and cultivated at 28 °C, 200 rpm for 2 hours and spraid on a LB plate containing 50 mg/L of rifampicin (Rifampicin) and 100 mg/L of kanamycin (Kanamycin) until positive mono colonies appeared. The positive mono colonies were picked up and cultivated and the plasmids thereof were extracted. Recombinant expression vectors 5 DBN100033 and DBN100076 were verified with restriction enzymes AhdI and Xhol. The results showed that the recombinant expression vectors DBN100033 and DBN100076 were correct in structure, respectively.

Example 3: Obtaining of the transgenic plant

1. Obtaining of the transgenic corn plant

According to the conventional Agrobacterium transfection method, the corn cultivar Zong 31 (Z31) was cultivated in sterilized conditions and the young embryo was co-cultivated with the Agrobacterium strains constructed in part 3 of Example 2 so as to introduce T-DNAs in the recombinant expression vectors DBN100033 and DBN100076 constructed in part 2 of Example 2 (including CaMV35S gene promoter sequence, Cry2Ab nucleotide sequence, CrylFa nucleotide sequence, Hpt gene and Nos terminator sequence) into the com genome. Com plants containing Cry2Ab nucleotide sequence, corn plants containing Cry2Ab-CrylFa were obtained respectively and wild type com plant was taken as a control.

As to the Agrobacterium-mcdvauA transfection of maize, in brief, immature maize young embryo was isolated from coms and contacted with Agrobacterium suspension, in which the 20 Agrobacterium can deliver the Cry2Ab gene and/or Cry2Ab-CrylFa gene into at least one cell of one young embryo. (Step 1: infection step). In this step, preferably, young embryo was immersed in Agrobacterium suspension (Οϋβόο = 0.4-0.6, infection medium (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 68.5 g/L of sucrose, 36 g/L of glucose, 40 mg/L of Acetosyringone (AS), 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), pH=5.3)) to initiate the 25 inoculation. Young embryo and Agrobacterium vjqw cocultivated for a period (3 days) (Step 2:

cocultivation step). Preferably, the Young embryo was cultivated on a solid medium (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 20 g/L of sucrose, 10 g/L of glucose, 100 mg/L of Acetosyringone (AS), 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 8 g/L of Agar, pH=5.8) after the infection step. After this cocultivation step, a selective “recovery” step can be 30 preceded. In the “recovery” step, the recovery medium (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 8 g/L of Agar, pH=5.8) contains at least one kind of known Agro6acterzum-inhibiting antibiotics (cephamycin) without the selective agent for plant transfectants (Step 3: recovery step).

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Preferably, the young embryo was cultivated on a solid medium culture containing antibiotics but without selective agent so as to eliminate Agrobacterium and to provide a recovery period for the infected cells. Then, the inoculated young embryo was cultivated on a medium containing selective agent (Hygromycin) and the transfected callus was selected (Step 4: selection step).

Preferably, the young embryo was cultivated on a selective solid medium containing selective agent (4.3 g/L of MS salt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 50mg/L of hygromycin, 1 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3 g/L of Agar, pH=5.8), resulting in the selective growth of the transfected cells. Then, callus regenerated into plants (Step 5: regeneration step). Preferably, the callus was cultivated on a solid medium containing 10 selective agent (MS differentiation medium and MS rooting medium) to regenerate into plants.

The obtained resistant callus was transferred to the MS differentiation medium (4.3 g/L MS salt, MS vitamins, 300 mg/L of casein, 30 g/L of sucrose, 2 mg/L of 6-benzyladenine, 50mg/L of hygromycin and 3 g/L of Agar, pH=5.8) and cultivated and differentiated at 25 °C. The differentiated seedlings were transferred to the MS rooting medium (2.15 g/Lof MS salt, MS 15 vitamins, 300 mg/L of casein, 30 g/L of sucrose, 1 mg/L indole-3-acetic acid and 3 g/L of agar, pH=5.8) and cultivated to about 10 cm in height at 25 °C. Next, the seedlings were transferred to and cultivated in the greenhouse until fructification. In the greenhouse, the corn plants were cultivated at 28 °C for 16 hours and at 20 °C for 8 hours every day.

