Application of GbCG1 in improvement of soybean genetic transformation

Abstract

The invention discloses an application of GbCG1 in improvement of soybean genetic transformation. Relates to the technical field of biology. The invention further provides specific application. A novel transcription factor GbCG1 is cloned from sea island cotton, the gene CDS is cloned into an overexpression vector in a full-length manner, then agrobacterium tumefaciens is introduced, soybean is transformed by an agrobacterium tumefaciens-mediated method, compared with a WMV067 empty vector transformation contrast, more green buds can be screened out through overexpression of GbCG1, the efficiency of obtaining positive seedlings from soybean stem tip meristem tissue is improved, and the soybean stem tip meristem tissue has the advantages of high yield, high yield and the like. The conversion time is shortened. Therefore, overexpression of the gene and application of the gene in commercial soybean varieties with difficulty in genetic transformation contribute to improvement of genetic transformation efficiency, so that yield, quality and stress resistance of excellent soybean germplasm are improved, and the gene has important economic value and social benefit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, more particularly to the application of GbCG1 in improving the genetic transformation of soybean. BACKGROUND

[0002] Soybean is an important economic crop in the world, as a core raw material for vegetable oil and soybean meal, it supports the food and feed industry. Its industrial chain is related to agriculture, energy and other fields, and is a key variable in the global economy, playing an important role in food security and trade policy. However, the current main varieties face severe challenges in yield, protein content, oleic acid content and disease resistance, and traditional breeding techniques cannot meet the needs of industrial upgrading.

[0003] The current bottlenecks in soybean genetic transformation are mainly: genotype dependence: only a few cultivars (such as Williams 82) are suitable for transformation, and most of the main cultivars have poor regeneration ability, limiting the scope of application. Low transformation efficiency: Agrobacterium-mediated transformation efficiency is restricted by soybean tissue regeneration ability, and the positive plant acquisition rate is usually less than 1%, the cost of gene gun transformation is high and easy to cause multiple gene copies. Long regeneration cycle: from explants to complete plants, it takes 3-6 months, and chimeras or deformities often occur. Unstable gene expression: foreign genes are prone to silencing or random integration, resulting in low expression efficiency of target traits, which requires multiple screening and verification.

[0004] In recent years, China has made breakthrough progress in the field of transgenic technology of dicotyledonous plants (such as the "stem cell-based" transformation system developed by the Chinese Academy of Agricultural Sciences), according to this system, using regeneration key genes and combining the soybean stem cell in situ transformation system, a soybean genetic transformation technology without genotype restriction has been developed. Using regeneration key factors to develop high-efficiency genetic transformation technology for soybean, overcoming the bottleneck of genotype restriction, the research field has frontier and innovation, which can promote the industrialization process of biological breeding in China.

[0005] Therefore, to provide the application of GbCG1 in improving the genetic transformation of soybean, to overcome the above technical deficiencies is a problem that the skilled in the art need to solve. SUMMARY

[0006] Therefore, the present application provides the application of GbCG1 in improving the genetic transformation of soybean. Specifically, the application provides the application of transcription factor GbCG1 gene in accelerating the genetic transformation efficiency of soybean. To improve the efficiency of genetic transformation of soybean.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] The application of GbCG1 in improving the genetic transformation of soybean, the gene sequence of GbCG1 is shown as SEQ ID NO. 3.

[0009] Preferably, the amino acid sequence encoded by the GbCG1 gene is shown as SEQ ID NO. 4.

[0010] Preferably, the soybean is Zheng 1307, Qi Huang 34 or Zhongpin 661.

[0011] The application also provides an expression cassette, a recombinant vector or a recombinant bacterium containing the GbCG1 gene, and the sequence of the GbCG1 gene is shown as SEQ ID NO. 3.

[0012] The application also provides the use of the above-mentioned expression cassette, recombinant vector or recombinant bacterium containing the GbCG1 gene in improving the genetic transformation of soybean.

