响应地黄连作的关键lncRNAs鉴定及作用研究

Continuous monoculture problem seriously affects the production of traditional Chinese medicinal materials. According to the applicant previous researches, differential expressed lncRNAs were found in continuous monoculture Rehmannia glutinosa. To confirm lncRNA functions, R. glutinosa cultivar “Wen 85-5” will be planted as an experimental material, at the same time, its whole plants from normal growth (first year planting, FP) as control samples and continuous monoculture (second year planting, SP) as treatments. Total RNA from FP and SP roots, and their leaves (four samples) will be exacted at earlier tuberous root expansion stages, respectively. Using Solexa sequencing, bioinformatics, qPCR, Race and some other technologies, the project will be carried on four aspects as follows. At first, to obtain lncRNA candidates, we will construct with and without polyA RNA transcripts from mixed FP and SP R. glutinosa roots and their leaves, respectively. Secondly, we will separately construct sRNA and degradome libraries of the four samples. The candidates of lncRNAs in R. glutinosa will be identified by mapping to sRNA sequences to find their characteric structures and by searching in degradome libraries to check the degraded fragements of lncRNA target sequences. Based on complementary traits between the fragments and coding protein sequences, the spliced targets were identified. With functional analysis of their targets, we will find the relevance between lncRNAs and adversity stress. Thirdly, we will measure spatial and temporal expression patterns of these lncRNAs and gain key lncRNAs responding to continuous monoculture problem. At last, the full-length sequences of the key lncRNAs will be cloned and their expression patterns will further be verified and the key lncRNAs involving in continuous monoculture problem will be targeted. According to their target functions, we will elucidate lncRNAs important roles in R. glutinosa subjected to continuous monoculture. The study will be of significance to unravel the underlying molecular mechanism of R. glutinosa continuous monoculture problem and also provide a theoretical basis for solving the continuous monoculture problem of Chinese medicine materials production.

连作障碍严重制约着中药材的生产。申请者前期研究发现连作地黄体内有lncRNAs差异表达。为确证lncRNAs在连作中的作用，本项目拟以地黄品种“温85-5”为供试材料，头茬为对照、连作为处理，在块根膨大前期，分别提取根、叶总RNA，利用Solexa测序、生物信息学、qPCR和Race等技术，主要研究：①分别构建“只含”与“不含”polyA的转录本库，候选lncRNAs。②构建sRNAs库，根据与候选lncRNAs匹配特征，鉴定lncRNAs；构建降解组库，寻找lncRNAs序列中降解片段，鉴定剪切的靶基因；根据靶基因功能，筛选逆境胁迫的lncRNAs。③测定lncRNAs时空表达模式，分析其与连作症状是否吻合，确定响应连作的关键lncRNAs。④克隆lncRNAs全长，验证在连作中的特异表达，最终锁定关键的lncRNAs，解读lncRNAs在连作毒害中的作用，为克服连作障碍难题奠定基础。

连作障碍严重制约着中药材的生产。lncRNAs是真核生物基因表达的重要调控者，项目主持人前期研究发现：连作响应了地黄体内特异表达的lncRNAs。为确证lncRNAs在地黄连作障碍中的角色，本项目以怀地黄品种“温85-5”为材料，头茬为对照、连作为处理，在块根膨大前期，分别提取根和叶总RNA，利用Solexa测序、生物信息学、Race、基因组步移、qPCR等技术，主要进行了以下研究：①分别构建了“只含polyA”与“不含polyA”的转录组文库，合并拼接后构建了1个大容量的lncRNA文库。②以已构建的sRNAs库，根据与候选lncRNAs匹配特征，鉴定lncRNAs；以已构建的降解组库，寻找lncRNAs序列中的降解片段，鉴定剪切的靶基因；根据靶基因功能，筛选了与逆境胁迫相关的lncRNAs。③为进一步确证关键lncRNAs的真实性及其在连作地黄中的特异响应，本研究克隆了12条具有代表性的连作地黄体内与逆境响应的lncRNAs全长转录本序列和4条lncRNAs的DNA序列及其启动子序列，验证了这些lncRNAs确实为连作地黄中真实存在的ncRNAs；通过4条lncRNAs的启动子序列分析表明，其启动子序列中存在着许多与逆境响应相关的可调控元件，这些元件的获取为深入系统分析地黄体内在连作毒害的形成过程中lncRNAs的转录、剪切的分子调控网络奠定了基础。④构建了连作与头茬地黄lncRNAs表达差异谱，筛选了764条在连作中差异表达的lncRNAs；为深入揭示lncRNAs在连作毒害中的作用，本研究分析了这些差异表达的lncRNAs靶基因的功能，并对12条关键的lncRNAs及其调控靶基因的时空表达模式进行了分析，其结果发现：这些lncRNAs与其靶基因呈现出正向或负向协同表达趋势，并且这些lncRNAs与连作毒害症状的发生呈现一致的趋势，验证了关键lncRNAs在连作毒害发生过程中调控的重要角色，初步阐明了lncRNAs在地黄连作障碍的分子调控作用，为揭示地黄连作障碍形成的分子机理和开发其消减技术提供理论依据和技术支撑。