【作者】 李红霞；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2008， 博士

【摘要】 黏类小麦雄性不育系是指小麦黏型（K型）和偏型（Ven型）等一类既易保持又易恢复、且种子饱满、保持系与恢复系来源都分布在普通小麦推广品种（系）内的小麦雄性不育系的总称。黏类小麦细胞质雄性不育系育性基因单一、不育性彻底、易恢复性高,是一种很有应用潜力的优良雄性不育类型,为小麦雄性不育研究和杂种优势利用提供了极有利的材料基础。本研究利用上述材料,通过对黏型小麦细胞质雄性不育恢复基因的遗传分析和分子标记筛选,以确定其恢复基因的作用方式,并试图得到与恢复基因紧密连锁的SSR标记,进而为杂交小麦分子标记辅助选择育种奠定坚实基础;同时以SSH技术分别构建了不育和可育条件下基因特异表达的抑制差减杂交cDNA文库,通过对两个cDNA文库的克隆测序、EST序列BLASTx分析,探讨了黏型小麦细胞质雄性不育系在不育和可育条件下育性相关表达基因的种类、功能和参与相关生化代谢途径的异同,旨在探索黏型小麦细胞质雄性不育的分子机制。获得下述重要结果:1、本论文以细胞质雄性不育系ms（Kots）-90-110、恢复系rk5451及ms（Kots）-90-110/ rk5451组合的F₂,BF₁和BC₁F₁群体为材料,连续两年对分离群体的育性分离情况进行了统计,对恢复基因的遗传规律进行了分析。结果表明,黏型小麦雄性不育的育性恢复主要受一对主效显性恢复基因控制;鉴于F₂的育性恢复既有质量性状的特征,又有数量性状的特征,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基因的影响;利用杂交、回交和自交育成了关于小麦黏型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。2、对黏型小麦雄性不育系及杂种F₁配子传递方式分析,发现黏型小麦雄性不育系具有配子体不育的特征。供试ms（Kots）-90-110/RK5451//90-110的BC₁F₁分离群体明显偏离1:1的分离比,ms（Kots）-90-110与rk5451自交F2后代全都表现可育,无不育株,且自交结实率F₂明显高于F₁,进一步表明黏型小麦雄性不育系杂种F₁是以配子体不育方式传递的。3、为了进一步探索黏型小麦育性恢复基因的遗传机理。以ms（Kots）-90-110/ rK5451//90-110的BC₁F₁代分离群体为研究对象,分别用定位于1B短臂染色体上的18对和6B染色体上的47对SSR引物对黏型小麦雄性不育育性恢复基因进行分子标记。初步筛选出Wms273和Wmc406两对揭示育性恢复基因多态性的引物,用132株BC₁F₁回交群体对这2对引物进行进一步检测,得出Wmc406与黏型小麦细胞质雄性不育系1BS上的恢复基因连锁,遗传距离为7.6cM。标记Wmc406可直接用于黏型小麦育性恢复基因的分子标记辅助选择。4、利用抑制差减杂交技术构建了黏型小麦雄性不育育性相关基因的cDNA文库。文库质量检测表明,差减杂交效率较高,质量较好。在不育和可育cDNA文库中随机挑选200个阳性克隆进行测序,获得100余条高质量的表达序列标签（EST）。对序列进行BLAST比对及功能注释,比较了不育和可育cDNA文库的基因表达谱,发现在可育cDNA文库中参与能量代谢的基因出现频率较高,在不育cDNA文库中检测到了与花发育调控相关的MADS-box转录因子、细胞凋亡相关的泛素结合酶及阻遏淀粉合成的腺苷二磷酸葡萄糖磷酸化酶等相关基因,这些基因很可能与育性相关。5、根据标签序列的比对结果,选择了4个与育性相关的基因片段进行电子延伸,克隆了4个育性相关基因的cDNA全长序列,并设计特异性引物,进行了RT-PCR验证。对这些基因的氨基酸序列、结构域、疏水性及系统进化情况进行了分析,其生物学功能尚需进一步构建相应的表达载体进行验证。更多还原

【Abstract】 Male sterility of wheat with Aegilops kotschyi cytoplasm is the generic name of wheat K-CMS and Ven-CMS.They are not only heritable and restorable but also seed plump,theirs maintainer line and restorer line derive from popularized variety in wheat.More studies showed that male sterility of wheat with Aegilops kotschyi cytoplasm was ideal sterile lines.According to the research with plasmatic effect,fertile restoration and transfer mode of androgamete and megagamete in male sterility of wheat with Aegilops kotschyi cytoplasm, male sterility of wheat with Aegilops kotschyi cytoplasm had the character with single sterile gene,thorough sterility and high restoration,which provided advantageous material foundation for the study of male sterility in wheat and the utilization of heterozyous advantage.Using above-mentioned materials, Genetic Analysis of the Male Sterility of Wheat with Aegilops kotschyi Cytoplasm was researched and molecular markers linked to restoring gene was Screened, which may provide a basis for choosing and breeding assistance;this study constructed differential expression SSH cDNA library under sterile and fertile conditon through SSH. Through sequencing of ESTs and BLASTx analysis of ESTs sequences in the two libraries, this study discussed the type of expressed gene, gene function and different biochemical metabolic pathway in male sterility of wheat with Aegilops kotschyi cytoplasm between sterile library and fertile library so as to analyze molecular mechanism of male sterility of wheat with Aegilops kotschyi cytoplasm.The results were as follows:1. The experiment materials are CMS line ms（Kots）-90-110, restore line