[0013] Preferably, the GbCG1 gene is integrated into the soybean by Agrobacterium-mediated method for overexpression.

[0014] Preferably, the recombinant vector: the GbCG1 gene is constructed into the vector WMV067, and the nucleotide sequence of the WMV067 vector is shown as SEQ ID NO. 5.

[0015] Preferably, the GbCG1 gene is constructed into the BamHI and SacI enzyme digestion sites of the vector WMV067.

[0016] Compared with the prior art, the application discloses the use of GbCG1 in improving the genetic transformation of soybean, and the technical effects are as follows: a new transcription factor GbCG1 is cloned from Gossypium barbadense, the full-length CDS of the gene is cloned into an overexpression vector, then introduced into Agrobacterium, and the Agrobacterium-mediated method is used to transform soybean, compared with the transformation of the WMV067 empty vector, more green sprouts can be screened out by overexpressing GbCG1, the efficiency of obtaining positive seedlings of the stem tip meristem of soybean is improved, and the transformation time is shortened. Therefore, overexpression of the gene and application of the gene in the commercialized soybean variety which is difficult to be genetically transformed can help to improve the genetic transformation efficiency, and then improve the yield, quality and stress resistance of the excellent germplasm of soybean, and the application has important economic value and social benefits.

[0017] The gene is used as a regeneration key gene, and a soybean genetic transformation technology without genotype restriction is developed by combining a soybean stem cell in situ transformation system, and the bottleneck of genotype restriction is overcome. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0019] Figure 1 The accompanying drawings are schematic diagrams of overexpression vector structures provided by the present application.

[0020] Figure 2 The accompanying drawings are PCR detection results of GbCG1 gene of transgenic soybean plants provided by the present application, wherein 1-19 are different OE-GbCG1 transgenic lines; 20 is a non-transgenic control, 21 is a plasmid positive control, and 22 is a marker III molecular weight marker.

[0021] Figure 3 The accompanying drawings are comparison diagrams of GbCG1 significantly improving genetic transformation efficiency of soybean provided by the present application. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0023] The embodiments of the present application disclose application of GbCG1 in improving genetic transformation of soybean

[0024] The experimental principles involved in the embodiments include:

[0025] The T-DNA fragment of the recombinant plasmid WMV067-GbCG1 of Agrobacterium contains a transcription factor GbCG1 and a spectinomycin resistance gene AADA (a resistance gene sequence provided by the vector); the transcription factor GbCG1 and the spectinomycin resistance gene AADA are combined to transform soybean, which greatly improves the transformation efficiency and breaks the genotype restriction, especially improves the genetic transformation efficiency of the main soybean varieties Zheng 1307, Zhongpin 661 and Qihuang 34. Since the non-transgenic plants are white in leaf when the regenerated soybean plants are screened by using the endosmosis type spectinomycin, the transgenic plants are easy to identify, which makes the identification of transgenic soybean plants simple and efficient, and provides convenience for soybean gene function verification and soybean genetic breeding.

[0026] The existing soybean varieties Zheng 1307, Zhongpin 661 and Qihuang 34 in the embodiments are from Henan Academy of Agricultural Sciences (the selected above-mentioned varieties are relatively difficult to perform genetic transformation). The reagents involved in the embodiments are products of Sigma-Aldrich.

[0027] Infection medium CAB comprises: 10 ml / L CA stock, 30 g / L glucose, 4.2 g / L MES (morpholinoethanesulfonic acid), 0.1 ml / L B5 vitamins (G219), 1 mg / L 6-BA (6-benzyladenine), 0.1 mg / L NAA (naphthalene acetic acid), 0.2 mM acetosyringone, 200 mg / mL cysteine (CYS), pH 5.4.