2. Obtaining of the transformed soybean plant

According to the conventional Agrobacterium transfection method, the soybean cultivar ZhongHuang 13 was cultivated in sterilized conditions and the young embryo was co-cultivated with the Agrobacterium strains constructed in part 3 of example 2 so as to introduce T-DNAs into the recombinant expression vectors DBN100033 and DBN100076 constructed in part 2 of Example 2 (including CaMV35S gene promoter sequence, Cry2Ab nucleotide sequence, CrylFa 25 nucleotide sequence, Hpt gene and Nos terminator sequence) into the soybean genome. Soybean plants containing Cry2Ab nucleotide sequence, soybean plants containing Cry2Ab-CrylFa were obtained respectively and wild type soybean plant was taken as a control.

As for agrobacterium mediated soybean transformation, simply speaking, germinate mature soybean seeds on a soybean germination medium (B5 salt 3.1g/L, B5 vitamin, sucrose 20g/L, 30 agar 8 g/L, pH5.6), inoculate the seeds on the germination medium and culture them under the following conditions: 25±1°C temperature; photoperiod (light/dark) 16/8h. After 4-6 days’ germination, selecte swollen sterilized soybean seedlings at bright green cotyledonary nodes and cut off the hypocotyledonary axis from the location 3-4mm below the cotyledonary node, the

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2016228053 16 Oct 2018 cotyledon is cut open longitudinally and apical bud, lateral bud and seed root are removed. The back of a scalpel is used to wound the cotyledonary node. Allow Agrobacteria suspension to contact with the wounded cotyledonary node tissues. The agrobacteria has the ability to transfer Cry2Ab nucleotide sequence and/or Cry2Ab-CrylFa nucleotide sequence to the wounded 5 cotyledonary node tissues (step 1: infection step). In this step, cotyledonary node tissues are preferably infected into agrobacteria suspension (OD66o=0.5-0.8, infection medium (MS salt 2.15g/L, B5 vitamin, sucrose 20g/L, glucose lOg/L, acetosyringone (AS) 40mg/L, 2-morpholineethanesulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, pH5.3) to initiate inoculation. Cotyledonary node tissues and agrobacteria are co-cultured for a period of time (3 10 days) (step 2: co-culture step). Preferably, after this infection step, cotyledonary node tissues are cultured on a solid medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose lOg/L, 2-morpholineethanesulfonic acid (MES) 4g/L, zeatin 2mg/L, agar 8g/L, pH5.6). After this co-culture stage, there may be a selective “recovery” step. In the “recovery” step, the recovery medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 15 30g/L, zeatin (ZT) 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid lOOmg/L, aspartic acid lOOmg/L, pH5.6) contains at least one antibiotic (cephalosporin) known to inhibit the growth of agrobacteria, and no selective agent of plant transformant is added (step 3: recovery step). Preferably, the tissue blocks regenerated by the cotyledonary node are cultured on a solid medium containing antibiotic, but no selective agent, to eliminate agrobacteria and provide a 20 recovery period for the infected cells. Then, the tissue blocks regenerated by the cotyledonary node are cultured on a culture medium containing selective agent (hygromycin) and select the growing transformed calli (step 4: selection step). Preferably, the tissue blocks regenerated by the cotyledonary node are cultured on a screening solid medium containing selective agent (B5 salt 3. lg/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 30g/L, 25 6-benzyladenine (6-BAP) lmg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid lOOmg/L, aspartic acid lOOmg/L, hygromycin 50mg/L, pH5.6), resulting in selective growth of the transformed cells. Then, the transformed cells regenerate into plant (step 5: regeneration step). Preferably, the tissue blocks regenerated by the cotyledonary node grown on a culture medium containing selective agent are cultured on a solid medium (B5 differentiation medium and B5 30 rooting culture ) to regenerate plant.

