Soybean genetic transformation method for improving positive transformation rate

Abstract

The invention discloses a soybean genetic transformation method for improving positive transformation rate, which comprises the following steps of: infecting a soybean explant with a recombinant expression vector containing a target gene and a marker gene through agrobacterium liquid; an ALS inhibitor is added into a bud induction culture medium and a bud elongation culture medium to improve screening culture of the positive conversion rate of the explants, and a transformant is obtained. According to the soybean genetic transformation method which takes the gene cloned from the cynodon dactylon as the marker gene and the ALS inhibitor as the screening agent, the positive transformation rate is increased to 6% or above, the maximum is 12.05%, and the soybean transformation efficiency is remarkably improved.

Description

(I) Technical field

[0001] The invention relates to a plant transformation method, in particular to a soybean genetic transformation method for improving the positive transformation rate. (II) Background technology

[0002] Soybean (scientific name: Glycine max (Linn.) Merr.) provides humans with abundant plant protein and oil, and is one of the most important grain and oil crops in the world. In 2023, the global area of ​​genetically modified crops was 206.3 million hectares, of which genetically modified soybeans accounted for 48.9%, making it the most widely planted genetically modified crop. Whether used for industrialization or basic research, gene transformation is the key technology to obtain genetically modified soybeans. Transgenic plants with commercial application value are generally selected from a large number of transformants, but soybeans are still a crop with low transformation efficiency due to the influence of the transformation system and receptor system. An important factor causing low soybean transformation efficiency or failure to obtain transformants is the inefficiency of the screening system. Appropriate marker genes play a vital role in the transformation process of soybean heritage. At present, the marker genes used in Agrobacterium-mediated soybean genetic transformation are mostly antibiotic resistance genes (nptII gene and hpt gene, etc.) and herbicide tolerance genes (bar gene, pat gene and EPSPS gene, etc.).

[0003] Bermuda grass is one of the most widely used fine grass species in the south of the Yellow River Basin in China. In the middle and lower reaches of the Yangtze River, it is often used to build lawns, sports fields and football fields. At the same time, it can also be used for soil consolidation and slope protection in roads, railways, reservoirs, etc. Because the grass stems of Bermuda grass contain a lot of protein, the grass is soft, the taste is light and slightly sweet, the leaves are abundant, and the palatability is good, it is a high-quality green fodder for horses, cattle, sheep, rabbits, pigs, poultry, and herbivorous fish. In addition, Bermuda grass has the functions of relieving fever and diuresis, relaxing muscles and activating blood circulation, and detoxification, and can be used as a Chinese medicinal material. Therefore, Bermuda grass is very safe for both animals and humans.

[0004] Acetolactate synthase (ALS) inhibitor herbicides control weeds by inhibiting acetolactate synthase, which is the first key enzyme in the biosynthesis pathway of branched-chain amino acids (isoleucine, leucine and valine), which are essential for protein synthesis and plant growth. The ALS enzyme is only present in plants and microorganisms. Treating plants with ALS inhibitor herbicides will not affect the biosynthesis of branched-chain amino acids in humans and animals. Therefore, ALS inhibitor herbicides are biosafe for animals and humans.

[0005] It has been reported that the ALS inhibitor bensulfuron-methyl was applied externally during soybean transformation to obtain transformants, but the positive rate of plants was low. For example, Chinese patent CN117511987A disclosed a method for genetic transformation of corn using a gene cloned from Bermudagrass as a marker gene and flazasulfuron-methyl as a screening agent, but the transformation rate was only 4.4%. Therefore, it is necessary to find a method to improve the positive rate of soybean genetic transformation plants. (III) Summary of the invention

[0006] The purpose of the present invention is to provide a soybean genetic transformation method for improving the positive transformation rate, thereby overcoming the defects of low positive rate of plants obtained in the prior art.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The invention provides a soybean genetic transformation method for improving positive transformation rate. The method comprises the following steps: using a recombinant expression vector containing a target gene and a marker gene, transforming soybean explants by an Agrobacterium-mediated transformation method, and adding an ALS inhibitor to a bud induction medium and a bud elongation medium to improve the positive transformation rate of the explants for screening and culturing, so as to obtain transformants; the ALS inhibitor comprises a sulfonylurea herbicide and a triazolopyrimidine herbicide; and the concentration of the ALS inhibitor in the bud induction medium and the bud elongation medium is 0.025-0.4 mg ai / L.

[0009] Furthermore, the recombinant expression vector (denoted as pXA) contains a protein expression cassette encoding a target gene and a protein expression cassette encoding a marker gene. The protein expression cassette encoding the target gene includes a CP4 EPSPS protein expression cassette (the promoter is Arabiaposis ubi10 promoter, the terminator is nopaline synthase terminator NOS-ter, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO.2) and a Bar protein expression cassette (the promoter is the cauliflower mosaic virus 35S gene promoter, the terminator is the cauliflower mosaic virus 35S gene terminator, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO.3). The protein expression cassette encoding the marker gene is a CdP450 protein expression cassette (the promoter is the Scrophulariaceae mosaic virus promoter FMVpromoter, the terminator is the nopaline synthase terminator NOS terminator, the marker gene is derived from Cynodon dactylon, the nucleotide sequence of the marker gene is shown in SEQ ID NO.1, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO.4).

[0010] Furthermore, the sulfonylurea herbicides include fluazifop-methyl and pyrazosulfuron-methyl; and the triazolopyrimidine herbicides include penoxsulam.

[0011] Furthermore, the concentration of the ALS inhibitor in the bud induction medium is 0.1 mg ai / L or 0.2 mg ai / L; and the concentration in the bud elongation medium is 0.025 mg ai / L or 0.05 mg ai / L.