[0028] Co-cultivation medium CCM comprises: 10 ml / L CA stock, 30 g / L glucose, 4.2 g / L MES (morpholinoethanesulfonic acid), 0.1 ml / L B5 vitamins (G219), 1 mg / L 6-BA (6-benzyladenine), 0.1 mg / L NAA (naphthalene acetic acid), 0.2 mM acetosyringone, pH 5.4.

[0029] Bud induction and selection medium R1 comprises: 4.4 g / L MS salts and B5 vitamins mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 6 g / L Agar, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / L spectinomycin, pH 5.6.

[0030] Rooting medium E0 comprises: 4.4 g / L MS salts and B5 vitamins mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 6 g / L Agar, 1 mg / L IBA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, pH 5.6.

[0031] Example 1

[0032] Cloning of GbCG1 gene

[0033] Primers were designed according to GbCG1 gene (ID number: Gbar_D02G000650.1, source: CottonMD http: / / yanglab.hzau.edu.cn / CottonMD, Cotton Multi-omics Database, which was constructed by the Bioinformatics and Molecular Design Center of Cotton Research Institute, Chinese Academy of Agricultural Sciences, and the Information College of Huazhong Agricultural University and the Cotton Institute of Xinjiang Agricultural and Irrigation Bureau, etc.):

[0034] GbCG1-F: 5'-GAGAGAACACGGGGGACGTCGACATGTTTGTTCTCCTCTGATGTACGTTTG-3', as shown in SEQ ID NO. 1;

[0035] GbCG1-R: 5'-GAACATCGTATGGGTACATGGATCCCCTCCTGTACCACGCCCAAAAAC-3', set forth as SEQ ID NO. 2;

[0036] Figure 1 WMV067-GbCG1) was constructed and transformed into Agrobacterium competent EHA105, and positive clones were selected and stored in liquid medium.

[0037] Table 1 PCR reaction system

[0038]

[0039] Table 2 PCR reaction procedure

[0040]

[0041]

[0042] Table 3 enzyme digestion system

[0043]

[0044] Example 2

[0045] Agrobacterium-mediated genetic transformation of soybean

[0046] The Agrobacterium-mediated method was used to transform soybean, as follows:

[0047] 1. Preparation of Agrobacterium

[0048] 1) Routine activation culture: inoculate from glycerol stock to solid medium containing antibiotics, cultivate at 28°C for 3-4 days to prepare working plates.

[0049] 2) Preparation of bacterial solution: collect bacterial solution from the plates, suspend in infection medium CAB, adjust OD600 to 0.6, add acetosyringone (200 μM) and dithiothreitol (1 mM).

[0050] 2. Agrobacterium inoculation and co-culture

[0051] 3) Seed sterilization: select soybeans with smooth surface and complete seed coat, cover a petri dish, place in the fume hood, add 100 ml sodium hypochlorite and 4 ml concentrated hydrochloric acid in a beaker, and sterilize with chlorine overnight. The next day, take out the sterilized soybeans and place them in a clean bench, and blow to allow the chlorine to disperse completely.

[0052] 4) Seed germination: place the sterilized soybean seeds in germination medium (1 / 2MS basic medium) and germinate at room temperature for 24 h.

[0053] 5) Infection: Take the germinated soybean seeds with tweezers, peel off the seed coat, and carefully separate the cotyledons with a scalpel along the suture of the two cotyledons, trying to keep one cotyledon with the complete hypocotyl and radicle. Under a microscope, peel off the soybean shoot tip to expose it, and then place the soybean shoot tip explants in the prepared Agrobacterium solution, add Poloxamer 188 (10% solution), ultrasonic treatment for 40 seconds, and incubate at 25°C on a shaker (100 rpm) for 1 hour.

[0054] 6) Co-culture: Transfer the explants to filter paper containing co-culture medium CCM, and incubate at 23°C in the dark for 3-4 days.

[0055] 3. Bud induction and screening

[0056] 7) Screening treatment: After co-culture, insert the roots of the explants into bud induction and screening medium R1.

