ZHAO Le,MA Ligang,HAN Duwan,et al.Cloning and Bioinformatic Analysis and Prokaryotic Expression of LaSPS Gene From Lepidium apetalum[J].Northern Horticulture,2016,40(06):92-99.[doi:10.11937/bfyy.201606023]
独行菜LaSPS基因的克隆与生物信息学分析及原核表达
- Title:
- Cloning and Bioinformatic Analysis and Prokaryotic Expression of LaSPS Gene From Lepidium apetalum
- Keywords:
- Lepidium apetalum; solanesyl diphosphate synthase; gene cloning; sequence analysis; prokaryotic expression
- 文献标志码:
- A
- 摘要:
- 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1 269 bp，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。
- Abstract:
- Taking leaves of Lepidium apetalum as experimental material，using PCR method，the open reading frame (ORF) of LaSPS gene was amplified，and the prokaryotic expression vector pET-32a-LaSPS was constructed and then Escherichia coli BL21 (DE3) cells were transformed with the plasmid pET-32a-LaSPS in order to express recombinant LaSPS protein.The results indicated that the ORF of LaSPS gene was 1 269 bp (GenBank accession number KT318734)， which encoded a protein of 422 amino acid residues.The LaSPS protein which contained solanesyl diphosphate synthase domain was the member of ‘polyprenyl diphosphate synthase’ family.Bioinformatic analysis indicated that LaSPS protein which located in chloroplast had no transmembrane domain and signal peptide.The multiple alignment and phylogenetic analysis indicated that LaSPS protein showed the highest homology，91% similarity，with AtSPS2 protein from Arabidopsis thaliana.The prokaryotic expression vector pET-32a-LaSPS was constructed and the recombinant LaSPS protein was successfully expressed in E.coli BL21 (DE3) cells.This study provided the foundation for follow-up research of its function in plastoquinoe biosynthesis pathway.
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备注/Memo
第一作者简介：赵乐（1983-），男，博士，讲师，现主要从事药用植物分子生物学等研究工作。E-mail：zhaole1983@126.com.责任作者：郑晓珂（1961-），女，博士，教授，博士生导师，现主要从事中药活性成分及作用机制等研究工作。E-mail：zhengxk.2006@163.com.基金项目：国家重点基础研究发展计划“973 计划”资助项目（2013CB531802）；河南中医学院博士科研基金资助项目（BSJJ2011-07）。