野三七鲨烯环氧酶基因的克隆及原核表达

王宝婕; 朱灵英; 周青青; 张帅; 马晓惠; 徐福荣

doi:10.13422/j.cnki.syfjx.20192212

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野三七鲨烯环氧酶基因的克隆及原核表达

资源与质量评价 | 更新时间：2021-02-09

- 野三七鲨烯环氧酶基因的克隆及原核表达
- Cloning and Prokaryotic Expression of Squalene Epoxidase Gene from Panax vietnamensis var. fuscidiscus
- 中国实验方剂学杂志 2019年25卷第22期页码：147-153
- 作者机构：
  
  云南中医药大学　中药学院，昆明　 650500
- 作者简介：
  
  王宝婕，在读硕士，从事分子生药学研究，E-mail：wangbaojie1994@126.com
  马晓惠，博士，讲师，从事分子生药学研究，E-mail：maxiaohui1988@126.com；
  徐福荣，博士，研究员，从事中药资源质量与开发研究，E-mail: xfrong99@163.com
- 基金信息：
  
  国家自然科学基金地区科学基金项目(81760690;81460581);云南省科技厅科技计划项目(2018FB144);云南省教育厅科学研究基金项目(2017ZZX288)
- DOI：10.13422/j.cnki.syfjx.20192212
  中图分类号： R284.2; R289; R22; R2-031
- 收稿日期：2019-03-24，
  
  网络出版日期：2019-08-08，
  
  纸质出版日期：2019-11-20
- 稿件说明：
移动端阅览
王宝婕, 朱灵英, 周青青, 等. 野三七鲨烯环氧酶基因的克隆及原核表达[J]. 中国实验方剂学杂志, 2019,25(22):147-153.

Bao-jie WANG, Ling-ying ZHU, Qing-qing ZHOU, et al. Cloning and Prokaryotic Expression of Squalene Epoxidase Gene from Panax vietnamensis var. fuscidiscus[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(22): 147-153.
王宝婕, 朱灵英, 周青青, 等. 野三七鲨烯环氧酶基因的克隆及原核表达[J]. 中国实验方剂学杂志, 2019,25(22):147-153. DOI： 10.13422/j.cnki.syfjx.20192212.

Bao-jie WANG, Ling-ying ZHU, Qing-qing ZHOU, et al. Cloning and Prokaryotic Expression of Squalene Epoxidase Gene from Panax vietnamensis var. fuscidiscus[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(22): 147-153. DOI： 10.13422/j.cnki.syfjx.20192212.

摘要

目的：

对野三七中可能参与三萜皂苷合成的关键酶鲨烯环氧酶基因(

PvfSE

)进行克隆，并对其进行生物信息学分析和原核表达。

方法：

采用Trizol法提取野三七根的总RNA并反转录为cDNA第一链，基于野三七的转录组数据，设计

PvfSE

基因序列特异性引物，克隆基因全长，利用相关软件对该基因进行生物信息学分析，构建pMal-c2X-

PvfSE

原核表达载体，在大肠埃希菌中诱导表达重组蛋白。

结果：

PvfSE

开放阅读框为1 887 bp，编码628个氨基酸，蛋白相对分子质量为68.8 kDa，理论等电点为9.28，脂肪系数为95.18，亲水性系数为－0.060，不稳定指数为40.36，属于不稳定蛋白。生物信息学分析表明，PvfSE具有2个跨膜结构域，无信号肽，可能定位在叶绿体或细胞质膜上，具有FAD/NAD(P)结合域和鲨烯环氧酶结构域，属于混合型蛋白。PvfSE与西葫芦和芒柄花中参与三萜皂苷合成的鲨烯环化酶(CpSE1，CpSE3，OsSE1，OsSE2)亲缘关系较近。此外，在大肠埃希菌BL21(DE3)中成功表达PvfSE的重组蛋白。

结论：

克隆得到

PvfSE

基因，并在大肠埃希菌中成功表达其重组蛋白，为进一步研究PvfSE的功能和解析野三七中三萜皂苷生物合成途径奠定了基础。

Abstract

Objective:

To clone the squalene epoxidase genes of

Panax vietnamensis

var.

fuscidiscus

(

PvfSE

)

and perform bioinformatics analysis and prokaryotic expression.

Method:

Total RNA was extracted from root of

vietnamensis

var.

fuscidiscus

by trizol method

and reverse-transcribed into first stand of cDNA. Specific primers for

PvfSE

cloning were designed according to the transcriptome data of

vietnamensis

var.

fuscidiscus

and the cDNA sequence of

PvfSE

gene was isolated. Bioinformatics of

PvfSE

was analyzed by relevant software. The prokaryotic expression vector pMal-c2X-

PvfSE

was built to express recombinant protein in

Escherichia coli

cells.

Result:

The

PvfSE

gene contained a 1 887 bp open reading frame

encoding a predicted protein of 628 amino acids. The calculated molecular weight was 68.8 kDa

the theoretical isoelectric point was 9.28

the aliphatic index was 95.18

the grand average of hydropathicity was -0.060

and the instability index was 40.36. The protein was unstable. Bioinformatics analysis showed that PvfSE had two transmembrane domains and no signal peptide. PvfSE was most likely to be located in chloroplast or cytoplasmic membrane. PvfSE was a mixed protein with FAD/NAD(P) binding domain and squalene epoxidase domain. Sequence alignment and phylogenetic analysis demonstrated that PvfSE had a relatively close relationship with CpSE1

CpSE3

OsSE1 and OsSE2

which was involved in the biosynthesis of triterpene saponins in

Cucurbita pepo

and

Ononis spinosa

. In addition

PvfSE protein was expressed in

coli

Conclusion:

In this study

PvfSE

gene was cloned and expressed in BL21(DE3)

which lays a foundation for the further study on the gene functions of PvfSE and the biosynthetic pathway of triterpenoid saponins in

vietnamensis

var.

fuscidiscus

关键词

Keywords

references

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