[0057] 8) Bud regeneration and screening: Incubate at 26°C under light intensity of 2000 Lx with a light cycle of 16h / 8h for 3-5 weeks until the buds grow to more than 3 cm in length.

[0058] 4. Rooting and plant regeneration

[0059] 9) Rooting induction: Cut off the buds and transfer them to rooting medium E0, without screening, and after 2-3 weeks, the rooting rate reaches 95%.

[0060] 10) Transplanting: Transplant the regenerated plants to a greenhouse, and the entire process from infection to seedling takes about 2-3 months.

[0061] 11) PCR detection: According to the specific primers related to the sequence of WMV067-GbCG1 vector, perform regular amplification and agarose gel electrophoresis, and specific amplification can detect positive plants. The PCR target band is 435 bp, which can be determined as a positive plant (as shown in Figure 2 ). Through the above experimental detection, positive plants are obtained for later transplanting.

[0062] OE-GbCG1 F: 5'-CAGGGTGAGGACCACATTCC-3', as shown in SEQ ID NO. 6;

[0063] OE-GbCG1 R: 5'-TCCGACATCGATCTCCTGGT-3', as shown in SEQ ID NO. 7.

[0064] Example 3

[0065] Soybean with GbCG1 gene promotes transformation efficiency

[0066] The results of Agrobacterium-mediated genetic transformation are shown in Table 4 and Figure 3As shown in Table 1 (OE-GbCG1-1, OE-GbCG1-2 are part of the transformation lines in the experiment), compared with the transformation control empty vector WMV067 (WMV067-GFP), the treatment of WMV067-GbCG1 vector can greatly improve the genetic transformation efficiency of the same variety. Among them, the transformation efficiency of Zheng 1307 as the receptor transforming empty vector WMV067 is 2.5-4%, while the vector overexpressing GbCG1 can increase the transformation efficiency to 10%-15%; the transformation efficiency of Qihuang 34 as the receptor transforming empty vector control is 1.0-1.5%, while the vector overexpressing GbCG1 can increase the transformation efficiency to 12.5-15.5%, and Zhongpin 661 increases from 1.5-2.0% to 15.5-19.0%.

[0067] Transformation efficiency = (positive seedlings / soybean stem tip number) x 100%

[0068] It can be seen that the agrobacterium overexpressing GbCG1 gene can greatly improve the transformation efficiency of soybean stem tip, especially solving the problem of genotype restriction of some main soybean varieties with low transformation efficiency.

[0069] Table 4

[0070]

[0071] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0072] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of GbCG1 to improve genetic transformation of soybean, characterized in that, The gene sequence of the GbCG1 is shown as SEQ ID NO.

3.

2. Use according to claim 1, wherein The amino acid sequence encoded by the GbCG1 gene is shown as SEQ ID NO.

4.

3. Use according to claim 2, wherein the compound is ###0002### The soybean is Zheng 1307, Qi Huang 34 or Zhongpin 661.

4. An expression cassette, a recombinant vector or a recombinant bacterium containing the GbCGl gene, characterized in that, The GbCG1 gene sequence is shown as SEQ ID NO.

3.

5. Use of the expression cassette, recombinant vector or recombinant bacteria containing the GbCG1 gene in claim 4 in improving genetic transformation of soybean.

6. Use according to claim 5, wherein The GbCG1 gene is integrated into soybean by Agrobacterium-mediated method to overexpress.

7. Use according to claim 6, wherein The recombinant vector: the GbCG1 gene is constructed into the vector WMV067, and the nucleotide sequence of the WMV067 vector is shown as SEQ ID NO.

5.

8. Use according to claim 7, wherein the compound is ###0002### The GbCG1 gene is constructed into the vector WMV067 between the BamHI and SacI enzyme digestion sites. The GbCG1 gene is constructed into the vector WMV067 between the BamHI and SacI enzyme digestion sites.

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