水稻品种N22种子休眠性QTL qSdn-1的候选基因验证与功能分析

Seed dormancy in rice, an important agronomic trait related to rice quality and seed quality, is a quantitative trait controlled by the major gene and multiple genes, and is affected by environmental conditions. To date, the molecular mechanism regulating this trait is not yet understood. In the preliminary study, the applicant had already constructed near-isogenic line carrying the major seed dormancy QTL qSdn-1 (Nanjing 35 as receptor and N22 as donor). Then a secondary segregation population built by near-isogenic line is utilized for high-resolution mapping and the qSdn-1 was localized to a 29.8 kb region containing three intact open reading fragments.In this study, based on the method of bio-informatics and previous research, qSdn-1 gene will be isolated and proceed for function verification by sequencing, blasting, expression file and yeast two-hybrid assay, and transgene for overexpression and RNAi of candidate genes. The relationship between qSdn-1 and known seed dormancy genes such as OsDOG1、OsSdr4、OsVP1,and traits related to physiological processes of seed dormancy and germination will be analyzed using near-isogenic lines, transgenic plants and mutuants. These results will help us to understand the molecular mechanism of qSdn-1 in rice seed dormancy and preharvest sprouting resistance, as well as the metabolic network controlled by qSdn-1, and to promote the breeding program of PHS resistance by molecular assitant seletion in rice.

水稻种子休眠性是关系到稻米品质和稻种质量的重要农艺性状，是受主基因+多基因控制的数量性状，且受环境条件的影响，迄今对调控该性状的分子机制尚不清楚。在前期研究中，已构建了以南粳35为受体、N22为供体的，携带qSdn-1的近等基因系，利用近等基因系构建的次级分离群体，对qSdn-1进行了精细定位，将其限制在29.8kb范围内，含3个完整的ORF。本研究拟在此基础上，结合生物信息学的方法，分离克隆qSdn-1基因。对此区间进行测序，通过序列分析、同源性比较、表达谱分析、酵母双杂以及转基因研究进行功能验证，结合前人已克隆的水稻种子休眠性相关基因（（包括OsDOG1、OsSdr4、OsVP1等）），利用近等基因系、转基因材料及突变体材料，分析qSdn-1与其他控制休眠性基因间的关系，阐明其在水稻种子休眠和萌发控制途径中的作用机制，同时获得紧密连锁或共分离分子标记为水稻适宜休眠性的分子育种提供基础。

水水稻种子休眠性是关系到稻米品质和稻种质量的重要农艺性状，是受主基因+多基因控制的数量性状，且受环境条件的影响，迄今对调控该性状的分子机制尚不清楚。在前期研究中，已构建了以南粳35 为受体、N22 为供体的，携带qSdn-1 的近等基因系，利用近等基因系构建的次级分离群体，对qSdn-1 进行了精细定位，将其限制在29.8kb 范围内，含3 个完整的ORF。在前期研究基础上，本研究进一步扩大群体进行精细定位，结合生物信息学方法和序列分析，分离克隆了qSdn-1基因，该基因编码一个磷酸烯醇式丙酮酸羧化酶（PEPCase）基因，通过连续多年多世代多种构建的转基因验证、基因亚细胞定位、基因表达谱分析、代谢组分析、基因蛋白表达及酶活测定，验证了基因的功能，利用近等基因系、转基因材料及突变体材料，分析qSdn-1与其他控制休眠性基因间的关系，表明其是通过对ABA的响应不同，影响种子代谢，进而影响种子的休眠。同时，对N22的两个弱休眠的突变体进行了转录组分析，分析了初步阐明了两个突变体休眠性减弱的分子机制。获得的近等基因系、转基因家系以及紧密连锁或共分离的分子标记，将为水稻适宜休眠性的分子育种提供基础。