CN115786365A - Cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant obtaining method and application - Google Patents

Abstract

The invention provides a cotton GhGT-3b _A04gene, protein, an expression vector, a transgenic plant acquisition method and application, belonging to the field of molecular biology and biotechnology, wherein the nucleotide sequence of the cotton GhGT-3b _A04gene is shown as SEQ ID No. 1. The GhGT-3b _A04gene can enhance the verticillium wilt resistance of arabidopsis, the obtained transgenic plants can normally bloom and bear fruits, and the transformation gene can be stably inherited to the next generation, so the gene can be further applied to the improvement of the verticillium wilt resistance, a new variety of verticillium wilt resistance is cultivated, and a new way is provided for the improvement of the verticillium wilt resistance of plants; the invention provides theoretical basis for the regulation and control of plant root system and epidermal hair development; the invention provides a new material for the improvement of verticillium wilt resistance and the research of plant root systems and epidermal hairs.

Description

A kind of cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant obtained methods and applications

technical field

The invention belongs to the field of molecular biology and biotechnology, and in particular relates to a cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application.

Background technique

my country is a big country of cotton production and consumption, and cotton plays an important role in our national economy. Verticillium wilt is a soil-borne fungal disease that seriously affects cotton yield and quality. The effective way to control Verticillium wilt is to cultivate and plant disease-resistant varieties. Therefore, identification of genes related to Verticillium wilt resistance and exploring its disease resistance mechanism are the keys to molecular breeding of cotton disease resistance. Salicylic acid is an important defense hormone that plays an important role in plant defense responses against pathogens and establishment of systemic acquired resistance. In cotton, the salicylic acid signaling pathway has been shown to be involved in the resistance process of Verticillium wilt.

Triple-helix Trihelix transcription factor, also known as GT transcription factor, its DNA binding domain contains 3 consecutive α-helices, which can specifically bind GT cis elements (GT1 box, 5'-GGTTAA-3'; GT2 box, 5' -GGTAAT-3'; GT3 box, 5'-GGTAAA-3'). According to the number of structural domains, GT transcription factors can be divided into three categories: GT-1, GT-2 and GT-3. According to the change of α-helical domain, it can be divided into five subfamilies: GT-1, GT-2, GTγ, SH4 and SIP1. Studies on the functions of some triple-helix Trihelix transcription factors have shown that Trihelix transcription factors are involved in the regulation of plant biotic and abiotic stress. Overexpression of rice OsGTγ-1, OsGTγ-2, OsGTγ-3 genes can improve the tolerance of plants to salt. Heterologous expression of soybean GmGT-2a and GmGT-2b genes in Arabidopsis plants has increased tolerance to salt, freezing and drought stress. ShCIGT gene overexpression tomato plants in wild tomato have higher cold tolerance. The Arabidopsis gtl1 mutant has significantly reduced stomatal density and number, and improved drought tolerance and water use efficiency. GTL1 interacts with MPK4, directly binds and regulates genes involved in salicylic acid biosynthesis, transport, and responses, positively regulates defense responses to Pseudomonas syringae strains and represses factors that regulate growth and development. In cotton, 102 Trihelix transcription factors were identified in the Upland cotton genome, among which Gh_A05G2067 (GT-2) was up-regulated under drought and salt stress conditions, which can improve the tolerance of cotton to drought and salt stress. However, the biological functions of other cotton Trihelix transcription factors, especially the regulatory factors that regulate cotton disease resistance defense responses through salicylic acid synthesis and signal transduction pathways, are still unclear.

Contents of the invention

In view of this, the object of the present invention is to provide a cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application, the cotton GhGT-3b_A04 gene can improve the resistance of plants to Verticillium wilt.

In order to realize the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a cotton GhGT-3b_A04 gene, the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown in SEQ ID No.1.

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in improving plant resistance to Verticillium wilt.

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in increasing the length of plant root hairs.

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in increasing the density of plant root hairs.

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in increasing the hair density of the plant epidermis.

The present invention also provides the protein encoded by the cotton GhGT-3b_A04 gene described in the above technical solution, the amino acid sequence of the protein is shown in SEQ ID No.2.

