棉花线粒体基因GhorfA调控细胞质雄性不育的分子机制

Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self-pollination, enabling hybrid crop breeding to increase yield. In the past decades, several kinds of cotton CMS lines, such as CMS-D2 (G. Harknessii), CMS-D8 (G. trilobum), 104-7A, JinA, XiangyuanA, were selected and exploited in the majority of “three-line” hybrid cotton production. However, the molecular mechanisms for generating the CMS remain to elucidate, and genes applicable for improving cotton CMS are very limited. P30A, a new type of G. hirsutum CMS, was discovered in the 2000s by our laboratory. Hybrid cotton bred by the three-line (CMS, maintainer and restorer) technology, which use CMS P30A lines, has strong heterosis that increases fiber yield about 20%. Genetic analysis indicated that the CMS in P30A might control by a mitochondrial locus. To identify the gene(s) responsible for CMS, we sequenced the mitochondrial genome of P30A and maintainer, P30B. Comparative analysis of P30A and P30B mitochondrial genome found a new mutant orf rearranged in P30A, and we designated this locus as GhorfA. Real time-PCR and northern blot analysis indicated that GhorfA specifically expressed in P30A but not in P30B. Significantly, the growth rate of E.coli harboring GhorfA gene was significantly inhibited compared with control. Taken together, we reasonably speculate that GhorfA might responsible for CMS in P30A. In this proposed study, we would like to investigate the mechanism of GhorfA involved in cotton CMS by using genetics, molecular biology, and biochemistry means, and explore its possible practical implication for future hybrid breeding in cotton.

细胞质雄性不育系是“三系”杂交育种的基础材料，不育系、保持系和恢复系“三系”配套的广泛应用，极大地提高了杂交棉制种效率、有效降低了生产成本。目前，国内外已育成多套应用于棉花育种的胞质不育系，然而对细胞质雄性不育调控机制的认识却非常的缺乏。P30A、P30B和Y18R是申请人实验室和合作单位联合研制的抗虫棉“三系”体系，利用该体系已成功培育多个棉花新品种。近10年来，申请人利用线粒体全基因组测序、生物信息学、RFLP、荧光定量PCR等生化和遗传手段初步证明：P30A线粒体基因组自然变异产生的新转录本GhorfA可能是控制其不育的原因，这一推测在大肠杆菌中已得到初步验证。本项目拟在此基础上，开展以下几个方面的研究：1）实验验证GhorfA是调控P30A棉花细胞质雄性不育的基因；2）阐明P30A细胞质雄性不育的分子机制以及和恢复基因Rf1核质互作机理；3）新型高产优质多抗的不育系材料的培育。

细胞质雄性不育系是“三系”杂交育种的基础材料，不育系、保持系和恢复系“三系”配套的广泛应用，极大地提高了杂交棉制种效率、有效降低了生产成本。目前，国内外已育成多套应用于棉花育种的胞质不育系，然而对细胞质雄性不育调控机制的认识却非常的缺乏。本研究利用实验室育成的P30A、P30B和Y18R三系亲本为材料，利用线粒体基因组测序、cRT-PCR、Northern blot、转基因功能验证等方法，对候选不育基因GhorfA开展工作，并取得如下重要进展：（1）组装出全长634,025bp的P30A线粒体基因组，包含39个编码蛋白基因，与网络公开的三裂棉、辣根棉、特纳氏棉等线粒体基因组亲缘关系较近；（2）结合P30A转录组数据，筛选获得230个差异表达ORFs,其中41个ORFs预测其编码蛋白含有跨膜结构域，通过qRT-PCR和northern blot实验最终确定GhorfA为控制棉花CMS性状的候选基因；（3）GhorfA全长1833bp，前1348bp与atpA基因保持一致，后485bp序列未能找到同源序列，属于新形成的嵌合基因； Southern、Northern以及Western Blot证明该基因在不育株系中特异表达，且在花器官中表达量最高；（4）异源表达GhorfA的拟南芥，出现花药不正常开裂、花粉量大幅度减少、花粉“染败”等雄性败育特征；过表达GhorfA的棉花植株，也出现雄性育性下降的性状，初步证明GhorfA的表达干扰了花粉的正常发育；（5）进一步分析线粒体基因组上GhorfA的上下游序列，发现其5’端相距约100bp的位置，还有一个951bp的转录本orf316，其表达模式与GhorfA相同，两者存在共转录现象；（6）原核细胞毒性实验表明，orf316和GhorfA都具有一定的细胞毒性，且两者共表达时毒性最强；（7）酵母双杂实验结果表明，ORF316和GhORFA之间直接互作；而且经过筛选，发现两者均可以与GhCOX11直接互作；（8）活性氧状态检测结果表明，P30A在幼蕾发育的小孢子母细胞时期，可以明确地检测到ROS信号，并同时检测到细胞凋亡 。以上实验结果初步明确了棉花CMS性状形成的分子机理：不育基因orf316和GhorfA在花器官中的上调表达，表达产物与GhCOX11互作，诱发了细胞中的活性氧大爆发，并触发绒毡层细胞过早死亡，从而导致花粉败育。