| Cotton is an important material that affects the national economy and people’s livelihood.The verticillium wilt caused by Verticillium dahliae seriously affects the improvement of cotton yield and quality.Exploring the genes for cotton resistance to verticillium wilt and creating new cotton germplasm resources are of positive significance for the sustainable development of cotton.Based on the previous research results of the research team,this study analyzed the differentially expressed proteins of sea island cotton after inoculation with Verticillium dahliae,and identified a stearyl acyl carrier protein desaturase(SACPD),whose amino acid sequence has 78% homology with Arabidopsis AtSSI2,so it was named Gb SSI2.Through sequence alignment,19 homologous genes of SSI2 were identified in the genome of upland cotton.This study systematically analyzed the SSI2 gene family and explored the function of GhSSI2s in cotton.The main research results are as follows:1.Using the protein sequence of AtSSI2(At2g43710)reported in Arabidopsis as a reference,19 highly homologous genes were identified in the upland cotton genome library.Candidate genes with more than 70% homology in upland cotton were selected as the objects of genome editing.CRISPR/Cas9 gene knockout was performed on candidate gene groups based on genetic evolution relationships,and four groups of gene knockout materials were obtained through cotton genetic transformation.2.Obtain GhSSI2s knockout cotton material.Knocking out the plants resulted in shorter growth compared to the control,more compact plant types,increased boll setting rate,and enhanced cotton’s resistance to verticillium wilt.The proportion of oleic acid in the knocked out plant seeds decreased significantly,while the total amount of fatty acids did not change significantly,indicating that this family of genes is involved in the metabolism of oleic acid in cotton.3.Obtain GhSSI2 overexpression cotton material.The transgenic plants grow robustly,with significantly higher plant height and more robust root systems compared to the transgenic receptor Jin668 and GhSSI2s knockout plants;At the same time,overexpressing plants exhibit abnormal growth,resulting in the heterogenesis of "two main stems" similar to the main stem under greenhouse conditions;Under field conditions,overexpression plants showed a significant increase in the number of leaf branches.Metabolic analysis showed that the proportion of oleic acid in overexpressed plants increased,while the proportion of stearic acid decreased,indicating that overexpression of this gene promoted the conversion of stearic acid to oleic acid in cotton.Overexpression of GH＿D05G3478has no significant effect on the resistance of cotton to verticillium wilt.4.The content of salicylic acid(SA)in the knockout plants was significantly higher than that in the control and overexpressed plants,and the expression level of the main disease resistance genes in the SA pathway was also significantly increased;After inoculation with Verticillium wilt,the SA related genes in the knockout plants were activated and upregulated,with a significantly higher degree of upregulation than the other two,which may be the main reason for their enhanced resistance.The content of jasmonic acid(JA)in overexpression plants was slightly lower than that in control plants,and the JA content in knockout plants was higher than that in control plants;After inoculation with Verticillium wilt,the expression level of JA related genes in the knockout plants was inhibited.Based on the above research results,we have preliminarily verified the negative regulation of cotton resistance to verticillium wilt by GhSSI2s,providing new germplasm resources and theoretical basis for cotton breeding against verticillium wilt;Overexpression of GhSSI2 can promote the nutritional growth of cotton,leading to the appearance of two "main stems" in cotton,which can provide reference for cotton plant type improvement. |
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