JP5190928B2 - Phospholipase D-deficient rice line - Google Patents

Description

  The present invention relates to a phospholipase D-deficient rice line.

  Brown rice prepared by removing rice husks from rice bran, and rice bran obtained by milling brown rice is an important picture that is rich in flavor and nutritional components when viewed from the perspective of functionality and health. However, it contains more lipids that are easier to oxidize than starch or protein. When the lipid is oxidized, the quality and flavor of brown rice and rice bran are reduced.

  In the oxidative degradation of lipids, phospholipase D (PLD) hydrolyzes phosphodylcholine in the spherosome (cell granule) membrane of rice nuka cells to disrupt the spherosome membrane, and neutral lipid accumulated in the spherosome is cytoplasmic. The neutral lipid is broken down into free fatty acids (FFA) by lipase. This FFA causes the quality and flavor of rice to deteriorate. In particular, in the milling process for whitening, sperm and flour, rice bran cells are destroyed, and lipid oxidation proceeds. Therefore, a method for suppressing the oxidation of brown rice and rice bran lipid has been desired.

  For example, several methods have been developed to increase the storability of rice by suppressing lipid oxidation and to stabilize rice bran. (I) A method of storing rice at a low temperature of about 15 ° C. (many storage warehouses (Ii) a method for inactivating a lipolytic enzyme by treating nuka with high-temperature and high-pressure steam (Patent Document 1), (iii) by adding a PLD inhibitor (such as corn) ) Method of preventing aging / degradation (Patent Document 2), (iv) Method of adding water and proteolytic enzyme to Nuka to inactivate lipolytic enzyme, followed by heating / dehydration treatment (Patent Document 3), (v ) A method of measuring the amount of FFA and further finening when the amount of FFA is high (Patent Document 4) can be mentioned.

However, in order to suppress the deterioration of rice, brown rice, or rice bran using these methods and the conventional methods, the following facilities or techniques are required. For example, in order to suppress the deterioration of rice (old rice), a cooling / cooling facility is required, or it is necessary to measure the amount of FFA and refine it. In addition, it is necessary to add an inhibitor of PLD in order to suppress the deterioration of the grain, or inactivate the lipolytic enzyme with heating or a proteolytic enzyme in order to suppress the deterioration of nuka. In order to perform these processes, equipment, time, and cost are required.
Therefore, it is desired to produce rice in which lipid oxidative degradation is suppressed.

  For example, Patent Document 5 discloses that rice in which expression of the PLD gene is suppressed was produced using an antisense vector in order to change the composition of phospholipids produced by cells. In this rice, the expression of the PLD gene is directly suppressed by introducing a gene encoding mRNA that hybridizes in the cell with the mRNA of the PLD gene in the host cell, targeting a known PLD gene. Has been.

Japanese Patent Laid-Open No. 11-9207 US Pat. No. 6,514,914 Japanese National Patent Publication No. 8-506720 Japanese Patent Laid-Open No. 10-4875 Republished WO97 / 31106

  An object of the present invention is to provide a novel rice line from which PLD is deleted and a method for determining the presence or absence of a PLD-deficient gene in a rice plant.

  As a result of intensive studies to solve the above problems, the present inventor treated rice seeds with a mutagen and cultivated the seeds, and found an individual lacking PLD. The present inventors have succeeded in establishing a PLD-deficient line by confirming the inheritance of PLD-deficient lines, identifying the PLD-deficient gene of the PLD-deficient rice line, and completing the present invention.

The features of the present invention are summarized as follows.
(1) A rice line (accession number FERM P-21231), progeny or a hybrid thereof that is PLD-deficient.
(2) A method for producing a PLD-deficient rice line, comprising treating rice seeds with a mutagen, cultivating the seeds into plant bodies, and screening the plant bodies for PLD deletion.
(3) A PLD-deficient rice line comprising crossing the rice line described in (1) with a rice wild-type strain, cultivating the seed into a plant body, and screening the plant body for PLD deletion. Production method.

(4) The cells obtained from the rice line described in (1) and the cells obtained from the rice wild-type strain are cell-fused, the obtained fused cells are cultured and regenerated into plants, To produce a PLD-deficient rice line.
(5) The method according to any one of (2) to (4), wherein the phospholipase D deletion is caused by a nonsense mutation of the phospholipase D gene.
(6) A rice line, a progeny thereof, or a seed of a hybrid thereof, which is PLD-deficient according to (1).

(7) Rice flour derived from the seed according to (6).
(8) Cooked rice derived from the seed according to (6).
(9) The cooked rice according to (8), which is cooked cooked rice.
(10) Rice oil derived from the seed according to (6).
(11) A method for improving the yield of rice oil, comprising obtaining rice bran from the seed according to (6) and squeezing the rice bran.

(12) A phospholipase D deletion gene which is any of the following (a) to (e):
(A) a phospholipase D-deleting gene comprising a DNA comprising a base sequence in which the guanine at the 2779th base is replaced with adenine in the base sequence shown in SEQ ID NO: 4 (b) 2779 in the base sequence shown in SEQ ID NO: 4 A phospholipase D deletion gene comprising a DNA having a base sequence in which the guanine of the 2nd base is substituted with adenine and one or several bases are deleted, substituted or added in the base sequence (c) SEQ ID NO: 4 A phospholipase D-deficient gene comprising a DNA comprising a nucleotide sequence having a guanine at the 2779th base in the nucleotide sequence shown in (2) replaced with adenine and having 95% or more identity to the nucleotide sequence (d) In the base sequence shown in No. 4, guanine at the 2779th base is substituted with adenine, and the salt Phospholipase D deletion gene containing DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the sequence (e) Phospholipase D deficiency containing DNA comprising the base sequence represented by SEQ ID NO: 22 gene

(13) A recombinant vector comprising the gene according to (12).
(14) A method for producing a transgenic plant lacking a phospholipase D deletion, which comprises introducing the gene according to (12) into a plant cell and carrying out homologous recombination to grow the plant.
(15) The method according to (14), wherein DNA is introduced using the recombinant vector according to (13).
(16) The method according to (14) or (15), wherein the plant is a gramineous plant.
(17) A method for determining the presence or absence of a phospholipase D-deficient gene according to (12), which comprises detecting a mutation of the 2779th base from guanine to adenine in the base sequence represented by SEQ ID NO: 4.
(18) The method according to (17), wherein the mutation is detected in the nucleic acid sample by any one of the CAPS method, the dot blot SNP method, the SSCP method, and the dCAPS method.
In the present specification, the rice wild-type strain includes all rice varieties / lines having PLD.

  Since the rice line of the present invention is substantially lacking in PLD activity (referred to herein as “phospholipase D deficiency” or “PLD deficiency”), there is little disruption of the spherosome membrane, and as a result There is little degradation of neutral lipids. Therefore, the seeds of the rice line of the present invention can be stored for a long period of time while maintaining the quality, can suppress the deterioration of the grain without adding a PLD inhibitor or the like, and improve the storage stability of rice. Energy saving and cost reduction can be promoted. In addition, brown rice and rice bran prepared from the seeds are inhibited from lipid oxidation, and the yield can be improved by producing rice oil from the rice bran. Furthermore, it is possible to produce rice flour, brown rice whole grain flour, rice oil and the like that are less prone to lipid oxidation and off-flavor generation.