The present invention also provides an overexpression vector, which contains the cotton GhGT-3b_A04 gene described in the above technical solution.

Preferably, the preparation method of the overexpression vector comprises the following steps:

1) The cotton GhGT-3b_A04 gene described in the above technical scheme is subjected to BP reaction with the entry vector pDONR-Zeo to obtain the pDONR-Zeo-GhGT-3b_A04 entry vector;

2) The pDONR-Zeo-GhGT-3b_A04 entry vector obtained in step 1) was subjected to LR reaction with the destination vector pEarleyGate 101 to obtain an overexpression vector.

The present invention also provides a method for obtaining transgenic plants, comprising the following steps:

a. Transform the overexpression vector described in the above technical scheme into Agrobacterium to obtain transformed bacteria;

b. Infecting plants with the transformed bacteria obtained in step a to obtain transgenic plants.

Preferably, the Agrobacterium includes Agrobacterium GV3101.

The beneficial effects of the present invention are:

1. The GhGT-3b_A04 gene can enhance the resistance of Arabidopsis thaliana to Verticillium wilt, and the obtained transgenic plants can flower and bear fruit normally, and the transformed gene can be stably passed on to the next generation, so this gene can be further used in the improvement of Verticillium wilt resistance , to breed new varieties resistant to Verticillium wilt, providing a new way for plant Verticillium wilt resistance improvement;

2. The invention provides a theoretical basis for the regulation and control of plant root system and epidermal hair growth.

3. The present invention provides new materials for the improvement of Verticillium wilt resistance and the research of plant root system and epidermis.

Description of drawings

Fig. 1 is the gene expression schematic diagram of the present invention cotton GhGT-3b_A04 gene sequence and its cloning and application;

Fig. 2 is the Arabidopsis plant identification and plant phenotype diagram of the cotton GhGT-3b_A04 gene sequence of the present invention and its cloning and application;

Fig. 3 is a schematic diagram of the gene subcellular localization of the cotton GhGT-3b_A04 gene sequence of the present invention and its cloning and application;

Fig. 4 is the gene sequence of cotton GhGT-3b_A04 of the present invention and its cloning and application gene Arabidopsis verticillium wilt treatment schematic diagram;

Fig. 5 is the DAB staining schematic diagram of the gene sequence of cotton GhGT-3b_A04 of the present invention and its clone and application after the treatment of Verticillium wilt in Arabidopsis plants;

Fig. 6 is the schematic diagram of root system development of Arabidopsis thaliana seedlings of cotton GhGT-3b_A04 gene sequence of the present invention and its clone and application;

Fig. 7 is a schematic diagram of the gene sequence of the cotton GhGT-3b_A04 gene of the present invention and its clone and application;

Fig. 8 is a schematic diagram of the gene sequence of cotton GhGT-3b_A04 of the present invention and its cloning and application of gene expression changes in Arabidopsis rosette leaf disease resistance-related genes.

Detailed ways

The invention provides a cotton GhGT-3b_A04 gene, the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown in SEQ ID No.1, specifically as follows:

