雷公藤贝壳杉烯酸氧化酶基因的全长cDNA克隆与表达分析

Cloning and expression analysis of kaurenoic acid oxidase gene in Tripterygium wilfordii

贝壳杉烯酸氧化酶(kaurenoic acid oxidase)是二萜赤霉素生物合成途径上的关键酶,参与植物生长发育等重要生物学过程。该文根据雷公藤转录组数据设计特异性引物,采用PCR技术克隆得到贝壳杉烯酸氧化酶全长c DNA序列,并进行生物信息学分析;使用实时定量PCR(qRT-PCR)研究基因的诱导表达水平。克隆得到Tw KAO长度为1 874 bp,编码487个氨基酸,蛋白相对分子质量56.02 k Da,理论等电点8.89;经茉莉酸甲酯(Me JA)诱导后,Tw KAO基因相对表达量在12 h达到峰值;经植株组织表达分析证实,Tw KAO基因在雷公藤的叶中表达量最高,根中最低。该研究首次克隆得到雷公藤KAO基因,并分析其mRNA表达特征,为深入研究雷公藤生长发育以及萜类活性成分次生代谢研究奠定基础。

Kaurenoic acid oxidase involved in biosynthesis pathway of gibberellin. According to the transcriptome database, the specific primers were designed and used in cloning the full-length eDNA of TwKAO, the bioinformatic analysis of the sequence was per- formed. The qRT-PCR were used to detect the expression level of TwKAO after MeJA treatment. The full-length eDNA of the TwKAO was 1 874 bp encoding a polypeptide of 487 amino acids. The calculate molecular weight was about 56.02 kDa, and the theoretical isoe- lectric point （pI） was 8.89. The relative expression level of TwKAO was deduced by MeJA and reached the highest at 12 h after the treatment. Plant tissue expression analysis indicated that, TwKAO expressed the highest in leaves, while lowest in roots. For the first time, we cloned and analyzed the expression characteristics of TwKAO, which laid a foundation for deep analysis of growing develop- ment and terpenoid secondary metabolites in T. wilfordii.