| Objective: Cotton Fusarium wilt is one of the most terrible diseases that affect the yield and quality of cotton in our country.Mining disease resistance related genes is the basis of using transgenic technology to cultivate cotton varieties resistant to Fusarium wilt.Besides,plant transcription factors play an important role in disease resistance signaling pathways,among which WRKY transcription factors are essential in plant disease resistance.In this study,a WRKY transcription factor gene Gh WRKY48,related to cotton Fusarium wilt resistance in upland cotton was cloned to analyze the gene function and expression characteristics,so as to provide genetic resources for Fusarium wilt resistance in cotton mocular breeding.Method: In this experiment,the WRKY gene fragment selected from the gene expression profile of the cotton root induced by Fusarium oxysporum was used as a probe,and the WRKY transcription factor gene Gh WRKY48 was cloned from upland cotton with high resistance to wilt disease by electronic cloning.Then,the expression of Gh WRKY48 gene under Fusarium oxysporum f.sp.,salicylic acid(SA),jasmonic acid(JA),ethylene(ET)stress was analyzed by real-time fluorescence quantitative PCR,and the function of this gene was studied by VIGS silencing technology.Results: 1.A WRKY transcription factor related to Fusarium wilt was cloned from the root c DNA of disease-resistant cotton variety “Zhongmiansuo 12”.Through multi-sequence alignment analysis,the gene named Gh WRKY48 belongs to the WRKY transcription factor family of type II.By sequence analysis,we found that full length c DNA of the gene was 1074 nucleotides,and encodeing 357 amino acids contains a WRKY domain and a C2H2 zinc finger structure.2.According to the bioinformatics analysis of Gh WRKY48 gene,it was found that the molecular weight of the encoded protein was 3.94 k Da,the fat index was 56.86,and the isoelectric point was 6.20,which belonged to hydrophobic protein.The protein secondary structure of the gene includes extended chain,alpha helix and random coil.What't more,three types of phosphorylation sites: Threonine(7),Serine(18)and Tyrosine(23)was included of the protein phosphorylation sites of Gh WRKY48 by predicting.3.It was found that the expression of Gh WRKY48 was significantly lower than that of the control group after treatment with Fusarium oxysporum by q RT-PCR analysis.Both SA and JA can induce Gh WRKY48 gene expression after treating the disease-resistant variety “Zhongmiansuo 12” with JA,SA and ET,respectively.Indeed,the expression of Gh WRKY48 was significantly lower than that of water treatment after SA treatment.While JA treatment found that the expression of Gh WRKY48 shows a fluctuation with increased at first and then decreased.Among them,there was a significant difference at 3h,Its expression level was significantly lower than that of the water-treated control,while at other time points,the relative expression level of the gene was lower than that of the control group.With ET treatment,the relative expression of this gene did not change significantly.4.The non-conserved segment was used to construct the interference fragment TRV2-Gh WRKY48-CO of the Gh WRKY48 gene,and the silencing efficiency was about 70%.The results of phenotypic observation and restoration culture showed that the cotton silenced with Gh WRKY48 gene was more sensitive to Fusarium wilt than control plants with empty vector,and display a higher wilting degree,number of diseased plants and disease index.In addition,it was found that the enzyme activities of CAT,PPO,POD and PAL in silent plants were significantly lower than those in control plants through the analysis of the activities of related enzymes.Conclusion: The cloned Gh WRKY48 gene was proved to be responsive to Fusarium wilt,salicylic acid and jasmonic acid by real-time fluorescence quantitative PCR.Besides,we confirmed that Gh WRKY48 gene was participated in cotton's resistance to Fusarium wilt by using VIGS technology to interfered its expression. |
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