  Moreover, a PLD deficient transformed plant can be produced by introducing the PLD deficient gene of the present invention into various plants.

  Furthermore, the presence or absence of a PLD deletion gene can be determined based on the nonsense mutation of the gene.

1. Rice system of the present invention and its production method The rice system of the present invention is the rice of the National Institute of Advanced Industrial Science and Technology patent biological deposit center (6th 1-1-1, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture) under the accession number FERM P-21231. A line, a progeny of the rice line by self-pollination, and a hybrid line obtained by crossing or cell fusion with a rice wild-type strain with the rice line as a parent, and also contains a rice line that is PLD-deficient . In the present specification, the rice line of the present invention is sometimes referred to as 03-s108.

  The rice line (03-s108) with the deposit number FERM P-21231 can be generated, for example, by artificial mutation.

  The rice used for mutagen treatment is not particularly limited. Since various varieties and lines of rice have been developed, they can be used.

  Mutagen treatment is performed by treating with various mutagenic chemicals (alkylating agents, nucleobase analogs, sodium azide, etc.) and various radiations (electromagnetic waves, ultraviolet rays, X rays, γ rays, particle rays, neutrons). Can be carried out by irradiating a plant with rays, α rays, β rays, electrons, ion beams, etc.). In the present invention, it is preferable to use sodium azide. For example, rice seeds may be immersed in a 1 mM sodium azide solution (pH 3) for about 3 to 6 hours.

  After treatment with sodium azide, seeds are sown and raised (M1 individual). For example, the M1 generation is cultivated for each strain, the M2 generation (seed) is cultivated, the PLD deletion of the M2 generation seed is confirmed, and the PLD deletion mutant is selected.

  Screening for PLD deletion can be performed, for example, by SDS electrophoresis, Western blotting using an anti-PLD antibody, enzyme activity measurement, or the like. For example, rice seed germ is ground in a protein extraction buffer containing SDS, mercaptoethanol, etc., and bathed in water (treatment at 100 ° C. for 3 minutes) to denature the extracted protein. Then, it centrifuges and uses a supernatant as a sample. Apply sample to polyacrylamide gel and perform electrophoresis. After electrophoresis, the separated protein is transferred to a membrane. An anti-PLD antibody labeled with biotin, horseradish-derived peroxidase, RI, fluorescein or the like is applied to the membrane to detect PLD. Moreover, you may use the method of determining the presence or absence of the below-mentioned PLD deletion gene.

  The M1 generation in which PLD was not detected can be selected, the M2 seeds can be sown and cultivated, and further progeny of PLD deficiency can be created by self-pollination.

  Also, the rice line with the deposit number FERM P-21231 was crossed as a parent with rice wild-type strains such as Koshihikari, Sasanishiki, Hitomebore, Hinohikari, Kirara 397, Nipponbare, Kinuhikari, Mutsuhorare, Kasaras, China No.183. Or cultivated by cell fusion, cultured and regenerated into plants, screened for PLD deficiency, and other traits (good taste, high yield, It is also possible to create a progeny having disease resistance, drought resistance, low temperature resistance, high temperature resistance, chemical resistance, etc.).

  In the crossing, seeing the day before the mating of the rice line of the present invention, the one with the tip of the pod reaching 1/2 or more of the bud is subject to male removal, and other buds are cut off. Cut the tip of the cocoon subject to removal with scissors, remove 6 buds with tweezers, lightly rub the cocoon taken from the rice wild type bud, and pollinate it. Pruning method). Alternatively, before mating, the ear just before flowering of the rice line of the present invention was immersed in warm water kept at 43 ° C. in a constant temperature bath for about 7 minutes, only pollen was sterilized, a mating bag was applied, and no flowering occurred on that day This is done by excising the spikelets and rubbing them lightly on the spiked buds removed from the spikes of wild rice.

  For cell fusion, callus is induced from the rice strain of the present invention and rice wild-type strains, scutellum, etc., protoplasts are prepared and fused by the polyethylene glycol (PEG) method or electroporation method.

  The callus induction medium includes, for example, sucrose as a carbon source in the Murashige Skog (MS) medium or Linsmeier Skog (LS) medium, and, for example, 2 mg / ml 2,4-D or NAA or Add auxin such as IAA and plant hormones such as kinetin.

The cultured cells transplanted to the liquid medium are treated with an enzyme solution containing cellulase, MES, CaCl ₂ , mannitol, etc. to isolate and wash the protoplasts.

  For cell fusion by the PEG method, for example, a 30% PEG 6000 solution is added to the protoplast suspension, mixed, and incubated at 30 ° C. for 10 minutes, for example, and then centrifuged to remove the supernatant. Add an alkaline washing solution containing 1% DMSO, centrifuge to remove the supernatant, and add a protoplast culture medium to wash.

  For cell fusion by electroporation, a protoplast suspension is placed in a cuvette and fused by applying a voltage under electrical conditions of, for example, 1000 μF, 30 msec, and 500 V / cm.

The fused protoplasts can be cultured by, for example, an agarose bead method or a nurse culture method. Specifically, the protoplast solution adjusted in concentration and a protoplast medium containing agarose (including pH 5.8, inorganic salts, MS vitamins, sucrose, 2,4-D, etc.) are mixed until the agarose is solidified. Cool to make an agarose block containing, for example, 1 × 10 ⁶ cells / ml protoplasts. Agarose block, protoplast medium, and nurse cells are placed in a petri dish and cultured.

  After about 10 days of culture, the nurse cells are removed and replanted in a protoplast medium. About 2 weeks later, agarose block is mixed with protoplast medium and preculture medium (pH 5.8, N6 modified medium with MS vitamin, proline, casamino acid, 2,4-D, sucrose, etc.) The plant is replanted with the prepared medium, and after another 2 weeks, it is replanted with the preculture medium.

The obtained protoplast-derived colonies are placed on a regeneration medium (containing inorganic elements, vitamins, iron, sucrose, sorbitol, etc.) and transplanted every 2 weeks. When the buds become about 3 cm, hormone-free medium (2, 4-D additive-free callus induction medium) is transplanted and cultivated, and the young plant is transplanted to the conditioned medium to cultivate the plant body.
Screening for PLD deletion in plants is performed in the same manner as described above.

2. Product Prepared from Seeds of Rice Line of Present Invention From rice seeds of the present invention, for example, rice flour, cooked rice, cooked cooked rice, rice oil and the like can be prepared.

  The rice flour is not particularly limited, and may be obtained by pulverizing brown rice, milled rice, etc. in the form of grains, or by pulverizing after heating.

