细极链格孢菌peaT2基因在毕赤酵母中的表达及蛋白功能确定

Expression of Alternaria tenuissima peaT2 gene in Pichia pastoris and its function

摘要:

从细极链格孢菌表达文库获得阳性克隆子,序列分析表明,克隆的DNA片段中含有完整的开放阅读框架,将该基因命名为peaT2(GenBank登录号为EF212880)。用PCR法扩增peaT2基因的编码序列并亚克隆到毕赤酵母表达系统的表达载体pPIC9K上,得到重组质粒pPIC9K/peaT2。重组质粒经SacⅠ线性化后用电穿孔法导入到毕赤酵母(Pichia pastoris)GS115中,采用MD、G418-YPD平板和PCR法筛选Mut+表型,获得了分泌表达的重组毕赤酵母。随机挑取一菌株作为表达菌,用甲醇诱导PeaT2蛋白表达。SDS-PAGE及Western blot检测结果均表明PeaT2在毕赤酵母中成功地分泌表达。用peaT2基因的表达蛋白处理小麦种子,生物测定表明,表达蛋白能明显促进小麦的生长,具有蛋白激发子作用。

Abstract:

A positive clone was screened from Alternaria tenuissima expression library . The result of sequencing and gene analysis indicated that the cloned DNA fragment has a complete ORF，which was named peaT2 (Protein Elicitor from Alternaria tenuissima 2) with the GenBank accession number EF212880. The gene was amplified by PCR and subcloned into the pPIC9K of Pichia pastoris expression system. The resulting recombinant plasmid pPIC9K/peaT2 was verified by sequencing and digested by SacⅠ. The linearized DNA was transformed into P. pastoris GS115 by electroporation. By means of MD and G418-YPD plates and PCR, the recombinant P. pastoris strains (his^- mut⁺) were obtained. A recombinant clone cultivated on YPD plate with high concentration of G418 was randomly selected as expression strain. The protein expression was induced by methanol and analyzed by 12% SDS-PAGE. The results of SDS-PAGE and Western blot indicated that this gene was expressed successfully in P. pastoris with the induction of methanol. In BMMY culture medium, the expressed protein reached its maximum amount at 72h, whereas no corresponding protein was detected in the negative control. Bioassay was performed with the expressed protein. After soaking with the expressed protein for 8h, wheat seeds were cultured, the height of seedlings was measured after 24h, 36h, 48h, 7d, respectively, and the root length was measured after 7 days. The results showed that the expressed protein can promote seeding growth and root length obviously in appropriate concentration. It is revealed that PeaT2 can act as protein elicitor.