TaPCNA调控小麦生理型雄性不育小孢子异常分裂的分子机理

In view of most crops male sterility,such as genetical and physiological male sterility,the microspore abortion have occurred from uninucleate to binucleate stage and no longer continual to divide, resulting in male infertility.In this study,the independent intellectual property gametocide SQ-1 is used as male sterility inducer. first of all, the male sterile and fertile physiological lines of same genotype will be constructed,Second, the cell cycle protein-TaPCNA obtained from previous research of this item applicant is used as targets,and the details of this scientific research is as follows:firstly,the inner relationships of TaPCNA and microspore nuclear division are determined using in situ hybridization and DAPI dyeing.Secondly,the specific cell cycle of pollen abortion induced by gametocide SQ-1 is analyzed via flow cytometry,and the key factors of regulating cell cycle are separated by co-immunoprecipitation.Finally,the expression pattern of cell cycle regulatory factors are detected by Western Blotting.The aims of these studies are to obtain the key factor of regulating cell cycle,aberrant sites,and molecular mechanism,which is of momentous scientific value and common practical significance in the aspect of elucidating the essential mechanism of pollen abortion in most crops directly from the results of male sterility ,and even exploiting newfashioned male gametocide or hybrid agent to utilize heterosis in wheat.

鉴于多数作物雄性不育，遗传型或生理型，其败育均发生在小孢子单核或二核期不再继续分裂，表现异常，进而导致不育。本研究以自主知识产权新型杀雄剂SQ-1为雄性不育诱导剂，首先，定向建拓出完全对等不育与可育等生理系；其次，以申请者前期研究小麦生理型不育所获细胞周期蛋白-TaPCNA为靶点，拟开展：（1）采用原位杂交和DAPI染色以确定TaPCNA与小孢子核分裂的内在关系；（2）利用流式细胞术以分析杀雄剂诱导后的花粉败育的具体细胞周期，并结合免疫共沉淀以分离调控细胞周期的关键因子；最终，（3）再以Westernblotting检测获得的细胞周期调控因子的表达模式。通过这些研究，旨在获得小麦生理型雄性不育小孢子败育的关键细胞周期调节因子，及败育起始位点与分子机理，这对多数作物直接从败育的结果最终探明败育的本质机理，甚或在开发新型杀雄剂以创制多途径利用作物杂种优势，具有重要科学价值和普遍的实践指导意义。

为研究TaPCNA调控小麦生理型雄性不育小孢子异常分裂的分子机理:利用RT-PCR克隆了小麦PCNA基因的cDNA，命名为TaPCNA。测序显示，小麦TaPCNA基因编码区为792 bp，编码263个氨基酸，分子量约为29.16kD，理论等电点为4.53。实时荧光定量PCR，TaPCNA在生理型不育系中表达量升高，而细胞周期G1/S期转换标志基因Histone H4基因在生理型不育系中表达下降。将得到的小麦TaPCNA基因片段连接到原核表达载体pET-32a，构建重组表达载体，转化大肠杆菌感受态细胞，用IPTG 进行诱导表达。经SDS-PAGE 分析，诱导的重组菌株与未经诱导的对照组相比，在相对分子量约50kD附近有明显的蛋白质表达条带。采用hiTAIL-PCR技术，获得TaPCNA基因起始密码子上游890bp的启动子序列。PlantCARE 启动子在线分析软件预测含有光应答调控元件（Box I）、脱落酸应答元件（ABRE）、花粉发育应答元件（GGTT motif，GTGA motif）及细胞周期转换结合位点（E2F-binding site）等。然后，用TaPCNA基因启动子序列替换植物表达载体pBI121 GUS基因前的CaMV35S启动子，并构建启动子融合表达载体，通过农杆菌EHA105介导转化烟草叶盘进行了瞬时表达分析，GUS作为报告基因研究在SQ-1胁迫下启动子活性。结果，SQ-1对启动子有明显的诱导效应。. 此外，还构建了酵母诱饵表达载体pGBK-TaPCNA，其在酵母细胞中没有毒性和自激活性。利用酵母双杂交系统筛选出6个与TaPCNA互作蛋白，其主要参与细胞周期调控以及DNA损伤修复。对TaPCNA互作蛋白基因的定量表达分析，DNA损伤修复基因TaKu70、TaXRCC1和TaFen-1在生理型雄性不育系中表达上调，而细胞周期调控基因TaICK1和TaCycD2的表达在生理型雄性不育系中下降。同时，采用双分子荧光互补技术在洋葱细胞中进一步验证TaPCNA与TaICK1之间的相互作用，结果在洋葱细胞的核内，TaPCNA相连的nYFP可与TaICK1相连的cYFP形成互补而产生荧光，证实它们之间在植物体内叶具有相互作用。. 综上结果，为揭示TaPCNA调控小麦不育小孢子异常分裂的机理奠定了理论基础。