Cloning And Functional Analysis Of Pectinase Methylesterase Inhibitorgene BnPMEI1 In Brassica Napus

Stem rot of Brassica napus caused by Sclerotinia sclerotiorum is a worldwide fungal disease that seriously harms the yield and quality of rapeseed.It is of great significance to study the resistance mechanism of sclerotinia sclerotinia and to explore and utilize the resistance genes to sclerotinia sclerotinia.Transcriptome sequencing technique was used to analyze the difference of gene expression at different time points after inoculation of Sclerotinia sclerotiorum in rapeseed.It was found that there was a significant difference in the expression of Bna C09g13100D gene between highly resistant and highly susceptible varieties.In this study,according to the sequence information of Bna C09g13100D in Gen Bank,the full length of the coding region（CDS）of the gene was cloned from Xiangyou 15,a highly resistant variety,and 98C40,a highly susceptible variety.Sequence analysis showed that the gene contained a signal peptide cleavage site and a PMEI domain,which was inferred to be a Pectinase methylesterase inhibitor gene,named BnPMEI1.Bioinformatics analysis showed that the full length of coding region of BnPMEI1 gene was 615bp,protein composition was C₉₈₆H₁₅₈₁N₂₆₃O₃₁₄S₁₀,molecular weight was 22.46k D,theoretical isoelectric point was 5.57.the protein was composed of 19 amino acid residues,of which serine accounted for 11.8%,followed by leucine and alanine,accounting for 9.8%and 9.3%,respectively.The instability coefficient is 42.47 and the average hydrophilic coefficient is-0.048.It is speculated that it is an unstable water-soluble protein,which is predicted to be located on the cell membrane and contains a transmembrane structure（7-29amino acids）.The signal peptide cut site is between 24 and 25 amino acids.Secondary structure analysis shows that it contains four secondary structure forms,the main isαhelix,accounting for 66.67%,followed by irregular crimping,accounting for 26.96%,extension chain andβangle accounting for 4.41%and 1.96%,respectively.Tissue expression analysis showed that BnPMEI1 was expressed in all organs of Xiangyou 15 and 98C40,but there were differences in expression characteristics between the two varieties.The expression of BnPMEI1 in various organs of Xiangyou 15 was leaf>stem>flower>grain>pod>root,which in 98C40 was leaf>grain>pod>stem>flower>root.The expression of BnPMEI1 in stem,leaf and flower of Xiangyou 15 was significantly higher than that of 98C40,which was 2.78,1.88 and 3.15 times that of 98C40,respectively.But the expression of BnPMEI1 in 98C40 pod was significantly higher than that of Xiangyou 15 and 5.98 times higher than that of Xiangyou 15,but there was no significant difference in root and grain.The expression analysis of BnPMEI1 in Xiangyou 15 at different time points after inoculation showed that the expression of BnPMEI1 was up-regulated by Sclerotinia sclerotiorum.The expression levels at 12h,24 h and 48h after inoculation were 3.40,15.03and 16.57 times higher than those before inoculation,respectively,and there were significant differences at different time points.The expression of BnPMEI1 in 98C40 was significantly down-regulated at 12h after inoculation,up-regulated at 24h after inoculation,and significantly higher at 48h after inoculation.The expression of BnPMEI1 at 24h and 48h after inoculation was 1.90 and 4.36 times higher than that before inoculation,respectively.The expression of BnPMEI1 in Xiangyou 15 was significantly higher than that of 98C40 at12h,24h and 48h after inoculation.The results of hormone treatment showed that the expression of BnPMEI1 in Xiangyou15 was up-regulated under SA treatment.The expression of BnPMEI1 at 24 h after SA treatment was significantly higher than that of before treatment and 12 h after treatment,while the expression of BnPMEI1 was inhibited,and the expression at each time point after inoculation was significantly lower than that before treatment in 98C40.Under Me JA treatment,the expression of BnPMEI1 gene in Xiangyou 15 showed a gradual upward trend,with the highest expression at 48h after treatment,and the expression at each time point after treatment was significantly higher than that before inoculation,while the expression of BnPMEI1 in 98C40 decreased at first,then increased and then decreased,and the expression at 12h after treatment was significantly lower than that before treatment,and the highest expression at 24h after treatment was significantly higher than that at 12h and 48h after treatment,but there was no significant difference from that before treatment.Under ethylene treatment,the expression of BnPMEI1 in both varieties was down-regulated,and the expression in 98C40 was significantly higher than that in Xiangyou 15.The overexpression vector of BnPMEI1 was constructed and seven positive transformed lines（0E1,OE2,OE3,OE4,OE5,OE6 and OE7）were obtained by transforming wild-type rapeseed Westar.The results of inoculation identification of Sclerotinia sclerotiorum showed that the disease spot of rape mutant overexpressing BnPMEI1 was significantly smaller than that of wild type leaf.The expression of Bn NPR1and Bn Lox2 in the OE5 strain with the highest expression of BnPMEI1 was significantly higher than that in the control wild-type Westar,while the expression of PDF1.2 and Bn CHB4 regulated by jasmonic acid had no significant change.Comprehensive analysis showed that BnPMEI1 is a pectin methylesterase inhibitor gene regulated by salicylic acid,jasmonic acid and ethylene.Under the infection of Sclerotinia sclerotiorum,it may participate in the interaction between rape and Sclerotinia sclerotiorum by inducing salicylic acid signal transduction pathway,jasmonic acid synthesis and signal transduction pathway,and positively regulate the resistance of rape to Sclerotinia sclerotiorum.The results can provide reference and candidate genes for molecular identification and molecular-assisted breeding of resistance to Sclerotinia sclerotiorum in rapeseed.