福建农林大学学报（自然科学版）2025，Vol.54Issue(4)：447-454,8.DOI:10.13323/j.cnki.j.fafu(nat.sci.).202408003
软腐病菌Dickeya fangzhongdai Onc5菌株vfmH和vfmI基因的克隆、原核表达及蛋白纯化
Cloning,prokaryotic expression and protein purification of genes vfmH and vfmI from soft rot pathogen Dickeya fangzhongdai strain Onc5
摘要
Abstract
[Objective]The two-component system(TCS)VfmH/VfmI is associated with the regulation of pathogenic factors and pathogenic processes in Dickeya.The cloning,prokaryotic expression and protein purification of the genes vfmH and vfmI from D.fan-gzhongdai strain Onc5 were studied to provide a basis for clarifying the regulation mechanism underlying VfmH/VfmI.[Method]The open reading frame(ORF)sequences of genes vfmH and vfmI from D.fangzhongdai strain Onc5 were cloned via reverse transcrip-tion-polymerase chain reaction(RT-PCR),then the coding sequences were cloned into the plasmid pET-32a for prokaryotic expres-sion,to construct of the recombinant expression plasmids pET-32a-VfmH and pET-32a-VfmI.After induced expression by isopropyl-β-D-thiogalactoside(IPTG),the recombinant proteins were identified through SDS-PAGE and Western blot.The VfmH and VfmI proteins were then purified using Ni2+-NTA agarose affinity chromatography.[Result]The ORF of the genes vfmH and vfmI were 1 401 bp and 1 320 bp in length,which encoded 466 and 439 amino acids,respectively.Homology analysis revealed high similarity in amino acid sequences between vfmH and vfmI from D.fangzhongdai strain Onc5 and those from other strains of genus Dickeya.SDS-PAGE and Western blot showed that the fused proteins with His-tag were over-expressed in the transformants with plasmids pET-32a-VfmH and pET-32a-VfmI under the induction of 0.5 mmol·L-1 IPTG.Additionally,the target proteins were purified suc-cessfully.[Conclusion]This study represented the first successful cloning of the two-component system genes vfmH and vfmI in D.fangzhongdai,and the successful purification of the proteins VfmH and VfmI,respectively.关键词
软腐病/双组分系统/狄克氏菌/vfmH基因/vfmI基因/原核表达Key words
soft rot disease/two-component system/Dickeya fangzhongdai/vfmH gene/vfmI gene/prokaryotic expression分类
植物保护学引用本文复制引用
王文婷,张晨燕,赵可冉,丁建华,荆恩荣,杨超..软腐病菌Dickeya fangzhongdai Onc5菌株vfmH和vfmI基因的克隆、原核表达及蛋白纯化[J].福建农林大学学报（自然科学版）,2025,54(4):447-454,8.基金项目
山西省科技厅基础研究计划项目(20210302124516) （20210302124516）
山西省高校科技创新计划项目(2021L462). （2021L462）
