水稻反光敏核不育基因rpms3-1的图位克隆与功能初步分析

Recently, the mostly used rice two-line male sterile lines were rice thermo-sensitive genic male sterile lines. The research and application of rice reverse photo-sensitive genic male sterile line was very few. In this study, the rice reverse photo-sensitive genic male sterile line D14S (temporary named) which was discovered in the process of selection of rice two-line male sterile lines was used as research material. D14S was fertile under the condition of long-day, but male sterile under short-day. Our research results showed that the sterile characteristic of D14S was controlled by single recessive nuclear gene. By now, the reverse photo-sensitive genic male sterile gene rpms3-1(temporary) was fine mapped on the long arm of chromosome 10 within an 18.5Kb interval. Bioinformatics analysis showed that there were 4 ORFs in the fine mapping interval. Gene expression analysis indicated that the expression of the first ORF（LOC_Os10g22080） under short-day condition was significant lower than that in long-day. Sequencing results indicated that there was a substitution of thymine to cytosine in the 608 base of CDS of LOC_Os10g22080, which caused the amino acid that encoded was proline not leucine. Therefore the first ORF（LOC_Os10g22080）was considered as the target gene of rpms3-1. Further the complementary, excessive expression and interference expression vectors will be built to complete the function complementary verification experiment. In the meaning time, the researches of pollen sterile cytology and gene function analysis will be conducted preliminarily. The research results will preliminarily clarify the sterile mechanism of rice reverse photo-sensitive genic male sterile line D14S, which will provide theoretical support to enrich and improve sterile mechanism of rice two-line male sterile lines.

目前我国水稻两系不育系以温敏核不育系为主，水稻反光敏核不育系的研究较少。本项目组在水稻两系不育系选育过程中发现一个水稻反光敏核不育系D14S（暂命名），该不育系在长日照与短日照条件下分别表现正常可育与花粉败育，其不育受1对主效隐性核基因控制；其反光敏核不育基因rpms3-1(暂定)被精细定位于第10染色体长臂18.5Kb的区域内，在该区域内共有4个ORF；其中第1个ORF（LOC_Os10g22080）在短日照条件下表达显著下调。测序分析显示LOC_Os10g22080的CDS的第608位处发生T - C的碱基替换，导致其编码的氨基酸由亮氨酸突变成脯氨酸。下一步将构建LOC_Os10g22080的互补，过量表达以及干扰载体完成功能互补验证。同时对D14S花粉败育细胞学以及基因功能开展初步研究。研究结果将初步阐明水稻D14S反光敏核不育机理，为丰富和完善水稻两系不育系不育机理提供理论支撑。

D14S是当前我国发现种类较少水稻反光敏核不育系材料。本研究以D14S为材料，对其花粉败育的细胞学、不育基因的精细定位与克隆进行了研究。花粉败育细胞学观察表明，D14S花粉败育起始于小孢子早期，一直持续至花粉成熟期，由于绒毡层细胞解体迟缓，不能提供其发育所需的营养物质而导致花粉败育。D14S花粉败育花药壁亚显微结构观察进一步证实了这一研究结果。.RPMS3-1不育基因精细定位结果表明，D14S反光敏核不育基因位于第10染色体短臂上，定位于标记RM6388与In-Del标记D37之间的18.5Kb的区域内。生物信息学分析表明，在该18.5Kb的区域内存在4个候选基因（ORF），根据各候选基因在D14S可育和不育条件下的表达以及各候选基因测序，最终第1个候选基因（LOC_Os10g22080）被认为最有可能是D14S不育目标基因。.对目标基因（LOC_Os10g22080）在D14S不同的日照长短和温度处理条件下基因表达分析表明，LOC_Os10g22080 的表达量在D14S可育时期和不育时期差异极显著，因此，将LOC_Os10g22080 确定为目标基因。Rpms3-1 Crisp/Cas9 基因敲除转基因T0植株夏季杭州表现花粉败育，Rpms3-1互补载体转基因T0植株与Crisp/Cas9 基因敲除转基因T1植株，均种植于海南，之后需对转基因后代进行育性追踪观测，确证目的基因，完成转基因功能互补验证。.利用与Rpms3-1紧密连锁的分子标记RM6388与In-Del标记D37，在D14S/泰丰A，D14S/隆638S 杂交组合后代进行分子标记辅助选择，最终在BC4F2群体中筛选出综合性状优良的水稻反光敏核不育系材料40余份，并对这40余份不育系材料进行配合力和杂种优势筛选，有望筛选出综合性状优良，配合力强的新的水稻反光敏核不育系。