[0012] Furthermore, it is preferred that the concentration of the ALS inhibitor in the bud induction medium is 0.2 mg ai / L; and the concentration in the bud elongation medium is 0.025 mg ai / L.

[0013] Furthermore, the soybean varieties include Tianlong No. 1 and Wandou 28.

[0014] Furthermore, more preferably, when the soybean explant is derived from Tianlong No. 1, the ALS inhibitor is selected from pyrazosulfuron-methyl or pyrazosulfuron-methyl, and the concentration in the bud induction medium is 0.1 or 0.2 mg ai / L, and the concentration in the bud elongation medium is 0.025 mg ai / L; when the soybean explant is derived from pea 28, the ALS inhibitor is selected from pyrazosulfuron-methyl, and the concentration in the bud induction medium is 0.1 or 0.2 mg ai / L, and the concentration in the bud elongation medium is 0.025 mg ai / L.

[0015] Furthermore, the soybean genetic transformation method of the present invention specifically comprises the following steps:

[0016] (1) transforming Agrobacterium with a recombinant expression vector containing a target gene and a marker gene obtained from Bermudagrass, and soaking soybean explants in the Agrobacterium resuspension to obtain infected soybean explants;

[0017] (2) placing the soybean explants infected in step (1) in a dark incubator at 21-22° C. for co-cultivation for 2-5 days to obtain co-cultivated explants;

[0018] (3) The explants after co-cultivation in step (2) are transferred to a recovery medium without ALS inhibitors and cultured at 26-28° C. for 7-9 days with a light:dark cycle (16 h:8 h).

[0019] (4) transferring the explants in step (3) into a bud induction screening medium containing an ALS inhibitor, culturing at 26-28° C. for 14 days with the same illumination time as in step (3), to obtain explants with new buds; the ALS inhibitor is fluazifop-methyl, pyrazosulfuron-methyl or penoxsulam;

[0020] (5) The explants with newly formed shoots in step (4) are transferred into a shoot elongation medium containing an ALS inhibitor, cultured at 26-28° C., and subcultured every 3 weeks until the transformed cells regenerate into plants or the regenerated plants are able to survive. The illumination time is the same as in step (3).

[0021] Furthermore, the concentration of fluazifop-methyl, pyrazosulfuron-methyl or penoxsulam in step (4) is 0.025-0.4 mg ai / L, preferably 0.1 mg ai / L or 0.2 mg ai / L; the concentration of fluazifop-methyl, pyrazosulfuron-methyl or penoxsulam in step (5) is 0.025-0.4 mg ai / L, preferably 0.025 mg ai / L or 0.05 mg ai / L.

[0022] Further, the infection medium in step (1) is based on B5 salt as a basic medium, and also includes B5 vitamin, 2-morpholineethanesulfonic acid, sucrose, glucose, dithiothreitol, 6-benzylaminopurine, gibberellin, and acetosyringone; the concentration of B5 salt can be 1-10 g / L, the concentration of B5 vitamin can be 0.1-5 mL / L, the concentration of 2-morpholineethanesulfonic acid can be 0.1-5 g / L, the concentration of sucrose can be 5-100 g / L, the concentration of glucose can be 5-100 g / L, the concentration of dithiothreitol can be 50-300 mg / L, the concentration of 6-benzylaminopurine can be 100-200 mg / L, and the concentration of 2-morpholineethanesulfonic acid can be 0.1-5 g / L. The concentration of gibberellin can be 0.1-5 mg / L, the concentration of gibberellin can be 0.05-1 mg / L, and the concentration of acetosyringone can be 10-100 mg / L; the above ingredients can be arbitrarily combined within their concentration ranges, and the preferred infection medium is: B5 salt 3.21 g / L, B5 vitamin 1 mL / L, 2-morpholineethanesulfonic acid 2.5 g / L, sucrose 20 g / L, glucose 10 g / L, dithiothreitol 154.2 mg / L, 6-benzylaminopurine 1.67 mg / L, gibberellin 0.25 mg / L, acetosyringone 40 mg / L, pH 5.4.

[0023] Further, in step (3), the recovery medium is based on MS salt as the basal medium, and also includes B5 vitamin, 2-morpholineethanesulfonic acid, sucrose, 6-benzylaminopurine, timentin, and agar; the concentration of MS salt can be 1-10 g / L, the concentration of B5 vitamin can be 0.1-5 mL / L, the concentration of 2-morpholineethanesulfonic acid can be 0.1-5 g / L, the concentration of sucrose can be 5-100 g / L, and the concentration of 6-benzylaminopurine can be 0.1-5 mg / L. / L, the concentration of timentin can be 100-300 mg / L, and the concentration of agar can be 5-10 g / L; the above ingredients can be arbitrarily combined within their concentration ranges, and the preferred recovery medium is: MS salt 4.33 g / L, B5 vitamin 1 mL / L, 2-morpholineethanesulfonic acid 1 g / L, sucrose 30 g / L, agar 8 g / L, 6-benzylaminopurine 1.67 mg / L, timentin 200 mg / L, agar 8 g / L, pH 5.6.

[0024] Further, in step (4), the bud induction screening medium is based on MS salt as the basic medium, and further comprises B5 vitamin, 2-morpholineethanesulfonic acid, sucrose, 6-benzylaminopurine, timentin, a screening agent, and agar; the concentration of MS salt can be 1-10 g / L, the concentration of B5 vitamin can be 0.1-5 mL / L, the concentration of 2-morpholineethanesulfonic acid can be 0.1-5 g / L, the concentration of sucrose can be 5-100 g / L, the concentration of 6-benzylaminopurine can be 0.1-5 mg / L, the concentration of timentin can be 100-300 mg / L, and the concentration of A can be 100-300 mg / L. The concentration of LS inhibitor (fluazuron-methyl, pyrazosulfuron-methyl or penoxsulam) can be 0.1-1 mg / L, and the concentration of agar can be 5-10 g / L. The above ingredients can be arbitrarily combined within their concentration ranges; the preferred bud induction screening medium includes: MS salt 4.33 g / L, B5 vitamin 1 mL / L, 2-morpholineethanesulfonic acid 1 g / L, sucrose 30 g / L, agar 8 g / L, 6-benzylaminopurine 1.67 mg / L, timentin 200 mg / L, ALS inhibitor (fluazuron-methyl, pyrazosulfuron-methyl or penoxsulam) 0.1 mg ai / L or 0.2 mg ai / L, agar 8 g / L, and pH 5.6.