ATGGAGGGACATCATCGCCATCATCATCATCAGCAGCAGCAACATCTGCAGCAACAACAACAGCCTGTAAGTGTCAACGTTGAAGCTGATAGGTTTCCTCAATGGAGTGTTCAAGAGACAAAGGATTTTTTAATGATCAGAGCAGAGCTGGATCAAAGTTTCATGGAGACCAAAAGGAACAAGCTGCTTTGGGAAGTTATCTCCACCAGGATGAGGGAAAAGGGTTATAATCGAAGCGCTGAACAGTGCAAGTGCAAGTGGAAAAACCTCTTTACTCGTTACAAGGGATGTGGAACGGTTGACGCAGAAATTGTGCGGCAACAGTTCCCGTTTTACGACGAGTTGCAGGCCATATTCACGGCGAGGATGCAAAGTGTTCTATGTTCGGAAAGTGAAGGCGGAGGAGCTACGGGGTCGAAAAAAAAGGCAGCACAGGCGCAGCTATCTTCCGACGAGGAAGACGATACGGAGGAAAACGAGTACAGCTTTAGGAAGAAGAAGAAAGGGAAAACTGGTGGTGGGAGTAGTAGTACGAGCGGCAGCATTAATATAAAGGAAATGTTAGCGAACTTCATGAGGCAACAGTTGCAGATGGAAATGCAATGGAGGGAAGCTTTGGAGTCGAGAGAAAACGAGAGGCGGATGAAGGAAATGGAGTGGAGGCAAACCATGGAAGCCCTTGAAAACGAGAGGATAATGATGGAGAGAAGGTGGAGGGAAAGGGAAGAACAAAGGAGAATAAGGGAAGAAGCTAGGGCTGAGAAGAGAGATGCTCTCATTACTGCACTTCTAAACAAGCTTAGAAGAGAAGATCAAATGTAG。

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in improving plant resistance to Verticillium wilt.

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in increasing the length of plant root hairs.

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in increasing the density of plant root hairs.

The present invention also provides the application of the cotton GhGT-3b_A04 gene described in the above technical solution in increasing the hair density of the plant epidermis.

In the present invention, the plant preferably includes Arabidopsis.

HIA of the present invention provides the protein encoded by the cotton GhGT-3b_A04 gene described in the above-mentioned technical scheme, is characterized in that, the aminoacid sequence of described protein is as shown in SEQ ID No.2, specifically as follows:

MEGHHRHHHHQQQQHLQQQQQPVSVNVEADRFPQWSVQETKDFLMIRAELDQSFMETKRNKLLWEVISTRMREKGYNRSAEQCKCKWKNLFTRYKGCGTVDAEIVRQQFPFYDELQAIFTARMQSVLCSESEGGGATGSKKKAAQAQLSSDEEDDTEENEYSFRKKKKGKTGGGSSSTSGSINIKEMLANFMRQQLQMEMQWREALESRENERRMKEMEWRQTMEALENERIMMERRWREREEQRRIREEARAEKRDALI TALLNKLRRE DQM。

The present invention also provides an overexpression vector, which contains the cotton GhGT-3b_A04 gene of claim 1.

In the present invention, the preparation method of the overexpression vector preferably includes the following steps:

1) The cotton GhGT-3b_A04 gene described in the above technical scheme is subjected to BP reaction with the entry vector pDONR-Zeo to obtain the pDONR-Zeo-GhGT-3b_A04 entry vector;

2) The pDONR-Zeo-GhGT-3b_A04 entry vector obtained in step 1) was subjected to LR reaction with the destination vector pEarleyGate 101 to obtain an overexpression vector.

In the present invention, the cotton GhGT-3b_A04 gene described in the above technical scheme is preferably subjected to BP reaction with the entry vector pDONR-Zeo to obtain the pDONR-Zeo-GhGT-3b_A04 entry vector. In the present invention, the entry vector pDONR-Zeo is preferably purchased from Invitrogen. In the present invention, the conditions of the BP reaction preferably include: the reaction system is 1 μl of pDONR-Zeo vector, 1 μl of PCR product, 0.5 μl of BP reaction enzyme (Invitrogen, California, USA), and react at 25° C. for 2 hours.

In the present invention, the obtained pDONR-Zeo-GhGT-3b_A04 entry vector is preferably subjected to LR reaction with the target vector pEarleyGate101 to obtain an overexpression vector. In the present invention, the target vector pEarleyGate 101 is preferably purchased from Youbao Biological Company. In the present invention, the conditions of the LR reaction preferably include: the reaction system is 1 μl of pDONR-Zeo-GhGT-3b_A04 carrier, 1 μl of PCR product, 0.5 μl of LR reaction enzyme (Invitrogen, California, U.S.), and reacted for 1 hour at 25° C. .

The present invention also provides a method for obtaining transgenic plants, comprising the following steps:

a. Transform the overexpression vector described in the above technical scheme into Agrobacterium to obtain transformed bacteria;

b. Infecting plants with the transformed bacteria obtained in step a to obtain transgenic plants.

