一年生三七地上茎的黄金分割式紫化及其与皂苷积累的关系

It has been found in previous researches that the aerial stems of one-year-old Panax notoginseng plants hold three colors, i.e. green, purplish green and purple, and the Purpling of P. notoginseng organs results from the anthocyanin accumulation. The applicant of this project proved the Purpling and the contents of the total saponins of the purplish green aerial stems of one-year-old P. notoginseng plants are both golden section-typed, which is regarded as the initial point of the studies of this project. By measuring the longitudinal changes of the contents of the saponin monomers and the transcription intensities of the structural genes (i.e. chalcone synthase, chalcone isomerase, flavanone-3-hydroxylase, flavonoid-3′-hydroxylase, dihydroflavonol-4-reductase, anthocyanidin synthase and UDP-glucose：flavonoid-3-O-glucosyltransferase genes) and the regulatory genes (i.e. R2R3-MYB, bHLH and WD40) of anthocyanin biosynthesis, and the structural genes (i.e. 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase and dammarenediol synthase genes) and the regulatory genes (i.e. PqWRKY1-like) of triterpenoid saponin biosynthesis, this project first identifies the key structural and regulatory genes, and the target structural genes of the regulatory genes which determine the longitudinally uneven synthesis and accumulation of the anthocyanins and triterpenoid saponins in the aerial stems. Then, this project clones the promoters of the key structural and regulatory genes and analyzes the functions of the promoters, constructing the regulation net of the anthocyanin and triterpenoid saponin synthesis in the aerial stems. This project would elucidate the tissue-specificity of P. notoginseng anthocyanin synthesis and further reveal the ecophysiological functions of P. notoginseng anthocyanins, including the saponin accumulation-enhancing, and also create the possibilities for the molecular breeding and the development of the anthocyanin-related new products of P. notoginseng.

研究发现，一年生三七地上茎有绿、绿紫过渡和紫三种颜色，三七器官紫化是源于花色苷的积累。申请人证实，一年生三七绿紫过渡色地上茎的紫化和总皂苷含量均呈黄金分割式，本项目以此为研究契入点，检测一年生三七绿紫过渡色地上茎中皂苷单体含量、花色苷合成结构基因（CHS、CHI、F3H、F3′H、DFR、ANS和3GT）和调节基因（R2R3-MYB、bHLH和WD40）、三萜皂苷合成结构基因（HMGR、FPS、SS、SE和DS）和调节基因（类PqWRKY1）转录强度的纵向变化，以明确决定地上茎花色苷、皂苷纵向不均匀合成、积累的关键结构基因和调节基因及其靶结构基因，再克隆关键结构基因和调节基因启动子序列并完成其功能分析，以构建地上茎花色苷、皂苷合成的调控网络。本项目将阐明三七花色苷合成的组织专一性特征、进一步揭示三七花色苷包括促进皂苷积累在内的生态生理作用，并为三七的分子育种和花色苷类新产品开发创造可能。

研究表明，一年生三七绿紫过渡地上茎的紫化和总三萜皂苷含量具黄金分割式。本项目研究发现：在过渡地上茎中，花色苷的主要径向组织定位点为皮层薄壁组织外的2~3层细胞，皂苷的为维管束；在纵向上，花色苷主要积累在茎中上部的黄金分割点处，皂苷在中下部。茎的主要皂苷单体为Rb1。从顶端向基部，茎的总花色苷、总皂苷和Rb1含量总体上分别表现为一条“单峰曲线”、“V形曲线”和“降-升-降三段式曲线”，总花色苷含量的最高值及总皂苷含量和Rb1的最低值均在黄金分割点处；总花色苷和总三萜皂苷的含量间的相关系数约为-0.767**；花色苷合成主要关键结构基因CHS、CHI、F3H、DFR、ANS及3GT和转录因子基因PnbHLH、PnMYB1、PnMYB2、PnWD40-1、PnWD40-2和PnWD40-3的转录水平表现为不同的波动模式；对于茎的花色苷合成，贡献最大的结构基因和转录因子基因分别为ANS和PnWD40-1，PnWD40-1的靶结构基因可能是ANS、CHS、3GT及CHI；三萜皂苷合成主要关键结构基因HMGR、FPS、SS、SE和DS和转录因子基因PnWRKY1的转录水平表现为不同的“多峰曲线”；对于茎的三萜皂苷合成，贡献最大的结构基因应为FPS，PnWRKY1应是决定皂苷合成的关键转录因子基因，PnWRKY1的靶结构基因可能是SS、DS、SE和FPS；茎中花色苷和三萜皂苷合成的结构基因和转录因子基因均未组建成共表达群。克隆到的ANS、PnWD40-1、FPS和PnWRKY1的启动子分别长1960、1240、2109和1050 bp，均含真核生物启动子的核心元件及低温等响应元件；FPS启动子还含WRKY识别、结合的W-box，这是FPS被PnWRKY1调控的基础。本项目阐明了三七花色苷合成的组织专一性、揭示了三七花色苷的生态生理作用，为三七的分子育种和花色苷类新产品开发创造了可能。

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