摘要
沙冬青是我国北方内蒙古荒漠地区特有的常绿阔叶灌木,具有很强的抗低温、干旱、盐碱能力,是重要的抗逆基因资源植物.将从低温诱导的沙冬青幼苗中克隆获得的巯基蛋白酶抑制剂基因AmPI亚克隆后构建到原核表达载体pTWIN1上,得到重组表达载体pT-PI,双酶切及测序结果证明pT-PI已经构建成功.将pT-PI转入大肠杆菌ER2566中,经异丙基β-硫代半乳糖苷(IPTG)诱导后,SDS-PAGE电泳检测发现有Mr35×103的目的融合蛋白表达,与预测结果一致,其中以在37℃诱导3～4h条件下表达量最高,表明目的基因AmPI在大肠杆菌中得到了成功表达.分别测定了低温(0℃)和热胁迫(50℃)条件下AmPI宿主菌的生存能力,结果表明AmPI的表达能提高宿主菌的存活率,可能是AmPI对细胞具有保护功能.
Ammopiptanthus mongolicus is the only evergreen broad-leaved shrub endemic to the Alashan desert in Inner Mongolia,China,and can survive at-30 ℃ or even lower in winter.One cold-induced cysteine proteinase inhibitor gene AmPI from A.mongolicus was introduced into Escherichia coli using the IMPACT-TWIN system to analyze the possible function of this cysteine proteinase inhibitor under heat and cold stresses.SDS-PAGE showed that a Mr 35×103 fusion protein was expressed after 3～4 hour IPTG induction at 37 ℃.Compared with control cells,the E.coli cells expressing AmPI fusion protein showed the improved chilling（0 ℃） and heat（50 ℃） resistance,illuminating the protein might play a protective role in cells under the stresses.The research results also indicated that the AmPI gene might have the function of protecting A.mongolicus from cold and heat injury.
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2010年第3期341-346,共6页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(Nos.30671476
30700559)资助~~
关键词
沙冬青
AmPI
大肠杆菌
融合蛋白
过量表达
非生物胁迫
Ammopiptanthus mongolicus
AmPI
Escherichia coli
fusion protein
heterologous expression
abiotic stress
