杭菊CHI基因的克隆及原核表达

Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.’Hangju’

从杭菊转录组数据库中筛选并通过PCR技术成功克隆得到3条杭菊査尔酮异构酶基因(Cm CHI)分别命名为:CmCHI1,Cm CHI2,Cm CHI3。生物信息学分析表明:Cm CHI1~3开放阅读框的碱基数分别为708,633,681 bp分别编码235,210,226个氨基酸。利用原核表达技术成功诱导得到3条30 k Da左右的融合蛋白,同时使用Ni-NTA树脂柱分离纯化得到相应的重组融合蛋白。聚类分析表明,筛选得到的3条Cm CHI与菊科植物同源性较高,Cm CHI1与Cm CHI3属于Ⅰ型CHI;Cm CHI2属于Ⅳ型CHI。以β-actin为内参基因,使用RT-qPCR检测并分析Cm CHI1~3基因在杭菊中的表达量差异,结果表明Cm CHI1,Cm CHI3表达量较高,Cm CHI2表达量较低;淹水处理能显著促进Cm CHI1,Cm CHI3基因的表达,而对Cm CHI2无调控作用,由此得出CmCHI1,Cm CHI3是主要参与杭菊黄酮类化合物合成的查尔酮异构酶基因,Cm CHI2为辅助基因。以上研究结果为进一步研究CHI基因在杭菊黄酮类化合物代谢中的分子调控机制提供依据,从而为提高杭菊黄酮含量及最终提高杭菊药用品质奠定基础。

Three Chrysanthemum-chalcone-isomerase genes(CmCHI)were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively.Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively.Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column.Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to typeⅠCHI.CmCHI2 belongs to typeⅣCHI.Usingβ-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju.The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower.It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene.Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2.The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.