摘要
目的为研究GGPPS基因在三七中的功能,从三七中克隆GGPPS基因CDS序列,并进行原核表达。方法以3年生三七叶组织为材料,依据Genbank中已报道的GGPPS基因序列设计引物,利用RT-PCR技术,克隆得到编码区序列;克隆片段连接到p ET-30α(+)表达载体,转入大肠杆菌BL21后,在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下进行表达。结果三七GGPPS基因CDS序列全长1 032 bp,编码343个氨基酸。SDS-PAGE结果表明,所构建的原核表达载体表达的融合蛋白大小在29 000~44 000,且表达产物主要以不溶性包涵体形式存在。结论成功克隆了三七GGPPS基因CDS序列,建立了稳定的原核表达体系,为进一步研究其在三七中的生物学功能奠定了基础。
Objective In order to study the function of geranylgeranyl pyrophosphate synthase（GGPPS） gene, the CDS nucleotide sequence of GGPS was cloned from Panax notoginseng, and its prokaryotic expression was performed. Methods The primers were designed according to the reported GGPPS gene sequence in Genbank, and the coding sequence was obtained by RT-PCR. The prokaryotic expression vector was constructed and transformed into Escherichia coli BL21 for the expression under the induction of isopropyl β-D-1-thiogalactopyranoside（IPTG）. Results The CDS of GGPS gene had a full length of 1 032 bp coding for 343 amino acids. Results of SDS-PAGE showed that a 29 000—44 000 protein was achieved and the recombinant protein was mainly in the form of insoluble inclusion body. Conclusion The CDS nucleotide sequence of GGPPS gene was successfully cloned, and the stable prokaryotic expression was established. This study will provide a foundation for the further functional researches of GGPPS gene in P. notoginseng.
作者
唐美琼
闵丹丹
赵以民
胡营
李刚
TANG Mei-qiong;MIN Dan-dan;ZHAO Yi-min;HU Ying;LI Gang(Guangxi Key Laboratory of Medicinal Resources Conservation and Genetic Improvement,Guangxi Botanical Garden of Medicinal Plants,Nanning 530023,China;Third People＇s Hospital of Jiujiang,Jiujiang 332000,China)
出处
《中草药》
CAS
CSCD
北大核心
2018年第15期3667-3671,共5页
Chinese Traditional and Herbal Drugs
基金
广西自然科学基金面上项目(2015GXNSFAA139097)
广西自然科学青年基金项目(2013GXNSFBA19087)
关键词
三七
GGPPS基因
CDS
克隆
原核表达
Panax notoginseng （Burk.） F. H. Chen
geranylgeranyl pyrophosphate synthase （GGPPS） gene
coding sequence
clone
prokaryotic expression
