《植物生理学报》 2012, 48(7): 676-682

四翅滨藜AcDHN基因的克隆及其抗逆功能分析

李敬涛*, 余刚*, 陈宣明, 范臻, 刘金亮, 潘洪玉**

吉林大学植物科学学院, 长春130062

通信作者：潘洪玉；E-mail: panhongyu@jlu.edu.cn；Tel: 0431-87836251

摘　要：

脱水素(dehydrin, DHN)是一类胚胎发育后期丰富蛋白(LEA), 在植物脱水条件下能保护细胞内蛋白质和膜结构免受 破坏。本研究中, 从四翅滨藜(Atriplex canescens) cDNA文库克隆得到逆境胁迫相关蛋白基因AcDHN的全长cDNA (登录号: JN974246), 并进行序列分析。将AcDHN分别插入到原核表达载体pET28a和双元表达载体pYES-DEST52中, 通过转化大肠 杆菌和酿酒酵母进行原核表达分析和真核表达分析。结果表明: AcDHN序列全长为1 408 bp, 完整的开放阅读框长为1 017 bp, 由338个氨基酸残基组成, 预测蛋白质分子量为38.3 kDa, 理论等电点为6.47, AcDHN与仙人掌中DHN蛋白同源性为55%, AcDHN基因在大肠杆菌BL21 (DE3)中诱导表达出分子质量约45.2 kDa的融合蛋白。重组酵母菌株能表现出良好抗逆 性, 特别是对NaCl、低温、Na2CO3和NaHCO3胁迫的抗逆性, 其中抗碱胁迫能力表现最强。

关键词：四翅滨藜; AcDHN; 酵母表达; 非生物胁迫抗性

收稿：2012-03-20   修定：2012-04-09

资助：农业部转基因生物新品种培育重大专项(2009ZX08009-062B)。

Cloning and Stress Risistance Functional Characterization of an AcDHN Gene from Atriplex canescens

LI Jing-Tao*, YU Gang*, CHEN Xuan-Ming, FAN Zhen, LIU Jin-Liang, PAN Hong-Yu**

College of Plant Science, Jilin University, Changchun 130062, China

Corresponding author: LI Jing-Tao; E-mail: panhongyu@jlu.edu.cn; Tel: 0431-87836251

Abstract:

Dehydrin (DHN) present a distinct biochemical group of late embryogenesis abundant (LEA) proteins. It is proposed that dehydrins can protect proteins and cell membranes against unfavourable structure changes caused by dehydration in the plant cells. In this research, the full sequence of cDNA sequence of AcDHN gene was cloned from Atriplex canescens. The accession number of AcDHN in GenBank was JN974246. The AcDHN gene was introduced into the prokaryotic expression vector pET28a and the binary vector pYES-DEST52, and transformed into Escherichia coli BL21 (DE3) and Saccharomyces cerevisiae INVScl, respectively. The results revealed that: the full sequence of AcDHN is 1 408 bp in length and contains an open reading frame (ORF) of 1 017 bp, encoding a protein of 338 amino acid residues, with protein molecular weight of 38.3 kDa and PI 6.47. The AcDHN had 55% amino acid sequence homology to the sequence of DHN from Opuntia streptacantha. AcDHN was expressed its fusion protein (about 45.2 kDa) in E. coli BL2l (DE3). The recombinant yeast cells have a higher stress resistance than control cells, especially exhibit the capability to cope with the NaCl, low temperature, NaCO₃ and NaHCO₃, and the tolerance to the stress of alkali is the most highly.

Key words: Atriplex canescens; AcDHN; yeast expression; abiotic stresses resistance