  The cooked rice may be prepared by cooking brown rice, polished rice, etc. in the usual manner, and also includes barley rice containing wheat and red rice containing azuki bean.

  Cooked cooked rice is cooked and seasoned cooked rice, for example, medicinal rice cooked with ingredients such as seafood, meat, and vegetables, cooked rice cooked with gomoku rice, etc., mixed with cooked rice and ingredients after cooking Rice, pilaf, fried rice, sushi rice, rice cake etc. can be mentioned.

  Rice oil is generally produced by extracting rice bran with hexane and purifying it through processes such as omission, degumming, deoxidation, and decolorization (Oil and oil handbook, supervised by Abe Yoshiro, Koshobo 1988 version).

3. Method for Improving Yield of Rice Oil If rice bran obtained from rice seeds of the present invention is used as a raw material for rice oil, the yield of rice oil can be improved.

  In the production of rice oil, when using conventional rice seeds as a raw material, the fat contained in rice bran is decomposed and oxidized, which is about 15-20% in summer and about 5-5 in winter. A 10% loss occurs. When seeds of the PLD-deficient rice line of the present invention are used as raw materials, lipid degradation and oxidation can be suppressed, so that loss is reduced.

4). PLD deficient gene The PLD deficient gene of the present invention is a gene in which a nonsense mutation has been introduced into the phospholipase D1 (PLD1) gene. In addition, the PLD1 gene can clarify the chromosome which sits from the rice genome information published by International Rice Genome Sequencing Project (Nature436: 793-800,2005). Located at 0 cM. More specifically, it is between the markers RG472 and RG246 (see FIG. 6).

The PLD deletion gene of the present invention includes the following (a) to (e):
(A) a phospholipase D-deleting gene comprising a DNA comprising a base sequence in which the guanine at the 2779th base is replaced with adenine in the base sequence shown in SEQ ID NO: 4 (b) 2779 in the base sequence shown in SEQ ID NO: 4 A phospholipase D deletion gene comprising a DNA having a base sequence in which the guanine of the 2nd base is substituted with adenine and one or several bases are deleted, substituted or added in the base sequence (c) SEQ ID NO: 4 A phospholipase D-deficient gene comprising a DNA comprising a nucleotide sequence having a guanine at the 2779th base in the nucleotide sequence shown in (2) replaced with adenine and having 95% or more identity to the nucleotide sequence (d) In the base sequence shown in No. 4, guanine at the 2779th base is substituted with adenine, and the salt Phospholipase D deletion gene containing DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the sequence (e) Phospholipase D deficiency containing DNA comprising the base sequence represented by SEQ ID NO: 22 One of the genes.

  The base sequence shown in SEQ ID NO: 4 is the DNA sequence of the PLD1 gene of ND0052, which is a mutant strain in which Tos17 is inserted into the PLD5 gene of Nipponbare (rice variety). ND0052 is available from the National Institute for Agrobiological Resources (2-1-2 Kannondai, Tsukuba City, Ibaraki Prefecture 305-8602). SEQ ID NO: 5 shows the amino acid sequence encoded by the DNA sequence.

  The 2277th to 2779th bases of the base sequence shown in SEQ ID NO: 4 are TGG, and this codon encodes tryptophan. However, the gene of the present invention is nonsense suddenly from the codon (TGG) to the stop codon (TGA). It has a mutation and exhibits PLD deficiency. In the amino acid sequence (SEQ ID NO: 5) encoded by the base sequence shown in SEQ ID NO: 4, the 80th amino acid is tryptophan (Trp) encoded by the codon (TGG). The PLD1 gene of the rice line 03-s108 of the present invention is shown in SEQ ID NO: 22. In SEQ ID NO: 22, the 2774th to 2776th bases correspond to the above stop codon (TGA). The amino acid sequence encoded by the base sequence shown in SEQ ID NO: 22 is shown in SEQ ID NO: 23.

  The above “base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 4” means, for example, 1 to 10 base sequences shown in SEQ ID NO: 4, preferably 1 to 5 bases may be deleted, 1 to 10, preferably 1 to 5 bases may be added to the base sequence shown in SEQ ID NO: 4, or shown in SEQ ID NO: 4 It means that 1 to 10, preferably 1 to 5 bases in the base sequence may be substituted with other bases.

  The “identity” of the “base sequence having 95% or more identity to the base sequence shown in SEQ ID NO: 4” is 95% or more, preferably 98% or more, more preferably 99% or more.

  The “stringent conditions” refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not substantially formed. For example, a highly complementary nucleic acid, that is, a complementary strand of DNA comprising a base sequence having 95% or more, preferably 98% or more, more preferably 99% or more identity with the base sequence shown in SEQ ID NO: 4 hybridizes. And a condition in which a complementary strand of a nucleic acid having lower identity does not hybridize. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C., formamide The condition is that the concentration is 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%. Further, under stringent conditions, the washing conditions after hybridization are usually such that the sodium salt concentration is 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 40 to 70 ° C., preferably 50 to It is 70 degreeC, More preferably, it is 60-65 degreeC. The cleaning liquid preferably contains 0.1 to 0.2% SDS.

  Examples of the above genes (b) to (d) include substitution of 2048th base guanine (G) to adenine (A) in the base sequence shown in SEQ ID NO: 4 (amino acid sequence shown in SEQ ID NO: 5) For example, a 17th alanine (Ala) to threonine (Thr) mutation) is not limited thereto. The above substitution is the 2045th base (adenine) in SEQ ID NO: 22 of the rice strain 03-s108 of the present invention, and the mutation is the 17th threonine in SEQ ID NO: 23.

  The PLD-deficient gene of the present invention uses a primer designed based on the base sequence of any of the genes (a) to (e) above as a template for a nucleic acid derived from a cDNA library or a genomic DNA library. By carrying out PCR amplification as described above, it can be obtained as a nucleic acid fragment. The gene can be obtained as a nucleic acid fragment by performing hybridization using a nucleic acid derived from the above-described library or the like as a template and a DNA fragment that is a part of the gene as a probe. Alternatively, the gene can also be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

  In addition, the deletion, addition and substitution of the base can be performed by modifying the gene by a technique known in the art. Mutation can be introduced into a gene by, for example, the Kunkel method or the Gapped duplex method. Mutation introduction kits using site-directed mutagenesis (Mutan-K (Takara Bio Inc.), LA PCR Mutation can be introduced using in vitro mutagenesis kit (Takara Bio Inc.).

5. Recombinant vector A recombinant vector used for plant transformation can be constructed by introducing any of the PLD-deficient genes (a) to (e) described above into an appropriate vector. Although it does not specifically limit as a vector, The vector of pBI type | system | group which can introduce | transduce a target gene into a plant via Agrobacterium, pPZP type | system | group, pSMA type | system | group, etc. are used suitably. In particular, pBI binary vectors or intermediate vector systems are preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3, and pBIG2113. Other vectors include pUC vectors that can directly introduce genes into plants, such as pUC18, pUC19, and pUC9. Furthermore, plant virus vectors such as cauliflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), tobacco mosaic virus (TMV) and the like can be mentioned.