[0025] Further, in step (5), the bud elongation medium is based on B5 salt as the basal medium, and also includes B5 vitamin, 2-morpholineethanesulfonic acid, aspartic acid, glutamine, sucrose, gibberellin, indoleacetic acid, zeatin, timentin, ALS inhibitor, and agar; the concentration of B5 salt can be 1-10 g / L, the concentration of B5 vitamin can be 0.1-5 mL / L, the concentration of 2-morpholineethanesulfonic acid can be 0.1-5 g / L, the concentration of aspartic acid can be 10-100 mg / L, the concentration of glutamine can be 10-100 mg / L, the concentration of sucrose can be 5-100 g / L, the concentration of gibberellin can be 0.1-5 mg / L, the concentration of indoleacetic acid can be 0.05-1 mg / L, and the concentration of zeatin can be 0.1- 5mg / L, the concentration of timentin can be 100-300mg / L, the concentration of ALS inhibitor (flysulfuron-methyl, pyrazosulfuron-methyl or penoxsulam) can be 0.01-0.1mg / L, the concentration of agar can be 5-10g / L, and the above ingredients can be arbitrarily combined within their concentration ranges; preferably, the bud elongation medium: B5 salt 3.21g / L, B5 vitamin 1mL / L, 2-morpholineethanesulfonic acid 1g / L, aspartic acid 50mg / L, glutamine 50mg / L, sucrose 30g / L, agar 8g / L, gibberellin 0.5mg / L, indoleacetic acid 0.1mg / L, zeatin 1mg / L, timentin 200mg / L, screening agent (flysulfuron-methyl, pyrazosulfuron-methyl or penoxsulam) 0.025mg ai / L or 0.05mg ai / L, agar 8g / L, pH 5.6.

[0026] The present invention adopts CP4 EPSPS rapid test paper strip to detect soybean positive transformants.

[0027] Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

[0028] 1. The present invention provides a soybean genetic transformation method for improving the positive transformation rate of plants. Using a specific ALS inhibitor herbicide as a screening agent, at a specific screening concentration, the positive transformation rate is increased to more than 6%, up to more than 12%, and the positive plant rate is more than 87%, which is significantly higher than the existing method.

[0029] 2. The transgenic soybean obtained by the present invention has a certain tolerance to ALS inhibitor herbicides, glyphosate and glufosinate ammonium, and can be directly used for the development of products tolerant to multiple herbicides. At the same time, ALS inhibitor herbicides and glufosinate ammonium can be used in rotation or in combination with glyphosate to alleviate the problem of increasing glyphosate-resistant weeds. (IV) Description of the drawings

[0030] Figure 1 This is a map of the pXA transformation vector.

[0031] Figure 2 The flowchart of soybean transformation. a: Sterilization of soybean seeds; b: Germination of soybean seeds; c: Infection of explants with Agrobacterium; d: Co-cultivation of explants with Agrobacterium; e: Recovery; f: Screening; g, h: Elongation; i: Transplantation.

[0032] Figure 3 The phenotypes of soybean explants treated with different effective concentrations of flazasulfuron-methyl. a: phenotype of soybean explants after 2 weeks; bf: phenotype of soybean explants after 4 weeks.

[0033] Figure 4 Analysis of herbicide tolerance of T1 transgenic soybean plants. (a) T1 transgenic soybean plants treated with flazasulfuron-methyl for 10 days, with a dosage of 30 g ai ha -1 (b) T1 transgenic soybean plants treated with glyphosate 10 days later at a dose of 900 g ae ha -1 (c) T1 transgenic soybean plants treated with glufosinate for 10 days at a glufosinate dosage of 600 g ai ha -1 .

[0034] ai=(active ingredient); ae=(acid equivalent)

[0035] Figure 5 is the target protein expression level in T1 generation transgenic soybean plants. (V) Specific implementation methods

[0036] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0038] The present invention soaks soybean explants in an infection solution including recombinant Agrobacterium to obtain infected explants, and transforms exogenous genes into soybean explants using recombinant Agrobacterium. Agrobacterium infection is a conventional method in the art, and the parts not described in the present invention can be carried out according to conventional means.

[0039] In the present invention, the method for constructing the recombinant Agrobacterium can preferably be carried out according to the following method: cloning the P450 gene and other target genes (such as herbicide resistance genes, insect resistance genes, drought resistance genes, etc.) into the same expression vector to construct a recombinant expression vector; and introducing the recombinant expression vector into Agrobacterium to obtain the recombinant Agrobacterium. The present invention does not specifically limit other target genes.

[0040] In the present invention, the soybean explants include but are not limited to soybean embryo tip growth point explants or soybean callus. In the present invention, the varieties of the soybean explants include but are not limited to Tianlong No. 1 and Wandou 28.

