番茄核雄性不育基因ms-10的功能研究及调控分析

Nowadays, the hybrid seeds of tomato are widely popularized and applied in production, which mainly produced by the artificial emasculation. Due to the overly complicated process of male sterile lines trans-breeding, the highest cost of seed production has been become one of the main factors that limited the heterosis utilization of tomato. Previously, we have obtained the candidate genes by fine mapping in ms-10, which is a mutant line of nuclear male sterile. Then the male sterile gene of ms-10 was cloned and validated by the functional complementation to use the virus-induced gene silencing. Based on these critical progress, compared protein group technology will be carried to investigate the differences expression profile of protein among the Ms-10, ms-10 and others genes that all related to male sterile. Moreover, the downstream candidate genes which are regulated by bHLH transcription factor Ms-10 will be screened by ATAC-Seq and ChIP-Seq. In addition, the specific recognition sites of Ms-10 and protein-protein interactions among Ms-10 and others genes will be investigated, which respectively by the yeast hybrid system and bimolecular fluorescence complementation. Finally, comprehensive analysis the above results, the metabolic model of all the male fertility-related genes which were regulated by Ms-10 will be established. The results of this project will not only help us to understand the molecular mechanism of the male sterile strain of ms-10 in biochemical processing, such as organ development, signal transduction and expression regulation, but also provide the theory for the male sterile further application in tomato.

由于目前番茄雄性不育系转育程序繁复，生产上大面积推广的杂交种，仍然以人工去雄为主，制种成本过高已成为番茄杂种优势利用的主要限制因素之一；我们前期利用一个核雄性不育突变体材料ms-10，通过精细定位获得了该基因的候选基因；并利用病毒诱导的基因沉默技术进行功能互补试验，完成了该雄性不育基因的克隆和验证。本项目拟在此研究基础上，通过比较蛋白组学检测Ms-10、ms-10基因及其育性相关基因在蛋白水平的差异表达；并运用ATAC-Seq和ChIP-Seq技术筛选bHLH类转录因子Ms-10所调控的下游候选基因；再利用酵母单杂交系统与双分子荧光互补技术，分析Ms-10基因的特异识别位点及其与育性相关基因间的互作关系；最后通过综合分析，建立Ms-10基因所调控的育性相关基因的代谢模型。从器官发育、信号传导及表达调控等生化进程，阐明ms-10系不育的分子机制，为番茄雄性不育的进一步应用提供理论依据。

番茄是自花授粉作物，杂种优势十分明显，杂交种比普通品种增产20%-30%以上，而且整齐度高，抗逆性强。截止目前，已报道的番茄自然雄性不育突变体有50多个，其中大部分属于核雄性不育。迄今为止，由于育种和应用上的缺陷，番茄遗传雄性不育的发生机制尚不清晰。. 本研究利用一个由ms-10基因导致的核雄性不育系2-517和它的野生型可育系VF-11。首先，利用iTRAQ定量技术，鉴定和分析可育系与不育系之间的差异表达蛋白；通过鉴定分析，一共鉴定了8272个在蕾期和花期差异表达的蛋白，此鉴定结果通过蛋白平行反应监测（PRM）、荧光定量PCR（qRT-PCR）和各类生理指标实验进行了验证；进一步分析发现这些蛋白主要参与C16和C18脂肪酸的ω-碳羟基化、三羧酸循环等代谢途径；也同时利用生物信息学将核雄性不育基因蛋白ms-10与其他蛋白的互作进行了分析。其次，由于已证明ms-10的等位基因Ms-10是bHLH类转录因子，所以利用染色质免疫共沉淀（ChIP-Seq）检测与Ms-10蛋白抗体结合的DNA序列，解析Ms-10调控的下游基因；实验检测到1124个和1257个序列含有GAG/AACCT/ACAT/GCC或其反向互补序列，再通过DNA转座酶研究开放染色质的可进入性的高通量测序技术（ATAC-seq）进行关联分析，获得4个候选调控区，初步判断这4个区域为转录因子MS-10的下游调控区域。最后，通过酵母单杂获得了76阳性克隆，从76个阳性克隆中筛选到了2个转录因子，初步推断这2个转录因子是MS-10的上游调控基因。. 基于以上研究，本研究推断不育基因ms-10中绒毡层的PCD推迟，致使脂类物质的代谢紊乱，使得花粉的正常发育受到了严重影响，从而导致花粉完全不育。同时，根据孢粉素和角质层合成突变体的细胞学研究和生化分析，本研究构建了一个关于花粉发育过程中花粉外壁和花药角质层生物合成的模型。