  In order to insert a target gene into a vector, first, a method in which the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA, and ligated to the vector is employed. The

  When a binary vector plasmid is used, the target gene is inserted into the boundary sequence (between LB and RB) of the binary vector, and this recombinant vector is propagated in E. coli. Subsequently, the amplified recombinant vector is introduced into Agrobacterium tumefaciens GV3101, C58, LBA4404, EHA101, EHA105, Agrobacterium rhizogenes LBA1334, etc. by electroporation or the like, and the Agrobacterium is used to plant. Introduce the target gene.

  Alternatively, the gene sequence present in the nuclear DNA may be incorporated on both sides of the target gene so that the target gene is homologously recombined with the nuclear DNA of the plant to be transformed. Furthermore, in the vector, upstream, inside or downstream of the target gene, promoter (cauliflower mosaic virus (CaMV) 35S promoter, rice-derived actin promoter, etc.), enhancer (enhancer region including upstream sequence in CaMV35S promoter, etc.), Terminators (such as nopaline synthase (NOS) gene terminator, CaMV 35S RNA gene terminator, etc.), replication origins for using binary vector systems (such as replication origins derived from Ti or Ri plasmids), selectable marker genes (hygroglomerates) A mycin resistance gene, an ampicillin resistance gene, etc.) can be linked.

6). Method for producing PLD-deficient transformed plant The PLD-deficient transformed plant of the present invention is obtained by introducing the gene of any one of (a) to (e) above or the above recombinant vector into a target plant and performing homologous recombination. Can be created by performing. The target plant is not particularly limited as long as it is a plant that wants to impart PLD deficiency, but is preferably a gramineous plant (rice, corn, wheat, barley, etc.), and, in addition, legumes (soybean, peanuts, etc.) Examples include cruciferous plants (eg, rapeseed).

  Plant materials to be transformed include plant organs and plant tissues such as stems, leaves, seeds, embryos, ovules, ovary, shoot tips, buds and pollen, sections of these, undifferentiated calli, and enzymes Any of plant cultured cells such as protoplasts, which are treated to remove the cell wall, may be used. In the case of the in planta method, water-absorbing seeds or the whole plant can be used.

  Examples of gene or recombinant vector introduction include known methods such as the Agrobacterium method, PEG-calcium phosphate method, electroporation method, liposome method, particle gun method, and microinjection method. In the Agrobacterium method, there are cases where protoplasts are used, tissue pieces are used, and plants themselves are used (in planta method). When using protoplasts, co-culture with Agrobacterium having Ti plasmid or Ri plasmid (Agrobacterium tumefaciens or Agrobacterium rhizogenes, respectively), fusion with spheroplasted Agrobacterium (spheroplast method), tissue When using a piece, it can be carried out by a method of infecting a sterile cultured leaf piece (leaf disc) of a target plant or a callus (undifferentiated cultured cell). In addition, when the in planta method using seeds or plants is applied, it can be carried out by direct treatment of Agrobacterium to water-absorbing seeds, seedlings (seedlings), potted plants and the like. These plant transformation methods can be carried out according to the description such as “Experimental protocol of new model plant from genetic method to genome analysis (supervised by Isao Shimamoto, Kiyotaka Okada, Shujunsha, 2001)”.

  Whether or not a gene has been incorporated into a plant can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method, a method for determining the presence or absence of a PLD-deficient gene described below, and the like. Alternatively, various reporter genes such as beta glucuronidase (GUS), luciferase (LUC), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), beta galactosidase (LacZ), etc. This can also be confirmed by preparing a vector ligated to A, transforming a plant in the same manner as described above using Agrobacterium introduced with the vector, and measuring the expression of the reporter gene.

  In selecting a transformant in which a PLD deletion gene is incorporated and growing a plant, when a plant cultured cell is a subject of transformation, the transformant is regenerated from the obtained transformed cell. For regeneration, a known tissue culture method may be used, and those skilled in the art can easily perform the regeneration. Regeneration from plant cells to plants can be performed, for example, as follows.

  First, when plant tissues or protoplasts are used as plant materials to be transformed, these are inorganic elements, vitamins, carbon sources, sugars as energy sources, plant growth regulators (auxin, cytokinin, gibberellin, abscisic acid, The plant is cultured in a callus formation medium sterilized by adding plant hormones such as ethylene and brassinosteroid, etc. to form dedifferentiated callus that grows indefinitely (hereinafter referred to as “callus induction”). The callus thus formed is transferred to a new medium containing a plant growth regulator such as auxin, and further grown (subcultured).

  When callus induction is performed in a solid medium such as agar and subculture is performed in, for example, liquid culture, each culture can be performed efficiently and in large quantities. Next, callus grown by subculture is cultured under appropriate conditions to induce organ redifferentiation (hereinafter referred to as “redifferentiation induction”), and finally a complete plant body is regenerated. Redifferentiation induction can be performed by appropriately setting the type and amount of various components such as plant growth regulators such as auxin and carbon sources, light, temperature, etc. in the medium. By such redifferentiation induction, somatic embryos, somatic roots, somatic shoots, adventitious shoots and the like are formed, and further grown into complete plants. Alternatively, storage or the like can be performed in a state before becoming a complete plant body (for example, encapsulated artificial seeds, dried embryos, freeze-dried cells and tissues).

  The PLD-deficient transformed plant of the present invention is the whole plant into which any of the genes (a) to (e) is introduced, or a part of the plant (for example, leaves, petals, stems, roots, pollen, etc.) , Plant cultured cells (eg, callus, protoplast, etc.) and seeds are included. Moreover, the plant body of the progeny obtained by the sexual reproduction or asexual reproduction of this plant body, a part of the progeny plant body, a cultured cell, and a seed shall also be included. The transformed plant of the present invention can be mass-produced by obtaining propagation materials such as seeds and protoplasts from the transformed plant and cultivating or culturing them.

7). Method for Determining Presence / absence of PLD Deletable Gene The method for judging the presence / absence of the PLD-deficient gene of any one of the above (a) to (e) of the present invention is represented by SEQ ID NO: 4 in Gramineae plants. This includes detecting a mutation from guanine (G) to adenine (A) at the 2779th base in the base sequence, or a nonsense mutation of the base at the corresponding position.

  Nucleic acid samples were prepared by known nucleic acid extraction and purification methods (Murray and Thompson (Nucleic Acid Res. 8: 4321-4325, 1980; or Sambrook, J. et al. (1989): “Molecular Cloning: A Laboratory Manual (2nd Ed.) "Cold Spring Harbor Laboratory, NY, etc.) For example, a nucleic acid sample is obtained by adding CTAB buffer to a sample obtained by pulverizing leaves and incubating, followed by extraction with chloroform / isoamyl alcohol. Commercially available nucleic acid extraction / purification kits may also be used.

