一份矮败玉米突变体核雄性不育基因的克隆及功能分析

Male sterility is very important for plant heterosis application.There are many advantage when using male sterile line as female parent for hybrid seed production，such as labour-saving, cost-saving and ensuring the hybrid seeds identity. So fine mapping and cloning of the male sterile gene which derived from spaceflight in maize is necessary and important for the continuous improvement of maize yield in the future in China. In our previous study, we have mapped the male sterile gene on the long arm of chromosome 3 by SSR technology with the F2 segregated population, with the aid of developing new molecular markers, we have narrowed the distance of two linked markers to 362Kb. Based on these results, the following works will be conducted: Fine mapping the male sterile gene with large back-cross population through constantly designing new molecular markers within the target region. Based on the data from maizegdb, predicting the gene models located within the target region, speculating the probable candidate genes. The candidate genes will be identified by the DNA sequence blast and different expression analysis between male sterile and male fertile. The function of the candidate genes will be validated by functional complementation assay and target gene modified with CRISPR-cas9 technology. The expression model of candidate gene, in situ hybridization, subcellular localization and RNA-seq were conducted to reveal the express feature of candidate gene. Furtherly, Yeast two hybrid was also conducted for the selection of interacted proteins with the target candidate gene. The implementation of the project will not only provide a theoretical basis for uncovering the candidate gene and reveal the mechanism of target gene for the male sterility obtained by spaceflight in maize, but also supply the male sterile gene resource for hybrid seed propagating in China and further enrich the research content for the maize reproductive biology.

精细定位和克隆具有自主知识产权的玉米核雄性不育基因，对促进我国不育化制种的发展具有重要意义。课题组以太空诱变玉米核雄性不育为材料，利用F2群体作为基因定位群体，目前已将该核不育基因定位在第3染色体362Kb的区域内。在此基础上，本研究拟开展以下工作：1、建立大规模回交后代分离群体，对核不育基因进行精细定位；预测目标区域内包含的基因并进行功能注释。2、结合基因组DNA水平序列差异和RNA转录水平差异，确定关键候选基因。3、通过候选基因的功能互补以及基因靶向修饰技术，验证候选基因的功能。4、通过表达模式分析、RNA组织原位杂交、亚细胞定位及RNA-seq分析候选基因的表达特征。5、利用酵母双杂筛选目标基因的互作蛋白。该研究结果为明确玉米太空诱变核不育性状的候选基因，探明其调控雄花败育的作用机理提供理论依据；为我国利用核不育进行玉米不育化制种提供基因资源，并丰富了玉米生殖发育生物学的研究内容。

课题组以太空诱变玉米核雄性不育突变体ms39为材料，利用（ms39×Mo17）F2群体作为基因定位群体，成功定位并克隆了关键候选基因Zm00001d043909（ZmCals12 ），通过序列比对发现突变体中的ZmCals12基因在第一外显子缺失了4个碱基，导致蛋白翻译提前终止。利用CRISPR-Cas9技术对玉米自交系KN5585中的ZmCals12基因进行敲除，得到11个稳定编辑株系，等位性测定结果显示11个敲除株系与核不育突变体ms39均等位。11个敲除株系的花粉败育彻底，不育性表现稳定，敲除系的株高相对于野生型KN5585均表现显著降低。这一结果证实了ZmCals12基因突变会导致玉米雄花败育和株高降低。通过组织细胞学观察，发现突变体花药的败育主要发生在减数分裂时期，由于ZmCals12基因编码胼胝质合成酶，对ms39突变体以及敲除系的花药进行苯胺蓝染色，发现四分体时期不育花药的胼胝质含量显著低于可育材料，暗示胼胝质的合成受到影响。电镜观察发现，相对于野生型材料，不育系减数分裂后期的花药内壁缺乏乌氏体结构，且绒毡层PCD提前，致使小孢子不能正常发育。花药转录组数据表明不育系的糖代谢过程异常，结合电镜观察发现在不育花药中存在较多的自噬体，推测糖代谢异常引起的碳饥饿可能是促使自噬现象增强的主要因素。通过构建不同遗传背景下的ms39染色体片段代换系，证实了当ms39基因片段导入到其他自交系后植株依然表现出完全败育和植株高度的降低。同时利用11个玉米自交系分别与ms39姊妹交后代的不育株和可育株配制杂交组合，发现不育基因ms39对杂交一代的杂种优势和主要产量性状无不良影响，可用于玉米不育化制种。本课题组获得的ms39不育突变体，花粉败育彻底，不育性状表现稳定，同时伴随着株高的降低，因此不育基因zmcals12的一因多效赋予了该矮败突变体更多的研究和利用价值。