Cloning and Expression Analysis of PaAACT Gene from Phytolacca americana

Ya-lin LIU; Yun-hao ZHU; Zhi-wei SHENG; Jing-yu CHENG; Wei-sheng FENG; Xiao-ke ZHENG; Le ZHAO

doi:10.13422/j.cnki.syfjx.20192113

您当前的位置：

首页 >

文章列表页 >

Cloning and Expression Analysis of PaAACT Gene from Phytolacca americana

Resource and Quality Evaluation | 更新时间：2021-02-09

- Cloning and Expression Analysis of PaAACT Gene from Phytolacca americana
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 21, Pages: 124-131(2019)
- 作者机构：
  
  1.河南中医药大学　药学院，郑州　 450046；
  2.河南中医药大学　呼吸疾病诊疗与新药研发河南省协同创新中心，郑州　 450046
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20192113
  CLC： R289; R284.1; R22; R2-031
- Received：02 May 2019，
  
  Published Online：19 July 2019，
  
  Published：05 November 2019
- 稿件说明：
移动端阅览
Ya-lin LIU, Yun-hao ZHU, Zhi-wei SHENG, et al. Cloning and Expression Analysis of PaAACT Gene from Phytolacca americana[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(21): 124-131.
DOI：

Ya-lin LIU, Yun-hao ZHU, Zhi-wei SHENG, et al. Cloning and Expression Analysis of PaAACT Gene from Phytolacca americana[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(21): 124-131. DOI： 10.13422/j.cnki.syfjx.20192113.

摘要

目的：

从垂序商陆根中克隆了商陆皂苷甲生物合成过程中关键酶乙酰乙酰辅酶A转移酶(acetoacetyl-CoA transferase，AACT)基因，进行生物信息学分析和原核表达。

方法：

提取垂序商陆根的总RNA，然后逆转录合成cDNA，在分析垂序商陆转录组数据的基础上，设计

PaAACT

基因的特异性引物，聚合酶链式反应(PCR)扩增

PaAACT

基因的cDNA序列，通过构建原核表达载体pET-32a-PaAACT，诱导表达并且纯化目的蛋白。

结果：

PaAACT

基因开放阅读框为1 254 bp，编码417个氨基酸。生物信息学分析显示PaAACT蛋白的分子式为C

1 914

3 120

538

576

，推测其相对分子质量为43.43 kDa，理论等电点8.90，不稳定系数32.27，属于稳定蛋白质。根据生物信息学分析结果，PaAACT蛋白属于硫解酶家族成员，在C末端含有硫解酶家族的1个保守位点和1个活性位点。PaAACT蛋白可能位于细胞质中、不含信号肽、没有跨膜区。系统进化分析显示PaAACT蛋白与甜菜等廖科植物AACT蛋白亲缘关系较近。经IPTG诱导，在大肠埃希菌BL21(DE3)菌株中表达了PaAACT重组蛋白，利用Ni

2＋

亲和色谱获得了纯化的目的蛋白。

结论：

该研究从垂序商陆中克隆

PaAACT

基因，为下一步测定PaAACT酶催化活性、制备抗体奠定基础，也为研究其在商陆皂苷甲生物合成途径中的作用提供理论依据。

Abstract

Objective:

To clone the key enzyme gene involved in the biosynthesis of esculentoside A(EsA)

acetoacetyl-CoA transferase(AACT) gene was cloned from

Phytolacca americana

for bioinformatics analysis and prokaryotic expression.

Method:

Total RNA was extracted from the root of

americana

and then cDNA was synthesized through the reverse transcription. Based on analysis of the transcriptome data of

americana

the specific primers of

PaAACT

gene were designed

and the cDNA sequence of

PaAACT

gene was amplified by polymerase chain reaction(PCR) method. Prokaryotic induction

expression and purification of the target protein were induced through the construction of the prokaryotic expression vector pET-32a-PaAACT.

Result:

The open reading frame (ORF) of

PaAACT

gene was 1 254 bp

and encoded 417 amino acid residues. Bioinformatic analysis showed that the molecular formula of PaAACT protein was C

1 914

3 120

538

576

inferring that its molecular weight was 43.43 kDa

the theoretical isoelectric point was 8.90

and the instability index of PaAACT protein was 32.27

which was a stable protein. According to bioinformatics analysis

PaAACT protein was a member of the thiolase family and contained one conserved site and one active site of the thiolase family at the C-terminal. PaAACT protein may be located in the cytoplasm

without a signal peptide or transmembrane domain. The phylogenetic analysis indicated that PaAACT protein showed the highest homology with AACT protein from polygonaceae plants (such as

Beta vulgaris

). The recombinant PaAACT protein was successfully expressed in

Escherichia coli

BL21(DE3) strain through IPTG induction

and the purified target protein was obtained by Ni

affinity chromatography.

Conclusion:

In this study

the

PaAACT

gene was cloned from

americana

which lays a foundation for further determination of enzyme activity assay of PaAACT and preparation of antibody

and provides the theoretical basis for studying its role in the biosynthesis pathway of EsA.