  In the base sequence shown in SEQ ID NO: 4, a nonsense mutation such as a mutation from guanine (G) to adenine (A) at the 2779th base may be detected using a known DNA mutation (polymorphism) detection method, Although not particularly limited, for example, CAPS method, dot blot SNP method, SSCP method, dCAPS method and the like can be mentioned.

  In the CAPS (Cleaved Amplified Polymorphic Sequence) method, a specific base sequence portion of DNA is amplified by a PCR (Polymerase Chain Reaction) method, the amplified product is cleaved by a restriction enzyme, and the mutation is caused by the difference in length of the cleaved DNA fragment. (See Konieczny and Ausubel Plant J. 4: 403-410, 1993).

  In the dot blot SNP method, a DNA-containing sample is dot-blotted onto a nylon membrane or the like, and a detection probe (a probe having a base sequence that specifically hybridizes to a DNA mutation and labeled with an appropriate labeling substance) Probe) and a competitive probe (probe having a base sequence that specifically hybridizes to a wild-type base sequence against a DNA mutation and not labeled) to the detection probe This is a method of detecting DNA mutation by applying the probe for competition in an appropriate excess ratio (for example, detection probe: competition probe = 1: 1 to 10) (Shirasawa et al. 2006 Theor Appl Genet 113, 147 -155).

  In SSCP (Singl Strand conformation Polymorphism) method, a specific nucleotide sequence of DNA is amplified by PCR method, the amplified product is heat denatured (heated and quenched), and the heat denatured amplified product is electrophoresed on polyacrylamide gel. In this method, DNA mutation is detected by a difference in mobility in electrophoresis (due to a change in DNA higher order structure after heat denaturation caused by a slight difference in base sequence) (Orita et al., Proc. Natl. Acad. Sci. USA86: 2766-2770).

  The dCAPS (derived CAPS) method is a method in which a primer is designed in a region close to a substituted base, and a new restriction enzyme site is created in the amplification product by introducing a mismatch into a part of the primer. Yes (see Neff et al., Plant J. 14: 387-392, 1998).

  In the method for determining the presence or absence of the PLD-deficient gene of the present invention, for example, when the CAPS method is used, the primers of SEQ ID NO: 16 and SEQ ID NO: 17 are used, and the 2779th position of the nucleotide sequence shown in SEQ ID NO: 4 is determined by PCR method. Amplify a DNA fragment containing the base G-to-A substitution, cleave the resulting DNA fragment with restriction enzyme PsrI, perform polyacrylamide gel electrophoresis, and stain to detect the band. These patterns are compared to determine the presence or absence of a PLD deletion gene.

The G-to-A mutation at the 2779th base of the base sequence shown in SEQ ID NO: 4 is caused by the recognition site “5 ′... (N) 7 G TA (N) 6GTTC (N) 12. .3 ′ ”(SEQ ID NO: 24) occurs in the underlined G, PsrI is replaced with G at the 2779th base of the base sequence shown in SEQ ID NO: 4 (A in the PLD deletion gene) It is suitable as a restriction enzyme used for DNA fragments containing

  In the method for determining the presence or absence of the PLD deletion gene of the present invention, for example, when using the dot blot SNP method, the primers of SEQ ID NO: 16 and SEQ ID NO: 17 are used, and the 2779 of the nucleotide sequence shown in SEQ ID NO: 4 by PCR method is used. A DNA fragment containing the G-to-A substitution part of the 2nd base is amplified and dot blotted onto a nylon membrane. Hybridization is performed using a probe of SEQ ID NO: 18 as a wild type (PLD gene) detection probe and a mixture of SEQ ID NO: 19 as a competitive probe in a ratio of 1: 5, for example. Further, hybridization is performed using a mixture of the probe of SEQ ID NO: 20 as a probe for detecting a mutant type (PLD deletion gene) and the probe of SEQ ID NO: 21 as a competitive probe in a ratio of 1: 5, for example. After hybridization, the dot blot signal is compared between the wild type and the mutant type to determine the presence or absence of the PLD deletion gene.

  Wild-type homozygous individuals, heterozygous individuals, and homozygous individuals having a PLD-deficient gene can be identified from the band pattern by the CAPS method and the signal by the dot blot SNP method, so that selection of PLD-deficient transformed plants can be easily performed. It can be carried out.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the present invention.

[Generation of PLD-deficient rice lines]
As rice seeds to be treated with mutagen, air-dried seeds of rice mutant line ND0052 were used.

  The seed stock and sterilized grains are removed from the seeds, a mutagen solution (0.1 M phosphate buffer containing 1 mM sodium azide, pH 3.0) is prepared, and 200 mL of the mutagen solution is added to 25 g of seeds. Shake for 6 hours at ° C. After the seeds were washed 3 times with tap water, the seeds were germinated.

  The seedlings were grown by the usual method of raising rice seedlings, and the seedlings were transplanted and cultivated one by one. The ripened M1 strain was harvested in stock units.

  Two M2 seeds per strain were randomly selected, and the embryos were taken out and ground at a rate of 40 μL per embryo with an extraction buffer for SDS electrophoresis. SDS electrophoresis was performed using the milled solution as a sample, and the separated protein was transferred to a PVDF membrane. PLD was detected by Western blotting using an anti-PLD antibody. The antibody used was distributed under the courtesy of Japan Tobacco Inc. If PLD by Western blotting can not detect an M1 strain was found was examined whether the PLD further prospect up to 10 grains. If there were 2 or more grains in which PLD could not be detected, M2 seeds were sown. Here, the M1 strain in which PLD could not be detected was named 03-s108.

  The M2 strain of 03-s108 was cultivated and M3 seeds were sown and the M3 seed of 03-s108 was sown and cultivated to obtain M4 seeds. And the presence or absence of PLD in M3 seed and M4 seed was detected by Western blotting using an anti-PLD antibody (FIGS. 1 and 2, arrows indicate PLD). In FIG. 1, lanes 1 and 7 represent Nipponbare, and lanes 2 to 6 represent 03-s108 M3 seeds. In the case of 03-s108 M3 seed, 10 grains were analyzed, but no PLD could be detected in all grains. In FIG. 2, lanes 1 and 4 are Nipponbare, lane 2 is ND0052, lane 3 is 03-s108 M4 seed, and again, 03-s108 M4 seed PLD was not detected.

  From the above results, it was considered that PLD in brown rice was deleted in 03-s108. In 03-s108, a PLD deletion trait was confirmed even in progeny, and a novel PLD deletion rice line (Accession No. FERM P-21231) was used.

[Measurement of PLD enzyme activity of PLD-deficient rice line 03-s108]
PLD enzyme activity was measured according to the method of Ueki et al. (Plant Cell Physiol. 36: 903-914, 1995) using the mechanism by which PLD decomposes phosphodylcholine as an indicator.

  First, a nuka-germ fraction was collected from ripe seeds / brown rice of Nipponbare, ND0052, and 03-s108 using a small rice milling machine. These were stored at −30 ° C. until needed.

