JP5062616B2 - Method for improving expression efficiency of introduced target gene in wheat - Google Patents

Description

  The present invention relates to a method for improving the expression efficiency of a target gene introduced in wheat.

  Generally, a plant transformation method introduces a target gene into a plant cell or tissue, cultures it, and regenerates the plant body.

  However, it is known that in wheat it is difficult to obtain a transformed plant using such a general method.

  Regarding the transformation of wheat, for example, a direct introduction method by Vasil et al. (Non-patent Document 1) and an Agrobacterium method by Cheng et al. (Non-patent Document 2) have been reported. Many examples of successful direct introduction methods have been reported, but the success rate expressed by gene introduction efficiency is very low. In wheat, even if the target gene is introduced into a cell or tissue, tissue culture is difficult, and it is difficult to confirm transformation by regenerating a plant.

  In wheat, immature embryos are the most commonly used tissues (explants) for introducing genes, but the transformation efficiency depends greatly on the state of the explants depending on the growth environment of the plant. It is difficult to reproduce the transformation system established in Japan in Japan.

  In wheat, the particle gun method is often used as a gene transfer method. In such a direct transfer method, a transformant in which the transgene is normally expressed can be obtained by reconstitution of the transgene or the like. Hateful.

  Regarding the regeneration efficiency in the transformation of wheat, for example, in Non-Patent Document 3, the scutellum of the ear collected on the 12th day after flowering and stored at 4 ° C. for 17 to 19 days was added with about 2-3% sucrose. It has been disclosed that the redifferentiation efficiency increased when cultured in a medium containing the same.

  Regarding the transformation efficiency of wheat, for example, in Non-Patent Document 4, when an explant of durum wheat was treated with high temperature or drought stress and transformed with a plasmid containing a herbicide resistance gene in an immature embryo, It has been disclosed that efficiency has improved.

  However, these non-patent documents disclose that the regenerative efficiency is improved or the gene transfer efficiency is improved by examining the expression of the selection marker gene, but the expression efficiency of the target gene is not disclosed.

When producing a transformant, even if the target gene can be introduced, it is meaningless unless the target gene is actually expressed. Therefore, improving the expression efficiency of the target gene is important for the production of transformants.
Vasil et al., Bio / Technology 10: 667-674 (1992). Cheng et al., Plant Physiology 115: 971-980 (1997) In Vitor Cell. Dev. Biol. Plant 39: 20-23, January-February 2003 Plant Cell Rep (2002) 20: 955-960

  An object of the present invention is to provide a method for improving the expression efficiency of an introduced target gene in wheat.

  The present inventor was introduced by low-temperature treatment of explants for transforming wheat, introducing the target gene into the explants, and selectively culturing the explants in a medium having a higher osmotic pressure than before. It is found that the expression efficiency of the target gene can be improved, and by using a medium with high osmotic pressure, the number of copies of the transgene is smaller than that of the conventional low osmotic pressure medium, and the probability that the transgene is expressed is increased. The present invention has been completed.

The features of the present invention are summarized as follows.
(1) Low temperature drying of immature embryos of wheat, introduction of the target gene into immature embryos or cells thereof by transformation operation, and medium containing 0.25 to 1.0 M osmotic pressure regulating substance in the immature embryos or cells thereof A method for improving the expression efficiency of a target gene introduced in an immature wheat wheat cell or a cell thereof, which comprises culturing in a cell.
(2) The method according to (1), wherein the transformed immature embryo of wheat or the cell thereof contains one or two copies of the introduced target gene.
(3) The method according to (1) or (2), wherein the low-temperature drying is performed at 0 to 10 ° C.
(4) The method according to any one of (1) to (3), wherein the osmotic pressure regulating substance is selected from the group consisting of maltose, sucrose, mannitol, sorbitol, polyethylene glycol, percoll and ficoll.
(5) The method according to any one of (1) to (4), wherein the transformation operation is a particle gun method.
(6) Expression efficiency of the introduced target gene, including regenerating a plant body using a transformed wheat immature embryo produced by the method according to any one of (1) to (5) or a cell thereof, Improved plant production method.
(7) The method according to (6), comprising transferring a target gene introduced into a plant body to a progeny.

  ADVANTAGE OF THE INVENTION According to this invention, the expression efficiency of the target gene in the transformant of wheat can be improved. Moreover, it becomes easy to reproduce the transformation system without being greatly affected by the state of the explants depending on the growth environment of the wheat plant. Furthermore, a wheat plant and its progeny with improved expression efficiency of the introduced target gene can be obtained.

  The method for improving the expression efficiency of a target immature embryo of wheat of the present invention or a cell thereof is obtained by drying a wheat immature embryo at low temperature, and transforming the immature embryo or its cells (callus and other cultured cells) by transformation. The target gene is introduced, and the immature embryo or its cells are cultured in a medium containing an osmotic pressure regulating substance such as 0.25 to 1.0 M sugar or sugar alcohol.

  Wheat immature embryos are used as explants for transformation. The immature embryo is preferably obtained by collecting ears 10 to 17 days after flowering of wheat (Triticum aestivum). Although it will not specifically limit if it is an immature embryo, For example, American Journal of Botany vol. 70, pp 308-311, immature embryos at the end of stage II with morphological observations are preferred.

  Preferably, the ears of wheat are collected together with the stems of the wheat and left in a low-temperature dark place without giving moisture from the outside, followed by drying treatment. The temperature is preferably 0 to 10 ° C, more preferably 4 to 6 ° C, and preferably 1 to 20 days, more preferably 5 to 7 days. If it is lower than 0 ° C, it freezes, and if it is higher than 10 ° C, ripening and drying of immature embryos proceed rapidly, which is not preferable. Moreover, since the development of immature embryos is slowed at low temperatures, there is an advantage that the development stage of immature embryos can be easily controlled. Drying may be accelerated or adjusted by methods such as air drying or dehydration with silica gel in a desiccator.

