摘要
以野生型拟南芥总RNA为模板,逆转录PCR反应获得拟南芥肌醇-1-磷酸合酶类蛋白基因cDNA(AtMIPS1),开放读码框为1533 bp,编码510个氨基酸.与已报道物种MIPS基因氨基酸序列比较分析表明,AtMIPS1与植物MIPS基因的氨基酸同源性和相似性较高达86%～90%与95%～96%,并含有MIPS基因的保守区域'334SYNHLGNNDG'.将该cDNA序列不改变阅读框架克隆到pET28a(+)原核表达载体上,SDS-PAGE结果表明:在0.12g/L IPTG诱导2 h的条件下得到最佳的蛋白表达效果,AtMIPS1的成功表达为其功能研究打下基础.
The Myo-inositol lphosphate synthase-like protein gene （AtMIPS1） of the Arabidopsis thaliana was amplified by RT- PCR from the total RNA. Sequence analysis revealed that when comparing the cDNA full length of 1 533 bp encoding a protein of 510 amino acids and the deduced amino acid sequences with homologous sequences from other organisms, AtMIPS1 was found to share much higher sequence identities and similarities （86% -90%and 95% -96% ） with plant MIPS genes than with other organisms. In addition, AtMIPS1 also contains a highly conservative motif “334SYNHLGNNDG”, which is identical to those of other MIPS from plants. The cDNA fragment was inserted into _expression vector pET28a（ ＋ ） and the resulting plasmid was expressed in E. coli BL21 （DE3） by IPTG induction. The results of SDS PAGE analysis indicated that fusion protein was highly expressed （0 12 g/L IPTG, 2 h）, which laid the foundation for further research.
出处
《哈尔滨工业大学学报》
EI
CAS
CSCD
北大核心
2005年第12期1641-1643,1657,共4页
Journal of Harbin Institute of Technology
基金
国家转基因植物开发与研究专项资助项目(J99-A-001)
国家自然科学基金资助项目(GN.30221120261)
关键词
拟南芥
肌醇-1-磷酸合酶类蛋白基因
克隆
原核表达
Arabidopsis thaliana
myo inositol 1 phosphate synthase -like protein gene
cloning
expression in E. coli
