Cloning And Functional Verification Of Fusarium Wilt Resistance Genes GbLEA3,GbPR10 And GbMPK16 In Gossypium Barbadense L.

Gossypium barbadense L has advantages of fiber length and fineness over other cultivated cotton species,which plays an important role in agricultural production and national economy.However,the spread of cotton Fusarium wilt has seriously affected the yield and quality of the island cotton,and brought negative effects on farmers' economic level.It has been one of the effective ways to cultivate new varieties of island cotton and reduce the risk of Fusarium wilt by studying the resistance gene and its functional verification at the molecular level.In this study,we cloned and analyzed the gene sequences chosen from transcriptional sequencing and disease resistance spectrum data and q RT-PCR analysis were taken on newly cloned resistance genes by using different resistant island cotton varieties induced by Fusarium wilt,ethylene and salicylic acid;The gene functions were Verifyied by VIGS technology.Overall results achieved are as follows:1.Based on the early transcriptional sequence and disease resistance expression data,three genes related to resistance to Fusarium wilt cloned from root tissue of resistance island cotton variety “06-146” and named resprctively Gb LEA3,Gb PR10,Gb MPK16,ORF sequences are 318 bp,480 bp,1665 bp,amino acid sequences are 105,159,554 respectively,belonging to LEA-3,PR10,D group MAPK gene family respectively,corresponding proteins are samely hydrophilic,distributed in mitochondria,cytoplasm,nucleus respectively.The conservative domain analysis showed that Gb LEA3 protein has a typical LEA-3 family conservative domain,Gb PR10 protein has a pathogenic Bet-v1 functional area and Glysine ring(GXGGGGXG)associated with acid enzyme activity,and Gb MPK16 protein has a protein kinase(Protein-Kinase-Dom)domain.It is speculated that Gb PR10 and Gb MPK16 genes are may closely related to plant disease resistance.2.qPCR analysis induced by Furasium oxysporum f.sp showed that the expression of Gb MPK16 gene is higher than that of susceptible cotton cultivars,and the expression of Gb LEA3 and Gb PR10 genes are alomost higher than that of susceptible cultivars.The results showed that the expression levels of Gb PR10 and Gb MPK16 genes increase.3.qPCR analysis induced by Ethylene showed that The expression level of Gb LEA3 gene begin to increase at 4 h,but the expression level of susceptible cultivars are lower than that before treatment;Gb PR10 gene increases and then decreases in the resistant cultivars,while the expression level of Gb PR10 gene increases in the susceptible cultivars at the end;The expression level of Gb MPK16 gene is gradually raised in resistant and sensitive varieties.After SA-induced,the expression level of Gb LEA3 increases in resistant cultivars at 2 h and 4 h,and then decreases rapidly,the expression level in the susceptible cultivars is lower than that before treatment except for 4 h;Gb PR10 showes a trend of upward and downward expression level in disease resistant cultivars,while few expression level in susceptible cultivars;The Gb MPK16 gene decreases slowly after the highest expression of 2 h in resistant cultivars,but increases and decreases in susceptible cultivars.The expression level of Gb LEA3,Gb PR10,Gb MPK16 genes in the resistant cultivators are almost higher than that of susceptible cultivars.4.Functional verification of each gene detected by the VIGS technique,and q PCR analysis showed that Gb LEA3,Gb PR10,Gb MPK16 genes are all successfully silenced,and Gb LEA3,Gb PR10 have a higher silence rate than Gb MPK16;The disease index of 15-days induced by Fusarium oxysporum f.sp showed that TRV2-Gb LEA3,TRV2-Gb PR10,TRV2-Gb MPK16 are higher than the control,and TRV2-Gb PR10,TRV2-Gb MPK16 are higher than TRV2-Gb LEA3.It is speculated that Gb LEA3,Gb PR10,Gb MPK16 genes may have resistant to Fusarium wilt in varying degrees.