辣椒素生物合成途径基因pal克隆及原核表达分析

以辣椒品种"益都红"胎座RNA反转录的cDNA为模板,根据pal基因保守序列设计引物,PCR扩增得到1条全长2157bp的目的片段。该基因包含有完整的开放阅读框架,编码718个氨基酸,预测的分子质量为73ku。与多种植物PAL的氨基酸序列有一定的同源性,其中与同科同属植物朝天椒的同源性最高。应用分子克隆的方法成功地构建了pET-32a-pal重组表达质粒,优化得到诱导表达的最佳条件为:IPTG0.3mmol·L-1,温度28℃,时间6h,经SDS-PAGE检测6×His-PAL在E.coli中得到了高效表达,重组蛋白分子质量约为93ku。因此,该基因的克隆与原核表达为深入研究辣椒PAL的基因结构、生物活性、表达调控机制以及辣椒素的生物合成机制奠定了重要基础。

Cloning and prokaryotic expression of pal gene referring to the pathway of pungency biosynthesis of Capsicum annuum

A target fragment with a full length of 2 157 bp was amplified with cDNA of Capsicum annuum placenta as the templates and the conservative sequences of pal gene as the primers.This sequence had a full open reading frame,encoding 718 amino acids.Putative protein was about 73 ku in molecular weight.The putative protein shared high identity to other homologues,especially Capsicum chinses.In the experiments,a recombinant plasmid pET-32a-pal was constructed.This recombinant prokaryotic expression vector(pET-32a-pal) was used to transform E.coli BL21(DE3).SDS-PAGE analysis confirmed that the recombinant 6 ×His-PAL with the predicted molecular weight of about 93 ku was successfully expressed in E.coli BL21(DE3) strain with the optimal condition:0.3 mmol·L-1 IPTG,28 ℃,shaking 6 h.Accordingly,the cloning and prokaryotic expression of pal gene of Capsicum annuum is the basis for further studying the genetic structure,bioactivity,expression and regulation mechanism of PAL,and the biosynthesis mechanism of capsaicin.