关键词

Keywords

references

国家药典委员会．中华人民共和国药典．一部 [M]．北京：中国医药科技出版社， 2015 ： 324 - 325 ．

李一飞，姚广涛．商陆药理作用及毒性研究进展 [J]．中国实验方剂学杂志， 2011 ， 17 ( 13 )： 248 - 251 ．

王鹏程，王秋红，赵珊，等．商陆化学成分及药理作用和临床应用研究进展 [J]．中草药， 2014 ， 45 ( 18 )： 2722 - 2731 ．

杜琳，王洁雪，陈聪地，等．商陆中皂苷类化学成分研究 [J]．中国中药杂志， 2018 ， 43 ( 12 )： 2552 - 2556 ．

抗晶晶，刘晓宁．商陆皂苷甲治疗炎症性疾病机制的研究进展 [J]．山东医药， 2017 ， 57 ( 42 )： 111 - 113 ．

裴莉昕，纪宝玉，陈随清，等．河南不同产地商陆药材质量分析 [J]．中国实验方剂学杂志， 2017 ， 23 ( 2 )： 48 - 52 ．

赵乐，朱畇昊，张莉，等．基于转录组测序挖掘商陆皂苷甲生物合成相关基因 [J]．药学学报， 2017 ， 52 ( 9 )： 1471 - 1480 ．

赵启红，张皓，刘海珍，等．垂序商陆抗坏血酸过氧化物酶(APX1)基因克隆及表达分析 [J]．基因组学与应用生物学， 2016 ， 35 ( 6 )： 1479 - 1486 ．

王校，赵会君，刘慧敏，等．商陆金属硫蛋白基因PaMT的表达分析 [J]．中国科学院研究生院学报， 2012 ， 29 ( 4 )： 449 - 454 ．

黄超，张丽丽，邓柳红，等．海南五指山美洲商陆抗病毒蛋白PAP-Ⅰ基因的原核表达及活性验证 [J]．热带生物学报， 2016 ， 7 ( 1 )： 82 - 88 ．

Vranová E , Coman D , Gruissem W . Network analysis of the MVA and MEP pathways for isoprenoid synthesis [J]. Annu Rev Plant Biol , 2013 , 64 ( 1 ): 665 - 700 .

Lichtenthaler H K . The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants [J]. Annu Rev Plant Physiol Plant Mol Biol , 1999 , 50 ( 4 ): 47 - 65 .

Dyer J H , Maina A , Gomez I D , et al . Cloning, expression and purification of an acetoacetyl CoA thiolase from sunflower cotyledon [J]. Int J Biol Sci , 2009 , 5 ( 7 ): 736 - 744 .

Germain V , Rylott E L , Larson T R , et al . Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid β-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings [J]. Plant J , 2001 , 28 ( 1 ): 1 - 12 .

CHEN Q , YAN J , MENG X , et al . Molecular cloning, characterization, and functional analysis of acetyl-CoA C-acetyltransferase and mevalonate kinase genes involved in terpene trilactone biosynthesis from Ginkgo biloba [J]. Molecules , 2017 , 22 ( 1 ): 74 .

赵瑜君，张萌，刘雨佳，等．雷公藤乙酰CoA酰基转移酶基因全长cDNA克隆及表达分析 [J]．中国中药杂志， 2015 ， 40 ( 5 )： 847 - 852 ．

赵乐，马利刚，杨泽岸，等．独行菜幼苗叶片乙酰CoA酰基转移酶基因克隆、序列分析与原核表达 [J]．中国实验方剂学杂志， 2017 ， 23 ( 11 )： 34 - 39 ．

朱畇昊，苏秀红，董诚明，等．冬凌草AACT基因的克隆与表达分析 [J]．中药材， 2016 ， 39 ( 1 )： 37 - 41 ．

刘娟，徐艳红，杨勇，等．白木香乙酰乙酰基辅酶A硫解酶基因(AsAACT)的克隆与表达分析 [J]．中国中药杂志， 2014 ， 39 ( 6 )： 972 - 980 ．

姚元枝，黎晓英，魏麟，等．鱼腥草乙酰辅酶A酰基转移酶基因克隆、表达及生物信息学分析 [J]．中草药， 2015 ， 46 ( 1 )： 107 - 111 ．

赵乐，马利刚，俎梦航，等．地黄RgGGPPS2基因克隆、生物信息学分析及表达分析 [J]．中草药， 2017 ， 48 ( 11 )： 2269 - 2278 ．

赵乐，马利刚，张金燕，等．独行菜LaF3H基因克隆、序列分析及原核表达 [J]．中草药， 2018 ， 49 ( 23 )： 5626 - 5632 ．

Soto G , Stritzler M , Lisi C , et al . Acetoacetyl-CoA thiolase regulates the mevalonate pathway during abiotic stress adaptation [J]. J Exp Bot , 2011 , 62 ( 15 ): 5699 - 5711 .

Ahumada I , Cairó A , Hemmerlin A , et al . Characterisation of the gene family encoding acetoacetyl-CoA thiolase in Arabidopsis [J]. Funct Plant Biol , 2008 , 35 ( 11 ): 1100 - 1111 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Cloning and Prokaryotic Expression of Glycosyltransferase Genes from Paris polyphylla var. yunnanensis

Cloning and Prokaryotic Expression of Squalene Epoxidase Gene from Panax vietnamensis var. fuscidiscus

Cloning, Sequence Analysis, and Prokaryotic Expression of LaAACT Gene From Leaves of Seedlings

Cloning and Functional Characterization of Farnesyl Diphosphate Synthase Gene in Biosynthesis of Terpenoid Components in Chinese Materia Medica

Cloning and Expression Analysis of Squalene Epoxidase Gene in Poria cocos

Related Author

Ying-hui SHI

Yan YIN

Si-yuan GUO

Jia LI

Wei GAO

Xia-nan ZHANG

Fu-rong XU

Xiao-hui MA

Related Institution

School of Traditional Chinese Medicine，Capital Medical University

School of Chinese Materia Medica， Beijing University of Chinese Medicine

School of Pharmaceutical Sciences，Capital Medical University

College of Pharmaceutical Science, Yunnan University of Chinese Medicine

Institute of Chinese Materia Medica，China Academy of Chinese Medical Sciences

AI问答

Postal code：100700
Tel：010-84076882 Email：syfjx_2010@188.com
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备11006657号-10 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