[0041] The reagents and sources involved in the following examples are as follows: B5 basic salt (PhytoTech, G398), MS basic salt (PhytoTech, M524), N6 basic salt (PhytoTech, C416), B5 vitamin (PhytoTech, G219), agar (Sigma, A7921), sucrose (Shanghai Test, 10021418), 2-morpholineethanesulfonic acid (Sigma, M3671), acetosyringone (PhytoTech, A104), aspartic acid (Sigma, A7219), glutamine (Sigma, G8540), 6-benzylaminopurine (Sigma, B3408), indoleacetic acid (Sigma, 13750), gibberellin (Sigma, 488 70), zeatin (Sigma, Z0375), timentin (Yeasen, T6315180), flazasulfuron-methyl (ISK, PD390), penoxsulam (Corteva, PD20170671), pyrazosulfuron-methyl (Lianyungang Liben Pesticide Chemical Co., Ltd., PD20086251), diquat (Shandong Shengpeng Technology Co., Ltd., PD20160776), glyphosate (Bayer, PD73-88), glufosinate (BASF, PD20096847), sodium chloride (Shanghai Trial, 10019308), yeast extract (Sigma, Y1625), peptone (Sigma, P5905), kanamycin (PhytoTech, K378), tetracycline (PhytoTech, T859).

[0042] Example 1: Using Tianlong No. 1 as the soybean explant source, according to Figure 2 Process, establish soybean genetic transformation method using ALS inhibitor herbicide fluazifop-methyl as screening agent

[0043] 1. Choice of receptor material

[0044] In order to maintain the vitality and stability of soybean seeds, Tianlong No. 1 seeds harvested from the field are stored in a cold storage. A certain number of seeds are taken out each time they are used, and the seeds that are shriveled, have disease spots on the surface, and are cracked are removed. The remaining seeds are used for explant infection.

[0045] 2. Sterilization and Germination of Recipient Materials

[0046] Place the selected mature soybean seeds in a plastic culture dish, and place it in a desiccator in a fume hood. Place a 250mL beaker in the middle of the desiccator, which contains 100mL of a 30% sodium hypochlorite aqueous solution. Add 8mL of concentrated hydrochloric acid dropwise along the wall of the beaker, close the desiccator and seal it, and close the glass of the fume hood at the same time, so that the soybean seeds are exposed to the chlorine in the desiccator for sterilization for 16 hours. Inoculate 15-18 sterilized soybean seeds per dish in soybean germination medium (3.21g / L B5 salt, 20g / L sucrose, 8g / L agar, pH 5.8), insert the hilum side of the soybean seeds into the medium, wrap the culture dish with plastic wrap after inoculation, and place it in an incubator for germination for 16 hours, and the culture condition is 26±2℃. After germination of soybean seeds for 16 hours, the imbibed soybean seeds were cut horizontally at 3-5 mm near the cotyledon node on the hypocotyl, and then cut longitudinally along the hilum to remove one cotyledon, retain the other cotyledon with the embryo and hypocotyl attached, remove the seed coat and primary leaves, and thus obtain explants.

[0047] 3. Screening Agent Sensitivity Determination

[0048] In order to better distinguish transformed cells from untransformed cells in the screening stage, the sensitivity test of explants to flazasulfuron was first conducted. A flazasulfuron mother solution with an effective concentration of 0.5 mg / mL was prepared with deionized water. A certain amount of flazasulfuron mother solution was added to the prepared soybean germination medium cooled to about 45°C, so that the final effective concentration of flazasulfuron was as follows: 0 mg / L, 0.025 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L and 0.4 mg / L, and the solid culture medium plate was formed by standing. Explants were prepared according to step 2. The prepared explants did not need to be infected with Agrobacterium, but were directly inserted into the solid culture medium plate (the end with the hypocotyl facing down), and cultured at 26°C, 16h light / 8h dark conditions for 4 weeks, and the growth was observed and recorded every week. After 4 weeks, the number of surviving explants in each treatment was counted, and the regeneration frequency was calculated based on the total number of explants. The calculation method of regeneration frequency was referred to Paz (Paz et al., 2006). Explants with one or more buds were considered to have successfully regenerated. The results showed that compared with the blank control, 0.025 mg ai / L of flazasulfuron had a certain inhibitory effect on the growth of explants. With the increase of flazasulfuron concentration, the inhibitory effect showed an upward trend. When the flazasulfuron concentration was 0.4 mg ai / L, the explant survival rate was 0 ( Figure 3 and Table 1).

[0049] Table 1 Effects of different concentrations of flazasulfuron on explant embryo growth

[0050]

[0051] *Indicates that a shoot with a height greater than 1 cm was counted as a viable explant.

[0052] 4. Preparation of infection solution

[0053] The recombinant expression vector pXA( Figure 1 ) contains a CdP450 protein expression cassette (nucleotide sequence shown in SEQ ID NO.4), a CP4 EPSPS protein expression cassette (nucleotide sequence shown in SEQ ID NO.2) and a Bar protein expression cassette (nucleotide sequence shown in SEQ ID NO.3). The exogenous T-DNA containing the target gene (CP4 EPSPS, Bar) and the marker gene (CdP450) is connected into the cloning vector pCAMBIA1300 (GeneBank: AF234296.1) to obtain a recombinant expression vector. The recombinant expression vector is introduced into Agrobacterium EHA105 by electroporation, and after verification, a recombinant Agrobacterium containing the transformation vector is obtained.

[0054] The recombinant Agrobacterium was inoculated on YEP solid medium containing 15 μg / mL tetracycline and 50 μg / mL kanamycin, cultured at 28°C for 48 h, and the colonies were scraped into infection solution (B5 salt 3.21 g / L, B5 vitamin 1 mL / L, 2-morpholineethanesulfonic acid 2.5 g / L, sucrose 20 g / L, glucose 10 g / L, dithiothreitol 154.2 mg / L, 6-benzylaminopurine 1.67 mg / L, gibberellin 0.25 mg / L, pH 5.4), suspended and mixed, and the B5 liquid medium was used as blank control, and the OD was detected by spectrophotometer. 660 nm, use B5 liquid medium to adjust the concentration of Agrobacterium tumefaciens to OD 600 nm is 0.5-0.6, and then 40 mg / L acetosyringone is added to obtain the Agrobacterium infection solution.