The resistant tissue blocks obtained from screening are transferred to the B5 differentiation medium (B5 salt 3. lg/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 30g/L, zeatin (ZT) lmg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin lmg/L, auxin lmg/L, hygromycin 50mg/L, pH5.6) and cultured them

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2016228053 16 Oct 2018 to differentiate at 25°C. The seedlings obtained from differentiation are transferred to the B5 rooting culture (B5 salt 3.1g/L, B5 vitamin, 2-morpholineethanesulfonic acid (MES) lg/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indol-3-butyric acid (IBA) lmg/L), and cultured on the rooting medium at 25 °C till a height of about 10cm, then transferred to a 5 greenhouse and cultured till fructification. In the greenhouse, they are cultured at 26°C for 16 h and then at 20°C for 8 h each day.

Example 4. Verification of transgenic com plants using TaqMan technique

100 mg of leaves from com plant transfected with Cry2Ab nucleotide sequence and corn plant transfected with Cry2Ab-CrylFa nucleotide sequence was taken as sample respectively.

Genomic DNA thereof was extracted using DNeasy Plant Maxi Kit (Qiagen) and the copy numbers of Cry2Ab gene and CrylFa gene were quantified through Taqman probe-based fluorescence quantitative PCR assay. Wild type com plant was taken as a control and analyzed according to the processes as described above. Experiments were carried out in triplicate and the results were the mean values.

The specific method for detecting the copy numbers of Cry2Ab gene and CrylFa gene was described as follows.

Step 11: 100 mg of leaves from every transfected corn plant (com plant transfected with nucleotide sequence of Cry2Ab and Cry2Ab-CrylFa, respectively) was taken and grinded into homogenate in a mortar in liquid nitrogen respectively. It was in triplicate for each sample.

Step 12: the genomic DNAs of the samples above were extracted using DNeasy Plant Mini Kit (Qiagen) following the product instruction thereof.

Step 13: the genome DNA concentrations of the above samples were determined using NanoDrop 2000 (Thermo Scientific).

Step 14: the genome DNA concentrations were adjusted to the same range of 80-100 ng/μΐ.

Step 15: the copy numbers of the samples were quantified using Taqman probe-based fluorescence quantitative PCR assay, the quantified sample with known copy number was taken as a standard sample and the wild type maize plant was taken as a control. It was carried out in triplicate for every sample and the results were the mean values. Primers and the probes used in the fluorescence quantitative PCR were shown as below.

The following primers and probe were used to detect Cry2Ab nucleotide sequence:

Primer 1: CTGATACCCTTGCTCGCGTC (as shown in SEQ ID NO:8 in the sequence listing);

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Primer 2: CACTTGGCGGTTGAACTCCTC (as shown in SEQ ID NO:9 in the sequence listing);

Probe 1: CGCTGAGCTGACGGGTCTGCAAG (as shown in SEQ ID NO: 10 in the sequence listing)

The following primers and probe were used to detect CrylFa nucleotide sequence:

Primer 3: CAGTCAGGAACTACAGTTGTAAGAGGG (as shown in SEQ ID NO: 11 in the sequence listing);

Primer 4: ACGCGAATGGTCCTCCACTAG (as shown in SEQ ID NO: 12 in the sequence listing);

Probe 2: CGTCGAAGAATGTCTCCTCCCGTGAAC (as shown in SEQ ID NO: 13 in the sequence listing);

PCR reaction system was as follows:

Jump Start™ Taq ReadyMix™ (Sigma)10μ1

OX primer/probe mixture1 μΐ

Genomic DNA3μ1

Water (ddEEO)6μ1

The 50X primer/probe mixture contained 45 μΐ of each primer (1 mM), 50 μΐ of probe (100μΜ) and 860 μΐ of 1XTE buffer and was stored in an amber tube at 4 °C.

PCR reaction conditions were provided as follows:

20 Step	Temperature	Time
21	95 °C	5 min
22	95 °C	30s
23	60 °C	lmin
24	back to step 22 and repeated 40 times

Data were analyzed using software SDS 2.3 (Applied Biosystems).

The experimental results showed that all the nucleotide sequences of Cry2Ab and

Cry2Ab-CrylFa have been integrated into the genomes of the detected com plants, respectively.

