Application of cotton GbCG1 gene in improvement of plant genetic transformation efficiency

Abstract

The invention discloses an application of a cotton GbCG1 gene in improvement of plant genetic transformation efficiency. Relates to the technical field of transgenosis and provides specific application. According to the invention, a somatic embryo regeneration candidate gene GbCG1 is screened out. The gene is only expressed in sea island cotton, overexpression of GbCG1 in upland cotton can significantly improve the callus differentiation efficiency and shorten the somatic embryo generation period, only 4-5 months are needed from transformation to seedling emergence, the time is 3-5 months faster than that of a control group, and the transformation efficiency is 3.5 times that of the control group. The invention discloses application of a GbCG1 gene in promoting somatic embryogenesis of cotton and improving the gene transformation efficiency, and the callus differentiation efficiency can be remarkably improved by overexpressing the GbCG1 gene. And valuable gene resources are provided for genetic breeding. The method can effectively solve the problem of cotton transformation genotype limitation, and broadens the variety types of cotton genetic transformation receptors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of transgenic technology, and more particularly to application of cotton GbCG1 gene in improving plant genetic transformation efficiency. BACKGROUND

[0002] Cotton is one of the most important natural fiber sources and economic crops in the world, and cotton industry accounts for about 56 billion US dollars of global social economic value. Cotton textile processing industry and planting industry have become an important part of the national economy. Using genetic engineering to improve cotton varieties is a quick and effective way. The genome editing technology emerging in recent years provides a simple and precise gene improvement method, and becomes a revolutionary breakthrough in the field of life science. Editing candidate functional genes in cotton can verify gene function more quickly and conveniently, and create mutant and excellent allele materials, and gene editing needs a mature, stable and efficient somatic cell regeneration system.

[0003] However, the current cotton varieties planted are severely limited by genotype, and it is difficult to perform genetic transformation by means of Agrobacterium mediation; even if the varieties that can be genetically transformed exist, there are problems such as long transformation cycle, high somatic embryo malformation rate and small number of normal regenerated seedlings, which seriously limit the application of genetic engineering in cotton variety improvement. In order to solve this "short board problem", it is necessary to screen key genes that can improve the cotton genetic transformation system, analyze the regulation network of cotton somatic embryogenesis, and then optimize the cotton somatic embryogenesis system, so as to lay a foundation for efficient creation of new cotton varieties.

[0004] The genetic analysis of cotton regenerable traits is mainly carried out on upland cotton, and few studies use Gossypium barbadense, which is generally considered to be more limited by genotype than upland cotton and more difficult to differentiate. Therefore, providing application of cotton GbCG1 gene in improving plant genetic transformation efficiency, overcoming the above technical deficiencies is a problem that the skilled in the art need to solve. SUMMARY

[0005] In view of this, the application provides application of a cotton GbCG1 gene in improving plant genetic transformation efficiency. Tissue culture is carried out by using a sea-land introgression line population, and phenotype data of somatic embryo regeneration ability of different materials are collected. The introgression line is resequenced to obtain genotype data of the population, introgression fragments at the whole genome level are identified, and an introgression map is obtained. The genotype and phenotype data are combined to carry out whole genome association analysis of somatic embryo regeneration ability, and key QTLs sites related to somatic embryo are obtained. The GWAS results and introgression results are further associated, and key QTLs sites of introgression are screened. By annotating SNPs and InDels on the introgression fragment and analyzing the expression amount of orthologous genes with sequence difference in upland cotton, the GbCG1 gene is finally locked, which is a key candidate gene for regulating somatic embryogenesis, and only expresses in sea-island cotton. Overexpression of GbCG1 in upland cotton can significantly improve the efficiency of callus differentiation and accelerate the speed of somatic embryogenesis. Through the innovative work of introducing the sea-island cotton stress resistance gene into upland cotton to significantly improve the transformation efficiency, important insights are provided for establishing a high-efficiency cotton molecular breeding genetic transformation system.

