地黄转录组文库的构建及响应连作障碍关键基因的鉴定

The shortage of R. glutinosa genetic background has seriously limited the further researches on its underling mechanism of consecutive monoculture problem and others. The project will construct transcriptome libraries of R. glutinosa roots and leaves and identify consecutive monoculture problem-related genes using Solexa/Illumina sequencing technique. In the experiment, consecutive monoculture (the second year planting, SP) R. glutinosa will be taken as treatment samples, normal growth ones (the first year planting, FP) as control samples. Its roots and leaves will be collected at the seedling, tuberous root extension and earlier tuberous root expansion stages, respectively. The main details are as follows:.At first, total RNA of roots and leaves mixed FP and SP R. glutinosa, respectively, will be extracted and then the root and leave transcriptome libraries were established by Solexa/Illumina sequencing. Secondly, with isolation and sequencing of these sRNA from different-stage roots and leaves, miRNAs differential expression profiles of R. glutinosa responding to consecutive monoculture problem will be constructed, resulting in identifying key miRNA and its targets. At the same time, using Digital Gene Expression Profiling (DGE) analysis, we will establish consecutive monoculture R. glutinosa DGE expression profiles, collecting candidates for consecutive monoculture problem-related genes. Thirdly, exploring qRT-PCR and Northern blot technologies, we will confirm termpitial and spatial expression profiles of candidate genes and verify key genes. The study will unravel the underling molecular mechanism of R. glutinosa consecutive monoculture problem and also provide a theoretical basis for solving the consecutive monoculture problem of Chinese medicine materials production.

地黄遗传信息的极度匮乏，严重影响了对其连作障碍机制及其他重要生理生化过程的深入研究。为突破这一瓶颈，本项目拟构建地黄根和叶转录组文库并利用转录组鉴定响应连作的关键基因及miRNAs。试验设正茬和连作（重茬2年）2个处理，分别于地黄苗期、块根伸长初期和膨大前期取其根与叶样品。主要研究：①利用Solexa测序，分别构建根和叶不同生育时期的转录组文库6个。②提取不同时期2个处理的根与叶总RNA，分离小RNA、测序，构建小RNA库，匹配转录组，建立地黄响应连作的miRNAs差异表达谱，确定地黄响应连作的miRNAs及其靶基因；并利用编码基因序列标签测序技术，构建连作地黄编码基因数字表达谱，筛选响应连作障碍的"候选基因"。③利用qRT-PCR、Northern blot等验证候选基因的时空表达，最终锁定响应连作障碍的关键基因。以揭示地黄连作障碍的分子机制，为解决中药材生产中连作障碍难题提供理论依据。

地黄是遭受连作毒害最为严重的药用植物之一，危害表现出严重性、特异性和持续性的典型特点，使其成为研究作物连作形成机制的典型材料。而地黄遗传信息极度匮乏，严重影响了对其连作障碍机制及其他中药生理生化过程的深入研究。为了突破这一瓶颈，彻底揭示地黄连作障碍的分子机制，本项目运用高通量技术开展地黄转录组构建和响应连作毒害关键基因的鉴定，主要研究结果如下：. ①构建和整合了地黄根叶转录组。利用Illumina技术对连作毒害关键期连作和头茬地黄根混合样品、叶混合样品分别测序构建毒害期根叶转录组。同时，对不同生育时期连作和头茬根混合样品、叶混合样品测序构建全生育期根叶转录组。以此为基础，利用生物信息方法对上述4套转录组进行整合，搭建了转录信息更全面的全株转录组。. ②筛选了响应连作毒害基因并解析了连作伤害分子机制。利用DGE技术构建了毒害关键期连作和头茬地黄根叶差异表达谱，筛选了敏感期的毒害响应基因；利用RNA-Seq技术构建了连作和头茬地黄不同毒害阶段差异表达谱，解析了连作不同伤害阶段的响应基因。同时，构建连作和不同胁迫下的差异表达谱，鉴定了连作的特异响应基因。通过上述不同层次、不同角度毒害基因的解读和整合，发现钙信号和MAPK信号感知了连作伤害，诱导ROS大量产生，破坏了植物免疫系统，阻碍了核心代谢途径，使植株全方位受害。. ③确证了miRNAs在连作毒害中的作用机制。利用Illumina技术构建了连作和头茬关键伤害期的sRNA差异表达谱及连作年限间的sRNA差异表达谱，筛选了响应连作的特异miRNAs。同时，通过构建降解组文库鉴定了差异miRNAs的靶向信息。结果发现miRNAs通过降解连作地黄体内与激素响应、细胞分裂、根系吸收等代谢途径核心基因，破坏了植物正常代谢途径，加重连作障碍危害。. 本项目通过地黄大容量转录组构建和整合，弥补了地黄遗传信息匮乏的局面，为深刻揭示地黄连作障碍形成分子机制、地黄生长发育机理、药用成分合成机制提供了重要信息基础。同时，通过对连作毒害感知、传递和响应分子证据链的确证，为进一步从分子入手开发出彻底消减连作危害的有效技术奠定了理论基础。