  In addition, by adjusting how the moisture is given to the plant body before pretreatment of the explant (immature embryo), the ripening temperature, and gently and moderately proceeding the intracellular change of the immature embryo by the drying treatment, It is also possible to adjust the pretreatment period.

  Under general greenhouse cultivation conditions (25/20 ° C.) for wheat, the cell activity is high, ripening is likely to proceed, and drying is also likely to proceed. Since water is most needed from heading and flowering to the early stage of ripening, drying progresses rapidly with a shortage of water, and the developmental stage of the embryo also progresses rapidly. For example, when grown in a greenhouse at 18/10 ° C., it is harvested 15-16 days after flowering and dried for 5 days at 5 ° C. (20 days), but in a greenhouse at 25/20 ° C., treatment begins after 10 days of flowering. It is too early, and it may be too late after 11 days, and if it is raised in a greenhouse at 18/10 ° C. and refrains from moisture, and drained after 14 days of flowering, it will be harvested 16-17 days after flowering. Since embryos in the same state can be obtained by drying at 5 ° C. for 3 days (19 days), the schedule for carrying out the introduction treatment can be adjusted by adjusting the way of giving temperature and moisture in this way. Also, depending on the size of the ears and seeds and the position of the seeds in the ears, the way water drains is different (large seeds are less likely to drain water, the upper part of the ear is more likely to drain water, and the lower part is less likely to drain). Therefore, it is preferable to adjust according to the growth state of the plant.

  As described above, immature embryos of wheat are dried at low temperature, pre-cultured in a callus induction medium for 3 to 6 hours, and then the target gene is introduced into the immature embryo or its cells.

  For the introduction of the target gene, a known genetic engineering technique can be used, and it is not particularly limited. In general, a vector containing the target gene is prepared and immature embryos or cells thereof, or callus derived from them, Agrobacterium method, electroporation method, particle gun method, PEG-calcium phosphate method, liposome method The vector is introduced by a microinjection method or the like. In wheat, a method of introducing it into an immature embryo using a particle gun method is preferable from the viewpoint of regeneration efficiency into a plant body. The particle gun method is a method in which a metal fine particle is coated with a gene and driven into a cell tissue, and is considered to be useful for plants having low redifferentiation ability such as wheat.

  A vector containing the target gene can be prepared, for example, as follows.

  The vector is not particularly limited, and pAL system (pAL51, pAL156, etc.), pUC system (pUC18, pUC19, pUC9 etc.), pBI system (pBI121, pBI101, pBI221, pBI2113, pBI2113, etc.), pPZP system, pSMA system, Intermediate vector systems (pLGV23Neo, pNCAT, etc.), cauliflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), tobacco mosaic virus (TMV) and the like can be used.

  In order to insert a target gene into a vector, for example, a method in which purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and ligated to the vector is used. it can.

  The target gene is not particularly limited as long as expression of the gene is desired, and may be an endogenous gene or a foreign gene of a target plant. Examples of such genes include sugar metabolism-related genes, useful substance (pharmaceuticals, pigments, aromatic components, etc.) production genes, plant growth control (promotion / suppression) genes, disease and insect resistance (insect feeding damage resistance, fungi) And bacterial disease resistance, virus (disease resistance, etc.) genes, environmental stress (low temperature, high temperature, drying, salt, light damage, UV) resistance genes, transporter genes, flour milling characteristics, baking characteristics, noodle characteristics Related genes and the like.

  In addition to the target gene, for example, a promoter, enhancer, intron, terminator, poly A addition signal, selection marker gene and the like can be linked to the vector.

  The promoter does not have to be derived from a plant as long as it functions in a plant body or a plant cell and can induce expression in a specific tissue of a plant or a specific developmental stage. Specific examples include cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase gene promoter (Pnos), corn-derived ubiquitin promoter, rice-derived actin promoter, tobacco-derived PR protein promoter, and the like.

  The terminator may be any sequence that can terminate transcription of the gene transcribed by the promoter, and examples thereof include nopaline synthase (NOS) gene terminator, octopine synthase (OCS) gene terminator, CaMV 35S terminator, and the like. It is done.

  Examples of selection marker genes include herbicide resistance genes (bialaphos resistance genes), drug resistance genes (tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, hygromycin resistance genes, spectinomycin resistance genes, chloramphenicol resistance) Genes, neomycin resistance genes, etc.), fluorescent or luminescent reporter genes (luciferase, β-galactosidase, β-glucuronidase (GUS), green fluorescence protein (GFP), etc.), neomycin phosphotransferase II (NPT II), dihydrofolate reductase, etc. These enzyme genes are mentioned.

  The vector containing the target gene is shot into an immature wheat wheat cell or a cell thereof (or a cultured cell such as a callus) by a particle gun method.

First, gold particles are washed and sterilized, and gold particles, vector, CaCl ₂ , and spermidine are added with stirring with a vortex mixer or the like, the vector is coated on the gold particles, and washed with ethanol. The gold particles are applied to a macro carrier film and dried. Then, the macro carrier film, the target immature embryo or its cells are set in a particle gun apparatus, and high-pressure helium gas is emitted from the gas acceleration tube toward the macro carrier film. The macro carrier film stops at the stopping plate, but the gold particles pass through the stopping plate, penetrate into the target placed under the stopping plate, and the target gene is introduced.

