雷公藤乙酰CoA酰基转移酶基因全长cDNA克隆及表达分析

Cloning and expression analysis of a acetyl-CoA C-acetyltransferase gene(TwAACT) from Tripterygium wilfordii

研究根据雷公藤悬浮细胞转录组数据库设计引物,对雷公藤悬浮细胞乙酰辅酶A酰基转移酶(acetyl-Co A C-acetyltransferase)基因Tw AACT进行全长c DNA(Gene Bank:KP297934)克隆以及生物信息学分析和基因表达分析。克隆得到Tw AACT c DNA全长为1 704 bp,编码405个氨基酸,等电点为6.10,相对分子质量为41.20 k Da,在线预测表明Tw AACT具有2个催化活性位点。雷公藤悬浮细胞经茉莉酸甲酯(Me JA)外源诱导后,通过荧光定量PCR表明,Tw AACT相对表达量明显增加,在24 h达到最高。研究首次克隆得到雷公藤AACT基因,为进一步阐述雷公藤萜类生物合成途径奠定基础。

In this study, based on the transcriptome data, we cloned the full-length cDNAs of TwAACT gene from Tripterygium wil- fordii suspension cells, and then analyzed the bioinformation of the sequence, detected the genetic differential expression after being in- duced by methyl jasmonate （MeJA） by RT-PCR. The full-length cDNA of the TwAACTwas 1 704 bp containing a 1 218 bp open read- ing frame （ORF） encoding a polypeptide of 405 amino acids （GeneBank accession No. KP297934）. The deduced isoelectric point （pI） was 6. 10, a calculated molecular weight was about 41.20 kDa, and online prediction showed that TwAACT had two catalytic ac- tive sites. After the induction of MeJA, the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly, that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.