黏类小麦育性相关基因SSH文库的构建  

Construction on cDNA Library from Fertility-Related Genes of Male Sterile Wheat with Aegilops kotschyi Cytoplasm by SSH

作　　者：李红霞[1] 张龙雨[1] 张改生[1] 牛娜[1] 朱展望[1] 

机构地区：[1]西北农林科技大学陕西省作物杂种优势研究与利用重点实验室/小麦育种教育部工程研究中心,陕西杨凌712100

出　　处：《作物学报》2008年第6期965-971,共7页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(301705760);国家高技术研究发展计划(863计划)重大专项(2002AA207004);陕西省13115科技创新工程重大科技专项(2007ZDKG-02);国家杨凌农业生物技术育种中心专项基金项目(99-1A);西北农林科技大学拔尖人才支持计划项目

摘　　要：为了深入研究黏类小麦雄性不育的分子遗传机制,利用抑制差减杂交技术构建了黏类小麦育性相关基因的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120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。Male sterility with Aegilops kotschyi cytoplasm has a great application potential in hybrid wheat （Triticum aestivum L.） breeding for its stable sterility and broad-spectrum of restoring gene resources. 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.

关 键 词：小麦 育性基因 抑制差减杂交(SSH) 表达序列标签(EST) 

分 类 号：S512.1[农业科学—作物学]