The present invention transforms the overexpression vector described in the above technical scheme into Agrobacterium to obtain transformed bacteria. In the present invention, the Agrobacterium preferably includes Agrobacterium GV3101. The present invention has no special limitation on the transformation method, and those skilled in the art can follow the routine operations.

The present invention infects plants with the transformed bacteria obtained in step a to obtain transgenic plants. The method of infection is not particularly limited in the present invention, and those skilled in the art can use conventional operations, which will not be repeated here.

In the present invention, the plant preferably includes Arabidopsis. In the present invention, the GhGT-3b_A04 gene can enhance the resistance to Verticillium wilt of Arabidopsis thaliana, and the obtained transgenic plants can normally flower and bear fruit, and the transformed gene can be stably inherited to the next generation.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1

Cloning of Cotton GhGT-3b_A04 Gene and Construction of Expression Vector

(1) Primer design

According to the cotton genome sequence reported by cottonFGD, by bioinformatics comparison, the cDNA sequence (SEQ ID No.1) of the GhGT-3b_A04 gene was determined, and the primers for the full-length sequence of the coding frame (ORF) were designed according to the sequence, and the primer sequences were as follows:

GhGT-3b_A04-F (SEQ ID No.3):

5'GGGGACAAGTTTTGTACAAAAAAGCAGGCTTAATGGAGGGACATCATCGC3';

GhGT-3b_A04-R (SEQ ID No.4):

5'GGGGACCACTTTGTACAAGAAAGCTGGGTACATTTGATCTTCTCTTCT3'.

(2) Extraction of Total RNA from Upland Cotton TM-1 Cotton Leaves

The young leaves were ground into powder with liquid nitrogen, and then the total RNA of the leaves was extracted using the kit R FastPure Plant Total RNAIsolation Kit (Novazyme, Nanjing, China). The RNA concentration and quality were detected by a spectrophotometer, the concentration was about 600ng/ul, OD260/OD280=2.0, OD260/OD230=2.0, and the extracted total RNA was immediately used in the next experiment.

(3) RNA reverse transcription

Take 1 μg RNA into a 0.2ml RNAase-free centrifuge tube, use the kit HiScriptⅢRT SuperMixfor qPCR (Novizan, Nanjing, China) to reverse-transcribe the RNA into cDNA, and detect the DNA concentration and quality by spectrophotometer, the concentration is about 1000ng /μl, OD260/OD280=1.8, OD260/OD230=2.0, the obtained cDNA was used for the next experiment.

(4) PCR amplification and purification

Use high-fidelity enzymes for PCR amplification, as follows: PCR reaction system: 1000ng/μl cotton cDNA 1μl, 10uM primers F and R each 2μl, 2×Phanta Master Mix: 25μl, ddH ₂ O 20μl, the total system is 50μl . PCR amplification conditions are as follows: 95°C pre-denaturation for 2 minutes; 95°C denaturation for 15 seconds, 55°C annealing for 15 seconds, 72°C extension for 40 seconds, 35 cycles; 1% agarose gel electrophoresis separation and Gelred staining detection amplification result. Omega PCR purification and recovery kit was used for PCR purification and recovery, the concentration reached 100ng/μl, and ligation reaction was carried out.

(5) PCR fragment connection expression vector

The Gateway system was used to construct the expression vector. First, the PCR product was subjected to BP reaction with the entry vector pDONR-Zeo. °C for 2 hours to obtain the entry vector pDONR-Zeo-GhGT-3b_A04. The entry vector was sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. The vector contained the nucleotide sequence shown in SEQ ID No.1, and its protein sequence contained a typical triple helix domain. Then the entry vector and the destination vector pEarleyGate101 were subjected to LR reaction, the reaction system: 1 μl of pDONR-Zeo-GhGT-3b_A04 vector, 1 μl of PCR product, 0.5 μl of LR reaction enzyme (Invitrogen, California, U.S.), and reacted for 1 hour at 25°C to obtain Overexpression vector pEarleyGate 101-GhGT-3b_A04. The pEarleyGate 101-GhGT-3b_A04 vector was transformed into GV3101 Agrobacterium, and positive colonies were identified by PCR.