  Next, wheat immature embryos or cells thereof into which the gene of interest has been introduced are cultured in a callus induction medium. The callus induction medium has a osmotic pressure of 0.25 to 1.0 M, preferably 0.3 to 0.65 M, more preferably 0.4 to 0.5 M in the medium so that the osmotic pressure is higher than that of the conventional medium. Add pressure regulator. If the osmotic pressure-regulating substance is less than 0.25 M, the cytoplasm will flow out and the gene transfer treatment cannot be tolerated, and if it exceeds 1.0 M, the cell activity is excessively suppressed, which is not preferable. Further, the osmotic pressure regulating substance is not particularly limited, but for example, sugar or sugar alcohol is preferable. Specifically, maltose, sucrose, mannitol, sorbitol, polyethylene glycol (PEG), percoll (trade name, Amersham Biosciences, Polyvinyl) Colloidal silica products having a pyrrolidone film) and Ficoll (trade name, Pharmacia, a water-soluble synthetic product obtained by copolymerizing sucrose and epichlorohydrin) can be used. These may be used singly or in combination with a plurality of sugars, but maltose is usually preferably used in wheat.

  Moreover, the composition used as the base of a callus induction medium will not be specifically limited if it is suitable for the cultivation of wheat, Agar, an inorganic element, a vitamin, a carbon source, and 2,4-D of high concentration (for example, 2 mg / L) Plant hormones (plant growth regulators) such as auxin, cytokinin, and the like. In the callus induction medium, culture is performed, for example, for about 1 to 3 days after gene introduction.

  The formed callus is transferred to a new callus maintenance medium containing a plant growth regulator such as auxin such as 2,4-D (for example, 0.5 mg / L) having a concentration lower than that of the callus induction medium. To do. It is preferable to add an osmotic pressure-regulating substance (preferably maltose) having the above concentration to the callus maintenance medium so as to achieve a high osmotic pressure.

  For selection, it is preferable to add a herbicide or a drug corresponding to the selection marker introduced into the vector to the medium. In the callus maintenance medium, culture is performed, for example, for about 2 to 4 weeks after callus induction.

  In addition, the conventional culture medium contains 0.088-0.12M maltose, and is a lower osmotic pressure condition than the culture medium used by this invention. When selective culture is performed under the high osmotic pressure condition of the present invention, the number of copies of the normal expression unit of the transgene contained in the cultured cell or the regenerated plant body is as small as about 1 or 2 compared with the low osmotic pressure condition. It is understood (see 5. Estimation of copy number of transgene in Example 2), and it is considered that the probability that the transgene is expressed increases.

  By further culturing the cultured callus under appropriate conditions, organ regeneration can be induced, and finally a complete plant can be regenerated. Redifferentiation induction is performed by appropriately setting the types and amounts of various components such as plant growth regulators such as auxin such as 2,4-D and cytokinin such as zeatin and carbon sources in the medium, light, temperature, etc. be able to. Adventitious embryos, adventitious buds, adventitious stems and leaves are formed by redifferentiation induction. Furthermore, it is transplanted to a rooting medium not containing auxin or cytokinin, and grown to a complete plant body.

  By the above method, a plant with improved expression efficiency of the introduced target gene can be produced. In addition, the plant produced in this way has a small copy number of the introduced target gene, stably expresses the target gene, and is normally inherited (transmitted) to progeny.

  The gene introduction efficiency into wheat and the target gene expression efficiency (transformation efficiency) can be examined as follows.

  For example, the efficiency of gene introduction can be determined by cultivating and redifferentiating while selecting in a medium containing a herbicide-resistant gene corresponding to a herbicide-resistant gene as a foreign gene introduced into the vector, extracting DNA from the redifferentiated individuals, and performing PCR. The gene can be detected by the method and electrophoresis, and the gene transfer efficiency is calculated from the number of explants used for the transfer and the number of redifferentiated individuals having the foreign gene.

  Regarding the expression efficiency of the target gene, the presence or absence of the protein expressed from the target gene is examined for the redifferentiated individuals in which the introduction of the gene has been confirmed. The presence or absence of protein can be confirmed, for example, by staining plant fragments, electrophoresis, Western blotting, or the like. Then, the target gene expression efficiency (transformation efficiency) is calculated from the number of explants used for introduction and the number of redifferentiated individuals in which the presence of the protein expressed from the target gene is confirmed.

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

Preparation of reagents and working medium Reagent (1) Maltose stock solution (0.5 g / ml)
To 600 ml of distilled water, 500 g of maltose monohydrate (Wako) was added and dissolved while heating with a hot stirrer. The solution was filled up to 1 L with distilled water and sterilized with a tissue culture filter unit (0.2 μm PES) (NALGENE). This was used as a stock solution and stored at 4 ° C.
(2) 1000 × MS vitamin stock solution In 50 ml of distilled water, 200 mg of glycine (Wako), 50 mg of nicotinic acid (Wako), 50 mg of pyridoxine-HCl (Wako), 100 mg of thiamin-HCl (Wako), myo-inositol 10 g of (Wako) was added and dissolved. Then, distilled water was added to fill up to 100 ml, which was used as a stock solution, divided into 1 ml portions in a 1.5 ml tube, and stored at −30 ° C.

(3) 2,4-D stock solution (1 mg / ml)
2,4-D (2,4-Dichlorophenic acid acid) (Wako) 50 mg was dissolved in 99.5% EtOH (Wako), filled up to 50 ml, and Sterilip Filter Unit (0.22 μm PES) (MILLIPORE) Filter sterilized with. This was used as a stock solution and stored at −30 ° C.
(4) picloram stock solution (2 mg / ml)
In 5 ml of 1N KOH (Wako), 100 mg of picram (4-amino-3,5,6-trichloro-pyridine-2-carboxylic acid) (SIGMA) was added and dissolved. Then, distilled water was added to fill up to 50 ml, and the filter was sterilized with a Steliplip Filter Unit (0.22 μm PES) (MILLIPORE). This was used as a stock solution, aliquoted into 1 ml sterilized 1.5 ml tubes and stored at -30 ° C.