  The Nuka-germ fraction was suspended in a predetermined buffer and then ground to obtain a crude enzyme fraction and an ammonium sulfate fraction. The protein amount of the crude enzyme fraction and the fraction fractionated with ammonium sulfate was determined using a Bio-Rad protein quantification reagent.

  PLD activity comparable to that of Nipponbare was observed in the crude enzyme fraction derived from the ND0052 Nuka-germ fraction, but almost no PLD activity was observed in 03-s108 (Table 1, FIG. 3).

  Numbers 1, 5 and 7 of the tube in FIG. 3 are Nipponbare, numbers 2, 6 and 8 are ND0052, number 3 is M3 seed of 03-s108, and number 4 is M4 seed of 03-s108. In addition, the protein concentrations of tubes 1 to 4 are 5 mg / ml, numbers 5 to 6 are 0.05 mg / ml, and numbers 7 to 8 are 0.5 mg / ml.

  The above results indicate that 03-s108 brown rice lacks PLD as both protein and enzyme activity, and it is thought that 03-s108 lacks PLD. .

[Genetic analysis of PLD deletion]
In order to clarify what genetic characteristics are governed by PLD deficiency, Koshihikari was crossed with 03-s108, and the presence or absence of PLD in the F2 seed was analyzed by Western blotting.

  In Koshihikari seeds, PLD is “Yes”, whereas in 03-s108 seeds, it is “No”. Therefore, in Table 2, Koshihikari type is “With PLD” and 03-s108 type is “No PLD”. Show. In F2 seeds, the presence / absence of PLD protein was 96:32, and the PLD protein was separated into 3: 1 (Table 2). Therefore, it was considered that the deletion of PLD is a recessive one-gene rule.

  DNA was extracted from the protein extract which investigated the presence or absence of PLD protein in F2 seed. Using this extracted DNA as a template, PCR was performed to test whether Tos17 was inserted into the PLD5 gene.

  The primers used were AGTGATTTTGCCGTTGTTC (SEQ ID NO: 1), CATTTTCAAGCAGCAGGTCA (SEQ ID NO: 2), and GAGAGCATCATCGGTTACACTCTTCTC (SEQ ID NO: 3). PCR conditions were 98 ° C. (2 minutes) -60 ° C. (1 minute) -60 ° C. (1 minute) -60 ° C. 2 minutes) -72 [deg.] C. (2 minutes)] and 30 cycles [72 [deg.] C. (10 minutes) -10 [deg.] C. (end stop in this state)]. PCR products were separated with agarose, and the presence or absence of Tos17 insertion was analyzed from the mobility of the PCR products.

In Koshihikari seeds, insertion of Tos17 was “absent”, whereas in 03-s108 seeds, it was “present”. In F2 seeds, there is no Tos17 insertion: hetero: Yes = 36 grains: 56 grains: 33 grains (χ ² = 1.495, p = 0.473), and insertion of Tos17 is a recessive gene rule of one gene (Tos17 (-) in Table 3, no Tos17 insertion, Tos17 (±) is hetero with respect to Tos17 insertion, Tos17 (+) indicates Tos17 insertion)).

  Next, the genetic relationship between the presence or absence of Tos17 insertion into the PLD5 gene tested by the PCR method and the presence or absence of PLD determined by the Western blotting was examined. About these two traits (presence / absence of Tos17 / presence / absence of PLD) No / Yes: No / No: Yes / Yes: Yes / No separated at about 9: 3: 3: 1, so characteristics of Tos17 insertion and no PLD protein Was estimated to be dominated by two independent recessive genes. That is, it was considered that the presence or absence of the PLD protein was determined regardless of the insertion of Tos17 (Table 4, T in the table had no Tos17 insertion, t had Tos17 insertion, P had PLD, p had no PLD, Showing).

[Microscopic observation of spherosomes]
A spherosome fraction and a crude enzyme fraction were prepared from the Nuka germ fraction of each variety line (Nipponbare, ND0052 and 03-s108). The preparation was carried out according to the method of Katsumi Takano et al. (Journal of the Japan Food Industry Association, 36: 468-474, 1987).

  The crude enzyme fraction was added to the spherosome fraction and observed using an optical microscope at 35 ° C., 0 hours, 6 hours and 24 hours after the treatment.

  FIG. 4 shows the crude enzyme fraction prepared from Nipponbare on the spherosome prepared from the Nuka-germ fraction of Nipponbare (Panels A, B, C) and 03-s108 (Panels D, E, F). Add the crude enzyme fraction prepared from 03-s108 to the spherosomes of panels C and F, or add the buffer solutions to the spherosomes and shake for 6 hours at 35 ° C. The structure of the spherosome is shown (observed at 200 times using an optical microscope).

  In the Nipponbare spherosome, fusion of the spherosome membrane was observed even when the crude enzyme fraction was added (0 hour after treatment) or when the buffer solution was added (panel A) (the arrow in FIG. 4 indicates the fused spherosome). The extent of the Nipponbare Spherosome membrane fusion by adding the buffer or crude enzyme fraction was in the order of “Nipponbare Enzyme >> 03-s108 Crude Enzyme> Buffer” (Panel B >> Panel C> Panel A). .

  Even when a buffer solution or a crude enzyme fraction was added to 03-s108 spherosomes, the fusion effect of each added sample was “Nippon Hare >> 03-s108> buffer solution” (panel E >> panel F> panel D). there were.

  Further, when a buffer solution was added to the Nipponbare and 03-s108 spherosome fractions, the degree of fusion of the spherosomes was “Nipponbare → 03-s108” (panel A >> panel D). When the Nipponbare crude enzyme fraction was added to the Nipponbare and 03-s108 spherosome fractions, the degree of fusion of the spherosome membrane was "Nipponbare >> 03-s108" (panel B >> E). When the crude enzyme fraction of 03-s108 was added to the Nipponbare and 03-s108 spherosome fractions, the degree of fusion of the spherosome membrane was “Nippon Hare >> 03-s108” (panel C >> panel F). In addition, also in 24 hours after a process, the result similar to 6 hours after a process was obtained (data not shown). In addition, regarding the fusion of the Nipponbare spherosome and the ND0052 spherosome membrane, there was no significant difference between cultivars and strains.

  The above results indicate that by adding the crude enzyme fraction of Nipponbare or ND0052 containing PLD to the spherosome fraction, PLD decomposes the phosphatidylcholine of the spherosome membrane, resulting in fusion of the spherosome membrane, and the spherosome of Nipponbare. It is considered that PLD is highly likely to be adsorbed.

[Linkage analysis with microsatellite markers]
The polyclonal antibody used to find the PLD-deficient line 03-s108 in Example 1 is an antibody against the PLD1 protein of Koshihikari seeds. Since the PLD1 gene is registered under NCBI accession number AB001920, Koshihikari and 03-s108 were crossed to clarify whether the PLD deletion mutation was derived from the PLD1 gene mutation. Linkage analysis was performed using the F2 population.

