芡实药材品质形成的相关基因克隆及分子机理研究

Size is an important quality character of herbs, which is one of the basis for the traditional classification of medicinal materials’ commercial specifications. Also, it determines the yield and quality of medicinal materials.Size is determined by genetic factors and regulated by environmental factors.However, which genes determine the size of medicinal materials, and the environment is how to affect the size, which is not clear now.The functional genome research of plant seed (like rice and arabidopisis) formation has made remarkable progress. These research provides a reference for the study of gene related to the quality of medicinal materials.We use Euryale ferox Salisb’s kernel as medicine, therefore seed formation process determines the quality of Semen (as medicinal materials).This paper puts forward the hypothesis of“Key genes of Semen seed formation is similar to that of rice, and the formation of seeds is regulated by plant hormones and environmental factors”.In this paper, the key genes of particle size control in rice were used as primers,the gene was located in Euryale ferox Salisb by FISH (Fluorescence In Situ Hybridization）, the sequence of the gene was cloned by map-based cloning,and verify the functional description of the target gene for the locator.At the same time, carrying out systematic biology research of the key gene in seed development,and analyzing plant hormones, environmental factors and herbs quality,and clarifying the molecular mechanism of the formation of Euryale ferox Salisb,which aims to provide a scientific basis for the Euryale ferox Salisb breeding and ensure the quality of its cultivation.

大小是重要的药材品质性状，是药材传统商品规格分类的依据之一，决定药材的产量与质量，大小由遗传基因决定，受环境因素调控。但药材大小由哪些基因决定，环境又是如何影响药材大小的形成，目前知之甚少。而以水稻为代表的作物及模式植物拟南芥的种子形成的功能基因组学研究已取得了显著进展，为药材品质相关功能基因研究提供了借鉴和可能。芡实为种仁入药，种子的形成过程决定了药材的品质，本课题提出的假说是决定水生植物芡实种子的关键基因与水稻相关基因相似，种子品质形成受植物激素和环境因子调控。本课题以水稻中控制颗粒大小的关键基因为引物，利用荧光原位杂交技术对芡实中这一基因进行定位，通过图位克隆技术对定位基因序列进行克隆，并对定位的目标基因序列进行功能注释；同时开展种子发育过程中关键基因的系统生物学研究，并分析植物激素、环境因子及药材品质，阐明芡实品质形成的分子机制。为芡实育种、保证栽培芡实药材品质提供科学依据。

本项目从水稻穗粒数、株高和抽穗多效性控制基因（GHD7）出发，挖掘芡实中候选基因EfGHD7，该基因在芡实种子幼嫩时期表达量高于成熟时期，在刺芡生长全程中表达量低于苏芡，或因此致刺芡果实籽粒数低于苏芡。不同栽培品种芡实中生长素分布不同，自由态生长素在杂交芡和刺芡中分别集中分布在子房附近和表皮，通过转录组学及分子生物学技术筛选芡实中EuSAUR基因并进行克隆和功能验证，研究了其表达的分子机制。杂交芡种子较刺芡大，生长素含量高，其子房周围细胞较长，利于生成大子房。通过在水稻中过量表达EuSAUR62，可产生较大颖壳和籽粒，增加颖壳细胞长度。EuSAUR62通过与生长素外排载体蛋白PIN-FORMED9结合，调控植物子房周围生长素的积累。EubZIP55在杂交芡中高表达，直接与EuSAUR62启动子结合，激活EuSAUR62。我们认为，EuSAUR62在调控芡实种子大小方面起重要作用，且EubZIP55可以被光照诱导并控制果实中生长素的分布。通过分析芡实淀粉性质结构，联合分析转录组和蛋白组，成功找到与芡实抗性淀粉合成相关的基因ACCI、CACI、PPDK、SBEII、GBSSI、SSIII、SSII及对应蛋白，系统分析了功能基因表达谱和功能基因蛋白谱的差异。从非靶标代谢组学和靶标代谢组学角度，对比各产区芡实药材品质。刺芡种仁小，环境适应力优，其大小受纬度、海平面气压、环境温度影响。苏芡生长全程中生长素含量高于刺芡，其累积模式差别可能导致种仁大小差异。芡实产地土壤重金属含量符合种植标准，肥力较好，水体存在富营养化。产地土壤中部分元素差异显著，部分环境因子间显著相关。苏芡和刺芡部分成分与生长环境水质富营养程度相关，但趋势相反。土壤氮和磷对芡实品质影响较大。本项目确定了七个参与抗性淀粉的生物合路径的关键基因和一个控制芡实种仁大小的关键基因，为揭示芡实抗性淀粉合成分子机制和种仁大小形成机制提供依据，为下一步建立芡实抗性淀粉代谢工程和培育芡实新品种提供依据。已形成论文8篇，发表论文6篇，其中SCI收录2篇；另有2篇SCI审稿中。