(5) ascorbic acid stock solution (100 mg / ml)
2 g of ascorbic acid (Wako) was added to 18 ml of distilled water and dissolved. Then, distilled water was added to fill up to 20 ml, and the filter was sterilized with Sterilip Filter Unit (0.22 μm PES) (MILLIPORE). This was used as a stock solution, aliquoted into 1 ml sterilized 1.5 ml tubes and stored at -30 ° C.
(6) Bialaphos stock solution (5 mg / ml)
250 mg of bialaphos (Shinyo Sangyo Co., Ltd.) was added to 40 ml of distilled water and dissolved. Then, distilled water was added to fill up to 50 ml, and the filter was sterilized with a Steliplip Filter Unit (0.22 μm PES) (MILLIPORE). This was used as a stock solution, aliquoted into 1 ml sterilized 1.5 ml tubes and stored at -30 ° C.

(7) L medium KH ₂ PO ₄ stock solution 20 g of KH ₂ PO ₄ (Wako) was added to 200 ml of distilled water and dissolved. Thereafter, distilled water was added to fill up to 250 ml and autoclaved. This was stored at 4 ° C. as a stock solution.
(8) L medium CaCl ₂ stock solution 15 g of CaCl ₂ .2H ₂ O (Wako) was added to 90 ml of distilled water and dissolved. Thereafter, distilled water was added to fill up to 100 ml and autoclaved. This was stored at 4 ° C. as a stock solution.
(9) L medium MgSO ₄ stock solution 25 g of MgSO ₄ .7H ₂ O (Wako) was added to 90 ml of distilled water and dissolved. Thereafter, distilled water was added to fill up to 100 ml and autoclaved. This was stored at 4 ° C. as a stock solution.

(10) L medium Fe / Na / EDTA stock solution 4 g of Fe / Na / EDTA (Dojin) was dissolved in 90 ml of distilled water. Thereafter, distilled water was added to fill up to 100 ml and autoclaved. This was stored at 4 ° C. as a stock solution.
(11) L medium micro inorganic salt stock solution of distilled water 90ml to MnSO ₄ · _5H 2 O and _{(Wako) 1120mg, ZnSO 4 ·} 7H 2 O (Wako) to _{860mg, H 3 BO 4 (Wako} ) to 620 mg, KI ( Wako) (83 mg), CuSO ₄ .5H ₂ O (Wako) (2.5 mg), and CoCl ₂ · 6H ₂ O (Wako) (2.5 mg) were added and dissolved. Thereafter, distilled water was added to fill up to 100 ml and autoclaved. This was stored at 4 ° C. as a stock solution.

(12) 1000 × L vitamin stock solution In distilled water 40 ml, thiamin-HCl (Wako) 500 mg, pyridoxine-HCl (Wako) 50 mg, nicotinic acid (Wako) 50 mg, Calcium (+)-pantothenate (Wako) 50 mg of 50 mg of ascorbic acid (Wako) was added and dissolved. Then, distilled water was added to fill up to 50 ml, and the filter was sterilized with a Steliplip Filter Unit (0.22 μm PES) (MILLIPORE). This was used as a stock solution, aliquoted into 1 ml sterilized 1.5 ml tubes and stored at -30 ° C.
(13) Zeatin stock solution (5 mg / ml)
50 mg of zeatin (6- (4-hydroxy-3-methylbut-2-ethylamino) purine) (SIGMA) was added to 2 ml of 1N HCl (Wako) and dissolved. Then, distilled water was added to fill up to 10 ml, and the filter was sterilized with Sterilip Filter Unit (0.22 μm PES) (MILLIPORE). This was used as a stock solution, aliquoted into 1 ml sterilized 1.5 ml tubes and stored at -30 ° C.

2. Medium (1) Callus-derived mannitol-containing medium (CI-0.2Man: Callus Induction-0.2M Mannitol)
650 ml of distilled water, 1 package of mixed salt for Murashige-Skoog medium (Nippon Pharmaceutical), 7.3 g of D (-)-mannitol (Wako), 500 mg of L (+)-glutamine (Wako), L (-) -Plorine (Wako) 115 mg, L-asparagine monohydrate (Wako) 150 mg, casein hydrates (DUCHEFA) 100 mg, MES (2- (N-morpholino) ethanesulphonic acid 1 1 ml of the stock solution was added, and 5N KOH was added to adjust the pH to 5.8. Filled up to 700 ml with distilled water, 2 g of phytagel (SIGMA) was added. Immediately after autoclaving, add 300 ml of maltose stock solution in a clean bench, stir well, then cool to about 50 ° C., then add 2 ml of 2,4-D stock solution, 1 ml of picorram stock solution, and 1 ml of ascorbic acid stock solution. did.

(2) Callus maintenance selection medium (CM-5B: Callus Maintenance-5 mg / L Bialapos)
It differs from CI-0.2Man medium in that mannitol is not added, maltose concentration and 2,4-D concentration are low, and bialaphos, which is a selective drug, is added.