[0006] Overexpression of the GbCG1 gene can not only improve the efficiency of somatic embryogenesis and shorten the differentiation time, but also greatly improve the efficiency of Agrobacterium transformation. Compared with normal zygotic embryogenesis, in vitro tissue culture has very complex growth conditions, and callus cells are exposed to oxygen, which often leads to oxidative stress and brown callus formation. Oxidative browning is a common problem in plant tissue culture, and many transformed cells die before reaching the redifferentiation stage, resulting in a decrease in transformation efficiency. After overexpression of the GbCG1 gene, the transformed cells can resist the damage caused by oxidative stress, quickly recover from the damage, and quickly enter the redifferentiation stage. The growth rate of the transformed cells is greater than that of the non-transformed cells, thereby improving the genetic transformation efficiency of cotton.

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

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

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

[0010] Preferably, the plant is upland cotton.

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

[0012] The application also provides application of the expression cassette, the recombinant vector or the recombinant bacteria containing the cotton GbCG1 gene in improving genetic transformation efficiency of Gossypium hirsutum.

[0013] Preferably, the GbCG1 gene is integrated into Gossypium hirsutum by Agrobacterium-mediated method to overexpress.

[0014] Preferably, the recombinant vector is that the cotton GbCG1 gene is constructed into the vector pCAMBIA2300.

[0015] Preferably, the cotton GbCG1 gene is constructed into the vector pCAMBIA2300 between Xba1 and Kpn1 enzyme cutting sites.

[0016] Compared with the prior art, the application discloses application of the cotton GbCG1 gene in improving genetic transformation efficiency of plants, and the following technical effects are achieved: the application screens a somatic embryo regeneration candidate gene GbCG1. The gene is expressed only in Gossypium barbadense, and overexpression of the GbCG1 gene in Gossypium hirsutum can significantly improve callus differentiation efficiency and shorten a somatic embryogenesis cycle, so that only 4-5 months are needed from transformation to emergence, 3-5 months are shortened compared with a control, and the transformation efficiency is 3.5 times that of the control. The application discloses application of the GbCG1 gene in promoting cotton somatic embryogenesis and improving gene transformation efficiency, overexpression of the GbCG1 gene can significantly improve callus differentiation efficiency, and provides valuable gene resources for genetic breeding. The application can effectively solve the genotype restriction problem of cotton transformation and widen types of cotton genetic transformation receptors. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the provided drawings.

[0018] Figure 1 The accompanying drawings are schematic diagrams of the overexpression vector structure provided by the present application.

[0019] Figure 2 The accompanying drawings are diagrams of GbCG1 promoting differentiation of cotton embryogenic callus provided by the present application, wherein A-C in the drawings are phenotype observation of GbCG1 transgenic materials and control materials cultured for 30 days; D-F in the drawings are phenotype observation of GbCG1 transgenic materials and control materials cultured for 60 days, the red arrow is a differentiated somatic embryo; the upper right diagram is expression quantity analysis of the GbCG1 gene in the OE-GbCG1 strain; and the lower right diagram is embryogenic callus induction rate of the transgenic materials and the control materials, i.e. EDR.

[0020] Figure 3 The attached drawings provide a comparison chart of transformation efficiency and time according to the present application. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in 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.

[0022] The present application discloses the application of cotton GbCG1 gene in improving plant genetic transformation efficiency.

[0023] The existing upland cotton (Gossypium hirsutum L.) variety in the embodiments: Zhongmiansuo 24 is derived from the germplasm bank of the Cotton Institute of Chinese Academy of Agricultural Sciences, and is referred to as CCRI24 hereinafter. The hormones involved in the following embodiments are all products of sigma company; the culture medium and other products are from reagent company;

[0024] The components of the basic culture medium MSB are shown in Table 1:

[0025] Table 1

[0026]

[0027] Preparation of commonly used culture media and solutions

[0028] Callus induction medium MSB1: 2,4-D and KT are added to the basic culture medium MSB, so that the content of 2,4-D and KT is 0.5 mol / L.

[0029] Embryogenic callus induction medium MSB2: IAA and KT are added to the basic culture medium MSB, so that the content of IAA and KT is 0.05 mol / L.

[0030] Embryoid induction medium MSB3: IBA and KT are added to the basic culture medium MSB, so that the content of IBA and KT is 0.01 mol / L.

[0031] Regeneration seedling induction medium MSB4: 6BA and NAA are added to the basic culture medium MSB, so that the content of 6BA and NAA is 0.01 mol / L.