rk5451and F₂ ,BF₁ and BC₁F₁ population ms（Kots）-90-110/rk5451 combination. The fertile isolations data of continuous two years were analysed. The analysis result showed there was one pair of dominant major gene. The fertility restoring exits quantity character and quality character. The results indicated that the fertility restoring of K-type CMS was controlled by the one pair 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.2.The manner of gametes transmission of male sterile wheat with Aegilops kotschyi cytoplasm and seed set of F₁ is analyzed in this paper. Obviously, male sterile wheat with Aegilops kotschyi cytoplasm have a characteristic of gametophte sterility; Fertility Separation ratio of ms（Kots）-90-110/rk5451//90-110 depatured seriously 1:1. In F₂ population of ms（Kots）-90-110/ rk5451 completely appear fertile without sterile.Test on hybridization F1 and F₂ indicates that average seed set of F2 is higher than F1 These results prove again that the hybridization F₁ of male sterile line with Ae. Kotschyi cytoplasm.Bicornis cytoplasms inherits in mode of gametophyte sterility.3.In order to probe into the fertility characteristics of male sterility with Aegilops kotschyi cytoplasm, SSR markers which were located on the chromosome 1BS and 6B were employed to tag the major restorer gene in BC₁F₁ of ms（Kots）-90-110/ rk5451//90-110. Two primer pairs（Wms273 and Wmc406） displayed polymorphism of the restorer gene . By analyzing the polymorphic markers in these segregating populations, the microsatellite locus Wmc406 located on chromosome 1BS was found to be linked to the restorer gene with the estimated genetic distance of 7.6cM. Wmc406 can be used in MAS directly.4.To further reveal the genetic mechanism of male sterility with Aegilops kotschyi cytoplasm, a male sterile line ms（Kots）-90-110（A） and its near isogenic line BC4F1 （fertility restored by rk5451） were employed to construct sterility and fertility cDNA libraries by suppression subtractive hybridization （SSH）. Wheat anthers with pollen mother cells at dikaryophase were sampled for RNA extraction via microscopic examination. The two cDNA libraries were evaluated to be of good quality with high efficiency of SSH. Total 120 positive clones were randomly selected and sequenced from the 2 libraries, and 100 high quality sequences were obtained. According to BLAST screening and functional annotation, energy-related genes were identified with high frequencies in fertility cDNA library, while MADS-box transcriptional factor TaAGL7, ubiquitin-conjugating enzyme, and adenosine diphosphate glucose pyrophosphatase were detected in sterility cDNA library. The MADS-box transcription factor TaAGL7 plays important roles in regulating flower development. Immature spike ubiquitin-conjugating enzyme is likely to relate to apoptosis. The expression of adenosine diphosphate glucose pyrophosphatase directely affectes starch synthase and normal supply of energy. The genes detected in our study are probably important in fertility of wheat with Aegilops kotschyi cytoplasm.5.The four differentially expressed genes, Which were supposed to involve in fertility of wheat, were selected and tested by means of PT-PCR. The amino acid sequence, structure domain, hydrophobicity, multiple alignment and homology phylogenetic tree of the genes were studied.更多还原