  In 900 ml of distilled water, 1 package of mixed salt for Murashige-Skoog medium (Nippon Pharmaceutical), 40 g maltose monohydrate (Wako), 500 mg L (+)-glutamine (Wako), L (-)-plorine (Wako) 115 mg, 150 mg of L-asparagine monohydrate (Wako), 100 mg of casein hydrolysate (DUCHEFA), 1.95 g of MES (2- (N-morpholino) ethanesulfonic acid) (titanium), 1000 ml MS vitamin stock 5N KOH was added to adjust the pH to 5.8. Filled up to 1 L with distilled water and added 2 g of phytagel (SIGMA). After autoclaving and cooling to about 50 ° C., 0.5 ml of 2,4-D stock solution, 1 ml of picloram stock solution, 1 ml of ascorbic acid stock solution and 1 ml of bialaphos stock solution were added.

(3) High Osmotic Callus Maintenance Selection Medium (CM-5B-150 Mal: Callus Maintenance-5 mg / L Bialapos-150 g / L Maltose)
Only the maltose concentration is different from the CM-5B medium.

  650 ml of distilled water, 1 package of mixed salt for Murashige-Skoog medium (Nippon Pharmaceutical Co., Ltd.), 500 mg of L (+)-glutamine (Wako), 115 mg of L (-)-plorine (Wako), L-asparagine monohydrate (Wako) ) 150 mg, casein hydrolysate (DUCHEFA) 100 mg, MES (2- (N-morpholino) ethanolsulfonic acid) 1.95 g, 1000 × MS vitamin stock solution 1 ml, and 5N KOH added to adjust pH Adjusted to 5.8. Filled up to 700 ml with distilled water, 2 g of phytagel (SIGMA) was added. Immediately after autoclaving, add 300 ml of maltose stock solution in a clean bench, stir well, then cool to about 50 ° C., then 0.5 ml of 2,4-D stock solution, 1 ml of picramram stock solution, 1 ml of ascorbic acid stock solution 1 ml of bialaphos stock solution was added.

(4) Redifferentiation medium (SG-2B: Shot Growth-2 mg / L Bialabos)
In 900 ml of distilled water, 1.4 g of KNO ₃ (Wako), 300 mg of NH ₄ NO ₃ (Wako), 2.5 ml of L medium KH ₂ PO ₄ stock solution, 3 ml of L medium CaCl ₂ stock solution, L medium MgSO ₄ ml of the ₄ stock solution, 1 ml of the L medium Fe / Na / EDTA stock solution, 1 ml of the L medium micro inorganic salt stock solution, 30 g of maltose monohydrate (Wako), 200 mg of myo-inositol (Wako), and 1N KOH was added to adjust the pH to 5.7. Filled up to 1 L with distilled water and added 5 g of Agar (Powder) plant medium (Wako). After autoclaving and cooling to about 50 ° C., 0.1 ml of 2,4-D stock solution, 1 ml of 1000 × L vitamin stock solution, 1 ml of zeatin stock solution, and 0.4 ml of bialaphos stock solution were added.

(5) Rooting medium (RG-5B: Root Growth-5 mg / L Bialaphos)
It differs from SG-2B medium in that it does not contain 2,4-D and zeatin and has a high bialaphos concentration and pH.

In 900 ml of distilled water, 1.4 g of KNO ₃ (Wako), 300 mg of NH ₄ NO ₃ (Wako), 2.5 ml of L medium KH ₂ PO ₄ stock solution, 3 ml of L medium CaCl ₂ stock solution, L medium MgSO ₄ ml of the ₄ stock solution, 1 ml of the L medium Fe / Na / EDTA stock solution, 1 ml of the L medium micro inorganic salt stock solution, 30 g of maltose monohydrate (Wako), 200 mg of myo-inositol (Wako), and 1N KOH was added to adjust the pH to 5.8. Filled up to 1 L with distilled water and added 5 g of Agar (Powder) plant medium (Wako). After autoclaving and cooling to about 50 ° C., 1 ml of 1000 × L vitamin stock solution and 1 ml of bialaphos stock solution were added.

Gene transfer to immature wheat embryos Plant material As a plant material, the strain name “Bobwhite SH 98 26” (Pellegrineschi et al., 2002 Genome 45: 421-430) selected by CIMMYT as a strain particularly suitable for transformation in the wheat variety “Bobwhite” is used. Using.

2. Plant material cultivation Four seeds were sown in 2 pots every week to prepare plant material. Two types of soil, Sakata soil mix (Sakata Seed): Kureha Horticultural soil (Kureha Chemical) is mixed at a ratio of 2: 1 and sifted. Prepared. Two pieces of 5 cm square gauze were placed in the bottom hole of a polypot having a diameter of 18 cm and a height of 15 cm, and 450 ml of soil that did not pass through the sieve was spread by volume under the pot, and 1650 ml of the sieved soil was placed for 1650 ml. Four seeds were placed on a circle with a center of 9 cm, and 450 ml of soil that had been sieved was covered with 250 ml of soil that had not passed through the sieve. The soil was not pressed down strongly, and was grown for about 12 weeks in a short-day greenhouse (15 ± 2 ° C., 8 hours long day (natural light)). After 4 weeks, the pot soil was pressed from above. After 6 weeks, the pot was placed on the vat so as not to dry the soil, and after 8 weeks, additional fertilization was carried out until flowering began. In the 8th and 9th weeks, 500 ml of Hyponex stock solution 6: 10: 5 (Hyponex) was given, and 500 ml of 333-fold diluted solution was given thereafter. Moved to a long-day artificial weather room (18 ° C / 10 ° C, 16-hour day length (400W metal halide lamp Mitsubishi Neo BOC lamp 800μmol / m ² s), humidity 50-70%) 12 weeks before flowering. Ripened.

