【作者】 赵宝存；

【导师】 黄占景； 沈银柱；

【作者基本信息】 河北师范大学 ， 植物学， 2005， 博士

【摘要】 目前,小麦杂种优势利用并未取得突破性进展。原因之一是小麦是异源六倍体自花授粉作物,具有庞大的基因组,加之长期人工选择的结果,遗传多样性较差,品种间杂种优势不够理想,增产幅度不十分突出;原因之二是生产上应用的T型细胞质雄性不育系保持容易恢复难。所以,培育新型恢复系,加强对小麦新型强优势组合的筛选有非常重要的作用。 本论文就T型恢复系7269—10恢复基因的遗传规律进行分析,并进一步克隆育性相关基因,为推进小麦杂种优势在生产中的应用奠定理论和实验基础。以细胞质雄性不育系75-3369A、恢复系7269-10及75-3369A/7269-10组合的F₂群体和F₃群体为材料,连续三年对F₂的育性分离情况进行了统计,并结合F₃家系的育性分离情况对恢复基因的遗传规律进行了分析。结果表明,恢复系7269-10育性恢复的遗传主要受两对显性恢复基因控制,且非等位恢复基因之间存在加性效应。鉴于F₂的育性恢复即有质量性状的特征,又有数量性状的特征,并且易受环境因素的影响,推测该育性恢复性状还受微效恢复因子的影响。利用杂交、回交和自交育成了关于小麦T型CMS育性恢复基因的拟近等基因系（INIL）和近等基因系（NIL）,可用于恢复基因定位及分离等研究。 以细胞质雄性不育系75-3369A和恢复系7269-10的F₂群体为材料,利用扣除杂交结合BSA法分离育性相关基因。将F₂中结实率为0%的10株材料的单核发育期花粉等量混合,提取其总RNA构建不育RNA池;结实率为100%的10株材料的单核发育期的花粉等量混合,提取其总RNA构建可育RNA池。在扣除杂交中不育RNA池和可育RNA池分别做Driver和Tester。扣除杂交共做三次,使扣除更充分。扣除终产物与T-Vector连接,转化大肠杆菌DH-5α,构建扣除文库。通过Reverse Northern鉴定,去除假阳性,对阳性克隆进行测序、BLAST比较、PCR鉴定及功能分析。 本研究克隆了5个在可育花药中特异表达的基因,其克隆编号分别是:B4,92,314,398和A34。测序分析结果表明,B4和314所含片段为全长序列,其他3个则仅含有基因的3′端片段。对上述5个基因的BLAST查询和生物信息学分析表明,B4所含片段与植物的遍在蛋白缀合酶（UBC）基因有很高同源性,而且含有85位Cys保守氨基酸,推断该基因为小麦的UBC基因,命名为TαUBC2,已经登录GenBank,登录号为:AY952317。生物信息学分析表明,314号克隆所含片段为一未知基因,该基因可能定位于线粒体中,与能量供应有关,推测其与育性有密切的关系。该基因命名为TaRF1,已更多还原

【Abstract】 At present, heterosis application can’ t make great progress in wheat. Firstly, wheat is allopoly hexaploid and self-pollination plant. Furthermore, wheat genome is very huge. As the result of artificial selection long ages the heterosis isn’ t obvious between varieties. Secondly, T-type (T. timopheevii) CMS line is maintained easily but is restored difficultly. So it is important to culture the new restore line and strengthen screening the hybrid combination in wheat. In the paper the genetic rule of restore gene was analysed and the fertil i ty relative gene were cloned. This would establish the theory and experiment base for wheat heterosis applying in agriculture production.The experiment materials are CMS line 75-3369A, restore line 7269-10 and F2 population and F3 family of 75-3369A/7269-10 combination. The F2 fertile isolations data of continuous three years were analysed. The analysis result showed there were two pairs of dominant major genes and there was additive effect between non-allele genes. The fertility restoring exits quantity character and quality character. The results indicated that the fertility restoring of T-type CMS was controlled by the two pairs of major genes together with some minor genes. The imitate near-isogenic lines (INIL) and near-isogenic lines (NIL)of Rf gene were developed utilizing crossing, backcrossing and self-pollinating.The fertility relative genes were cloned from the single-nuclear pollen of F2 fertility plants using subtractive hybridization. Bulked segregant analysis (BSA) method was used to weaken the inherit background. Two bulks representing extremely fertile (100%) and sterile (0%) plants were constructed. The fertile bulk is the Tester, the sterile bulk is the Driver. Hybridizations were taken three times in order to hybrid adequately. The final hybrid production amplified by LD-PCR was inserted into T-vector to construct the subtractive library. Screening the false positive recombination by reverse northern. The positive clones were used to sequence, BLAST analysis, PCR identify and function analysis.更多还原