【作者】 于春霞；

【导师】 李景富；

【作者基本信息】 东北农业大学 ， 蔬菜学， 2006， 硕士

【摘要】 细胞质雄性不育(Cytoplasmic Male Sterility)是作物杂种优势利用的基础,另一方面,它又是研究细胞质遗传、核质互作和花粉发育的极好材料。因此,成为当前分子生物学和基因工程学的热点之一。已有大量的试验证实,细胞质遗传虽然独立于核基因组,但细胞核和细胞质之间存在着不可分割的依存关系,其间的相互作用对生物的生长和发育起着重要的作用。目前由于对胡萝卜核质互作雄性不育(G-CMS)的产生及不育机理缺乏深入研究和了解,在很大程度上阻碍了胡萝卜CMS在实际育种中的充分利用,胡萝卜的杂种优势利用仍然比较落后。因此,有必要进一步分离与胡萝卜雄性不育密切相关的基因,为搞清核质互作不育机理提供理论基础,最终利用基因工程手段创造胡萝卜雄性不育新材料,使其尽快应用到胡萝卜的杂种生产当中。本研究采用cDNA-AFLP技术结合分离群体分组混合分析法(Bulked segregant analysis, BSA),研究瓣化型胡萝卜核质互作雄性不育系和保持系的花器官发育过程中基因转录表达差异,克隆了不育相关基因,并对其进行了序列相似性比较分析。此外,还通过CAPS标记的筛选,分别获得不育系和保持系的atp6基因片段,分析了二者的差异以及对瓣化型胡萝卜雄性不育发生的影响。主要研究结果如下:1.分别以不育系和保持系的cDNA混合池为模板,选用128对E+2/M+3的选择性扩增引物进行育性差异和时期差异的筛选,在此基础上分析得到了3条与胡萝卜雄性不育基因相关的差异表达cDNA片段。2.序列分析表明,BY1949 cDNA片段长度为364bp,推导的ORF编码120个氨基酸,与来源于拟南芥第五号染色体的质子依赖性的类肽转运蛋白基因和膜蛋白基因有58%的同源性,与水稻Nef1突变体蛋白也有40%的同源性。相关文献的查询发现,该基因的编码产物影响花粉膜状层和孢子外壁脂质的积累导致了拟南芥的雄性不育。3. BY2562 cDNA片段长度为432bp,编码88个氨基酸的开放阅读框,与雄性不育胡萝卜、向日葵、油菜和拟南芥等多种作物的线粒体功能蛋白高度同源,同源性达90%以上,并具有线粒体膜蛋白YMF19的保守结构域。通过GenBank BLAST的查询发现,YMF19参与了芸苔属作物不育性的形成。4. BY1162 cDNA片段长度为637bp,编码143个氨基酸的开放阅读框,具有glyA基因及其编码的丝氨酸羟甲基转移酶(serine hydroxymethyl- transferase,SHMT)的保守结构域。SHMT为一种光呼吸的关键酶,在线粒体和叶绿体中行使功能。5.基于同源序列的候选基因法,通过检索GenBank的DNA和cDNA数据库获得atp6基因的保守序列,经生物学软件分析,根据保守区设计特异PCR扩增引物,在瓣化型胡萝卜雄性不育系和保持系之间进行扩增。结果显示,不育系和保持系间的特异引物扩增产物未表现多态性。6.利用5种在基因组中出现切割位点频率较高的限制性内切酶EcoR I,Mse I ,AdeI,Hind III和Bgl II对PCR产物进行限制性酶切,对酶切后表现多态性的PCR产物进行测序。更多还原

【Abstract】 CMS (Cytoplasmic Male Sterility) is the base of crop heterosis. It is excellent material on studies of cytoplasm inheritance, G-CMS and pollen growth that has become the one of prevalence in field of molecular biology and gene engineering today. Lots of experimentations proved that they depend on each other for existence and their reciprocity take on important effect on development though cytoplasm is nucleus–independent. At present sufficient utilization of CMS has been hindered in carrot breeding due to be lacking in in-depth study and understand about form and mechanism of CMS in carrot and that utilizing of carrot heterosis has still been laggard. Therefore, it is indispensable to clone more gene related to CMS in carrot so that the basal theory would be provided to research mechanism of G-CMS. The new materials of sterility in carrot that created with gene engineering would be applied to production ultimately.cDNA-AFLP and BSA were dveloped to study differential expression in course of flower development on male sterile line and fertile line in CMS carrot . The genes related to CMS were cloned and analyzed comparably. In addition to, the atp6 genes of male sterile line and fertile line were obtained by CAPS marker respectively and their difference together with effect on CMS in carrot were analyzed. The main results were as follows:1. Three obvious cDNA differential fragments related to CMS were obtained successfully from selective PCR amplification with 128 pairs E+2/M+3 primer combinations by screening out differences of sterility and fertility together with phases.2. Sequence analysis showed that cDNA fragment of BY1949 is 364bp that coding 120 amino acid residues. It has 87% identity with proton-dependent peptide transporter-like protein and membrane protein from Arabidopsis thaliana as well as 40% identity with Nef1 from Oryza sativa. The retrieval of available literature indicated that its coding product affects lipid accumulation in the plastids of the tapetum and exine formation of pollen that resulting in male sterility in Arabidopsis thaliana.3. cDNA fragment of BY2562 is 432bp that coding 88 amino acid residues. It is over 90% identity with mitochondrial function protein of many vegetable consist of CMS carrot, CMS common sunflower, rape and Arabidopsis thaliana etc. And it has mitochondrial membrane protein YMF19 motif, which be involved in cytoplasmic male sterility (CMS) in Brassica by GenBank database.更多还原