Furthermore, com plants transfected with nucleotide sequences of Cry2Ab and Cry2Ab-CrylFa

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2016228053 16 Oct 2018 respectively contained single copy of Cry2Ab and Cry2Ab-CrylFa gene respectively.

The transgenic soybean plants were tested and analyzed according to the method of testing transgenic corn plants mentioned above.The result shows that all the nucleotide sequences of Cry2Ab and Cry2Ab-CrylFa have been integrated into the genomes of the detected soybean 5 plants, respectively. Furthermore, soybean plants transfected with nucleotide sequences of

Cry2Ab and Cry2Ab-CrylFa respectively contained single copy of Cry2Ab and Cry2Ab-CrylFa gene respectively.

Example 5: Detection of pesticidal protein contents in transgenic plants

The com plants transformed with the Cry2Ab nucleotide sequence, the com plants transformed 10 with the Cry2Ab-CrylFa nucleotide sequence; the soybean plants transformed with the Cry2Ab nucleotide sequence, the soybean plants transformed with the Cry2Ab-CrylFa nucleotide sequence; corresponding wild-type com plants and soybean plants, as well as non-transgenic com plants and soybean plants which were tested by Taqman are being tested for insect-resistance to Spodoptera litura.

1. Detecting the insecticidal effect of transgenic com plants

Fresh leaves are taken from the corn plant transformed with Cry2Ab nucleotide sequence, the corn plant transformed with Cry2Ab-CrylFa nucleotide sequence, wild corn plant and non-transgenic corn plant as identified by Taqman (V3-V4 stage) respectively and washed with sterile water. The water on the leaves is sucked dry by gauze. Meanwhile the leaves 20 are cut into squares of about lcmx4cm. Three cut square leaves are put on the filter paper at the bottom of a round plastic culture disk. The filter paper is moistened with distilled water.

artificially fed Pest Spodoptera litura (newly hatched larvae) are put into each culture dish. The culture dishes with pests are covered and then put into a square box with wet gauze at its bottom and rest for 3 days under the conditions of 25-28°C, RH 70%-80% and 25 photoperiod (light/dark) 16: 8. Based on three indexes: development progress and mortality of Spodoptera litura larvae and leaf damage rate, the total score of resistance is obtained: Total score=100xmortality+[100xmortality+90^x(number of newly hatched larvae/total number of inoculating larvae)+60^x(number of newly hatched larvae - number of pests in negative controls/total number of inoculating larvae)+10x(number of pests in negative 30 controls/total number of inoculating larvae)]+100^x(l-leaf damage rate). There are 3 strains (SI, S2 and S3) transformed with Cry2Ab nucleotide sequence, 3 strains (S4, S5 and S6) transformed with Cry2Ab-CrylFa nucleotide sequence, a strain identified by Taqman to be non-transgenic (NGM1) and a wild strain (CK1); three plant are selected from each strain 27

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2016228053 16 Oct 2018 to do test and each plant is tested six times. The results are shown in Table 1 and FIG. 3.

Table 1 Results of insecticidal experiments of transgenic corn plant inoculated with Spodoptera litura

Plant

Leaf damage rate (%)

Development progress of

Spodoptera litura (single strain)

Newly

Newly hatched hatched .

negative control

Death situation of

Spodoptera litura (single strain) >negat ive control

Total

Mortal number of • 1 · ^ity inoculating , (%) larvae

Total score (single strain)

Average total score

SI	12.5	0.5	2.5	0	10	70	247
S2	15	0.5	2.5	1	10	60	226	242
S3	15	0	1	1	10	80	252
S4	10	0.5	1.5	0	10	80	264
S5	12.5	0.5	1.5	0.5	10	75	252	265
S6	5	1.5	0	0	10	85	279
NGM1	60	0	1	8	10	10	74	74
CK1	80	0	1	7	10	20	73	73

ίο

The results in Table 1 indicate: The corn plant transformed with Cry2Ab nucleotide sequence, the corn plant transformed with Cry2Ab-CrylFa nucleotide sequence both has good insecticidal effect on Spodoptera litura, the death rate of the Spodoptera litura are 60% above, the total scores are around 250 points; The total scores of non-transgenic corn plant identified by Taqman and wild com plant in bioassay are around 70 points in general.

