Identification Of GhLAC Gene Family And Functional Analysis Of Candicate GhLAC Genes From Gossypium Hirsutum In Verticillium Wilt Resistance

Cotton（Gossypium spp）is an important economic crop worldwide.Verticillium wilt（VW）is the most destructive disease in cotton yield and fiber quality.Looking for the resistance to Verticillium wilt genes,clarifying gene function,and further analyzing the molecular mechanism of cotton resistance to Verticillium wilt remains a necessary problem in cotton disease resistance breeding.High-quality cotton reference genome is necessary to improve the accuracy of gene family analysis.In this study,the laccase（LAC）family genes in the update genome of G.hirsutum L.cv.TM-1 were identified by bioinformatics,and its physical and chemical properties,gene structure,chromosome location and expression pattern under V.dahliae stress were analyzed.Four new genes homologous to Arabidopsis thaliana were selected and named GhLAC4,GhLAC7,GhLAC11 and GhLAC12,combined with anti-disease transcriptome data and full-length cDNA library screening and functionally unreported premise.In order to clarify the function and molecular mechanism of candidate genes against Verticillium wilt,this study further studied the function of four candidate genes from gene expression and VIGS gene function.The main findings are as follows:1.The 83 LAC family members of the G.hirsutum L TM-1 genome were identified and distributed on 24 chromosomes.All LAC proteins were located extracellularly and shared the same/similar conserved motif.Phylogenetic analysis showed that the members of the LAC genes family were divided into seven subgroups,Bioinformatics analysis showed that GhLAC4,GhLAC7,GhLAC11 and GhLAC12 encoded 558,564,563 and 510amino acid proteins respectively.GhLAC4,GhLAC7,GhLAC11 had one signal peptide respectively.GhLAC12 had no signal peptide.GhLAC11 had a transmembrane domain,and the other three genes had no transmembrane domain.Subcellular localization results showed that four laccase genes were localized outside the cell.2.Based on the transcriptome data of disease-resistant G.hirsutum L 601 under the stress of Verticillium wilt,the expression of each member of the LAC gene family was divided into three modes.The first type of genes showed up-regulation after pathogen induction,including 24 members including GhLAC79,GhLAC80 and GhLAC43.The second type of genes were down-regulated by pathogens,including 56 members including GhLAC07,GhLAC40,GhLAC45 and GhLAC72.The expression of these genes was significantly inhibited after inoculation,suggesting that these genes play a negative regulatory role in cotton resistance to Verticillium wilt.The third type of genes do not respond to V.dahliae treatment,suggesting that they do not participate in the cotton resistance to Verticillium wilt,including GhLAC11,GhLAC27 and GhLAC49.3.The expression of four laccase genes induced by V.dahliae was identified by real-time fluorescent quantitative PCR.It was found that GhLAC4,GhLAC11 and GhLAC12 were up-regulated by V.dahliae,which was consistent with the expression of transcriptome.GhLAC7 was inhibited by V.dahliae.The results of tissue-specific expression showed that the four genes were expressed in roots,stems and leaves;GhLAC4was the highest in stems and the second in roots;GhLAC7,GhLAC11 and GhLAC12 were the highest in roots,followed by stems.The expression level in leaves was the lowest.4.Analysis of promoter components indicated that the GhLAC4 gene contains cis-acting elements in response to methyl jasmonate（JA）and salicylic acid（SA）,GhLAC11 contains cis-acting elements in response to SA,the leaflets were sprayed with SA or JA cotton seedlings to detect the expression of two genes.The results showed that GhLAC4 and GhLAC11 were induced by SA signal;GhLAC4 was induced by JA signal.5.The VIGS vector was constructed with the target fragments of GhLAC4,GhLAC7,GhLAC11 and GhLAC12,which were cloned from the resistant breed 601,and the length was 340 bp.After 25 days of treatment with V.dahliae,the diseased index of the silenced cotton plant was lower than the mock,and the disease resistance was enhanced.The H₂O₂,SOD and NO content of the silenced cotton plant were higher than that of the mock;the POD activity of the silenced cotton plant was lower than that of the mock.6.The lignin staining and determination results of silencing cotton plants and mock with 25d inoculation showed that the lignin staining of silencing cotton plants became lighter,indicating that lignin accumulation decreased after laccase gene silencing;The lignin content indicated that the percentages of lignin in silencing cotton plants GhLAC4,GhLAC7,GhLAC11 and GhLAC12 were 15.34%,7.58%,14.78%,23.22%,respectively,and the percentage of lignin in mock was 33.31%.7.Using qRT-PCR to analyze the transcriptional changes of phenylpropanoid pathway and its flavonoids and gossypol synthesis pathway genes in silencing plants.The lignin synthesis pathway genes PAL,4CL,COMT,HCT,CCoAOMT and CAD expression levels were higher than the mock,indicating that the lower lignin content has negative feedback on the transcription level of the lignin pathway synthetase gene;the flavonoids and gossypol synthesis related genes CHS,CHI,DFR,F3H,F3’H,LAR,ANR and TBS expression levels were higher than mock.In summary,this study identified 83 GhLAC members of upland cotton,and clearly defined the expression patterns of the members under the induction of V.dahliae.The functions of four genes GhLAC4,GhLAC7,GhLAC11 and GhLAC12 were identified and the mechanism of resistance to Verticillium wilt of four members was initially identified.