[0055] 5. Infection and Co-cultivation

[0056] Soak the explants from step 2 in the Agrobacterium infection solution from step 4 and place at room temperature for 3 hours. Take out the infected explants, dry the liquid on the surface of the explants with sterile filter paper, and transfer them to sterile plastic culture dishes with the cotyledons facing up, 15-18 cotyledons per dish. Seal with parafilm and culture in a constant temperature dark incubator at 21-22°C for 3 days.

[0057] 6. Resume Culture

[0058] The elongated hypocotyls of the explants after co-cultivation were cut off, and then the explants with the cut hypocotyls were transferred to recovery medium (MS salt 4.33 g / L, B5 vitamin 1 mL / L, 2-morpholineethanesulfonic acid 1 g / L, sucrose 30 g / L, agar 8 g / L, 6-benzylaminopurine 1.67 mg / L, timentin 200 mg / L, pH 5.6), and cultured at 26-28°C for 9 days to eliminate Agrobacterium and provide a recovery period for the infected cells.

[0059] 7. Screening Culture and Shoot Elongation Culture

[0060] According to the number of explants that survived under different concentrations of pyrazosulfuron selection pressure, 0.1 mg ai / L and 0.2 mg ai / L were selected as the concentrations of pyrazosulfuron in the bud induction screening medium. After the recovery period, the explants were transferred to the bud induction screening medium containing the screening agent (MS salt 4.33 g / L, B5 vitamin 1 mL / L, 2-morpholineethanesulfonic acid 1 g / L, sucrose 30 g / L, agar 8 g / L, 6-benzylaminopurine 1.67 mg / L, timentin 200 mg / L, pyrazosulfuron 0.1 mg ai / L or 0.2 mg ai / L, pH 5.6), thereby causing the transformed cells to selectively grow, and the screening culture was carried out for 2 weeks at a temperature of 26-28°C and a photoperiod (light / dark) of 16:8. The resistant tissue treated with the selection agent was removed from the bud induction selection medium, the dead tissue and the cotyledons attached thereto were cut off, and the resistant tissue was transferred (obliquely inserted) to a bud elongation medium containing the selection agent (B5 salt 3.21 g / L, B5 vitamin 1 mL / L, 2-morpholineethanesulfonic acid 1 g / L, aspartic acid 50 mg / L, glutamine 50 mg / L, sucrose 30 g / L, agar 8 g / L, gibberellin 0.5 mg / L, indoleacetic acid 0.1 mg / L, zeatin 1 mg / L, timentin 200 mg / L, flazasulfuron 0.025 mg ai / L or 0.05 mg / L). ai / L, pH5.6), screen and culture at 26-28°C and a photoperiod (light / dark) of 16:8, and transfer to a new elongation medium every 3 weeks. If the buds elongate, the lid of the culture dish is replaced with a high lid or transferred to a larger plastic box. When the height of the buds in the plastic box grows to a position close to the lid, wash off the substrate at the bottom of the buds and transplant them to pots for growth.

[0061] The infected, co-cultivated and recovered explants were inserted into the bud induction screening medium and cultured at 26-28°C for 2 weeks. After 2 weeks, the explants with new buds were transferred to the elongation medium containing 0.05 mg ai / L flazasulfuron. As can be seen from Table 2, the results of the first three batches of tests showed that the elongation frequencies of the two combinations were relatively low. Then the concentration of flazasulfuron in the subsequent elongation medium was adjusted to 0.025 mg ai / L. The results showed that the elongation frequencies of the combinations of 0.2 mg ai / L and 0.025 mg ai / L, 0.1 mg ai / L and 0.025 mg ai / L were higher than those of the combinations of 0.2 mg ai / L and 0.05 mg ai / L, and 0.1 mg ai / L and 0.05 mg ai / L.

[0062] Table 2 Combinations of concentrations of the screening agents used in the bud induction screening medium and the bud elongation medium

[0063]

[0064]

[0065] Elongation frequency (%) = number of bud elongation / total number of explants infected × 100

[0066] Positive rate (%) = number of positive plants / number of bud elongation × 100

[0067] 8. Conversion Rate

[0068] All T0 generation transformants were tested with CP4 EPSPS immune test strips. CP4 EPSPS test strips were purchased from Shanghai Youlong Biotechnology Co., Ltd., item number: AA0832-LSGB. 50-100 mg of leaves were taken from each transformant, and non-transgenic soybeans were taken as controls. They were placed in 2 mL centrifuge tubes, a steel ball was placed in the tube, and the tubes were frozen with liquid nitrogen and crushed on a grinder. Be careful not to cross-contaminate between samples. 300 μL of protein extraction buffer was added to each centrifuge tube, and the test strips were placed in the centrifuge tube according to the instructions. The results were read after 5 minutes. The non-transgenic soybean rapid test paper only showed C line, and the rapid test paper of the positive T0 generation transformant showed C line and T line. According to the color development results, the conversion rate was statistically calculated. The results showed that the combination of 0.2 mg / L and 0.025 mg / L had the highest conversion rate, and the average positive conversion rate reached 12.05% (Table 3).