The results in FIG. 3 indicate: compared with wild com plant, the corn plant transformed with Cry2Ab nucleotide sequence, the com plant transformed with Cry2Ab-CrylFa nucleotide sequence can cause massive death of newly hatched Spodoptera litura larvae within three days and significantly inhibit the development progress of the small portion surviving larvae. 3 days later, the larvae are basically in a newly hatched state. Moreover, the com plant transformed with Cry2Ab nucleotide sequence, the com plant transformed with Cry2Ab-CrylFa nucleotide sequence basically suffer mild damage only and the leaf damage rates are all around or below 15%, almost invisible by naked eyes.

Thus it is proved that the corn plant transformed with Cry2Ab nucleotide sequence, the corn plant transformed with Cry2Ab-CrylFa nucleotide sequence both show high activity against

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Spodoptera litura and this activity is enough to generate harmful effect on the growth of

Spodoptera litura, so as to control it on farmland.

2. Detecting the insecticidal effect of transgenic soybean plants

2016228053 16 Oct 2018

Fresh leaves are taken from the soybean plant transformed with Cry2Ab nucleotide sequence, the soybean plant transformed with Cry2Ab-CrylFa nucleotide sequence, wild soybean plant and non-transgenic soybean plant as identified by Taqman (V3-V4 stage) respectively and washed with sterile water. The water on the leaves is sucked dry by gauze. Meanwhile the leaves are cut into squares of about lcmx4cm. A cut square leaf is put on the filter paper at the bottom of a round plastic culture disk. The filter paper is moistened 10 with distilled water. 10 artificially fed pest Spodoptera litura (newly hatched larvae) are put into each culture dish. The culture dishes with pests are covered and then put into a square box with wet gauze at its bottom and rest for 3 days under the conditions of 25-28°C, RH 70%-80% and photoperiod (light/dark) 16:8. Based on three indexes: development progress and mortality of Spodoptera litura larvae and leaf damage rate, the total score of 15 resistance is obtained: Total score=100^xmortality+[100^xmortality+90^x(number of newly hatched larvae/total number of inoculating larvae)+60^x (number of newly hatched larvae number of pests in negative controls/total number of inoculating larvae)+10^x(number of pests in negative controls/total number of inoculating larvae)]+100^x(l-leaf damage rate). There are 3 strains (S7, S8 and S9) transformed with Cry2Ab nucleotide sequence, 3 strains 20 (S10, Sil and S12) transformed with Cry2Ab-CrylFa nucleotide sequence, a strain identified by Taqman to be non-transgenic (NGM2) and a wild strain (CK2); three plant are selected from each strain to do test and each plant is tested six times. The results are shown in Table 2 and FIG. 4.

Table 2 Results of insecticidal experiments of transgenic soybean plant inoculated with

Spodoptera litura

Development progress of Death situation of

Spodoptera litura (single Spodoptera litura (single

Plant

T eaf	strain)
damage		Newly
rate (%)	Newly	hatched -
	hatched	negative control

strain)

Total >negat Mortal number of ity ^lve inoculating , (%) control i_arvae

Total score (single strain)

Average total score

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S7	12.5	0	1	1.5	10	75	245
S8	35	0.5	1.5	2	10	60	201	234
S9	20	0	1	0.5	10	85	257
S10	5	1	0.5	0	10	85	277
Sil	7.5	0	1.5	1	10	75	253	256
S12	15	0	2	1	10	70	238
NGM2	too	0	0	9	10	10	29	29
CK2	too	0	0	10	10	0	10	10

The results in Table 2 indicate: The soybean plant transformed with Cry2Ab nucleotide sequence, the soybean plant transformed with Cry2Ab-CrylFa nucleotide sequence both have good insecticidal effect on Spodoptera litura, the death rate of the Spodoptera litura are 60% above, the total scores are around 250 points; The total scores of non-transgenic soybean plant identified 5 by Taqman and wild soybean plant in bioassay are both below 30 points.

