地黄块根发育比较转录组学研究

The tuberous root of Rehmannia glutinosa, the medicinal part of this plant, is one of the most widely used herbal medicines in China. The development of tuberous root of R. glutinosa is easily affected by natural conditions such as virus diseases and replanting problem, which cause decline on yield and tuberous root quality. Presently the molecular mechanism underlying tuberous root development is undiscovered. Aimed to identify candidate genes involved in tuberous root development, we had carried out deep sequencing the trancriptome of R. glutinosa. Subsequently, we plan to use RNA-seq to discover diffentially expressed genes in tissues of R. glutinosa, which will allow us to identify tuberous root development related genes. In another hand, due to genetic variation and human selection, present rehmannia cultivar has evolved and has larger tuberous roots than wild type. Then we are going to carry out comparative tanscriptome analysis between a R. glutinosa cultivar and its wild type. By comparing their orthologous transcripts, we could identify protein-coding genes that have likely experienced strong positive selection (Ka/Ks>1), from which we will identify genes that is most likely to be involved in tuberous root bulking selection through comprehensive analysis. In addition, we also plan to decipher the common molecular mechanism underlying the sweet potato and R.glutinosa tuberous root development by analyzing their orthologous genes. Our study will establish the basis for better understanding of the molecular mechanism underlying the tuberous root development in R. glutinosa and increase the potential to manipulate the medicinal crop by gene engineering.

地黄为我国传统的大宗中药材，以块根入药。地黄在生产过程易受自然条件如病毒病、连作障碍等的影响，严重药材的产量与质量。目前地黄块根发育的分子机制不明，项目拟在地黄块根转录组深度测序的基础上，利用数字表达谱分析技术对地黄不同组织和不同发育阶段的差异表达基因进行分析，鉴定、筛选与地黄块根发育相关的差异表达基因，分析块根发育相关基因的表达调控机制。同时，根据栽培地黄和野生地黄块根膨大程度的差异，对栽培地黄与野生地黄的转录组进行比较分析，研究不同种质的地黄块根膨大程度差异的分子基础。此外，项目还拟根据当前甘薯块根发育分子生物学的研究进展，对地黄、甘薯两种植物的块根发育相关基因进行横向的比较分析，筛选、鉴定在两种植物的块根内表达、表达模式一致或接近的同源基因，探索两种植物块根发育的共同分子机制。综合以上手段筛选出地黄块根发育相关的候选基因，并通过转基因等实验手段对候选基因进行功能分析，为地黄块根发育分子机制的阐明和品种改良奠定理论基础。

地黄为我国传统的大宗中药材，其药用部位块根的膨大机制不明。项目围绕地黄块根发育，在地黄块根转录组深度测序的基础上，利用多种手段筛选、鉴定地黄块根发育相关的候选基因，获取地黄块根发育的分子线索。项目首先利用RNA-seq分析技术对栽培种85-5的地黄块根形成发育不同阶段的差异表达基因进行了分析，筛选出8,393个unigenes在地黄块根内富集，1,048个unigenes在地黄块根发育的4个阶段中表达量下调。我们还首次利用比较转录组学分析技术对2个栽培种与5份野生种质（块根细长，产量极低）进行了比较转录组学分析，鉴定出在栽培地黄块根内特异表达的差异基因160个，这些基因包括转录因子、信号转导、蛋白合成以及代谢相关的酶类。此外，我们还对地黄不同种质的SNP进行了鉴定，并根据Ka/Ks值筛选出16个在栽培地黄中受到正向筛选的基因。通过将地黄与甘薯块根发育相关同源基因之间的比较，发现木质素合成代谢相关的同源基因在2个物种块根发育中表达模式相近，预示去木质化是块根形成的一个重要步骤。在这些地黄块根发育（进化）候选基因中，我们克隆了其中部分基因的全长。以上研究使我们对地黄块根发育有了更深入的认识，为全面解释地黄块根形成发育（及栽培地黄块根定性进化）机制和指导生产实践提供了大量翔实的数据和理论基础。