关键酶基因启动子和编码区甲基化对刺五加皂苷含量的作用机制

Farnesyl diphosphate synthase (FPS), squalene synthase (SS) and squalene epoxidase (SE) genes which catalyzed the biosynthesis of Eleutherococcus senticosus saponins all have single nucleotide polymorphism (SNPs) and gene family, but the difference of E. senticosus saponins content can not be fully revealed merely by the variation of the pure DNA sequence. DNA methylation is a common phenomenon in most organisms, among which the methylation of promoter and coding region plays an important role in the regulation of gene expression. This study is planned to find out the DNA methylation sites of promoters and coding regions of three cloned and analyzed key enzyme genes through bisulfite sequencing PCR (BSP). Penicillium minioluteum P116-1a is used to induce E. senticosus clones with various states of DNA methylation without changing the genotype of E. senticosus, thus the disadvantages of sexual reproduction that possibly lead to a complex genetic background can be avoided and the asexual reproduction E. senticosus had the same DNA methylation. Based on the results of saponins content and expression content of key enzyme genes under different states of methylation, we can screen out the specific methylation status that closely is related to saponins content. According to the confirmed key DNA methylation sites in wild E. senticosus, we can theoretically explain the molecular mechanisms of how DNA methylations of the key enzyme gene’s promoter and coding region lead to the variation of saponins content in E. senticosus.

催化刺五加皂苷生物合成的法尼基焦磷酸合酶、鲨烯合酶和鲨烯环氧酶基因具有单核苷酸多态性和基因家族现象，但单纯的DNA序列变异无法全面揭示刺五加皂苷含量差异的分子机制。生物体中广泛存在DNA甲基化现象，其中启动子和编码区的甲基化对基因的表达调控等具有重要的作用。本项目拟在克隆和分析3个关键酶基因启动子和编码区的基础上，通过重亚硫酸盐测序法找到其中存在的DNA甲基化位点。进而利用不改变刺五加基因型，但可改变宿主DNA甲基化程度的内生青霉P116-1a诱导相同基因型刺五加形成不同程度的DNA甲基化。从而避免有性繁殖刺五加遗传背景复杂、无性繁殖刺五加DNA甲基化相同的弊端。结合各种甲基化状态下关键酶基因表达量及皂苷含量的测定结果，筛选出与皂苷含量密切相关的DNA甲基化位点，并将关键甲基化位点在野生刺五加中进行验证，从而基于关键酶基因启动子和编码区DNA甲基化解析刺五加皂苷含量差异形成的分子机制。

法尼基焦磷酸合酶（farnesyl diphosphate synthase，FPS）、鲨烯合酶（squalene synthase，SS）和鲨烯环氧酶（squalene epoxidase，SE）是催化刺五加皂苷合成的关键酶，其编码基因的改变可影响皂苷的含量，但单纯的DNA序列变异无法全面揭示刺五加皂苷含差异的分子机制。本研究分析了FPS、SS和SE基因启动子和编码区的DNA甲基化情况及其对基因表达和皂苷含量的影响。研究结果表明：1）获得长7952、10228和6307 bp的FPS、SS和SE基因的编码区和启动子序列，证实各启动子均具有启动功能；2）刺五加FPS、SS和SE基因的编码区中均不存在DNA甲基化位点。重亚硫酸盐测序法分析发现刺五加FPS基因启动子中存在19个DNA甲基化位点，8种DNA甲基化类型，DNA甲基化率在2.68%-3.40%之间。SS基因启动子中有9个DNA甲基化位点，2种DNA甲基化类型，DNA甲基化率在0%-0.27%之间。SE基因启动子中有16个DNA甲基化位点，7种DNA甲基化类型，DNA甲基化率在0.35%-5.61%之间；3）刺五加FPS基因的表达量与FPS基因启动子DNA甲基比率间不存在相关性，SS和SE基因启动子DNA甲基比率分别与对应基因的表达量和皂苷含量间呈极显著的负相关关系，且高DNA甲基化状态下，多数萜类物质的合成显著下调；4）SS基因起始密码子上游-558 bp处和SE基因起始密码子上游-847和-684 bp处的胞嘧啶C发生DNA甲基化后可分别引起SS、SE基因表达显著降低，进而减少刺五加皂苷的含量，是决定刺五加皂苷含量高低的关键位点。在FPS基因启动子区域整体DNA甲基化程度较低，SS基因启动子-558 bp位点和SE基因启动子-847和-684 bp位点均未被甲基化时，最有利于刺五加皂苷的积累。

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