【作者】 刘慧娟；

【导师】 杨仲南；

【作者基本信息】 上海师范大学 ， 遗传学， 2005， 硕士

【摘要】 花药及花粉发育是植物发育生物学的一个重要研究领域,花药及花粉发育异常会导致雄性不育。很多高等植物中都存在着雄性不育现象,从雄性不育突变体中克隆相应基因并研究该基因在花药及花粉发育过程中的作用是研究花药及花粉发育分子机理的一个重要方法。本研究以模式植物拟南芥的一个雄性不育突变体EC2-157 为材料,对突变体进行了遗传分析、细胞学观察、并对相应的基因进行了精细的定位和对候选MS157基因进行了预测。遗传分析表明,EC2-157 为单基因(MS157)隐性突变所导致。形态学观察发现该突变体花丝短、花药瘪,无花粉粒,果荚短小,不含种子,但雌蕊发育正常。细胞学观察表明,在花药发育的第6 期突变体绒毡层和中层细胞过度肥大,高度液泡化。利用胼胝质特异性荧光染料甲基蓝对花药进行染色,表明突变体胼胝质降解延迟, 导致小孢子不能从四分体中释放出来,最终没有花粉粒的形成。这一结果表明MS157 基因可能通过胼胝质降解途径,对雄配子的发育进行调控。利用图位克隆的方法将MS157 基因定位于第四条染色体上BAC 克隆T6K22上74kb 区间内。目前尚未见到该区间内有雄性不育基因的报道,因此ms157 是一个新的雄性不育基因.该区间共有11 个基因,大都是未知功能的基因。通过芯片数据库分析发现基因AT4G21350 和AT4G21370 是在花药中特异表达的基因,对其中一个基因(AT4G21350)进行了测序和分析。另外,我们通过www.arabidopsis.org 购买了在该区间中的11 个T-DNA 插入突变体。初步的表型分析表明,该区间的一个转录因子AT4G21330 的T-DNA 插入突变导致无花粉粒的形成,造成雄性不育。该区间的11 个基因中哪个为MS157,我们正在进一步的验证之中。本项工作为我们克隆花粉发育必需基因MS157 及其深入的功能分析奠定基础。更多还原

【Abstract】 Anther and pollen development is one of the most important areas in plantdevelopment biology. The defective of anther and pollen development will result in malesterility. Male-sterile mutants have been identified in many higher plants. It is an efficientstrategy to investigate the molecular mechanisms of plant anther and pollen develop bycloning genes from male-sterile mutants followed by functional analysis.In this study, the phenotype of an Arabidopsis male sterile mutant EC2-157 was desc-Ibed and the corresponding gene was fine-scale mapped.The mutant shows shorter filamentsthan wild type and there is no pollen grain in the anther. Cytological observation suggeststhat tapetum and middle layer become fatter and have more vacuoles than wild type in stage6 of anther development.Callose staining with particular fluorescence dye-Aniline blue indic-ated that the degradation of callose was delayed.There is no free microspores released fromthe tetrads and no pollen grains were formed in mutant anther.These results showed thatMS157 plays a vital role in anther development by regulating the degradation of the callosewall.ms157 has been mapped to a region of 74kb located in BAC clone T6K22 on chromo-some IV using a map-based cloning strategy. As no male sterile genes have been reportedin this region, we believe that ms157 could be a new male sterile gene.There are 11 genesin this region, but most of them are unknown proteins. As gene expression analysis throu-gh chip database indicated that AT4G21350 and AT4G21370 are specifically expressed inanther,we sequenced and analyzed AT4G21350.On the other hand,we also bought T-DNAtagging lines of these 11 genes from the www.arabidopsis.org web. Phenotype analysisindicated that T–DNA insertion mutant of transcription factor AT4G21330 result in malesteility. The further work to identify MS157 is being undertaken. The molecular cloningand further functional analysis will provide further insight of anther development.更多还原