3. Pre-drying treatment The flowering date was checked for each ear, and it was placed directly on the floor of the meteorological room 1 week after the ear bloomed, and water was given sparingly so that the leaves would not withstand (about 400 ml per day). . Water supply was stopped 12 days after flowering (3 days before treatment), and 15 days after flowering, it was cut from the base of the stem and cut at 5 ° C, 5-7 Hitachi in a dark room, and dried slowly at low temperature. As a control for which no pretreatment was performed, water that had been continuously given and that was 18 days after flowering was used.

4). Extraction of immature embryos In order to introduce genes into immature embryos, immature seeds were sterilized, and immature embryos were extracted and pre-cultured in CI-0.2 Man medium.

  Immature seeds were taken from the ears, wrapped in gauze and tied with a rubber band. Rinse with 70% ethanol for about 1 minute, dilute 15 ml of sodium hypochlorite solution (Wako) 10 times with distilled water (effective chlorine concentration 0.5%), polyoxyethylene (20) sorbitan monolaurate (Tween 20) (Wako) ) Was added to a solution containing 70 μl, and sterilized by stirring for 15 minutes with a stir bar. Thereafter, the aseptic operation was performed. The immature seeds were washed 3 times with 100 ml of sterile water. While immature embryos were cut out with a scalpel while observing with a stereomicroscope, the immature embryos were excised and placed on a circle of 1 cm in the center of CI-0.2Man medium with the scutellum side facing up, 32 per plate. Pre-cultured at 25 ° C. in the dark for 3 to 5 hours. FIG. 1 shows a photograph of an immature embryo placed on a medium.

5. Gene transfer Gene transfer into an immature wheat embryo was performed as follows using a particle gun method.
(1) Transgene In the examples, a plasmid DNA (pAL156) containing a herbicide tolerance gene (bar) and / or a reporter gene (GUS: β-gluconidase) was used as the transgene. pAL156 was developed by Dr. Harwood of John Inns Center, UK, and designed to express the bar gene and GUS gene under the control of the maize ubiquitin promoter and the first intron (PUbi + In), respectively (Fig. 2). FIG. 2 shows the DraI cleavage site, the size of the excised fragment, and the region used as a probe in Southern analysis.

  Bialaphos, the active ingredient of the herbicide, selects cells that express the bar gene to be resistant to bialaphos, but there is no guarantee that other genes on the same plasmid are expressed in cells that are resistant to bialaphos. . In other words, conventionally, in many cases where a gene (target gene) to be transformed (to be expressed) is introduced, cells expressing the target gene have not been selected. Therefore, assuming that the GUS gene that is easily confirmed to be expressed is the target gene, the ratio of the transgenic individuals into which the selected bar gene has been introduced, and the transformation in which the GUS gene is expressed. The ratio at which the body was obtained was used as an evaluation index.

(2) Sterilization of gold particles 40 mg of 1 μm gold particles were weighed, 1 ml of 99.5% EtOH was added, and the mixture was well suspended in vortex. Thereafter, the gold particles were precipitated by centrifugation, and ethanol was removed. Thereafter, 1 ml of sterilized water was added to obtain a sterilized gold particle solution.

(3) DNA coating Prior to use, the sterilized gold particle-containing solution was carefully suspended using an ultrasonic generator (Tomy Seiko UR-20P), and 10 μl per shot was placed in a 1.5 ml tube. Thereafter, a plasmid DNA solution (1 μg / μl) purified by Qiagen Midi Kit (Qiagen) was added to 1 μg per shot and stirred by pipetting. Next, put 10 μl of CaCl ₂ (nacalai test) (2.5 M) per shot on the lid of a 1.5 ml tube, add 4 μl of Supermidin (SIGMA) (0.1 M), and stir by pipetting on the tube lid. The lid was closed and all the liquids were mixed. All operations so far were performed on ice. Immediately after mixing, the suspension was vigorously suspended with voltex for 5 minutes. After standing at room temperature for 3 minutes, the supernatant was removed and washed once with 70% ethanol and twice with 100% ethanol dehydrated with Molecular Sieves 3A (Wako). The supernatant was removed and 10 μl of dehydrated 100% ethanol was added per 1 shot and well suspended. It was poured into the center of the macro carrier in a clean bench and allowed to air dry.

(4) Introduction The particle gun uses a Biolistic PDS-1000 / He Particle Delivery System (BIO-RAD), and the pressure at the time of shooting is about 63.3 kg / cm ² (900 psi), and the distance to the target tissue is 6 cm. It was. Shot twice for each petri dish.

6). Propagation and Selection of Transfected Cells A plastic petri dish (Iwaki) with a diameter of 9 cm and a thickness of 2 cm was used for the culture, and surgical tape (3M) was used for all the seals during the culture. After gene introduction with particle gun, callus was induced with CI-0.2Man medium in the dark at 25 ° C, and transplanted to CM-5B or CM-5B-150Mal medium containing bialaphos as a selective drug 3 days after introduction treatment The cells were selected and cultured in the dark at 25 ° C. In selective culture, the number of calli placed on a petri dish was limited to 16. Two weeks later, the callus was transplanted into a new selection medium and further selected and cultured for 2 weeks. FIGS. 3A and 3B show photographs of callus immediately before transplantation to a redifferentiation medium when selective culture is performed with CM-5B medium and CM-5B-150Mal medium. When selective culture is performed on CM-5B medium, the volume of callus is greatly increased, but the ratio of the submerged site is large (Fig. 3A, the arrow indicates the submerged site). I did not get the shot. In the case of selective culture in CM-5B-150Mal medium, the volume increase was small compared to CM-5B medium, but the ratio of the submerged site was small (FIG. 3B).