The results in FIG. 4 indicate: compared with wild soybean plant, the soybean plant transformed with Cry2Ab nucleotide sequence, the soybean plant transformed with Cry2Ab-CrylFa nucleotide sequence can cause massive death of newly hatched Spodoptera litura larvae within three days and significantly inhibit the development progress of the small portion surviving 10 larvae. 3 days later, the larvae are basically in a newly hatched state. Moreover, the soybean plant transformed with Cry2Ab nucleotide sequence, the soybean plant transformed with Cry2Ab-CrylFa nucleotide sequence basically suffer mild damage only and the leaf damage rates are all around 20%, especially, the damages on the leaves of soybean plants transformed with Cry2Ab-CrylFa nucleotide sequence are almost invisible by naked eyes.

Thus it is proved that the soybean plant transformed with Cry2Ab nucleotide sequence, the soybean plant transformed with Cry2Ab-CrylFa nucleotide sequence both show high activity against Spodoptera litura and this activity is enough to generate harmful effect on the growth of Spodoptera litura, so as to control it on farmland.

The above experimental results also indicate that the corn plant transformed with Cry2Ab 20 nucleotide sequence, the corn plant transformed with Cry2Ab-CrylFa nucleotide sequence, the soybean plant transformed with Cry2Ab nucleotide sequence, the soybean plant transformed with Cry2Ab-CrylFa nucleotide sequence can control/prophylaxis Spodoptera litura apparently because the plant themselves can generate Cry2Ab protein. Therefore, it is well known to those skilled in the art that based on the same toxic fuction of Cry2Ab protein to Spodoptera litura, 25 transgenic plant that may generate similar expressive Cry2Ab protein can be used to control the harm of Spodoptera litura. The Cry2Ab protein in the present application includes without

10744520_1 (GHMatters) P106611.AU

2016228053 16 Oct 2018 limitation the Cry2Ab protein given amino acid sequence in embodiments. Meanwhile, the transgenic plant may also generate at least a second-type insecticidal protein different from Cry2Ab protein, such as CrylFa protein, Cry 1A.105 protein or Vip3A protein etc.

To summarize, uses of insecticidal protein of the present application controls pest Spodoptera litura through generating Cry2Ab protein inside plant to kill Spodoptera litura; compared with agricultural control method, chemical control method and physical control method used in the prior art, the present application protects the whole plant throughout all growth period to control encroach of pest Spodoptera litura, furthermore, it causes no pollution and no residue and provides a stable and thorough control effect. Also it is simple, convenient and economic.

Finally what should be explained is that all the above examples are merely intentioned to illustrate the technical solutions of present invention rather than to restrict present invention. Although detailed description of this invention has been provided by referring to the preferable examples, one skilled in the art should understand that the technical solutions of the invention can be modified or equivalently substituted while still fall within the spirit and scope of the 15 present invention.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of 20 further features in various embodiments of the invention.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

Claims (18)

1. The claims defining the invention are as follows:

   1. A method for controlling Spodoptera litura, wherein the method includes contacting Spodoptera litura with insecticidal protein which is Cry2Ab protein, or a combination of

   5 Cry2Ab protein and a second protein which is CrylFa protein or Vip3Aa protein, the Cry2Ab protein comprises an amino acid sequence shown in SEQ ID NO:1.
2. 2. A method according to claim 1, wherein the Cry2Ab protein at least exists in a host cell expressing Cry2Ab protein, and Spodoptera litura at least contacts with Cry2Ab protein by ingestion of the host cell.

   10
3. 3. A method according to claim 2, wherein the Cry2Ab protein at least exists in bacteria or transgenic plant expressing Cry2Ab protein, and Spodoptera litura at least contacts with Cry2Ab protein by ingestion of the bacteria or a tissue of the transgenic plant; thereafter the growth of Spodoptera litura is inhibited and/or Spodoptera litura dies, so as to achieve controlling the damage of Spodoptera litura to the plant.

   15
4. 4. A method according to claim 3, wherein the tissues of the transgenic plant are leaves, stems, fruits, tassels, ears, buds, anthers or filaments.
5. 5. A method according to claim 3 or 4, wherein the plant is selected from the group consisting of corn, soybean, cotton, sweet potato, taro, lotus, tianjing, tobacco, sugar beet, cabbage and eggplant.