[0069] Table 3 Conversion rate of Tianlong No. 1 under treatment with different concentrations of flazasulfuron-methyl

[0070]

[0071] Positive transformation rate (%) = number of positive transformants / total number of explants infected × 100

[0072] Example 2: Herbicide tolerance test of T1 transgenic soybeans

[0073] The T1 transgenic plants XA-2, XA-17, XA-24 and the non-transgenic control Tianlong No. 1 selected in Example 1 were planted in a greenhouse and treated with 30 g ai ha -1 Fyazoxanthone, 900g ae ha -1 Glyphosate and 600g ai ha -1 Glufosinate treatment. Figure 4 The effect of herbicide spraying 10 days later. We found that all transgenic lines showed high tolerance to fluazifop-butyl after 10 days. Sensitive plants showed yellowing symptoms 4-5 days after fluazifop-butyl treatment, and then slowly withered and died ( Figure 4 (a). Transformants XA-17 and XA-24 grew normally after being treated with glyphosate, while the growth of XA-2 was slightly inhibited, while the growth of non-transgenic soybean Tianlong No. 1 was severely inhibited ( Figure 4 b). After 10 days of glufosinate treatment, the leaves of Tianlong No. 1 were severely withered. The transformant XA-17 had a high tolerance to glufosinate, while XA-2 and XA-24 were affected to varying degrees ( Figure 4 (c).

[0074] Example 3: Determination of target protein expression in T1 transgenic soybeans

[0075] The CdP450 kit used in this study was purchased from Zhongding Bio's protein-specific ELISA detection kit, and the CP4EPSPS and Bar enzyme-linked immunosorbent quantitative detection kits were purchased from Shanghai Youlong Biotechnology Co., Ltd.

[0076] 1.CdP450 kit operation steps:

[0077] 1) Take the test kit out of the refrigerated environment in advance and place it at room temperature (20-25°C) for more than 20 minutes to allow all reagents and required strips to return to room temperature. Shake each liquid reagent well before use.

[0078] 2) Take 40-50 mg soybean tissue (crush the seeds first), put it into a 2 mL centrifuge tube, add steel balls, freeze it in liquid nitrogen, and grind it. Add 600 μL PBS buffer, incubate it at 4 °C for 1 hour, then centrifuge it at 12000 rpm for 10 minutes, take the supernatant, dilute it a certain multiple and then test it, and calculate the OD 450 The value is controlled within the measurable range.

[0079] 3) Take 1mL of double distilled water and add it to the standard protein. Tighten the tube cap, turn it upside down several times, and mix it gently after it is fully dissolved (concentration 200ng / ml). Then use PBS buffer to make multiple dilutions (Note: do not make multiple dilutions directly in the reaction well, do it in the EP tube). It is recommended to prepare the following concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0ng / mL, and PBS buffer is directly used as the blank well 0ng / mL.

[0080] 4) Preparation of washing solution: dilute the concentrated washing solution in the kit with double distilled water at a volume ratio of 1:19.

[0081] 5) Biotinylated antibody: Take 20 μL of double distilled water and add it to the biotinylated antibody. After it is fully dissolved, take 1 μL of biotinylated antibody and dilute it with 10 mL of sample diluent, i.e., dilute it 1:10000.

[0082] 6) Streptavidin-HRP: dilute 1 μL of Streptavidin-HRP with 10 mL of sample diluent, i.e., dilute 1:10,000.

[0083] 7) Add 100 μL of PBS buffer (blank control) / standard / sample to each microwell of the ELISA plate, seal the ELISA plate with Parafilm membrane, and incubate at 37°C for 60 min.

[0084] 8) After the reaction is completed, pour out the liquid in the plate, spin dry, add 300 μL of washing solution to each well and wash thoroughly three times, and pat the liquid in the well dry on absorbent paper.

[0085] 9) Add 100 μL of diluted biotinylated antibody to each well, seal the ELISA plate with Parafilm membrane, and incubate at 37°C for 60 min.

[0086] 10) After the reaction is completed, pour out the liquid in the plate, spin dry, add 300 μL of washing solution to each well and wash thoroughly three times, and pat the liquid in the well dry on absorbent paper.

[0087] 11) Add 100 μL of diluted Streptavidin-HRP to each well, seal the ELISA plate with Parafilm membrane, and incubate at 37°C for 60 min.

[0088] 12) After the reaction is completed, pour out the liquid in the plate, spin dry, add 300 μL of washing solution to each well and wash thoroughly 4 times, and pat the liquid in the well dry on absorbent paper.

[0089] 13) Add 100 μL of substrate solution (TMB) to each well, seal the ELISA plate with Parafilm membrane, and incubate at 37°C for about 15 minutes (shorten the time as appropriate depending on the actual color development, but not more than 15 minutes. When a clear gradient appears in the standard well, terminate the process).

[0090] 14) Add 100 μL of stop solution to each well to stop the reaction. The blue color will immediately turn yellow. The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution. Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader. The ELISA reader should be powered on in advance, the instrument should be preheated, and the detection program should be set.

[0091] 15) According to the OD of the standard sample 450 The values ​​can be used to draw a standard curve. In order to eliminate the systematic errors between each measurement, a standard curve is prepared for each measurement sample.

[0092] 16) OD of the sample 450 Substitute the value into the standard curve, read the corresponding concentration of the sample from the standard curve, and calculate the CdP450 protein content (μg / g) = sample concentration (ppb) * dilution factor * sample extract volume (μL) / leaf weight (mg) / 1000.

[0093] 2.CP4 EPSPS kit operation steps:

[0094] 1) Take the test kit out of the refrigerated environment and place it at room temperature (20-25°C) for more than 30 minutes to allow all reagents and required strips to return to room temperature. Shake each liquid reagent before use.

[0095] 2) Take about 40-50 mg of soybean tissue (crush the seeds first), put it into a 2 mL centrifuge tube, add steel balls, freeze it with liquid nitrogen, and crush it with a grinder. Add 600 μL of sample extract, shake and mix for 5 minutes, let it stand at room temperature for 5 minutes, and centrifuge it at 12000 rpm for 10 minutes. Dilute the sample with sample extract as needed and use it for determination. 450 The value is controlled within the measurable range.