[0032] Rooting medium: 6BA and NAA are added to the basic culture medium MSB, so that the content of IAA and KT is 0.01 mol / L.

[0033] Example 1

[0034] Cloning of GbCG1 gene

[0035] According to the GbCG1 gene (Gbar_D02G000650.1, CottonMD, http: / / yanglab.hzau.edu.cn / CottonMD, Cotton Multi-omics Database, which is constructed by the Bioinformatics and Molecular Design Center of Cotton Research Institute, Chinese Academy of Agricultural Sciences, and many other units such as the Information College of Huazhong Agricultural University and the Cotton Institute of Xinjiang Agricultural and Cultural Institute) in cotton, primers were designed:

[0036] GbCG1-F:

[0037] 5'-AGAACACGGGGGACTCTAGAATGTTTGTTCTCCATCTGATGG-3'

[0038] (SEQ ID NO. 1)

[0039] GbCG1-R:

[0040] 5'-TAGTCAGGCGCGCCGGTACCTTACCTCCTGTACCACGCCCAAAC TCCA-3' (SEQ ID NO. 2)

[0041] Figure 1 The pCAMBIA2300-GbCGl was transformed into Agrobacterium EHA105 by the method described above. The positive clones were selected by kanamycin resistance and stored in liquid LB medium containing 50 μg / mL kanamycin, 25 μg / mL rifampicin, 50 μg / mL streptomycin. Meanwhile, the pCAMBIA2300 empty vector was transformed into Agrobacterium EHA105 by the method described above. The positive clones were selected and stored in liquid LB medium.

[0042] Table 2 PCR reaction system

[0043]

[0044] Table 3 PCR reaction procedure

[0045]

[0046] Table 4 Enzyme digestion system

[0047]

[0048] Example 2

[0049] Agrobacterium-mediated genetic transformation of cotton

[0050] Agrobacterium transformation of cotton process:

[0051] (1) Sterile seedling culture: Take a proper amount of sulfuric acid after the de-cotton of cotton variety CCRI24, soak in 3% hydrogen peroxide (H2O2) for disinfection overnight, then wash with sterile water for 3-5 times, put the seeds in sterile seedling culture medium, cultivate in dark at 28°C for 5-6 days, so that it germinates and grows into yellow sterile seedlings.

[0052] (2) Agrobacterium infection: Before infection, the Agrobacterium strain containing pCAMBIA2300-GbCGl described above was inoculated into LB liquid medium containing 50 μg / mL kanamycin, 25 μg / mL rifampicin, 50 μg / mL streptomycin; on the day of transformation of cotton, the activated bacteria were inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin, 25 μg / mL rifampicin, 50 μg / mL streptomycin at a ratio of 1:100, and cultured in a shaking incubator at 28°C, 190 rpm until the OD600 value reached 0.3.

[0053] (3) Take the yellow seedlings with good growth after 5-6 days of culture at 28°C, cut the hypocotyls into 0.5 cm segments on the clean bench, and cut about 6-8 segments from each yellow hypocotyl. Soak the cut hypocotyl segments in the Agrobacterium prepared in step (2) with an OD value of 0.3, soak for 3-5 min, then pour out the bacterial solution, use sterile filter paper to absorb the residual bacterial solution on the hypocotyl segments, then transfer the hypocotyls to MSB basic medium, cultivate in dark at 23°C for 48 hours.

[0054] (4) Callus screening: the hypocotyls after 48 hours of co-culture were cut into segments and subcultured into callus induction medium MSB1 containing kanamycin, at 28±2°C, light intensity 2000 Lx, light cycle 16h / 8h, for 2 months until large callus grew from both ends of the segments.

[0055] (5) Differentiation induction: the induced positive callus was subcultured into embryogenic callus induction medium MSB2, at 28±2°C, light intensity 2000 Lx, light cycle 16h / 8h, for 1 month until yellow-green millet-shaped embryogenic callus appeared.

[0056] (6) Embryoid induction: the induced embryogenic callus was subcultured into embryoid induction medium MSB3, at 28±2°C, light intensity 2000 Lx, light cycle 16h / 8h, subcultured once every 15-20 days until embryoids appeared.