   20
6. 6. A method according to any one of claims 3 to 5, wherein prior to contacting, the method includes a step of planting a seedling containing a polynucleotide encoding Cry2Ab protein.
7. 7. A method according to claim 6, wherein the polynucleotide encoding Cry2Ab protein comprises a nucleotide sequence shown in SEQ ID NO:2.

   25
8. 8. A method according to any one of claims 3 to 7, wherein the plant also contains at least a second nucleotide sequence encoding CrylFa protein or Vip3Aa protein.
9. 9. A method according to claim 1 or 8, wherein the amino acid sequence of CrylFa protein comprises an amino acid sequence shown in SEQ ID NO:3.
10. 10. A method according to claim 1 or 8, wherein the second nucleotide sequence comprises 30 a nucleotide sequence shown in SEQ ID NO:4.
11. 11. Use of insecticidal protein to control Spodoptera litura, wherein the insecticidal protein is Cry2Ab protein, or a combination of Cry2Ab protein and a second protein which is CrylFa protein or Vip3Aa protein, the Cry2Ab protein comprises an amino acid sequence shown in SEQ ID NO:1.

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12. 12. Use of a plant cell, a plant tissue, a plant or a bacterium which is transformed with a gene encoding insecticidal protein to control Spodoptera litura, the insecticidal protein is Cry2Ab protein, or a combination of Cry2Ab protein and a second protein which is CrylFa protein or Vip3Aa protein, the Cry2Ab protein comprises an amino acid sequence shown in

    5 SEQ ID NO: 1.
13. 13. A method of control of Spodoptera litura, comprising the steps:

    i) introducing a polynucleotide sequence encoding insecticidal protein into the genome of the plant, wherein the insecticidal protein is Cry2Ab protein, or a combination of Cry2Ab protein and a second protein which is CrylFa protein or Vip3Aa protein, the Cry2Ab

    10 protein comprises an amino acid sequence shown in SEQ ID NO:1; and ii) contacting the Spodoptera litura with the plant to achieve control of the Spodoptera litura.
14. 14. A method for producing a plant seed that controls Spodoptera litura, wherein the method includes a step of hybridizing a first plant produced by a method according to claim
15. 15 13 with a second plant, thus produces a seed containing a polynucleotide sequence encoding Cry2Ab protein.

    15. A method for cultivating a plant that controls Spodoptera litura, wherein the method includes the following steps:

    i) planting at least one seed whose genome contains a polynucleotide sequence encoding 20 insecticidal protein which is Cry2Ab protein, or a combination of Cry2Ab protein and a second protein which is CrylFa protein or Vip3Aa protein, the Cry2Ab protein comprises an amino acid sequence shown in SEQ ID NO: 1;

    ii) making the seed grow into a plant;

    iii) making the plant grow under the condition of artificial inoculation and/or natural 25 occurrence of Spodoptera litura and harvesting the plant with weakened plant damage and/or increased plant yield compared to other plant without the polynucleotide sequence encoding Cry2Ab protein.
16. 16. A plant cell, a plant tissue or a plant that controls Spodoptera litura, wherein the plant cell, the plant tissue, or the plant contains a polynucleotide sequence encoding insecticidal

    30 protein which is Cry2Ab protein, or a combination of Cry2Ab protein and a second protein which is CrylFa protein or Vip3Aa protein, the Cry2Ab protein comprises an amino acid sequence shown in SEQ ID NO:1, contacting the Spodoptera litura with the plant cell, the plant tissue or the plant to achieve control of the Spodoptera litura.

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17. 17. A plant cell, a plant tissue or a plant according to claim 16, wherein the nucleotide sequence encoding Cry2Ab protein comprises a nucleotide sequence shown in SEQ ID NO:2.
18. 18. A plant cell, the plant tissue or the plant according to claim 16 or 17, wherein the plant 5 cell, the plant tissue or the plant at least contains a second nucleotide sequence which encodes CrylFa protein or Vip3Aa protein.