【作者】 邱峰；

【导师】 赵云；

【作者基本信息】 四川大学 ， 遗传学， 2005， 硕士

【摘要】 为了从可能的差异表达基因入手研究甘蓝型油菜幼叶黄化突变体Cr3529黄化性状的成因,从本实验室构建的野生型3529——突变体Cr3529正向抑制性消减杂交(suppression subtractive hybridization, SSH)文库中选取了33个克隆进行序列分析。测序结果显示,33个克隆中,7、14号克隆为重复的克隆,二者大小相同,且只有一个碱基的差异;15、18号克隆为同一基因的不同片段;其余均为独立的克隆。BLASTn和BLASTx分析的结果表明,11、22、31号克隆与捕光色素chla/b结合蛋白同源,3、17分别与PSⅡ反应中心及其相关的蛋白同源,另外15和18号克隆与推测的chl P基因有着较高的同源性,该基因被认为参与叶绿素的合成。由此可以初步证实本实验室关于突变体的黄化性状与光合作用,特别是叶绿素的合成相关的推测。此外,对其余克隆的序列分析表明,这些克隆涉及到氨基酸代谢、糖代谢、脂肪酸代谢等多个代谢过程,初步说明了黄化性状还可能涉及到各个代谢途径。 为了研究反向消减文库中一个未知基因功能,利用RACE(rapid amplification of cDNA ends)技术,在已知片段的基础上,对该基因的5’端cDNA进行了扩增,经过两轮PCR得到了该基因5’端约1000bp的序列,将之与原有已知片段拼接得到一个大小为1343bp的片段,序列分析发现该片段的1～1299位碱基为基因编码区;BLAST分析表明,该基因与来自拟南芥的一个编码未知蛋白质的基因(GenBank登录号At4g01080)同源性达80.1%,但由于单个碱更多还原

【Abstract】 In order to realize the mechanism of the chlorophyll-reduced mutation of Brassica napus G3529 from the base of potential differently expressed genes, 33 clones from the forward suppression subtractive hybridization (SSH) library of Brssica napus 3529 (wild type) and Cr3529 (mutant) were analyzed by sequencing and BLAST. In all 33 clones, number 7 and 14 were determined as duplicated clone of each other, with only one different base. Number 15 and 18 were identified as the different fragments of a same gene. The others were individual clones. BLASTn and BLASTx analysis showed that number 11、 22、 31 clones were homologous with chlorophyll a/b-binding protein, number 3 and 17 clones were homologous with photosystem II reaction center W (PsbW) protein and its related protein, respectively, and number 15 and 18 clones were homologous with a putative chl P gene, which was supposed to be related with the synthesis of chlorophyll. These results have supported the former hypothesis that the chlorophyll-reduced mutation was related with the process of photosynthesis, especially with the synthesis of chlorophyll. The analysis of other clones indicated that they were involved in the metabolism of amino acid, glucide and fatty acid, et al, which indicated that the chlorophyll-reduced mutation might be related with complex processes.Base on a known fragment from the backward SSH library, RACE was applied to amplify its 5’ end for the purpose of studying its function. After two rounds ofPCR amplification, a 5’ end of about lOOObp was obtained, which was joined with the original piece and formed a 1343bp cDNA sequence. Sequence analysis of this cDNA identified its l~1299bp as the encode region. BLAST research showed it was homologous with a gene encoding an unknown protein from Arabidopsis thaliana at the ratio of 80.1%, but was deleted with 9 amino acid residues at the 3’ end because of its abrupt termination caused by a mutated base. Swiss-model was used to predict the structure of the induced protein, and a potential transmembrane helix from site 46 Leu to 66 Phe was identified. GLOBE prediction of globularity showed that this protein possessed a compact globular domain.To determine its relation to the formation of chlorophyll-reduced mutation, the expressions of this gene in the cotyledon, seedling and mature leaves, from both 3529 and Cr3529, were analyzed by Northern blot. The results showed that there was no notable expression difference between mutant and the wild type, but slightly lower expression in mature leaves than that in cotyledon and seedling leaves.更多还原