两极型小麦光温敏不育系337S短日低温败育的表达谱及育性相关的小RNA

The study of wheat photoperiod-thermo sensitive male sterile line is one of the important part of research about heterosis utilization, to explore the sterile mechanism of photoperiod-thermo sensitive male sterility and the regulation of sterile gene has become an extremely important issue for its reasonable utilization. 337S is the male sterile wheat line sensitive to both long day length/high temperature condition and short day length/low temperature condition. It has its own special features of the male sterile stability and the wide seed production areas. In order to explore the sterility mechanism of the line, this study will take anthers of male sterile line 337S and male fertile line 337F in pollen mother cell formation stage at short-day-length/low temperature condition, as material, sepereated and identified differentially expressed genes and miRNAs between both two lines using the Illumina technique. It will be made clear of coordinating function and regulated relationship between differentially expressed genes and miRNAs by further analysis the data. Whereafter integrating the differences between two lines in metabolism, the molecular speciality of sterility in 337S line could be understanded preliminarily. In addition, in order to identify the functions of differentially expressed genes in anther development stage, the Virus-Virus Induced Gene Silencing approach will be used for screening and cloning candidate genes. Then the key genes related to fertility can be obtain in 337S line. Combined with front research, we will elucidate the regulation and metabolism pathways for 337S line in fertility conversion stage of anther development, and then reveal the abortive molecular mechanism of 337S, the thermo-sensitive male sterile line, in short day length/low temperature condition.

光温敏雄性不育是小麦杂种优势利用的重要途径之一，研究其光温敏雄性不育机制及不育基因的调控方式对其的合理利用有重要意义。337S是对长日高温，短日低温均敏感的两极光温敏小麦雄性不育系，具有不育性稳定，制种区域广的特点。本申请项目拟以337S光温敏雄性不育系及可育材料337F在短日低温条件下花粉母细胞形成期的花药为材料，采用Illumina技术对其花药进行基因表达谱和小RNA分析，分离其差异表达基因和miRNAs，明确它们之间的调控关系或协调作用，并结合代谢层次的差异，明确337S的短日低温不育的分子特点。同时运用病毒诱导的基因沉默技术构建起小麦花药发育差异基因的高效克隆和筛选技术体系，获取337S育性调控的关键基因，以揭示其花药发育过程中育性转换的调控或代谢通路，解析337S在短日低温不育的分子机理。

利用植物的杂种优势是提高农作物产量的重要方式，光温敏雄性不育是杂种优势利用两系法中的关键。本项目通过细胞学鉴定与小RNA测序分析，筛选到几个与小麦光温敏不育系337S败育相关的候选miRNAs，并揭示它们在光温敏不育系育性转换过程中发挥的作用，进而为两系法小麦杂种优势利用的研究提供可靠的理论依据与基因资源。本项目主要研究内容:1.对337S不育系花药发育各时期的细胞学特征进行了详细的观察与研究，鉴定337S不育系在短日低温条件下发生败育的关键时期；2.构建小麦337S不育系正常可育条件和不育条件下关键时期的miRNA表达谱，筛选并获取337S不育系败育相关的特异miRNAs；3.通过qRT-PCR技术验证表达谱以及分析重要候选基因和候选miRNA的可靠性及表达特征。4.进行降解组测序，以获取miRNAs可靠的靶基因；5.对候选miRNA和靶基因进行克隆、进化、组织定位与遗传转化等分析，解析337S在短日低温环境下的败育机制。重要研究结果：1.本研究已对337S不育系败育的细胞学特征进行了详细的观察与研究，结果表明减数分裂时期是小孢子败育的高峰期；2.对小麦337S在短日低温不育和正常可育下的花药进行小RNA测序和降解组测序，初步筛选到在几个可能与337S花药败育有关的miRNAs；3.选择其中的miR1127b，miR2275和miR9652作为3个候选基因在不同小麦品种中进行了遗传转化，发现超量表达miR2275后转基因株系出现结实率下降的现象；4.对miR2275的靶基因CAF1的启动子进行克隆与遗传转化，GUS染色结果表明CAF1基因的表达部位为花药中；亚细胞定位分析的结果发现CAF1基因定位于细胞核中；CAF1蛋白功能的鉴定工作正在进行之中。5.对337S小麦光温敏雄性不育系中与不育相关的环状RNA进行鉴定，并发现circRNAs与上述研究中的候选miRNAs存在调控关系，初步形成了337S败育过程中的小RNA调控网络。总之本项目不仅获得几个与保障减数分裂正常有序运转有关的候选miRNAs，还在植物中初步建成了miR2275与其靶基因CAF1在花药发育过程中的调控通路。本项目为进一步发掘337S育性转换的“开关”，以及利用此“开关”对光温敏不育进行智能化控制的研究奠定了基础，对两系法改良有着重要的科学意义。