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以独行菜(Lepidium apetalum)幼苗叶片为试材,采用PCR方法,扩增得到LaSPS基因的开放阅读框(open reading frame,ORF),构建pET-32a-LaSPS原核表达载体并在大肠杆菌BL21(DE3)菌株中表达LaSPS重组蛋白。结果表明:LaSPS基因ORF长1 269bp,编码422个氨基酸,包含茄呢醇焦磷酸合酶结构域,是异戊烯基合成酶家族的成员;序列分析发现,LaSPS蛋白定位于叶绿体中,不含信号肽没有跨膜区;通过序列比对和系统进化分析,显示LaSPS蛋白与拟南芥(Arabidopsis thaliana)AtSPS2蛋白相似度为91%,亲缘关系最近;在大肠杆菌BL21(DE3)菌株中成功表达LaSPS重组蛋白,为研究LaSPS基因在独行菜质体醌生物合成途径中的功能奠定了基础。
The open reading frame (ORF) of LaSPS gene was amplified by PCR from the leaves of Lepidium apetalum seedlings. The prokaryotic expression vector pET-32a-LaSPS was constructed and expressed in Escherichia coli BL21 (DE3 ) Strain expressing LaSPS recombinant protein. The results showed that the ORS of LaSPS gene was 1 269 bp in length and encoded 422 amino acids, including solanesol pyrophosphate synthase domain, which was a member of isopentenyl synthase family. Sequence analysis revealed that LaSPS protein was located in chloroplast, The signal peptide had no transmembrane domain. Sequence alignment and phylogenetic analysis showed that the similarity of AtSPS2 protein between LaSPS protein and Arabidopsis thaliana was 91%, which was the most closely related to the others. The sequence was successfully expressed in Escherichia coli BL21 (DE3) strain LaSPS recombinant protein laid the foundation for the study of the function of LaSPS gene in the pathogen of quinoid body quinone biosynthesis.