陆地棉单隐性核不育系1355A恢复基因的克隆与功能验证

The recessive genic male sterile line 1355A of upland cotton is stable sterility and abnormal thicker nexine of pollen during the stage 8, which leads to male sterility. Recently biochemical analysis and preliminary mapping were used to investigate the 1355A male sterile mutant. However, relatively little has been reported on molecular mapping and molecular genetic mechanism of 1355A male sterility. In this study, the bulked segregant analysis (BSA) cloning based on next generation sequencing were performed, and identified a candidate region about ~630kb. To fine map the male sterile locus, new markers will be developed on the basis of the genome sequence. Futher candidate genes were identified by combined with the differentially expressed genes and proteins derived from the RNA-seq and proteomic analysis. To confirm the candidate genes, genetic complementation experiments are performed. Firstly, candidate genes were mutated by using CRISPR/CAS9 system. Secondly, candidate genes were genetically transformed using near-isogenic line Y1355AB, containing abortive genes and capable of genetic transformation. In order to understand the molecular mechanism of 1355A male sterility, the yeast two-hybrid system and other technology of protein interaction are performed to exploit the regulation network of 1355A male sterile gene. It is significant to detect the molecular mechanism of genic male sterility, which will benefit the utilization of genic male sterility system on heterosis of cotton and reduce the cost of the cotton hybrids seed production. At the same time, it is good for understanding the regulation of cotton pollen wall development.

陆地棉单隐性核不育系1355A败育彻底，细胞学观察发现1355A花粉外壁内层异常加厚。对1355A有简单的生化分析及初步定位，但对1355A恢复基因的克隆与功能验证及其分子机理研究仍未见报道。本研究利用BSA方法初步将1355A恢复基因定位在630kb范围内。进一步开发分子标记，在BC1分离群体中对恢复基因进行精细定位，结合前期1355A/B花药的RNA-Seq及蛋白组分析的差异表达基因和蛋白缩小候选基因的范围。利用遗传互补实验验证候选基因功能，一方面通过CRISPR突变候选基因，另一方面以含败育基因且可以进行遗传转化的近等基因群体Y1355AB为受体材料进行候选基因的遗传转化。并通过酵母双杂等蛋白互作技术及体外的生化实验，初步解析候选基因导致败育的分子机理。本研究对促进棉花细胞核不育系在杂种优势上的利用和降低制作杂交种的成本具有重要意义，同时可加深对棉花花粉壁发育调控的认识。

细胞核雄性不育系（GMS）是世界范围内杂种优势利用的重要种质资源。GMS育性恢复系在棉花上的研究报道有限，制约了棉花的遗传改良和育种。在本项目中，我们发现棉花无花粉壁刺突突变体（Ghnsp）的花粉具有花粉壁形成缺陷的特征，该特征具体表现为花粉外壁内层加厚，花粉外壁外层无刺突和缺少花粉内壁层。通过图位克隆，鉴定获得了控制无花粉壁刺突突变体表型的基因GhNSP，并通过CRISPR/Cas9系统创制该候选基因突变体。利用突变体材料完成共分离检测和等位检测，从遗传层面证实Ghir_D02G025140基因为GhNSP突变体育性恢复基因。通过原位杂交分析发现GhNSP基因在绒毡层中高量表达，该基因编码的是多聚半乳糖醛酸酶蛋白，具有多聚半乳糖醛酸酶活性。进一步的生化分析和免疫组织化学研究结果表明，未酯化的同型多聚半乳糖醛酸作为GhNSP的底物在Ghnsp突变体的绒毡层和外壁中过度积累，导致其花粉外壁无刺突和花粉外壁内层加厚，进而表现出败育表型。本研究结果表明，未酯化的同型多聚半乳糖醛酸的适度降解对花粉外壁内层和花粉外壁刺突的形成起着关键作用，最终间接的影响花粉活力，更新了对花粉外壁组成成分的认识，为进一步研究花粉外壁和刺突的形成和功能提供了材料。此外，通过CRISPR/Cas9系统获得的Ghnsp突变体为选育海岛棉雄性不育系提供了新途径，为棉花杂种优势利用提供了潜在的材料。

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