  Since Koshihikari homozygous seeds having a PLD gene contain PLD, signals are detected by Western blot analysis. On the other hand, since the PLD is not contained in the 03-s108 homozygous seed having the PLD deletion gene, no signal is detected by Western blot analysis. In addition, since seeds heterozygous for the PLD deletion contain PLD, signals are detected by Western blot analysis, but in the self-grown seeds, Koshihikari type homozygous, heterozygous, and 03-s108 type homozygous are separated. Therefore, it separates into seeds in which signals are detected by Western blot analysis and seeds in which signals are not detected.

  Phenotypes of F2 plants were determined by progeny tests by detecting the presence or absence of PLD by using a plurality of embryo extracts of F3 seeds grown in 44 F2 plants and detecting the presence or absence of PLD.

  For linkage analysis, among the microsatellite markers published by the International Rice Genome Sequencing Project (Nature436: 793-800, 2005), RM3453 that sits in the vicinity of the PLD gene (present at 25.0 cM from the short arm side) Was used. DNA was extracted from the leaves of 44 F2 plants by the method of Murray and Thompson (Nucleic Acid Res. 8: 4321-4325, 1980), and PCR was performed using this as a template. CTAAAATGACAAAAGATAGGCA (SEQ ID NO: 6) and AAATTCTGACTTGTATGCA (SEQ ID NO: 7) were used as primers. PCR conditions were 94 ° C (1 minute) for 1 cycle, [94 ° C (30 seconds)-55 ° C (30 seconds)-72 ° C (30 seconds)] for 40 cycles, [72 ° C (10 minutes)-10 ° C ( In this state, the end pause) was performed in one cycle. PCR products were separated on an 8% polyacrylamide gel (acrylamide: bis = 19: 1), and the genotype of each DNA marker was determined from the difference in mobility of the PCR products.

As a result of Western blot analysis, in F2 plants, the PLD-deficient phenotype was Koshihikari type homo: hetero: 03-s108 type homo: 12: 19: 13 (χ ² = 0.864, P = 0.649) The separation ratio is shown.

  In the analysis with the microsatellite marker RM3453, Koshihikari type homo: hetero: 03-s108 type homo showed a segregation ratio of 13:17:14, and there were 4 recombinant individuals with PLD deficiency (FIG. 5). Asterisk). The recombination value was 1 × 4 / (44 × 2) = 4.5 cM. That is, it was revealed that the PLD deletion gene sits in the vicinity of RM3453 and the genetic distance is 4.5 cM from RM3453 (FIG. 6).

[Determination of nucleotide sequence of PLD deletion gene]
In order to investigate whether the PLD deletion property is due to the mutation of the PLD1 gene, the base sequence of the exon part of the PLD gene of 03-s108 was determined. Since the length of the PLD1 gene is about 6000 bases and it is difficult to amplify the full length by PCR using one pair of primer pairs, the PLD gene was divided into four regions (PLD-1 to 4). PCR was performed using the genomic DNA of 03-s108 as a template, and the base sequence of each amplified fragment was determined. Primers are CGACGGACAGATACTCTCTACCC (SEQ ID NO: 8) and CAAACAAGAAATGGCCAAAGC (SEQ ID NO: 9) in the PLD-1 region, GTGTGTGATGTGTGCTCTGTGTTACTGACTGTGTGTCTGACTGATGTGTCTGACTGATGTGTCTGACTGATGTGTCTGACTG (SEQ ID NO: 12) and GGGAAGCATGAACTTCAACTTAG (SEQ ID NO: 13), and GAAGTCCATGCTCCCCTTTACTC (SEQ ID NO: 14) and ACGAGCCATAAACAACATACCACC (SEQ ID NO: 15) were used for the PLD-4 region. Furthermore, in order to determine the entire base sequence of the PLD1 gene (about 6000 bases), primers GGTGTGAGGCCTCAAAACCTAG (SEQ ID NO: 25) and AGAGCAAGAGCAAAGACGAGTA (SEQ ID NO: 26), primers CATTTCTCCATCACATCAACT (SEQ ID NO: 27) and AAGAAGAGGGGAGCAGA (G) (SEQ ID NO: 29) and CTCAGGGGTATCAGGGAACC (SEQ ID NO: 30), primers CTGTGTGGTGATGTGTCTT (SEQ ID NO: 31) and TGAACAATGCTGCCTTGAG (SEQ ID NO: 32), primers GGGATGTTCTTTACAATTTGC (SEQ ID NO: 33) and AAGAGATGATGA CCATGT (SEQ ID NO: 34), was used TCTTCTGCTCTCTAAATCTG (SEQ ID NO: 37) and primer CTGGCAAAGGAGAACAATG (SEQ ID NO: 35) CAACAACGCTAAACAGTAG (SEQ ID NO: 36). PCR conditions are 94 ° C (1 minute) for 1 cycle, [98 ° C (10 seconds)-68 ° C (1 minute)] for 35 cycles, [72 ° C (3 minutes)-4 ° C (stop in this state)] Was performed in one cycle. The obtained DNA fragment was purified with QIAquick Gel Extraction Kit (manufactured by QIAGEN). Reaction was performed by the dye terminator method using this as a template, and the base sequence was determined with a DNA sequencer manufactured by ABI. The obtained base sequence of 03-s108 is shown in SEQ ID NO: 22, and the amino acid encoded by the base sequence is shown in SEQ ID NO: 23.

  The base sequence of 03-s108 (SEQ ID NO: 22) was compared with the base sequence of PLD1 of ND0052 (SEQ ID NO: 4). The base sequence of PLD1 of ND0052 is the same as the base sequence of PLD1 of Nipponbare.

  The guanine (G) of the 2779th base in the base sequence of the ND0052 PLD1 gene (SEQ ID NO: 4) was mutated to adenine (A) in 03-s108 (corresponding to the 2776th base of SEQ ID NO: 22). This mutation was a nonsense mutation that mutates the codon (TGG) specifying the tryptophan of the third exon to a stop codon (TGA).

  In 03-s108, normal PLD protein was not synthesized by this nonsense mutation, which was considered to be a cause of PLD deficiency.

[Method for determining the presence or absence of a PLD deletion gene]
Example mutations from the found guanine (G) by 6 to adenine (A), the recognition site for the restriction enzyme PsrI (5 '... (N) 7 G TA (N) 6GTTC (N) 12 .. .3 ′), the mutation occurred in the first base (underlined G), and the mutation was detected by the CAPS method using the restriction enzyme PsrI. Mutations were also detected by the dot blot SNP method that can detect any single nucleotide polymorphism.

(i) CAPS method A DNA fragment containing the target mutation was amplified by PCR. CACGTGAGCTCATGTCAACAGTTTG (SEQ ID NO: 16) and GCAGGTAAGCCCTCCCAATATTCG (SEQ ID NO: 17) were used as primers. PCR conditions were 94 ° C (1 minute) for one cycle, [94 ° C (30 seconds)-60 ° C (30 seconds)-72 ° C (30 seconds)] for 35 cycles, [72 ° C (10 minutes)-10 ° C ( In this state, the end pause) was performed in one cycle. The obtained DNA fragment was cleaved with the restriction enzyme PsrI and electrophoresed on a 6% polyacrylamide gel. After electrophoresis, the gel was stained with ethidium bromide, and the band was detected by irradiating the gel with ultraviolet rays.

