乌腺金丝桃HaHYP1基因的克隆及原核表达

Cloning and Prokaryotic Expression of Hypericin Synthase Gene HaHYP1 in Hypericum attenuatum

目的:克隆乌腺金丝桃HaHYP1基因,并进行表达分析,为进一步探索金丝桃素生物合成的分子机制奠定基础。方法:根据已报道的金丝桃素合成酶Hyp-1同源基因设计兼并引物,利用qRT-PCR扩增金丝桃素合成酶Hyp-1基因,并进行序列分析和原核表达;同时利用qRT-PCR技术分析HaHYP1基因在不同组织部位的表达模式。结果:乌腺金丝桃HaHYP1基因的完整ORF为480 bp,编码159个氨基酸,推测的分子量为17.9 kDa,等电点为5.54。HaHYP1蛋白具有典型的P环结构和Bet v1家族特征性基序区,属于PR-10亚家族。乌腺金丝桃HaHYP1与贯叶连翘HpHYP1位于同一小的分支,表明两者亲缘关系最近。qRT-PCR检测结果表明,该基因在乌腺金丝桃叶中表达最高,其次是花和茎,根中表达最少。原核表达载体pQE-30-HaHYP1转化至大肠杆菌M15感受态细胞,在18 kDa附近表达出目的蛋白。结论:该研究为深入开展金丝桃素生物合成的分子机制和体外转化研究提供了理论依据。

Objective:To clone Hypericum attenuatum gene HaHYP1 and analyze its expression,so as to lay a foundation for further exploring the molecular mechanism of hypericin biosynthesis.Methods:The degenerate primers were designed according to the reported hypericin synthase Hyp-1 homologous gene,and the hypericin synthase gene Hyp-1 was cloned by qRT-PCR,sequence analysis and prokaryotic expression were performed.The expression patterns of HaHYP1 gene in different tissue sites were analyzed by qRTPCR.Results:The complete ORF of HaHYP1 gene of Hypericum attenuatum was 480 bp,encoding 159 amino acids,with a presumed molecular weight of 17.9 kDa and isoelectric point of 5.54.HaHYP1 protein,belonging to PR-10 subfamily,had typical P-ring structure and characteristic motional region of Bet v1 family.Hypericum attenuatum HaHYP1 and Hypericum perforatum HpHYP1 were located in the same small branch,indicating that the two were closely related.The qRT-PCR results showed that the gene expression was highest in the leaves of Hypericum attenuatum,followed by flowers and stems,and lowest in roots.The prokaryotic expression vector pQE-30-HaHYP1 was transformed into Escherichia coli M15 competent cell and the target protein was expressed near 18 kDa.Conclusion:This study provides a theoretical basis for further research on molecular mechanism and in vitro transformation of hypericin biosynthesis.