萝卜镉吸收累积性状关键基因鉴定与功能分析

To deeply elucidate the molecular genetic mechanism of cadmium (Cd) uptake and accumulation in radish, basing on the investigation of inheritance and QTL mapping of Cd uptake and accumulation, this study would be conducted to construct a secondary mapping population with CSSLs (Chromosome Segment Substitution Lines) and high-resolution mapping of the major QTLs for Cd uptake and accumulation Moreover, through selecting the BAC (Bacterial Artificial Chromosome) library with the tightly-linked markers to the target QTLs,the cloning contigs containing the target regions of the major QTLs would be constructed. Using radish advanced inbred lines which show significant differences in Cd accumulation, two dimensional electrophoresis (2-DE) with mass spectrometry (MS) and iTRAQ would be employed to isolate and identify the specific responsive proteins involving in Cd uptake and accumulation. Meanwhile, the full length cDNA of specific identified genes would be obtained with RACE (Rapid amplification of cDNA ends) or cDNA library screening. Solexa high-throughput sequencing technique would be adopted to isolate a set of miRNAs in response to Cd stresses, and related miRNA target genes would be validated by RLM-5'RACE and high-throughput degradome sequencing. Furthermore, the sense/anti-sense expression vectors are to be constructed for genetic transformation to validate and analyze the biological and molecular functions of specific Cd-responsive genes and miRNAs. These results could provide new insights into elucidating the molecular genetic characters of Cd uptake and accumulation, and provide a powerful theoretical basis for genetic improvement and germplasm enhancement of low-Cd content radish cultivars and genetic management of heavy metals in plants.

在对萝卜Cd吸收累积性状进行遗传分析与QTLs标记定位基础上，本项目利用染色体片段代换系构建次级作图群体对该性状主效QTLs进行精细定位，并利用紧密连锁标记筛选BAC文库，构建覆盖主效QTLs目标区域的克隆重叠群；以Cd吸收累积差异显著的萝卜高代自交系为材料，利用2-DE与iTRAQ技术研究Cd胁迫处理前后肉质根蛋白差异，采用生物质谱进行特异表达蛋白鉴定，对重要蛋白根据氨基酸信息分离cDNA片段，采用RACE或筛选cDNA文库分离克隆Cd胁迫处理下重要响应基因；利用高通量测序技术分离鉴定萝卜Cd胁迫响应miRNAs，应用RLM-5'RACE或降解组测序验证相关miRNAs靶基因；构建正义/反义表达载体，通过遗传转化进行Cd胁迫响应关键基因与miRNAs功能验证分析，以阐明Cd吸收累积性状的分子特征，为萝卜低Cd含量遗传改良与种质创新以及植物重金属有效遗传控制提供理论依据。

镉(Cd)是生物毒性极强的重金属之一。选育与推广优良低吸收累积品种是解决蔬菜生产上重金属污染问题的最经济有效途径之一。萝卜为原产我国的以膨大肉质直根为产品器官的重要根菜类蔬菜。萝卜Cd胁迫下重要差异表达的功能基因与起调控作用的miRNAs分离鉴定工作未见报道， Cd吸收累积与胁迫响应调控机制尚未阐明，极大限制了从分子遗传学角度进行蔬菜作物Cd吸收累积特性的遗传控制。.本项目在萝卜Cd吸收累积性状QTLs标记分析定位基础上，结合基因组序列信息，运用Cd吸收累积性状紧密连锁标记进行遗传基因组学分析，获得覆盖主效QTLs目标区域的候选多个功能基因；利用高通量测序技术分离鉴定出萝卜Cd胁迫响应miRNAs，应用降解组测序验证相关miRNAs靶基因；利用RNA-Seq进行了萝卜肉质根Cd胁迫响应基因表达谱分析，分离到Cd胁迫响应差异表达基因；利用2-DE与iTRAQ技术研究了Cd胁迫处理前后肉质根蛋白差异，采用生物质谱进行特异表达蛋白鉴定，对重要蛋白根据氨基酸信息分离出cDNA片段，分离克隆了Cd胁迫处理下重要响应基因。构建了功能基因正义/反义表达载体，在模式植物烟草中通过遗传转化初步进行了Cd吸收累积与胁迫响应关键基因功能分析。基于遗传与生物组学分析，构建了萝卜肉质根Cd吸收累积与胁迫响应的遗传调控网络，初步阐明了Cd吸收累积与胁迫响应的分子特征，研究结果为萝卜肉质根低Cd含量遗传改良与种质创新以及植物重金属有效遗传控制提供了理论依据与技术支撑。.项目实施期间，公开发表论文10篇，其中SCI论文9篇；获教育部科技进步二等奖1项，创制Cd低吸收萝卜新种质1个；项目组成员入选科技部中青年科技创新领军人才（2017），入选国家“万人计划”科技创新领军人才（2017.12公示），江苏省优秀博士学位论文指导教师（2014）；通过项目实施，共培养博士研究生3人，硕士研究生3人。