  As a result of CAPS analysis, in Koshihikari, bands showing 177, 145, 114, 82, and 32 bp sized DNA fragments were detected, whereas 259 bp sized DNA fragments were not cut in 03-s108. A band was detected (Figure 7). As a result of analyzing the F2 population consisting of 32 individuals, 9 individuals were Koshihikari type homo, 12 individuals were 03-s108 type homo, and 11 individuals were heterozygous. In all individuals, the genotype of the phospholipase D gene coincided with the phenotype by Western blot analysis (FIG. 7).

(ii) Dot blot SNP method A DNA fragment containing the target mutation was amplified by PCR. The primers and PCR conditions used are the same as in the CAPS method. According to the method of Shirasawa et al. (Theor. Appl. Genet. 113: 147-155), the obtained DNA fragment was dot-blotted on a nylon membrane, and hybridization using a competitive probe was performed. As a probe for detecting a Koshihikari type allele, an oligonucleotide labeled with digoxigenin at its 5 ′ end (CCTCCGCTGGTATGAGTC (SEQ ID NO: 18)) and an unlabeled oligonucleotide (CCTCCGCTGATATGAGTC (SEQ ID NO: 19)) as a competitive probe are used. A mixture with a concentration ratio of 1: 5 was used. On the other hand, the probe for detecting the allele of type 03-s108 includes an oligonucleotide labeled with digoxigenin at the 5 ′ end (CCCTCGCTGATATGAGTC (SEQ ID NO: 20)) and an unlabeled oligonucleotide (CCTCGCTGTGTATGAGTC (sequence) No. 21)) mixed at a concentration ratio of 1 to 5 was used. Hybridization was performed at 42 ° C. in 5 × SSC, 0.1% Sarcocyl, 0.02% SDS, 1% Blocking Reagent (# 1096176, Roche) buffer, and the wash solution was 0.5 × SSC at 42 ° C. /0.1% SDS was used.

  As a result of dot blot SNP analysis, when hybridization was performed with a probe that detects an allele of the Koshihikari type, a signal specific to Koshihikari was obtained, and a signal was detected in 31 of 44 F2 plants ( FIG. 8, A1 and A5 were blotted with 03-s108, and B1 and B5 were blotted with Koshihikari DNA. Conversely, when hybridization was performed with a probe that detects an allele of type 03-s108, a signal specific to 03-s108 was obtained, and a signal was detected in 31 of 44 F2 populations ( FIG. 8). In heterozygous signals were detected when both probes were used. Of these 44 individuals, 13 F2 plants that detected a signal only when using a probe that detects a Koshihikari type allele were used when a probe that detects a Koshihikari type homozygous, 03-s108 type allele was used. The 13 F2 plants that detected only the signal were 03-s108 homozygous, and the 18 F2 plants that detected the signal when both probes were used were heterozygous. Moreover, the genotype of PLD1 gene and the phenotype by Western blot analysis corresponded in all the individuals (FIG. 8).

  The results of (i) and (ii) confirmed that the PLD deletion property was due to the nonsense mutation of the PLD1 gene shown in SEQ ID NO: 4.

The western blotting which investigated the presence or absence of PLD of the rice system | strain of this invention is shown. The western blotting which investigated the presence or absence of PLD of the rice system | strain of this invention is shown. The PLD activity of the rice line of the present invention is shown. Panels A-C show the fusion of Nipponbare spherosome membranes. Panels D-F show the fusion of the 03-s108 spherosome membrane. An example of linkage analysis of F2 plants with the microsatellite marker RM3453 is shown. The linkage map containing the microsatellite marker RM3453 is shown. The example of the analysis by the CAPS method of F2 plant is shown. The example of the analysis by the dot blot SNP method of F2 plant is shown.

Claims (15)

  Rice line (Accession No. FERM P-21231), progeny or a hybrid thereof that is phospholipase D deficient.   Production of a phospholipase D-deficient rice line comprising crossing the rice line according to claim 1 with a rice wild-type strain, cultivating the seed to form a plant, and screening the plant for phospholipase D deletion Method.   A cell obtained from the rice line according to claim 1 and a cell obtained from a rice wild type strain are cell-fused, the obtained fused cell is cultured and regenerated into a plant body, and the plant body is screened for phospholipase D deletion. A method for producing a phospholipase D-deficient rice line.   The method according to claim 2 or 3, wherein the phospholipase D deletion is due to a nonsense mutation of the phospholipase D gene.   A rice line, a progeny thereof, or a hybrid strain thereof, which is phospholipase D-deficient according to claim 1.   The rice flour derived from the seed according to claim 5.   6. Seed-derived cooked rice according to claim 5.   The cooked rice according to claim 7, which is cooked cooked rice. The phospholipase D deletion gene which is any of the following (a) to (e).
(A) a phospholipase D-deleting gene comprising a DNA comprising a base sequence in which the guanine at the 2779th base is replaced with adenine in the base sequence shown in SEQ ID NO: 4 (b) 2779 in the base sequence shown in SEQ ID NO: 4 A phospholipase D deletion gene comprising a DNA having a base sequence in which the guanine of the 2nd base is substituted with adenine and one or several bases are deleted, substituted or added in the base sequence (c) SEQ ID NO: 4 A phospholipase D-deficient gene comprising a DNA comprising a nucleotide sequence having a guanine at the 2779th base in the nucleotide sequence shown in (2) replaced with adenine and having 95% or more identity to the nucleotide sequence (d) In the base sequence shown in No. 4, guanine at the 2779th base is substituted with adenine, and the salt Phospholipase D deficiency comprising DNA comprising the nucleotide sequence represented by phospholipase D deficient gene (e) SEQ ID NO: 22 containing the DNA complementary strand hybridizes to DNA having a base sequence having a sequence 95% or more identity Gene loss   A recombinant vector comprising the gene according to claim 9.   A method for producing a phospholipase D-deficient transgenic plant, comprising introducing the gene according to claim 9 into a plant cell, performing homologous recombination, and growing the plant.   The method according to claim 11, wherein DNA is introduced using the recombinant vector according to claim 10.   The method according to claim 11 or 12, wherein the plant is a gramineous plant.   The phospholipase according to claim 9, which comprises detecting a mutation of guanine at the 2779th base in the nucleotide sequence represented by SEQ ID NO: 4 from guanine to adenine, or a nonsense mutation at a position corresponding to the mutation in a grass family. A method for determining the presence or absence of a D-deficient gene.   The method according to claim 14, wherein the mutation is detected in the nucleic acid sample by any one of the CAPS method, the dot blot SNP method, the SSCP method, and the dCAPS method.

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