Cloning And Functional Verification Of Glycosyltransferase Related Genes Responding To Fusarium Wilt Resistance In Gossypium Barbadense L

The effective way to solve the problem of quality reduction and yield reduction caused by Fusarium wilt is to select and breed new varieties of cotton resistant to disease,and to explore the resistance genes and study their regulatory mechanism to provide an important theoretical basis for molecular breeding of cotton resistant to disease.In this paper,the research in the early of the transcriptome sequencing data enrichment to the glycosyl transferase differentially expressed genes related to genetic analysis（DEG）,use of resistant material and infected the real-time fluorescence quantitative expression（q RT-PCR）analysis,the appraisal to the key gene cloning and bioinformatics analysis,the method of using virus mediated gene silencing of validation of gene function,specific results are as follows:1.DEG analysis of the 9 glycotransferase-related genes enriched in the transcriptome showed that:After inoculation,GB＿A03G0575and GB＿A11G1787 showed a decreasing trend in resistant varieties,and the decreasing ratio in resistant varieties was greater than that in susceptible varieties.GB＿A13G1825 and GB＿D07G2156 showed an upward trend in resistant varieties,and the upward ratio in resistant varieties was higher than that in susceptible varieties.It was suggested that the different resistance of island cotton might be related to these 4 glycotransferases.2.QRT-PCR analysis of the 9 glycotransferase-related genes enriched in the transcriptome showed that:there were differences in the expression levels of glycotransferase-related genes between resistant and susceptible materials.After inoculation,the expression of GB＿A03G0575 would decrease first and then increase with the change of time,GB＿A13G1825and GB＿A11G1787 gene expressions in resistant materials were higher than those in susceptible materials,and it was speculated that these genes play a role in disease resistance.3.Cloning and bioinformatics analysis of GB＿A03G0575（GbGSTU7）and GB＿A13G1825（GbUGT73C1）genes showed that the length of GbGSTU7 gene CDS was 711 bp,encoding 236 amino acids.The encoded protein was s Tab,hydrophilic and fat-soluble protein with no transmembrane structure,including GST-N-Tau and GST-C-Tau domains,both belonging to the glutathione S-transferase family.The total length of GbUGT73C1 gene CDS is 1488 bp,encoding 496 amino acids.The encoded protein is hydrophobic,fat-soluble and uns Tab protein,without transmembrane structure,with GTB structure and PSPG motif,and belongs to the Glycosyltransferase＿GTB-type super family.Subcellular simulation localization of GbGSTU7 in cytoplasm and GbUGT73C1 in cell membrane.4.Verification of virus-mediated gene silencing function:Albino phenotype appeared in positive control TRV₂-CLA14-15 days after injection,and silencing efficiency of GbGSTU7 gene was 74.0%,and silencing efficiency of GbUGT73C1gene was 25.4%.The VIGS-GbGSTU7 transgenic plants developed more serious disease after inoculation,and the disease index was 31.4 higher than that of the control.The activity of glutathione transferase in vitro was 2 times lower than that of the control,which indicated that GbGSTU7 played an important role in the resistance of Island cotton to Fusarium wilt.5.Verification of overexpression function:PCMBIA3301-GbGSTU7 overexpression vector was constructed.（1）The wild-type plants of Arabidopsis thaliana were infected by flower impregnation method and transgenic plants were screened with BAST herbicide.So far,T2 generation of transgenic Arabidopsis seeds have been obtained.（2）Xinhai 14 and DJ-07-136 were transgenic by pollen tube pathway.Xinhai 14 treated 594 flowers and harvested 370 bolls,with an average boll formation rate of 62.3%;DJ-07-136 treated 469 flowers and harvested 380 bolls,with an average rate of 81.0%.