[0057] (7) Regeneration induction: the induced embryoids were subcultured into regeneration medium MSB4, at 28±2°C, light intensity 2000 Lx, light cycle 16h / 8h, subcultured once every 15-20 days until regeneration appeared.

[0058] (8) Rooting induction: the sprout tissue was subcultured into seedling medium, and after the seedlings grew, further transferred to the rooting medium of the regeneration seedlings for rooting culture, at 28±2°C, light intensity 2000 Lx, light cycle 16h / 8h, for 2 weeks, then the regenerated seedlings were taken out of the medium, soaked in tap water, at 28±2°C, light intensity 2000 Lx, light cycle 16h / 8h, for hardening, and transplanted into flowerpots after a week to obtain CCRI24 overexpression OE-GbCG1 plants.

[0059] (9) The positive regenerated seedlings after rooting were placed in vermiculite nutrient pots and placed in a cool and dry greenhouse, watered in time, transplanted to the test field after hardening for two weeks, and the T0 generation seeds were obtained by strict selfing.

[0060] Example 3

[0061] GbCG1 promotes cotton genetic transformation

[0062] The overexpression vector of GbCG1 gene was constructed for functional verification. (35S promoter driven GbCG1) overexpression vector pCAMBIA2300-GbCG1 and empty vector p2300 were transformed into Gossypium hirsutum CCRI24 to obtain regenerated seedlings.

[0063] As Figure 2qRT-PCR (see Tables 5-7 for system, procedure, and primers) results showed that the expression level of GbCG1 gene in transgenic lines was much higher than that in control line CCR124. The transgenic lines and the control were cultured on callus induction medium, and after 7 days of culture, histological observation was performed, and then the number of embryogenic callus differentiated from callus was counted at 30, 40, 50, and 60 days, and the embryogenic callus induction rate (EDR) was calculated.

[0064] After 30 days of culture of the transgenic lines GbCG1-7 and GbCG1-12 on callus induction medium, the callus started normally, the callus plasmid was loose, and presented small granular vitality, while the callus block of the empty vector was dense, and partially whitish. After subculture of GbCG1-7, GbCG1-12, and the control material on embryogenic callus induction medium, it was found that a large number of differentiated loose and light yellow embryogenic callus had been formed in the overexpression vector at 50 days of culture, while the number of differentiated blocks of the empty vector control was very small. After 60 days of culture, the embryogenic callus differentiated in the GbCG1 overexpression material had grown into tender green embryoid, while the empty vector material only began to form embryogenic callus. Compared with the empty vector, the overexpression of GbCG1 gene greatly shortened the time for the formation of embryogenic callus, increased the number of embryoid, thereby shortening the transformation period and improving the transformation efficiency. From transformation to emergence, only 4-5 months of time was needed, which was 3-5 months faster than the control, and the transformation efficiency was 3.5 times that of the control. Figure 3

[0065] It is shown that GbCG1, as an island cotton stress resistance gene, can promote somatic embryogenesis in upland cotton.

[0066] Table 5 qRT-PCR reaction system

[0067]

[0068] Table 6 qRT-PCR reaction procedure

[0069]

[0070] Table 7 qRT-PCR primer information

[0071]

[0072] Each of the embodiments in the specification is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the embodiments can be referred to each other.

[0073] ​The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of cotton GbCG1 gene in improving plant genetic transformation efficiency, characterized in that, The cotton GbCG1 gene sequence is shown as SEQ ID NO.

3.

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

4.

3. Use according to claim 2, wherein the compound is ###0002### The plant is Gossypium hirsutum.

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

3.

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

6. Use according to claim 5, wherein The GbCG1 gene is integrated into Gossypium hirsutum by Agrobacterium-mediated method for overexpression.

7. Use according to claim 6, wherein The recombinant vector: the cotton GbCG1 gene is constructed into the vector pCAMBIA2300.

8. Use according to claim 7, wherein the compound is ###0002### The cotton GbCG1 gene is constructed into the vector pCAMBIA2300 between the Xba1 and Kpn1 enzyme cutting sites. The cotton GbCG1 gene is constructed into the vector pCAMBIA2300 between the Xba1 and Kpn1 enzyme cutting sites.

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