7). Regeneration After 4 weeks of selective culture, the cells were transplanted to a regeneration medium. The culture conditions were moved to 25 ° C. in a bright place to promote redifferentiation and cultured for 3 weeks. In the redifferentiation medium, the number of calli placed on a petri dish was up to 8. There was a high probability that an individual (Fig. 3C) in which a regenerated shoot was formed and rooted in a redifferentiation medium was an individual into which a gene was introduced.

Only the regenerated shoots were transplanted to the rooting medium (FIG. 3D). In the rooting medium, the regenerated shoots were distinguished from each immature embryo from which they were derived, and the regenerated shoots placed on one dish were derived from 4 immature embryos. Individuals growing vigorously after 3 weeks in the rooting medium were selected and replanted in pots containing Kureha Horticultural soil (Kureha Chemical). Grow in a long-day artificial weather chamber (18 ° C / 10 ° C, 16-hour day length (400W metal halide lamp Mitsubishi Neo BOC lamp 800 μmol / m ² s), humidity 50-70%). A breeding bag was hung and self-bred. After about 5 months, self-grown seeds were harvested.

Confirmation of gene transfer and gene transfer efficiency and substantial transformation efficiency Confirmation of gene introduction In the obtained plant body, the presence or absence of the bar gene which is a selection marker gene was examined by PCR method. Genomic DNA was extracted from 100 mg of plant leaves 2 weeks after potting using DNeasy Plant Mini Kit (Qiagen), and PCR reaction was performed using primers prepared from sequences specific to the bar gene using this as a template. went.
Primer sequence: GGTCTGCACCATCGTCACC (SEQ ID NO: 1)
Primer sequence: GTCATGCCCAGTTCCCGTGCT (SEQ ID NO: 2)

  To the PCR reaction solution, add 10 pg genomic DNA, ExTaq (Takara) 0.25 U, 10 × attached buffer 1 μl, 2 mM dNTPs, 2.5 pmol each primer pair, 1 μl PCRx enhancer (Invitrogen), and fill to a total of 10 μl with sterile distilled water. Up.

  For PCR, TaKara PCR Thermal Cycler Dice (Takara) was used for 30 cycles of 95 ° C for 20 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. After the PCR reaction, 1.2% agarose gel (Agarose LO3 “TaKaRa”) electrophoresis was performed, and the PCR product was detected by staining with ethidium bromide, and the expected 421 bp bar gene fragment was detected. It was judged to be a transgenic individual.

2. Gene transfer efficiency Number of immature embryos that have been transferred from the number of transgenic individuals obtained in 4 combinations of the presence or absence of pre-drying treatment and maltose concentration (0.12M and 0.44M) in callus selection maintenance culture Per gene transfer efficiency (number of transgenic individuals / number of treated immature embryos × 100) was calculated. Table 1 shows the results of gene transfer efficiency in nine independent experiments under each condition.

  Regarding the gene transfer efficiency, the difference in the average value between the conditions was not significant due to large errors, but the efficiency was improved by performing the drying pretreatment regardless of the osmotic pressure conditions during callus selection maintenance culture. . Further, when the pretreatment for drying was not performed, the gene transfer efficiency was lowered due to the high osmotic pressure condition in callus selection maintenance culture.

3. Confirmation of transgene expression The expression of the GUS gene, which is the reporter gene, was examined by GUS staining of the leaves of the plant body that was determined to be a transgenic individual from the results of the PCR method. GUS activity (histochemical method) was performed as follows. About 10 leaf pieces of a width of several millimeters were added to 100 μl of GUS reaction solution (100 mM pH 7.0 sodium phosphate buffer, 1 mM X-gluc (5-bromo-4-chloro-3-indole-β-glucuronic acid), (5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% Triton X-100), the solution was infiltrated by aspirating with a vacuum pump for 5 minutes, and then incubated at 37 ° C. overnight. After completion of the reaction, it was decolorized with 70% ethanol, and those stained blue were judged as GUS expressing individuals. An example of GUS activity detection is shown in FIG.

4). Substantial transformation efficiency From the number of substantive transformants expressing the GUS gene in four combinations of the presence / absence of pre-drying treatment and maltose concentration (0.12M and 0.44M) in callus selection maintenance culture The substantial transformation efficiency per number of immature embryos subjected to gene transfer treatment (the number of GUS gene expressing individuals / number of treated immature embryos × 100) was calculated. The results of substantial transformation efficiency (GUS gene expression efficiency) in nine independent experiments under each condition are shown in Table 1 above, and a graph of Table 1 is shown in FIG. 6 (error bars are standard errors). . Substantial transformation efficiency was controlled by the conventional method (no pretreatment for drying, osmotic pressure condition (low) 0.12M) only when callus selection maintenance culture was performed under high osmotic pressure conditions after drying pretreatment. As a result, a significant difference was observed at the 5% level (* in FIG. 6), and the efficiency was improved about 7 times.

5. Estimation of transgene copy number The transgene copy number was analyzed by the Southern blotting method for the plants determined to be transgenic individuals from the results of the PCR method.

