摘要： 为探讨PGIP蛋白与PG的互作及pgip 基因在油菜抗黑胫菌病中的作用,根据GenBank中油菜pgip9 、pgip15 CDS序列设计引物,并去掉信号肽序列,以接种黑胫病病原菌Leptosphaeria biglobosa (菌株NM-1) 7 d后油菜叶片总RNA反转录的cDNA为模板,PCR扩增出pgip9 、pgip15 除信号肽以外的编码区片段,并克隆到巴斯德毕赤酵母表达载体pPICZαA中构建重组质粒pPICZαA-pgip9 、pPICZαA-pgip15 .重组质粒经单、双酶切、菌落PCR筛选鉴定后热激化法转化酵母细胞PMAD16,再经Zeocin^TM的YPD平板和菌落PCR筛选鉴定,经甲醇诱导表达,SDS-PAGE和Western Blot检测,均在36~37 kDa处出现单一的蛋白条带,蛋白分泌表达成功.通过对pgip 基因的体外表达,为探讨PGIP蛋白与PG的互作及pgip 基因在抗油菜黑胫菌病中的作用奠定基础.

关键词: Leptosphaeria biglobosa, pgip, 去信号肽, 毕赤酵母, 分泌表达

Abstract: In this study,according to the sequence of pgip9,pgip15 CDS in GenBank,the primers were designed,and removed the signal peptide sequence,the total RNA of rapeseed leaves inoculated with Leptosphaeria biglobosa (NM-1) after 7 days,were extracted and reversed transcription cDNA as template.The pgip9 and pgip15 coding region fragments were amplified excluding signal peptide by PCR,and cloned into Pichia pastoris expression vector constructing the recombinant plasmid pPICZαA- pgip9 ,pPICZαA- pgip15.The recombinant plasmids were identified by single,double enzyme digestion,colony PCR,and then were transformed into yeast cells PMAD16,then were identified by YPD including Zeocin^TM and colony PCR inducing by methanol,analyzed by SDS-PAGE and Western Blot,producing a single band of approximately 36-37 kDa,proteins were expressed and secreted successfully.The expression of PGIPs in vitro were conducted to provide a basis for the interaction between PGIP and PG,and the pgip gene function in canola resistant to phoma stem canker.

Key words: Leptosphaeria biglobosa, pgip, Deleted singal peptide, Pichia pastoris, Secretion expression

中图分类号: 

• S565.4

• Q78