(6) Real-time PCR

Extract the RNA from roots and leaves of upland cotton Jimian J11 and Zhongzhimian No. 2 ZZ2 verticillium wilt infection 0h, 6h, 12h, 24h, 48h, 72h and each tissue of upland cotton TM-1, reverse transcribe into cDNA, use SYBR qPCR Master Mix for Real-time PCR, the expression of GhGT-3b_A04 gene in the roots and leaves of J11 and ZZ2 were induced by Verticillium dahliae (A-B in Figure 1), and in the roots, stems, petals and leaves of TM-1 There is a relatively high expression in stamens (C in Figure 1).

Example 2

Obtaining transgenic Arabidopsis

(1) Arabidopsis transformation

Put the wild-type Arabidopsis Col0 in a refrigerator at 4°C for vernalization for 4 days, mix the nutrient soil and vermiculite at a ratio of 3:1, add water to moisten it, and divide it into small pots. Put the Col0 seeds directly with a gun The heads are sucked into small pots, 9 trees per pot. Arabidopsis was cultured in a greenhouse at 24°C with 16 hours of light and 8 hours of darkness. Cultivate for about 45 days, and transform when there are many flower buds. Get 200 μl of the positive Agrobacterium pEarleyGate 101-GhGT-3b_A04 obtained in Example 1 in 2ml LB+rif+kan culture medium, 28°C, shake culture at 200rpm overnight, take 1ml of bacterial liquid and expand culture in 100ml of corresponding medium, 28 ℃, 200rpm shaking culture for 12 hours, 4000rpm, 10min to collect the bacterial liquid, use the infection solution (1/2MS+5% sucrose+0.05% silwetl-77) to resuspend the bacterial liquid, and adjust the OD600 to 1.5. Invert the prepared infection solution into the culture dish, soak the Arabidopsis flocs in the infection solution for 1 min, place the transformed Arabidopsis plants horizontally, cover with plastic wrap to keep them moist, and place them in the greenhouse for 24 hours after dark treatment. After the Arabidopsis thaliana fruited, the seeds were harvested, and the transgenic positive plants were screened for resistance.

(2) Screening and identification of positive plants

The obtained transgenic Arabidopsis thaliana T0 generation seeds were evenly sown in the nutrient soil matrix. After the seeds germinated, the Basta herbicide was sprayed every 5 days for a total of 3 times. A total of 20 resistant plants were obtained. Genomic DNA and RNA of three of the resistant Arabidopsis thaliana were extracted by CTAB method, and PCR identification and semi-quantitative PCR of the target gene were carried out. All of them were identified as positive plants and highly expressed at the RNA level. They were named GhGT-3b_A04-YFP-1~3 respectively (A in Fig. 2). Due to the fusion expression of GhGT-3b_A04 and yellow fluorescent protein YFP, the anti-GFP antibody was further used to detect GhGT-3b_A04-YFP-1~3 by western blot. It was found that the GhGT-3b_A04-YFP proteins of the three strains were all There is expression (B in Figure 2). At the same time, using the empty transgenic line as a control, the phenotype of the Arabidopsis thaliana 28-day-old plant was observed. It can be seen that the overexpression line is smaller than the plant of the control material (C in Figure 2). Small. Confocal observation of Arabidopsis thaliana transgenic lines showed that the empty carrier was expressed in the cell membrane, nucleus and cytoplasm, while the fluorescence of the GhGT-3b_A04-YFP transgenic line was only distributed in the nucleus by DAPI staining (Figure 3) .

