三七中花色苷合成结构基因的克隆及表达分析

Cloning and expression analysis of anthocyanin biosynthesis structural genes in Panax notoginseng

目的克隆三七Panax notoginseng的花色苷合成结构基因(anthocyanin biosynthesis structural genes,ABSG),研究ABSG的表达模式及其与相关表型的关联性。方法利用RT-PCR技术克隆三七ABSG,进行生物信息学和表达模式分析;使用pH示差法分析三七紫根的花色苷含量,并通过qRT-PCR技术进行相关结构基因的关联性研究。结果克隆获得6类共8个三七ABSG,其开放阅读框(open reading frame,ORF)长度在600~1500 bp,均含有花色苷生物合成相关的保守结构域。进化树分析显示,8个基因被分为5个分支,且与其他物种有较高的同源性。顺式作用元件分析表明,三七ABSG启动子序列中含有多种元件,其中光响应和转录因子类元件的数量最多。表达模式显示,8个基因主要分为在花发育前期阶段(花蕾期)高丰度表达基因和在根部高量表达基因。此外,对三七紫根的分析发现,相比于普通型黄白根,其花色苷含量提高约3倍,PnF3’H1基因和PnUFGT基因表达显著增强,表明它们可能是参与紫根三七中花色苷形成的关键结构基因。结论克隆获得8个三七ABSG,为该类基因的功能机制研究和三七种质创新利用奠定基础。

Objective To clone Panax notoginseng anthocyanin biosynthesis structural genes(ABSG)and study their expression patterns.Methods The complete open reading frame(ORF)of P.notoginseng ABSG were cloned using RT-PCR technology,and the bioinformatic method and quantitative real-time PCR(qRT-PCR)were used to analyze these genes.By pH differential method,total anthocyanin content was determined in purple root(PR)and yellow white root(YWR)of P.notoginseng.The differential expression of related structural genes in PR and YWR were analyzed by qRT-PCR.Results Eight ABSG belonged to six types were cloned.The ORF length of these genes was from 600 bp to 1500 bp,and they all contained conserved domains related to anthocyanin biosynthesis.Phylogenetic tree analysis showed that eight genes were divided into five branches,and these genes in P.notoginseng had high homology with other species.The promoter analysis of eight genes indicated that there were multiple putative cis-acting elements,among which the number of cis-acting element of light and transcription factor MYB/MYC was the most.According to the expression pattern,the eight genes were divided into two types,one was mainly highly expressed in the flowers at the early development stages(budding stage),and the other was mainly highly expressed in the roots.Furthermore,the anthocyanin content was detected to increase about three times and the expression of PnF3’H1 and PnUFGT were also significantly increased in PR compared with YWR,indicating that these two genes may be the key ABSG involved in formation of anthocyanin in PR.Conclusion Eight ABSG were cloned in P.notoginseng,and its expressions were analyzed,which will provide basic knowledge for the further functional studies of these genes and the innovative utilization of Sanqi germplasm.