【作者】 曹英豪；

【导师】 刘国振；

【作者基本信息】 河北农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 真核生物基因组中尤其是植物中广泛存在着U-Box结构。U-Box结构高度保守,其构象与RING-finger极其相似,这两者都能发挥泛素连接酶E3的作用,促进底物蛋白质泛素化降解,对细胞内异常蛋白质的降解及质量控制方面发挥着重要的作用。本文通过全面的计算分析从水稻中确定了77个U-box基因,并对这类基因家族进行了一个完整的概述,包括基因结构,染色体定位和保守序列。此外,还通过对EST数据库的搜索分析了U-box的转录水平。用计算机软件预测抗原决定簇来设计多肽,通过合成多肽免疫动物制备抗体,检测水稻在不同发育时期的表达谱,对U-box蛋白质的表达水平进行分析。通过分析,发现水稻U-box基因可分为7类结构,其中含U-box和Arm结构的家族成员最多,为31个。对水稻的U-box保守结构域的内含子分布分析发现有一半的基因U-box结构域内都没有内含子。通过染色体定位发现2号,4号,6号染色体上U-box基因为37个,约占水稻整个U-box基因家族的50%。通过对NCBI的EST数据库中274个文库中超过100万条的水稻EST分析,发现在U-box和Arm结构基因中呈现部分富集现象,而只含有U-box结构的基因,检测到EST的数目并不多。整体来说水稻U-box基因家族的转录水平明显低于水稻平均转录水平。和其他16个水稻基因家族的EST比较表明基因家族的转录水平与家族成员的数目并不成正比,基因家族成员数目多的,转录平均水平较低。通过合成多肽制备的抗体有特异性,Western blotting检测可见U-box家族的蛋白质表达谱具有两种模式,一种为组成型表达,另一种为组织特异性表达。根据分析结果及已发表文献,讨论了U-box蛋白质家族的结构与功能的关系。并得出结论,该U-box家族在水稻中的扩增可能是由于染色体/段复制和串联复制。这些结果将有助于对U-box家族基因的进一步功能分析。更多还原

【Abstract】 U-box domain was a high conserved motif existing in eukaryotes, especially in plants. The domain of U-box was similar to that of the RING-finger. Both of them could function as E3 ubiquitin ligase which play a key role in the recognition and selection of abnormal proteins targeted for ubiquitination and subsequent degradation, a process for the maintenances and quality control of proteins exist in living cells. In this study, a comprehensive computational analysis identified 77 U-box family genes in rice. A complete overview of this gene family in rice is presented, including the gene structures, chromosome locations, and conserved motifs. In addition, the analysis with its transcription pattern was performed by searching EST database. Epitope prediction were carried out by computer software to synthesize polypeptides. Polyclonal antibodies were generated by rabbit immunization. Western blotting analysis were carried out for rice material collected at different developmental stages to detect expression pattern. As a result of these analyses, we found U-box gene family could be divided in to 7 groups, including the group with U-box and Arm domain representing the largest class.Analysis of U-box family conserved domain in Rice found that half of the members have no introns. There are 37 members located on the 2, 4, 6 chromosome, represented 50% percent of the rice U-box family. EST analysis based on 1 million ESTs derived from 274 libraries from NCBI EST database revealed that enrichment of ESTs were found in the U-box gene with Arm domain, while not many ESTs were found in the class with only U-box domain. After all, the transcription of U-box gene in rice was significantly lower than the average of all genes. The comparison with other 16 gene families shows that the transcription level of gene family is not directly proportional to the number of family members. The family with large members has a lower transcription level. Western blotting detection of U-box protein family has two profiles, one for the constitutive expression, and the other for the tissue-specific expression. We discuss the relationship between the structure and function of the U-box family proteins based on these results and published information. It was further concluded that the expansion of the U-box family in rice might have been due to chromosomal/segmental duplication and tandem duplication. These results will be useful for future functional analyses of the U-box family genes.更多还原