斑地锦Em4CL2a基因及启动子的克隆与分析OA

Objective:To clone and analyze the Em4CL2a gene and its promoter from Euphorbia maculata,providing a basis for understanding the formation mechanism of the superior qualities of Euphorbia maculata.Methods:The ORF region and promoter se-quence of the Em4CL2a gene were cloned using RACE and Tail-PCR techniques,and bioinformatics analysis was conducted.The ex-pression patterns of stem tissues of Euphorbia maculata at different growth stages were analyzed by qRT-PCR.The prokaryotic protein expression recombinant plasmid pET-32a(+)-Em4CL2a was constructed and identified.Results:The ORF length of the obtained Em4CL2a gene was 1 674 bp(Login number:OR944055),and the promoter length was 1 404 bp(login number:PP779128).This gene encoded 557 amino acids.Its encoded protein contained conserved domains BoxⅠ/Ⅱ and had two transmembrane structures.It was most closely related to Euphorbia peplus and Jatropha curcas.The Em4CL2a gene was highly expressed specifically from 20 to 60 days after flowering,which was the critical period for quercetin synthesis.The pET-32a(+)-Em4CL2a recombinant plasmid was successfully constructed and its successful expression in Escherichia coli was verified by SDS-PAGE.Conclusion:This study successfully clones and preliminarily analyzes the function of the Em4CL2a gene,enriches the resources of the 4CL gene family,and lays a foundation for further development and utilization of this gene.