油菜pgip基因去信号肽片段在毕赤酵母中的分泌表达

Secretion Expression of Brassica napus pgip Gene Removing Singal Peptide in Pichia pastoris

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Gen Bank中油菜pgip9、pgip15CDS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c DNA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p PICZαA中构建重组质粒p PICZαA-pgip9、p PICZαA-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 k Da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。

In this study,according to the sequence of pgip9,pgip15 CDS in Gen Bank,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 c DNA 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 p PICZαA-pgip9,p PICZαA-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 k Da,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.