【作者】 张在宝；

【导师】 杨仲南； 王全喜；

【作者基本信息】 上海师范大学 ， 水生生物学， 2004， 硕士

【摘要】 随着模式植物拟南芥基因组全序列测定的完成，基因功能研究成为当前生命科学的前沿领域。本论文以正向遗传学的方法研究花药及花粉发育过程的基因功能，主要利用图位克隆的方法从拟南芥雄性不育突变体中克隆相应的基因。 首先对19个经化学诱变剂EMS处理得到的雄性不育突变体进行背景纯化和遗传分析，从中筛选到四个单个隐性基因控制的雄性不育突变体(EC2-157、EC1-188、EC2-115和EC2-214)。细胞学观察表明，EC2-157花药中不能形成花粉粒，EC1-188突变体中只有少量的花粉粒，但花粉粒的内容物较少；EC2-115和EC2-214突变体中能够形成花粉粒。 为了从突变体中克隆基因，我们建立了一套分子标记(共24个)用于突变基因的初定位。其中本次实验发展了18个分子标记。利用这套分子标记，将四个不育突变体EC2-157、EC1-188、EC2-115、EC2-214相应的突变基因分别定位于拟南芥的第Ⅳ，Ⅴ，Ⅴ，Ⅰ号染色体上。这套分子标记比较稳定，容易检测，目前也用于拟南芥其他突变基因的定位。 然后对突变体EC1-188的突变基因(ms188)进行了精细定位。在与ms188连锁的分子标记MC015附近设计了8个InDel分子标记，对遗传群体中2135株不育植株进行基因型分析，最后将目的基因定位于第五条染色体分子标记MDA7和K24C1之间95.8KB的区间内。该区间共有28个基因，其中一个是转录因子AtMYB103。 利用PCR的方法从突变体中扩增AtMYB103基因并进行序列分析，结果表明突变体中AtMYB103基因第二个外显子上发生了点突变，由原来编码谷氨酰胺的CAA密码子突变为终止密码子TAA。因此，我们推测，EC-188突变体可能是转录因子AtMYB103基因发生突变的结果。进一步的遗传互补以及功能分析正在进行之中。更多还原

【Abstract】 With the completion of the model plant Arabidopsis genome sequencing in 2000, the emphasis of the life science has been into the functional genomics. In this dissertation we focused on the function of genes associated with anther and pollen development including map-based cloning of Arabidopsis genes from male sterile mutants.From 19 Arabidopsis male sterile lines isolated from an ethyl methanesulphonate-induced (EMS-induced) population, a total of four male sterile mutants were screened with each mutant controlled by a single recessive gene. Cross-section of these male sterile mutant anthers showed that there is no pollen formation in ms157 anther while msl88 anther contains a few pollen grains with pollen shape distorted and reduced cytoplasmic content. However, pollen was formed in both anthers of ms115 and ms214.In order to isolate these genes, we established a set of molecular markers for the first-pass mapping of Arabidopsis genes. Of the 24 InDel markers, a total of 18 were developed in this work. The mutated genes ms157, ms188, msl 15 and ms214 were mapped on chromosome IV, V, V, I, respectively, using the molecular markers developed above. This set of molecular markers is stable, and easy to use. They are also used for other gene mapping in Arabidopsis.The ms188 gene was finely mapped. A total of 8 new InDel markers were designed to map msl88 using a segregating population with a total of 2135 male sterile progenies. MS188 was finally mapped to a region of 95.8kb between the molecular marker MDA7 and K24C1. There are a total of 28 genes in this region including a transcription factor AtMYB103.PCR method was used to identify candidate MS 188. Sequence analysis indicated that a point mutation occurred in the second exon of the AtMYB103 gene in male sterile mutant with CAA(Gln) replaced by a stop codon TAA. Thus, we believe that AtMYB103 is the candidate gene for MS 188. The genetic complementation and gene function analysis are being undertaken.更多还原