水稻温敏核不育系育性转换调控基因的克隆和功能分析

At present,most of the two-line sterile lines used in production are the thermo-sensitive genic male sterile lines whose genic male sterile gene is allele with tms5. Critical sterile temperature is an important indicator whether this kind of male sterile lines can be used in the field production or not. If we can make clear the molecular regulation mechanism of the critical sterility temperature, it is of great application value to breeding and scientific utilization of thermo-sensitive genic male sterile lines, so as to reduce the propagation and hybrid seed production risk of these thermo-sensitive genic male sterile lines. Although tms5 has been cloned at present, the molecular mechanism of the critical sterility temperature is not yet clear. The sterility-to-fertility conversion regulation gene（B）and the critical sterile temperature regulation gene（C）have been found in the thermo-sensitive genic male sterile lines ZHS and Y58S respectively by the applicant of this project through genetic analysis of the lowermost critical sterile temperature characteristics of ZHS which was selected from the offspring of the hybrid between the thermo-sensitive genic male sterile lines C815S and Xiangling628S. Preliminary studies show that these two pairs of genes regulate the critical sterile temperature of the thermo-sensitive genic male sterile line under lower temperature conditions. At present, the sterility-to-fertility conversion regulation gene has been finely mapped and the only candidate gene has been also predicted. On this basis, this project is intended to carry out the following research on the only candidate gene of the sterility-to-fertility conversion regulation gene:(1)functional complementary verification;(2)gene expression pattern;(3)molecular action pathway;(4)the biochemical function of the gene product. These findings will help to reveal how the sterility-to-fertility conversion regulation gene regulates the sterility-to-fertility conversion and the critical sterile temperature of the thermo-sensitive genic male sterile line under lower temperature conditions.

目前生产上利用的绝大部分两系不育系均是不育基因与tms5等位的温敏核不育系，临界不育温度是这类不育系是否具有生产利用价值的重要指标。如能明确临界不育温度分子调控机制，这对不育系的选育和科学利用，降低其在生产上所面临的繁殖和制种风险具有十分重要的应用价值。目前tms5已克隆，但临界不育温度分子调控机制尚不明确。申请者利用C815S/湘陵628S后代分离出的具有极低临界不育温度不育株ZHS为材料，通过对其低临界不育温度特性遗传分析，在ZHS中发现了育性转换调控基因（B），在Y58S中发现了临界不育温度关键调控基因（C），低温下这两对基因共同作用调控了温敏核不育系临界不育温度。目前B已精细定位并遴选出了唯一候选基因。本项目拟对该候选基因进行如下研究：1）功能互不验证；2）基因表达模式；3）分子作用通路；4）基因产物生化功能。该结果将有助于揭示该基因低温下调控温敏核不育系临界不育温度的分子机制。

目前生产上大面积利用的两系不育系绝大部分是核不育基因为tms5的温敏核不育系，临界不育温度是这类温敏核不育系是否具有生产利用价值的关键指标。对临界不育温度分子调控机制开展研究，有助于安全性温敏核不育系的选育和科学利用，这对促进两系杂交稻的进一步发展，保障我国粮食安全具有重要意义。.ZHS是在C815S/湘陵628S杂交后代中发现的具有极低临界不育温度的不育株。通过对ZHS极低临界不育温度遗传分析，在ZHS中发现了温敏核不育系育性转换调控基因（B），而在临界不育温度较高的Y58S中发现了临界不育温度关键调控基因（C）。低温条件下，这两对基因相互作用调控了温敏核不育系的临界不育温度。.在此基础上，以ZHS、深08S为亲本，通过构建ZHS//深08S/深08SBC1F1分离群体，采用全基因组重测序技术将B定位在12号染色体上，并筛选出候选基因OsR498G1221912100.01。构建该基因过表达载体，采用农杆菌介导法导入深08S，低温条件下对转基因深08S的育性进行了观察，结果发现转基因深08S育性表现与深08S一致，表明该基因并非温敏核不育系育性转换调控基因。导致候选基因筛选失败，是由于定位群体单株表型鉴定采用稻篼移植法，最终导致少量单株表型鉴定有误所致。.于是再次构建ZHS//深08S/深08SBC1F1分离群体，将种子冬季播种在海南三亚，育性敏感期安排在低温条件下，依据实生苗低温条件下的育性表现对定位群体单株表型进行鉴定，采用全基因组重测序技术将B基因仍然定位于12号染色体上，但目标区域与第一次定位结果有差异，利用特异引物进一步将该基因精细定位在RM28404—Chr12MM2043之间，并筛选出候选基因OsR498G1221881400.01。目前已构建该基因过表达载体，后续将对该基因的功能进行互补验证和分子调控机制研究。该项目为临界不育温度分子调控机制研究提供了新的思路。