摘要
乌桕是一种重要的木本油料树种。SAD(stearoyl-acyl ACP desaturase)是油料植物中将饱和脂肪酸转变成不饱和脂肪酸的一种关键脱氢酶。为了进一步揭示乌桕SsSAD的功能,该研究在大肠杆菌中表达了该蛋白。结果表明:(1)通过RT-PCR的方法从乌桕种子中克隆出了SsSAD基因编码区全长序列,并将其克隆到低温诱导的原核表达载体pCold TF上,构建原核重组表达载体pCold TF/SsSAD,转化大肠杆菌BL 21star(DE3)并获得原核表达工程菌株。(2)通过IPTG法低温诱导表达融合蛋白。该重组质粒在大肠杆菌中得到了高效表达,融合蛋白分子质量约为101kD,且在上清液和包涵体中均有表达,可溶性部分经亲和层析纯化和Western blotting检测证实获得了重组蛋白,上述结果为进一步研究乌桕SsSAD的结构和功能奠定了基础。
Sapiurn sebiferurn is one of the most important oil trees. SAD（stearoyl-acyl ACP desaturase） protein is a dehydrogenase,which is a key factor for transforming saturated fatty acid to unsaturated fatty acid in oil plant. We expressed and purified SsSAD from Sapiurn sebiferum in E. coli to investigate its function. The result suggested that： （1）The full length cDNA encoding SsSAD was amplified by RT-PCR from Sapium sebiferum and cloned into cold shock inducible expression vector pCold TF. The recombinant prokaryotic expression plasmid was transformed into E. coli BL 21 star （DE3） strain for gaining of the ge- netic engineering strain. （2）The transformed strain was induced with IPTG（isopropylthio-D-galactoside） for expressing fusion protein at low temperature. Results showed that the recombinant protein with a mo- lecular mass 101 kD was highly expressed in E. coli and present both in the supernatant and the pellet part of E. coli lysates. The supernatant was further purified by affinity chromatography and detected by West- ern blotting,which demonstrated that high quality recombinant protein was obtained and laid the founda-tion for investigation on the structure and function of SsSAD.
出处
《西北植物学报》
CAS
CSCD
北大核心
2014年第3期444-448,共5页
Acta Botanica Boreali-Occidentalia Sinica
基金
中南林业科技大学青年基金(QJ2011046B)
中南林业科技大学人才引进项目(104-0182)
国家自然科学基金青年基金(31200516)
湖南省教育厅优秀青年基金(13B150)
关键词
SsSAD基因
pCold
TF
原核表达
融合蛋白
纯化
SsSAD（Sapium sebiferum stearoyl-ACP desaturase） gene
pCold TF
prokaryotic expressionfusion protein
purification