Example 3

Transgenic Arabidopsis more resistant to Verticillium wilt

Transgenic T3 generation homozygous lines were cultured in a greenhouse with 16 hours of light and 8 hours of darkness at 24°C for 35 days, and then infected with Verticillium dahliae. After 20 days, the phenotype of the transgenic lines was observed, and three overexpression transgenic lines could be observed The leaf yellowing degree of GhGT-3b_A04-YFP-1～3 was lower than that of the wild type, indicating that the transgenic lines were more resistant to Verticillium wilt than the wild type (Figure 4). DAB staining was performed on the rosette leaves of Verticillium dahliae infection for 24 hours and 48 hours, and it can be observed that the transgenic lines stained darker than the wild-type leaves (Figure 5), indicating that the transgenic lines can produce more after Verticillium dahliae infection. Active oxygen to fight Verticillium wilt.

Example 4

Changes of root hairs and epidermal hairs in transgenic Arabidopsis thaliana

In order to analyze the root hair changes of transgenic Arabidopsis thaliana, the seeds of transgenic homozygous lines and control Arabidopsis thaliana were sterilized, vernalized at 4°C, sown on 1/2MS solid medium containing 1% sucrose, and cultured in light for 10 days, and then photographed to observe the root length and root hair count. It can be seen that the root hair length and root hair density of the GhGT-3b_A04-YFP-1～3 transgenic lines are higher than those of the wild type ( FIG. 6 ).

The homozygous lines of the transgenic T3 generation were cultured for 28 days in a greenhouse at 24°C with 16 hours of light and 8 hours of light, and the trichome phenotype of the leaves of the transgenic lines was observed. It can be observed that the density of the trichomes of the transgenic lines is higher than that of the wild type (Fig. 7 ).

Example 5

Induced expression of SA signal and synthesis pathway and disease resistance-related genes in transgenic Arabidopsis thaliana

Transgenic T3 homozygous lines were cultured in a greenhouse for 28 days with 16 hours of light and 8 hours of darkness at 24°C. RNA was extracted from rosette leaves, reverse-transcribed into cDNA, and Real-time PCR was performed to detect the signal and synthesis of salicylic acid The expressions of pathway genes CBP60G, ICS1 and PBS3 were induced and expressed in the transgenic lines (A, B, G in Figure 8); at the same time, the resistance-related genes PR2, CHI, CYP71A13, FRK1, PNP- A. The expression of EARLI1, these six genes were induced to express in the transgenic lines (C-F, H-I in Fig. 8), indicating that GhGT-3b_A04 enhances the expression of yellow yellow by activating the salicylic acid signal and synthesis pathway and the expression of disease resistance genes. Wilt resistance.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (10)

1. A cotton GhGT-3b_A04 gene, characterized in that the nucleotide sequence of the cotton GhGT-3b_A04 gene is as shown in SEQ ID No.1. 2. the application of the cotton GhGT-3b_A04 gene according to claim 1 in improving plant resistance to Verticillium wilt. 3. the application of the cotton GhGT-3b_A04 gene described in claim 1 in improving the plant root hair length. 4. the application of the cotton GhGT-3b_A04 gene described in claim 1 in improving plant root hair density. 5. the application of the cotton GhGT-3b_A04 gene described in claim 1 in improving the density of plant epidermis. 6. The protein encoded by the cotton GhGT-3b_A04 gene of claim 1, wherein the amino acid sequence of the protein is as shown in SEQ ID No.2. 7. An overexpression vector, characterized in that the overexpression vector contains the cotton GhGT-3b_A04 gene of claim 1. 8. overexpression vector according to claim 7, is characterized in that, the preparation method of described overexpression vector comprises the following steps: 1) carrying out BP reaction with the cotton GhGT-3b_A04 gene described in claim 1 and the entry vector pDONR-Zeo to obtain the pDONR-Zeo-GhGT-3b_A04 entry vector; 2) Perform LR reaction with the pDONR-Zeo-GhGT-3b_A04 entry vector obtained in step 1) and the destination vector pEarleyGate101 to obtain an overexpression vector. 9. A method for obtaining transgenic plants, comprising the following steps: A, the overexpression vector described in claim 7 is transformed in the Agrobacterium, obtains transformed bacteria; b. Infecting plants with the transformed bacteria obtained in step a to obtain transgenic plants. 10. The obtaining method according to claim 9, wherein the Agrobacterium comprises Agrobacterium GV3101.

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