About 1 month after potting, 0.5 g of young leaves were sampled, and genomic DNA was extracted by the CTAB method. The DNA concentration was measured with a microspectral altimeter NanoDrop (scrum), and the genomic DNA (30 μg) of each gene-transferred individual was digested with the restriction enzyme DraI. As a standard for copy number estimation, the genome size of wheat is 16,000,000 kbp, the amount of introduced plasmid DNA (11 kbp) corresponding to 2, 4 and 8 copies to 30 μg of genomic DNA, 21 pg, 42 pg and 84 pg, respectively. A mixture of non-transformed genomic DNA (30 μg) and digested with the restriction enzyme DraI was prepared. After 0.9% agarose gel electrophoresis, In accordance with the standard method described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, A Laboratory Manual, 2nd Edition, it was subjected to alkali denaturation treatment and neutralization treatment, and blotted to a nylon membrane positive charge (Roche). A GUS gene-specific probe (region shown in FIG. 2) was prepared by PCR DIG probe synthesis kit (Roche). Primers used for probe preparation were as follows and labeled with digoxigenin using a PCR reaction.
Primer sequence: TTGAACTGCGTGATGCGAT (SEQ ID NO: 3)
Primer sequence: ACTGCCCTCTTCGCTGTACAG (SEQ ID NO: 4)

  The probe was hybridized overnight at 42 ° C. using DIG EasyHive (Roche) as the hybridization solution. Wash with 0.1 × SSC for 20 minutes twice at 65 ° C., DIG wash and block buffer set (Roche), AP-labeled anti-digoxigenin antibody, Fab fragment (Roche), CDP-star (Roche) as luminescent substrate The band was visualized by exposing to an X-ray film.

  The result of Southern analysis is shown in FIG. In FIG. 5, each condition and the presence or absence of GUS activity are shown on the upper side of the figure, and the position of the 4.63 kb band excised by DraI is shown on the right side of the figure, M is a λ HindIII molecular weight marker, NT is a non-introduced individual, 2C 4C and 8C show 2 copies, 4 copies and 8 copies of plasmid DNA mixed with genomic DNA of non-introduced individuals and digested with DraI, respectively.

  A band was not detected in the non-transformant, whereas some band was detected in all the transgenic individuals examined. In all individuals in which GUS activity was detected, a band at a position of 4.63 kbp indicating that the expression unit of the reporter gene was successfully introduced was detected. The other bands are considered to have been introduced by reconstitution of the transgene or part of the transgene cut out and incorporated. From comparison of the 4.63 kbp band density and the standard band density for copy number estimation, the copy number of the normal expression unit was determined to be callus under osmotic conditions of 0.44 M with or without pre-drying treatment. In the case of selective maintenance culture, the number of copies was about 1 copy, whereas in the case of 0.12M osmotic pressure condition, the number of copies was large, 3 to 10 copies.

  When callus selection maintenance culture was performed under high osmotic pressure conditions without drying pretreatment, the adverse effect on gene transfer efficiency was large. By performing the pre-drying treatment, this adverse effect is improved, and it is considered that the pre-drying treatment has an effect of acclimatization that can withstand callus selection maintenance culture under high osmotic pressure conditions. It is considered that the copy number of the gene introduced by callus selection maintenance culture under high osmotic pressure conditions is suppressed, and this increases the probability that the transgene is expressed. From these facts, it is considered that substantial improvement in transformation efficiency was achieved by combining the pretreatment for drying and selective culture under high osmotic pressure conditions.

6). Genetic isolation of the transgene The resulting transformants were examined for genetic isolation of the transgene. Twenty-four self-propagating seeds were sown on two lines in which the copy number of the transgene was small and the expression of the GUS gene was confirmed, and one line in which the copy number of the transgene was large and the expression of the GUS gene was confirmed. The presence or absence of the bar gene was examined by the PCR method. As a result, in the progeny of the lines with a small number of copies of the transgene, the separation status of individuals holding the bar gene: individuals not holding the bar gene was 15: 9 and 16: 7 (no single germination). It was. The separation ratio of 1 locus 3: conforming to 1 segregation ratio (chi ² test). Further, when the GUS activity was examined, the GUS activity was detected only in the individual carrying the bar gene. On the other hand, in the progeny of the strain with a large number of copies of the transgene, all 24 individuals retained the bar gene, but only 3 of them detected faint GUS activity.

  In this way, stable expression of the target transgene was normally inherited in lines with a small number of copies of the transgene obtained by pretreatment and selective callus culture under high osmotic pressure conditions. . In a transformation system, a transgene is stably expressed, and can be used for gene function analysis only when the transgene is stably inherited in accordance with the segregation of the transgene. Under improved conditions, transformants that can be used for such gene function analysis could be efficiently produced.

  The present invention makes it possible to increase the expression efficiency of a target gene introduced in wheat.

The wheat immature embryo immediately after gene transfer treatment placed on the medium is shown. The structure of plasmid pAL156 containing the transgene is shown. The callus after 4 weeks of selective culture in a callus maintenance selective medium (CM-5B medium) containing 0.12 M maltose is shown. The callus after 4 weeks of selective culture in a callus maintenance medium (CM-5B-150Mal medium) containing 0.44 M maltose is shown. The shoots regenerated after 3 weeks of culture in the regeneration medium are shown. The redifferentiated plant body after 3 weeks culture | cultivation in a rooting medium is shown. The detection of GUS activity in the leaves of redifferentiated plants is shown. The detection of the transgene by Southern blotting is shown. The gene transfer efficiency and the transgene expression efficiency per treated explant (immature embryo) are shown.

Claims (4)

The immature embryos obtained by collecting ears in 10-17 days after flowering of wheat 4-6 and 5-7 days cold dry in the dark at ° C., immature embryos or purpose to the cell by transformation operation by particle bombardment Introducing the gene and culturing the immature embryo or its cells in a medium containing 0.25 to 1.0 M maltose to improve the expression efficiency of the target gene introduced in the wheat immature embryo or its cells Method.   The method according to claim 1, wherein the transformed wheat immature embryo or a cell thereof contains one or two copies of the introduced gene of interest. A method for producing a plant with improved expression efficiency of an introduced target gene, comprising regenerating a plant body using a transformed wheat immature embryo produced by the method according to claim 1 or 2 , or a cell thereof. The method according to claim 3 , comprising transferring a target gene introduced into a plant body to a progeny.

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