Transgenic Germplasms Creation And Functional Identification Of Two Gossypium Hirsutum Genes GhIAA43 And Ghb561-8 Related To Verticillium Wilt Resistance

Cotton is an important cash crop and fiber crop as well as an important natural fiber and oil crop in the world.Verticillium wilt is a kind of soil-borne vascular disease that can seriously affect the growth and development of cotton.Because it has not been completely cured,Verticillium wilt has now become one of the main factors restricting cotton production.It is an effective method for cotton Verticillium wilt resistance by excavating and identifying cotton Verticillium wilt resistance genes,then creating cotton disease-resistant germplasm materials using genetic engineering technology.The AUX/IAA family is an important part of the auxin signal transduction pathway,which can regulate the expression of auxin-responsive genes by affecting the auxin signal transduction pathway,thereby affecting plant growth and development and disease resistance.The cytochrome b561 family is a type of intrinsic protein genes that mediate the electron transport during the formation of ascorbic acid,which can affect the process of plant disease resistance by mediating the formation of ascorbic acid.In the previous studies from our laboratory,through the released cotton genome data and gene family analysis,we have found that the gene GhIAA43 in the AUX/IAA family and the Ghb561-8 in the cytochrome b561 family,which was significantly induced by Verticillium dahliae.In this study,we used VIGS to conduct preliminary identification of disease resistance of GhIAA43 and Ghb561-8,then created the GhIAA43 transgenic germplasms through Agrobacterium-mediated genetic transformation.And combined with Ghb561-8 transgenic cotton materials which previously created in our laboratory,we completed the identification of disease resistance and growth and development-related phenotypes of GhIAA43 and Ghb561-8 transgenic materials.The main conclusions are as follows:1.Respectively silenced GhIAA43 and Ghb561 can improve the resistance to Verticillium wilt in Gossypium hirsutum.GhIAA43 is a gene in the Aux/IAA family that responds to the induction of Verticillium wilt,and its subcellular location shows that it is located on the nucleus.Ghb561-8 is a member of the cytochrome b561 family.The subcellular location results indicate that Ghb561-8 is mainly distributed outside the cell.GhIAA43 and Ghb561-8 were respectively silenced in Gossypium hirsutum TM-1 through VIGS technology,and then Verticillium dahliae infection experiment was carried out.The results showed that the incidence of gene-silenced plants was significantly reduced and showed stronger resistance to Verticillium wilt compared with control ones.It showed that GhIAA43 and Ghb561-8 genes are involved in the defense response of Verticillium wilt in cotton.2.Created transgenic materials that overexpress and interfere with the expression of GhIAA43.Firstly,in order to obtain cotton material with increased resistance to Verticillium wilt,we constructed p CAMBIA2301-GhIAA43 and p ART27-GhIAA43 vectors using cotton hypocotyls as receptors creating transgenic materials through Agrobacterium-mediated transformation.Then,we screened and identified for the progeny homozygous strains of transgenic materials.Finally,3progeny homozygous strains were identified from 4 independent transformation events of p CAMBIA2301-GhIAA43 vectors,and 2 progeny homozygous strains were identified from 7 independent transformation events of p ART27-GhIAA43.3.Interfering with the expression of GhIAA43 and Ghb561-8 genes can improve the resistance of Gossypium hirsutum to Verticillium wilt.In this study,Verticillium wilt resistance of GhIAA43 and Ghb561-8 transgenic materials was identified.And the results showed that the transgenic materials with GhIAA43 and Ghb561-8 gene interference expression showed stronger resistance to Verticillium wilt,which confirmed that the decreased expression of GhIAA43 and Ghb561-8 genes can improve the resistance of Gossypium hirsutum to Verticillium wilt.4.GhIAA43 can affect the growth of cotton.By scanning the roots of GhIAA43 transgenic materials,we found that the reduction of GhIAA43 expression could significantly promote the growth of cotton roots.The seed germination of GhIAA43 transgenic materials indicates that overexpression or interference of GhIAA43 can affect the germination rate and germination process of cotton seeds...