A Method Of Cotton Gene Transformation In Vivovia Agrobacterium And Its Application In Cottonseed Quality Improvement

Cotton is an important economic crop in China. Cotton fiber, a kind of high-quality natural fiber, is the important raw material for textile industry. Besides the cotton fiber, cotton is an important oil-bearing crop. The cottonseeds are rich in protein and oil. Because the cottonseed is the potential resource for the production of protein and cooking-oil, the nutritional quality of cottonseed gradually becomes also a hot research field. Meanwhile the improvement and utilization of nutritional quality traits of cottonseeds are another important goal of cotton breeding.In this research, the high oil gene, VgDGAT1a, was acted as the purpose gene, and glyphosate resistance gene, EPSPS-G6, which owes our independent intellectual property right, was used as a plant selection marker gene. By technology of Agrobacterium-mediated transformation, the transgenic cotton plants were obtaining after the target genes were transferred into its recepted cultivar, ZMS-49. After a series of selfcrossing purification, the purified genetically modified (GM) germplasms were obtained. The nondestructive analysis-near infrared spectral analysis technology was used to analysis the nutritional quality of GM plants seeds and the value of transgenic VgDGAT1a germplasms were evulated in this paper. The main results were as follows:1. Based on the plant expression vector of pCAMBIA1301-VgDGAT1a which had the plant screening marker gene, hygromycin resistance gene, and active expression vector was reconstructed by replaced hygromycin resistance gene with a herbicide resistant gene, EPSPS-G6, an owe independent intellectual property right gene.2. The restructuring plasmids, pCAM31A1301-VgDGATla+EPSPS-G6, were transferred into Agrobacterium EHA105. With the transferred Agrobacterium EHA105,1268grams of candidate transgenic cottonseeds were obtained via the method of Agrobacterium-mediated transformation techniques in vivo. 3. The candidate transgenic cotton seedlings with three leaves were screened with20mmol/1of glyphosate, a total of125transgenic plants were obtained a week after sparying. Continuous screening with glyphosate and strictly self-crossing, as well as PCR analysis and Southern blot test,51puried transgenic VgDGAT1a gene cotton lines were derived.4. The51puried transgenic line seeds, as well as the non-transgenic genetical background cotton cultivar seeds, were analyzed for oil contents, protein contents, and amino acids contens, used the nondestructive analysis technique developed by our lab. Compared with the control group, the cottonseed oil contents in the transgenic VgDGAT1a lines were increased and the protein content, as well as amino acids, were decreased significantly. The average oil content among the transgenic cotton lines was32.70%and their protein content was45.18%. The significant negative correlation relationship between protein content and oil content was not changed among the transgenic cotton lines. In other words, as the increasing of the cottonseeds oil due to the introduced of VgDGAT1a gene, the decreasing of cottonseed protein, as well as the amino acids will take place in the transgenic cotton line seeds.