Creation,Molecular Detection And Safety Evaluation Of Transgenic Drought-resistant Cotton Materials

With the transfer of Chinese main cotton producing areas to the northwest inland areas,drought stress has become the primary abiotic stress factor hindering the sustainable development the cotton industry.Therefore,it is of great significance to analyze the mechanism of cotton response to drought stress and to mine stress-resistant gene resources for the steady increase of cotton yield in China.In recent years,technologies such as omics technology,genetic engineering and gene editing have been widely used in the genetic improvement of cotton varieties,and a large number of stress resistance-related genetic loci and genes have been excavated and applied to the creation of transgenic stress-resistant cotton materials.However,biosafety issues brought about by transgenic technology have also attracted attention from all walks of life.Therefore,the State Council has formulated and promulgated the Regulations on the Safety Control of Genetically Modified Organisms in Agriculture of Genetically Modified Organisms(GMOs),aiming at regulating genetic modification technology and evaluate the safety of GMOs.In this study,vector construction and genetic transformation of genes relevant to drought resistance in cotton were carried out to create over-expression and gene editing materials,and molecular identification,genetic stability analysis and the biosafety analysis of exogenous proteins were carried out on the obtained transgenic plants,to complete the primary evaluation of the safety of genetically modified materials.The main results are as follows:1.Over-expression vectors and CRISPR/Cas9 vectors were constructed for the candidate genes(Gh AIP1、Gh PLT1、Gh GGT3、Gh D7450、Gh AT5G、Gh ACD22、Gh AEL3、Gh SRP、Gh AT1G、Gh YLS9、Gh WRKY6、Gh MYB108、Gh NREC)related to drought resistance screened by GWAS and drought resistance expression profiling analysis,and Agrobacterium was used to infect cotton hypocotyls for genetic transformation,successfully creating transgenic cotton.2.Molecular characterization of low-generation transgenic cotton(OE-Gh WRKY6,OE-Gh MYB108 and OE-Gh NREC)has been completed.Positive individual strains were screened out by PCR,indicating that the foreign gene had been successfully transformed into the recipient plants.Further,the integration of exogenous genes in the genome was analyzed,and the results of Southern blotting of OE-Gh NREC over-expression materials showed that NOE5-1,NOE5-3,NOE1-2,NOE1-4 and NOE1-8 were all single-copy insertion lines in the 7 materials tested;the results of Southern blotting of the Gh PYL8 overexpression line OE8 showed that the target gene Gh PYL8 was single-copy insertion in both the T3 and T4 generations,indicating that the target gene can be stably inherited in the overexpression line OE8.Finally,the expression level of the target gene was analyzed.RT-PCR and q RT-PCR results showed that the expression level of the target gene(Gh WRKY6,Gh MYB108,Gh NREC,Gh PYL8)in the screened transgenic materials was significantly increased,indicating that the target gene can be stably expressed in the transgenic material.In addition,according to the requirements of environment release,we analyzed the expression of the target protein of Gh PYL8 over-expressing transgenic lines.Western blotting results showed that the Myc-tagged target protein Gh PYL8 was expressed in roots,stems and leaves,and the protein expression was the highest in leaves,followed by stems,and the lowest in roots,it indicated that the target gene Gh PYL8 was expressed in multiple tissue parts,and a new protein was formed to function through transcription and translation.3.In accordance with the provisions of the Administrative Measures for the Safety of Agricultural Genetically Modified Organisms,we used bioinformatics technology to predict the toxicity,allergenicity and anti-nutrition of proteins translated from target genes(Gh TGA7,Gh ABF1,Gh WRKY6,Gh MYB108,Gh NREC,Gh PYL8)and marker genes(NPT II),and combined with the results of molecular identification,the safety assessment of transgenic cotton was carried out.The comparison results of NCBI,Uni Prot and T3 DB databases showed that the newly expressed protein had no sequence homology with known toxin proteins,allergenic proteins and anti-nutritional factors;it was preliminarily considered that the transgenic cotton material had high biosecurity.In summary,this study constructed the over-expression vector and CRISPR/Cas9 vector of drought resistance-related candidate genes,and created transgenic cotton,which laid a foundation for the investigation of phenotype and the analysis of drought resistance mechanism.Further,in combination with the Regulations on the Safety Control of Genetically Modified Organisms in Agriculture of Genetically Modified Organisms,we have analyzed genetic stability and biosecurity on the genetically modified materials that have obtained regenerated plants,and have successfully carried out intermediate tests and environmental release studies,it is of great significance for the approval and industrial application of security certificates of genetically modified materials.