萝卜耐盐性关键基因鉴定与生物学功能分析

Radish (Raphanus sativus L.) is an economically important root vegetable crop which originated in china. The secondary salinization in soil adversely affects the growth and development of the taproots in radish. Therefore, improving the salt tolerance of radish and developing highly salt-tolerant radish varieties would be a significant reseach work for current genetic improvement of vegetable crops. Based on successfully screening of elite radish genotypes of salt tolerance, in this project, the salt-responsive genes will be identified by digital gene expression profiling (DGE) technology using advanced inbred lines as materials which significantly exhibited difference in resistence to high salinity. Meanwhile, association analysis will be carried out between the transcriptome and miRNA expression profiling data obtained in our lab to identify key candidate genes involved in salt stress response. Furthermore, temporal and spatial expression profiles of these genes would be conducted with RT-qPCR. The biological functions of these key candidate genes will be validated by adopting Gateway system-based overexpression technique and artificial microRNAs (amiRNAs) which would silence the target gene efficiently. The regulatory network of salt stress response and molecular mechanism underlying the tolerance to salinity would be elucidated at the levels of transcription, miRNA-mediated post-transcriptional and from gene biological function validation. The results would provide an important theoretical foundation for genetic improvement of salt tolerance and germplasm enhancement in radish.

萝卜是起源于我国的重要根菜类蔬菜，土壤次生盐渍化严重制约了萝卜肉质根的产量和品质。提高萝卜品种的耐盐性、选育高耐盐的品种已成为当前蔬菜遗传改良领域的重要课题。本项目在成功进行萝卜耐盐性基因型筛选的基础上，以耐盐性差异显著的高代自交系为材料，拟利用基于转录组测序的数字基因表达谱(DGE)技术分离鉴定萝卜盐胁迫响应相关差异基因；对所测得的转录组和miRNA数据进行关联分析，鉴定萝卜耐盐性关键候选基因，并利用RT-qPCR技术分析其时空表达特征；通过基于Gateway体系的过表达技术和能高效引起体内靶基因沉默的人工microRNA(amiRNA)技术，验证耐盐性关键基因的生物学功能。本项目从转录水平、miRNA介导的转录后调控及基因功能验证等三个方面解析萝卜盐胁迫响应调控网络与耐盐性分子机制，研究结果将为萝卜耐盐性状遗传改良及种质创新提供重要理论基础。

萝卜是原产于我国的一种十字花科根菜类蔬菜，耐贮运且营养药用价值高，在世界范围内广泛种植。盐胁迫是作物生产中重要非生物胁迫之一，严重影响作物品质与产量。肉质直根为萝卜主要食用器官，不同萝卜基因型对盐害的敏感度不同，如何提高萝卜品种耐盐性、选育优质、高耐盐品种成为当前蔬菜遗传改良领域重要研究课题。然而，前人对萝卜盐胁迫响应的研究主要集中在结构形态和生理生化方面，萝卜盐胁迫响应关键基因分离鉴定及其调控网络解析相关工作尚未开展，导致萝卜盐胁迫响应分子机制仍未阐明。.本项目在进行萝卜耐盐性基因型筛选基础上，以耐盐性差异显著的萝卜高代自交系为材料，利用基于转录组测序的数字基因表达谱(DGE)技术分离鉴定不同盐胁迫处理前后差异表达基因，功能注释发现差异基因主要参与胁迫信号感知和转导、渗透调节、离子平衡和活性氧清除等方面。利用定量蛋白组学iTRAQ技术分理出肉质根不同盐胁迫下差异响应功能蛋白，基于蛋白组、转录组、miRNA多组学关联分析，初步提出萝卜肉质根盐胁迫响应分子调控网络；分离出关键侯选基因RsAPX1、RsSOS2、RsNHX1和RsNHX2等cDNA序列，利用qRT-PCR技术解析了其时空表达特性。基于萝卜基因组数据库，分离出60个阳离子质子逆向转运蛋白（CPA）家族基因，主要参与植株体内离子平衡与逆境响应调控过程，分析了萝卜盐胁迫下CPA家族基因时空表达特性，明确了CPA家族中Na+/H+转运蛋白NHX类相关基因为萝卜耐盐性关键侯选基因。由于萝卜再生与遗传转化体系困难，通过构建正义/反义表达载体，将关键侯选基因在拟南芥中进行了过表达与抑制表达功能验证，同时利用VIGS技术在萝卜中对部分基因功能进行了初步分析，从转录、转录后调控和基因功能验证水平系统解析萝卜盐胁迫响应调控网络与耐盐性的分子机制，研究结果将为萝卜耐盐性状遗传改良及种质创新提供重要理论基础。.项目实施期间，在Plant Cell Reports、Front Plant Sci、Sci Hort期刊发表SCI论文3篇，获教育部科技进步二等奖1项；通过项目实施，共培养博士研究生1人，硕士研究生2人。