独行菜法尼基焦磷酸合酶基因的克隆与原核表达

发布时间：2018-04-20 18:23

  本文选题：独行菜 + 法尼基焦磷酸合酶　； 参考：《河南农业科学》2017年09期

【摘要】：从独行菜(Lepidium apetalum)中克隆强心苷合成途径的法尼基焦磷酸合酶(FPS)基因,并在大肠杆菌中表达,为进一步研究独行菜强心苷的合成途径提供参考。分析前期获得的独行菜幼苗转录组数据,选择其中注释为FPS且具有完整开放阅读框的序列,从独行菜叶片c DNA中通过PCR克隆该序列,并进行序列分析。结果表明,克隆得到独行菜FPS基因的cDNA序列,Gen Bank登录号KY366218,命名为La FPS。序列长度为1 332 bp,含有1 161 bp的开放阅读框,推测编码386个氨基酸。La FPS蛋白可能不含跨膜结构,定位于线粒体中,含有聚异戊二烯合成酶结构域。La FPS蛋白氨基酸序列与拟南芥(Arabidopsis thaliana)FPS1蛋白相似性高达92%,进化树分析结果表明,La FPS与同为十字花科的拟南芥FPS1、遏蓝菜(Noccaea caerulescens)FPS1、高山南芥(Arabis alpina)FPS亲缘关系最近。构建的载体p ET-32a-La FPS可在大肠杆菌中成功诱导表达。表明成功克隆了独行菜FPS基因,并建立了其原核表达体系。
[Abstract]:The FPS gene of cardiac glycoside synthesis pathway was cloned from Lepidium apetalumin and expressed in E. coli, which provides a reference for further study on the synthesis pathway of cardiac glycoside. The transcriptome data obtained in the early stage were analyzed, and the sequence annotated as FPS with complete open reading frame was selected. The sequence was cloned by PCR from the leaves of C. DNA, and the sequence was analyzed. The results showed that the cDNA sequence of FPS gene was cloned into GenGen Bank accession number KY366218, named La FPS. The sequence length was 1 332 BP, containing an open reading frame of 1 161 BP, which suggested that the 386-amino acid .La FPS protein might not contain transmembrane structure and was located in mitochondria. The amino acid sequence of the polyisoprene synthase domain. La FPS protein was similar to Arabidopsis thaliana)FPS1 protein of Arabidopsis. The phylogenetic tree analysis showed that La FPS was closely related to FPS1 of Arabidopsis, Noccaea caerulescensus FPS1 and Arabis alpina)FPS of Alpine. The constructed vector p ET-32a-La FPS can be successfully induced in E. coli. The results showed that the FPS gene was cloned successfully and its prokaryotic expression system was established.
【作者单位】： 河南中医药大学药学院;呼吸疾病诊疗与新药研发河南省协同创新中心;黑龙江中医药大学药学院;
【基金】：国家重点基础研究发展(973)计划项目(2013CB531802) 河南中医学院博士科研基金项目(BSJJ2011-18)
【分类号】：S567.2
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本文编号：1778900