[重楼鲨烯合酶基因的分子克隆及其在大肠杆菌中的表达]

[Molecular cloning of squalene synthase gene form Paris polyphylla and its expression in Escherichia coli].

作者信息

Gao Fei, Luo Xiao-Peng, Tao Liang, Li Cheng-Lei, Ding Chun-Bang, Chen Hui, Wu Qi

机构信息

College of Life and Basic Sciences, Sichuan Agricultural University, Ya'an 625014, China.

出版信息

Zhongguo Zhong Yao Za Zhi. 2013 Jul;38(13):2086-91.

PMID:24079231

Abstract

OBJECTIVE

To clone the cDNA sequence of squalene synthase gene from Paris polyphylla, and characterize the biological features of the obtained SQS.

METHOD

Using homology cloning and RACE technique, a full-length cDNA sequence of PpSQS gene was isolated from P. polyphylla. The obtained sequence was analyzed by bioinformatics softwares. A plasmid [named pET-30b (+)-PpSQS] was constructed for prokaryotic expression the recombinant PpSQS.

RESULT

The full-length cDNA of PpSQS gene is 1 498 bp, which contains a 1 212 bp ORF. Sequence analysis indicated that PpSQS encoded 403 amino acids residues with a calculated molecular weight (MW) of 46.36 kDa and an isoelectric point (pI) of 6.83. SDS-PAGE results showed that the recombinant PpSQS was expressed in Escherichia coli BL21 (DE3) by inducing with 1 mmol x L(-1) IPTG.

CONCLUSION

The full-length cDNA sequence of PpSQS gene was obtained from P. polyphylla, and its molecular features were consisted with classic SQS in plant. The recombinant PpSQS was successfully expressed in E. coli.

摘要

目的

克隆七叶一枝花鲨烯合酶基因的cDNA序列，并对获得的鲨烯合酶（SQS）进行生物学特性分析。

方法

采用同源克隆和RACE技术，从七叶一枝花中分离出PpSQS基因的全长cDNA序列。利用生物信息学软件对所得序列进行分析。构建用于原核表达重组PpSQS的质粒[命名为pET-30b(+)-PpSQS]。

结果

PpSQS基因的全长cDNA为1498 bp，其中包含一个1212 bp的开放阅读框。序列分析表明，PpSQS编码403个氨基酸残基，计算分子量（MW）为46.36 kDa，等电点（pI）为6.83。SDS-PAGE结果显示，用1 mmol/L IPTG诱导后，重组PpSQS在大肠杆菌BL21(DE3)中表达。