[0096] 3) Dilute the standard concentration to 48 ppb, 24 ppb, 12 ppb, 6 ppb and 3 ppb. Dilute the 20× concentrated washing solution with deionized water to 1× washing working solution. Dilute the 11× concentrated enzyme label solution with enzyme diluent at a volume ratio of 1:10.

[0097] 4) Add 100 μL of sample extract (blank control) / standard / sample to each microwell of the ELISA plate, gently shake to mix, seal the ELISA plate with Parafilm film, and shake on a horizontal shaker in a light-proof environment at room temperature for 45 minutes.

[0098] 5) After the reaction is completed, pour out the liquid in the plate, add 250 μL of washing solution to each well and wash thoroughly 4-5 times, and pat the plate dry on absorbent paper.

[0099] 6) Add 100 μL of enzyme-labeled working solution to each well, gently shake to mix, seal the plate with Parafilm, and shake on a horizontal shaker in a dark environment at room temperature for 30 minutes. Repeat step 5.

[0100] 7) Add 100 μL of color developer to each well, gently shake to mix, seal the plate with Parafilm, and shake on a horizontal shaker in a dark environment at room temperature for 15 minutes.

[0101] 8) Add 100 μL of stop solution to each well, gently shake and mix, and measure OD in a microplate reader within 5 minutes. 450 .

[0102] 9) According to the OD of the standard sample 450 The values ​​can be used to draw a standard curve. In order to eliminate the systematic errors between each measurement, a standard curve is prepared for each measurement sample.

[0103] 10) OD of the sample 450 Substitute the value into the standard curve, read the corresponding concentration of the sample from the standard curve, and calculate the CP4 EPSPS protein content (μg / g) = sample concentration (ppb) * dilution factor * sample extract volume (μL) / leaf weight (mg) / 1000.

[0104] 3.Bar kit operation steps:

[0105] Steps 1), 2), 3), 4), and 5) are the same as those of the CP4 EPSPS kit, but the dilution concentrations of the standard are 3.2 ppb, 1.6 ppb, 0.8 ppb, 0.4 ppb, and 0.2 ppb.

[0106] 6) Add 100 μL of antibody working solution to each well, gently shake to mix, seal the plate with Parafilm, and shake on a horizontal shaker in a dark environment at room temperature for 30 minutes. Repeat step 5.

[0107] 7) Add 100 μL of enzyme labeling working solution to each well, gently shake to mix, seal the plate with Parafilm, and shake on a horizontal shaker at room temperature in a dark environment for 30 minutes. Repeat step 5. The color development and termination steps are the same as those of the CP4 EPSPS kit.

[0108] 8) A standard curve can be drawn based on the OD450 value of the standard sample. In order to eliminate the systematic error between each measurement, a standard curve is prepared for each measurement of the sample.

[0109] 9) Substitute the OD450 value of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and calculate the Bar protein content (μg / g) = sample concentration (ppb) * dilution factor * sample extract volume (μL) / leaf weight (mg) / 1000.

[0110] The expression of the target protein in the T1 transgenic soybean was determined according to the steps of the above three kits, and leaves were collected from the surviving plants of Example 2, with 6 plants collected from each transformant. The results of ELISA showed that the expression level of CdP450 protein was the highest in transformant XA-24 and the lowest in transformant XA-2; the expression level of CP4 EPSPS protein was the highest in transformant XA-17 and the lowest in transformant XA-2; the expression level of Bar protein was the highest in transformant XA-17 and the lowest in transformant XA-24 ( Figure 5 ). The expression level of the target protein in different transformants was positively correlated with herbicide tolerance.

[0111] Example 4: Using Pisum sativum 28 as a soybean explant source, establishing a soybean genetic transformation method using the ALS inhibitor herbicide flazasulfuron as a screening agent

[0112] The soybean variety in the above Example 1 was replaced with pea 28. According to the positive rate and transformation rate of the four combinations in Example 1, two combinations of 0.1 mg ai / L and 0.05 mg ai / L, and 0.2 mg ai / L and 0.05 mg ai / L were selected as the concentrations of flazasulfuron in the bud induction screening medium and the bud elongation medium. Each combination was infected with 300 explants, and the results showed that the transformation rates of the two combinations were 4.33% and 3.67%, respectively.

[0113] Table 4 Conversion rate of pea 28 treated with different concentrations of flazasulfuron

[0114]

[0115] Elongation frequency (%) = number of bud elongation / total number of explants infected × 100

[0116] Positive rate (%) = number of positive plants / number of bud elongation × 100

[0117] Positive transformation rate (%) = number of positive plants / total number of explants infected × 100

[0118] Example 5: Using Tianlong No. 1 as a soybean explant source, establishing a soybean genetic transformation method using the ALS inhibitor herbicide penoxsulam as a screening agent

[0119] According to the method of Example 1 above, fluazifop-methyl in the bud induction screening medium and the bud elongation medium was replaced with penoxsulam, and the final concentration of penoxsulam in the bud induction screening medium was selected to be 0.1 mg ai / L and 0.2 mg ai / L, and the final concentration in the bud elongation medium was selected to be 0.05 mg ai / L. Each combination was infected with 300 explants, and the results showed that the transformation rates of the two combinations were 3.00% and 3.33%, respectively.

[0120] Table 5 Conversion rate of Tianlong No. 1 under different concentrations of penoxsulam treatment

[0121]

[0122] Elongation frequency (%) = number of bud elongation / total number of explants infected × 100

[0123] Positive rate (%) = number of positive plants / number of bud elongation × 100

[0124] Positive transformation rate (%) = number of positive plants / total number of explants infected × 100

[0125] Example 6: Using Pisum sativum 28 as a soybean explant source, establishing a soybean genetic transformation method using the ALS inhibitor herbicide penoxsulam as a screening agent

[0126] According to the method of Example 1 above, the soybean variety was selected as Pea 28, and the fluazifop-methyl in the bud induction screening medium and the bud elongation medium was replaced with penoxsulam, and the final concentration of penoxsulam in the bud induction screening medium was selected to be 0.1 mg ai / L and 0.2 mg ai / L, and the final concentration in the bud elongation medium was selected to be 0.05 mg ai / L. Each combination was infected with 300 explants, and the results showed that the transformation rates of the two combinations were 4.67% and 3.67%, respectively.

