Functional Analysis Of GbOsmotin34 For Resistance To Verticillium Wilt In Fotton

Cotton is one of the most important economic crops in the world and an important source of natural fibers and oil.In cotton-growing areas,Verticillium dahliae poses a serious threat to cotton yield and fiber quality.Since the discovery of cotton verticillium wilt,it has been unable to be cured,and it has gradually become the number one disease in cotton production.Using bioinformatics and molecular biology methods to mine the key disease resistance genes of cotton provides a new way for cotton disease-resistant germplasm materials.After the pathogens attack,the defense signaling pathways,salicylic acid(SA)and jasmonic acid(JA),are activated,which further leads to the accumulation of PR proteins(Pathogenesis-related proteins),thereby causing pathogen loads or diseases in uninfected plant organs minimize.The PR5 gene plays an important role in the process of plant disease resistance.The PR5 gene mediated resistance to Verticillium wilt in cotton is rarely reported.In this study,the PR5 family genes in different cotton species were mined from the whole genome level through bioinformatics,combining the disease-resistant material Hai7124 and the disease-sensitive material TM-1 transcriptome data,found a key disease resistance gene in the cotton PR5 family,and further conducted a preliminary study on the function of this gene,the specific results are as follows:1).Genome-wide excavation of the PR5 gene family and identification on candidate genes related to resistance in cotton:The PR5 family has a thaumatin protein domain.Using the seed file PF00314 of this domain as a probe,the genome-wide identification of the PR5 gene in four cotton species with published sequences was carried out.There are 90 PR5 genes identified in the G.hirsutum acc.TM-1(ADI)and 91 in G.barbadense acc.Hai7124(AD2),48 in G.arboreum(A2),and 59 in G.raimondii(D5).By analyzing the transcriptome data,it was found that the four PR5 genes were predominantly expressed in the root tissue,and induced by Verticillium dahliae in Hai7124 but not induced by TM-1.Further qRT-PCR verification results were consistent with transcriptome expression.One of the PR5 genes expression levels is higher,and the expression difference is the highest in Hai7124 and TM1,the gene has the highest similarity to Osmotin34 in Theobroma cacao.Further analysis revealed that in tetraploid upland cotton and island cotton,which is located on the D subgenome.According to its cotton seed origin,the gene derived from upland cotton was named GhOsmotin34,and the gene derived from island cotton was named GbOsmotin34.2).Tissue and organ expression analysis of Osmotin34 and disease resistance studies:Through analysis of cotton transcriptome data,it was found that Osmotin34 is predominantly expressed in root,and the expression level in the disease-resistant material Hai7124 is significantly higher than that in the disease-sensitive material TM-1,and induced by Verticillium dahliae in Hai7124.The results of subcellular localization showed that GbOsmotin34 was mainly distributed extracellular,and the disease resistance of cotton was significantly reduced after VIGS technology silenced this gene in Hai7124.In vitro bacteriostasis experiments showed that the purified protein of GbOsmotin34 had a significant inhibitory effect on fungal spores during the growth of Verticillium dahliae,and the effect was more obvious in the early stage of the growth of Verticillium dahliae.3).Molecular mechanism of Osmotin34 resistance to Verticillium wilt:Based on the significant expression differences between Hai7124 and TM-1,the 1371bp Osmotin34 promoter sequence was cloned from Hai7124 and TM-1,respectively.Sequence analysis showed that PGhOsmotin34 and PGbOsmotin34 had a CCAAT/CCGAT motif mutation in the susceptible material TM-1 and the disease-resistant material Hai7124,and the CCAAT motif was the binding site of the transcription factor NFYA.The SNP marker at this site was further designed,and 239 natural island cotton populations were subjected to PCR amplification and detection.It was found that 129 materials were CCAAT and 110 materials were CCGAT.Screening of the NFYA family genes in TM-1,combined with TM-1 expression data at different time points of Verticillium dahliae treatment,it was found that the NFYA gene GhD07G2390 named GhNFYA5,significantly up-regulated after treatment with Verticillium dahliae.The results of qRT-PCR quantitative verification of the cDNA extracted from the root tissue of TM-1 induced by Verticillium dahliae showed that GhNFYA5 was up-regulated by Verticillium dahliae in upland cotton.By designing a yeast one-hybrid experiment in which different motifs bind to the GhNFYA5 transcription factor,it was found that GhNFYA 5 will specifically recognize the CCAAT motif and inhibit the expression of downstream genes.Tobacco transient expression found that simultaneous injection of the bacterial solution containing the CCAAT motif promoter and the CDS fragment of the GhNFYA5 gene would significantly inhibit the expression of the GUS gene downstream of the promoter,which also confirmed the results of the previous experiment.This shows that the GhNFYA5 transcription factor does indeed reduce the expression of this gene by specifically binding to the promoter motif CCAAT of GhOsmotin34 when Verticillium dahliae infects cotton,but can not bind to the promoter motif CCGAT of GbOsmotin34 in Hai7124,so that the island cotton material Hai7124 and the upland cotton material TM-1 show different resistance.