棉铃虫乙醇脱氢酶的表达规律

赵洁; 任苏伟; 马纪; 刘小宁

doi:10.13568/j.cnki.651094.2017.03.003

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棉铃虫乙醇脱氢酶的表达规律

研究精粹 | 更新时间：2026-01-21

- 棉铃虫乙醇脱氢酶的表达规律
- 棉铃虫乙醇脱氢酶的表达规律
- 新疆大学学报（自然科学版中英文） 2017年34卷第3期页码：267-272
- 作者机构：
  
  新疆大学新疆生物资源和基因工程重点实验室
- 作者简介：
- 基金信息：
  
  国家自然基金(31471781);自治区青年科技创新人才培养工程项目(qn2015yx001)资助
- DOI：10.13568/j.cnki.651094.2017.03.003
  中图分类号： Q966
- 纸质出版：2017
- 稿件说明：
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[1]赵洁,任苏伟,马纪,等.棉铃虫乙醇脱氢酶的表达规律[J],2017,34(03):267-272+352.
[1]赵洁,任苏伟,马纪,等.棉铃虫乙醇脱氢酶的表达规律[J],2017,34(03):267-272+352. DOI： 10.13568/j.cnki.651094.2017.03.003.

DOI：10.13568/j.cnki.651094.2017.03.003.

摘要

乙醇脱氢酶(Alcohol dehydrogenase

ADH)是一种在醇类代谢途径中起重要作用的脱氢还原酶.为了深入了解ADH对棉铃虫CYP6B6的调控

基于酵母单杂交的实验结果

采用RT-PCR的方法从六龄幼虫的中肠cDNA中扩增得到棉铃虫乙醇脱氢酶5(HaADH5)

构建重组菌株BL21-p ET32a-HaADH5

IPTG诱导表达后通过镍柱纯化获得目的蛋白

Western blot检测目的蛋白的表达和纯化结果

实时定量PCR检测棉铃虫不同发育阶段和组织中HaADH5的表达规律

最后检测2-十三烷酮处理下六龄幼虫中肠组织内HaADH5的变化规律.结果表明

克隆得到的HaADH5大小为1 002 bp

编码334个氨基酸

预测的蛋白质分子量和等电点分别是36.5k D和6.55.氨基酸序列分析表明HaADH5蛋白不含信号肽和跨膜结构域.HaADH5在大肠杆菌BL21中主要以可溶蛋白形式存在

Western blot结果显示融合蛋白His-HaADH5的大小与预期一致且纯度较高.HaADH5在棉铃虫的所有组织中都表达且中肠内表达量最高

在幼虫的所有龄期都表达且预蛹期的表达量最高.2-十三烷酮处理后HaADH5的表达量降低

且10 mg/g处理组中HaADH5和CYP6B6随着时间的延长表达规律相一致.本文将为后续利用HaADH5作为分子标记来研究棉铃虫蜕皮和变态发育过程奠定基础.

Abstract

Alcohol dehydrogenase(ADH) is a dehydrogenase/reductase family in cellular alcohol metabolic network. In order to better understand the role of ADH gene and its regulatory mechanism in the CYP6B6 expression of Helicoverpa armigera

we cloned the H. armigera ADH5 gene(HaADH5) c DNA sequence from midgut on the basis of yeast one-hybrid results. The open reading frame of the HaADH5 was 1002 bp

encoding 334 amino acid residues with the predicted molecular weight and isoelectric point of 36.5 k D and6.55

respectively. And no signal peptide and transmembrane helices. Then we constructed the recombinant expression plasmid p ET32a-HaADH5 and expressed it in Escherichia coli BL21. Western blot analysis indicated that there were predicted band and higher purity of the fusion protein purified using Ni2+affinity chromatography. Then we determined the HaADH5 expression profile on the different developmental stages and in the different tissues. The results showed that HaADH5 was not only expressed in different tissues but also highest in the midgut. The HaADH5 was also expressed in larval stage and highest in the pre-pupae.The HaADH5 expression was significantly decreased after 2-tridecanone treatment

while expression profile of HaADH5 and CYP6B6 was similar in 10 mg/g treatment group. Our results will provide very useful information for using ADH as a molecular target to understand H. armigera how to regulate molting and metamorphosis.

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