[0127] Table 6 Conversion rate of pea 28 treated with different concentrations of penoxsulam

[0128]

[0129]

[0130] Elongation frequency (%) = number of bud elongation / total number of explants infected × 100

[0131] Positive rate (%) = number of positive plants / number of bud elongation × 100

[0132] Positive transformation rate (%) = number of positive plants / total number of explants infected × 100

[0133] Example 7: Using Tianlong No. 1 as a soybean explant source, establishing a soybean genetic transformation method using the ALS inhibitor herbicide pyrazosulfuron-methyl as a screening agent

[0134] According to the method of Example 1 above, pyrazosulfuron-methyl in the bud induction screening medium and the bud elongation medium was replaced with pyrazosulfuron-methyl, and the final concentration of pyrazosulfuron-methyl in the bud induction screening medium was selected to be 0.1 mg ai / L and 0.2 mg ai / L, and the final concentration in the bud elongation medium was selected to be 0.025 mg ai / L. Each combination was infected with 300 explants, and the results showed that the positive transformation rates of the two combinations were 7.00% and 8.00%, respectively.

[0135] Table 7 Conversion rate of Tianlong No. 1 under different concentrations of pyrazosulfuron-methyl treatment

[0136]

[0137] Elongation frequency (%) = number of bud elongation / total number of explants infected × 100

[0138] Positive rate (%) = number of positive plants / number of bud elongation × 100

[0139] Positive transformation rate (%) = number of positive plants / total number of explants infected × 100

[0140] Example 8: Using Pisum sativum 28 as a soybean explant source, establishing a soybean genetic transformation method using the ALS inhibitor herbicide pyrazosulfuron-methyl as a screening agent

[0141] According to the method of Example 1 above, the soybean variety was selected as Pea 28, and the flazasulfuron in the bud induction screening medium and the bud elongation medium was replaced with pyrazosulfuron amide, and the final concentration of pyrazosulfuron in the bud induction screening medium was selected to be 0.1 mg ai / L and 0.2 mg ai / L, and the final concentration in the bud elongation medium was selected to be 0.025 mg ai / L. Each combination was infected with 300 explants, and the results showed that the transformation rates of the two combinations were 4.67% and 6.00%, respectively.

[0142] Table 8 Conversion rate of pea 28 under different concentrations of pyrazosulfuron-methyl treatment

[0143]

[0144] Elongation frequency (%) = number of bud elongation / total number of explants infected × 100

[0145] Positive rate (%) = number of positive plants / number of bud elongation × 100

[0146] Positive transformation rate (%) = number of positive plants / total number of explants infected × 100

[0147] Comparative Example 1:

[0148] According to the method of Example 1 above, no transformants were obtained when the screening agent was replaced with the photosynthesis inhibitor herbicide diquat.

[0149] The above results show that the soybean genetic transformation method established by the present invention using the gene derived from bermudagrass as a marker gene and ALS inhibitor herbicide as a screening agent has a positive transformation rate of 12.05%, and the genetic transformation method broadens the scope of soybean genetic transformation screening marker genes.

Claims

1. A soybean genetic transformation method for improving positive transformation rate, characterized in that: The method comprises: using a recombinant expression vector containing a target gene and a marker gene to transform soybean explants, and obtaining transformants by adding an ALS inhibitor to a bud induction medium and a bud elongation medium to improve the positive transformation rate of the explants through screening and culturing; the ALS inhibitor comprises a sulfonylurea herbicide and a triazolopyrimidine herbicide; and the concentration of the ALS inhibitor in the bud induction medium and the bud elongation medium is 0.025-0.4 mg ai / L.

2. The method according to claim 1, characterized in that The recombinant expression vector contains a protein expression frame encoding a target gene and a protein expression frame encoding a marker gene, wherein the protein expression frame encoding the target gene includes a CP4EPSPS protein expression frame and a Bar protein expression frame, and the protein expression frame encoding the marker gene is a CdP450 protein expression frame.

3. The method according to claim 1, characterized in that The sulfonylurea herbicides include fluazifop-methyl and pyrazosulfuron-methyl; the triazolopyrimidine herbicides include penoxsulam.

4. The method according to claim 3, characterized in that The concentration of the ALS inhibitor in the bud induction medium is 0.1 mg ai / L or 0.2 mg ai / L; the concentration in the bud elongation medium is 0.025 mg ai / L or 0.05 mg ai / L.

5. The method according to claim 1, characterized in that The soybean varieties include Tianlong No. 1 and Wandou 28.

6. The method according to claim 1, characterized in that The method comprises the following steps: (1) soaking soybean explants in an Agrobacterium infection solution containing a recombinant expression vector to obtain infected soybean explants; (2) placing the soybean explants infected in step (1) in a dark incubator at 21-22° C. for 2-5 days to obtain co-cultivated explants; (3) transferring the explants after co-cultivation in step (2) into a recovery medium without an ALS inhibitor and culturing at 26-28° C. for 7-9 days; (4) transferring the explants in step (3) into a bud induction screening medium containing an ALS inhibitor, and culturing at 26-28° C. for 14 days to obtain explants with new buds; (5) The explants with newly formed shoots in step (4) are transferred into a shoot elongation medium containing an ALS inhibitor, cultured at 26-28° C., and subcultured every 3 weeks until the transformed cells regenerate into plants or the regenerated plants are able to survive.

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