Multi-Resistant Cotton Mediated By Heterologous GNA?ACA?NhaD?HEWLl And CP4-EPSPS Genes

Cotton is an important economic crop in China.The production of cotton is always affected by pests,diseases,herbicides,salinized land and other factors.In order to obtain multi-functional transgenic cotton with insect resistance,stress resistance,disease resistance and herbicide resistance,we select five exogenous genes which contains Galanthus nivals agglutinin(GNA),Amaranthus caudatus agglutinin(ACA),Na~+/H~+antiporter(NhaD)and Hen Egg White Lysozyme(HEWL)and CP4-EPSPS to construct multi-functional gene expression vector.Agrobacterium mediated transformation of cotton was carried out in order to obtain new multi-functional transgenic cotton materials with resistance to aphids,verticillium wilt,salt and alkali,and herbicides.In this study,a plant expression vector containing five exogenous genes was constructed,in which GNA and ACA were connected by 2A polypeptide and mediated by 35S promoter and NOS terminator,NhaD,HEWL and CP4-EPSPS were also connected by 2A polypeptide and controlled by plastocyanin promoter and plastocyanin terminator.The transgenic lines 5g-1 and 5g-4 with 5 exogenous genes were obtained by Agrobacterium mediated transformation.PCR showed that the foreign genes were integrated into the cotton chromosome,and the exact insertion site of T-DNA in 5g-1 and 5g-4 transgenic lines was determined by Genome Walking:T-DNA was inserted into A02(11664124-11664203)of upland cotton in 5g-1,and T-DNA was inserted into A06(95514261-95514281)in reverse in 5g-4.The expression of foreign gene in cotton was confirmed by qRT-PCR,Western blot and ELISA.The aphid inoculation experiment on the transgenic plants showed that the transgenic plants had a certain inhibition on aphids,the inhibition rate was 36%?55.50%,which was significantly better than the control plants.In this paper,10 KM mice were immunized with commercial snowdrop lectin and amaranth lectin protein by subcutaneous injection.Western blot and ELISA showed that the antiserum of the mice met the requirements of detecting the target protein of transgenic cotton.qRT-PCR and Western blot of transgenic plants found that the expression of exogenous in plastocyanin promoter was better than that of 35S promoter in cotton,and the plastocyanin promoter could be used when the expression level was considered to be increased.The new transgenic cotton materials with five foreign genes were obtained in this study,which provided ideas and technical support for the cultivation of new multi-resistant cotton varieties.It was found that the expression of exogenous genes mediated by plastocyanin promoter were better than that of35S promoter.It provides the research idea and foundation for the high expression of exogenous gene in cotton.