【作者】 林长发；

【导师】 谈家桢； 杨金水；

【作者基本信息】 复旦大学 ， 遗传学， 2003， 博士

【摘要】 为了研究水稻叶组织特异基因及其表达，利用抑制消减杂交法（SSH）建立了水稻叶对根的差减文库。经筛选，获得171个差异克隆。其插入片段分别输入GenBank中进行同源搜索，发现其中水稻中已克隆基因有85个，未克隆的功能基因有45个，未知基因片段有41个。它们涉及水稻光合作用、代谢、膜运输、信号传导以及抗病性等生理活动。利用SSH分离得到的片段。我们分别克隆了水稻磷酸烯醇式丙酮酸羧化酶（PEPC）、蔗糖转化酶（invertase）及Lon蛋白酶等基因的全长cDNA，并对它们的基因组序列及功能进行了初步研究。同源性搜索发现Ospepc与植物许多已知的PEPC具有很高的同源性（84%－86%），它还含有许多PEPC特异的保守功能域。定量PCR分析证实Ospepc在叶中的表达明显强于在根中的表达。对Osinv的同源性搜索发现它与植物其它中性/碱性转化酶也具有较高的同源性（71%－80%）。RT-PCR分析，结果发现Osinv在叶中增强表达，而且盐处理和低温处理会提高它的表达水平。Oslon推测编码水稻线粒体Lon蛋白酶，可特异降解线粒体中的异常蛋白质。同源性搜索显示该蛋白质与其它Lon蛋白酶具有较高的同源性（77%－92%），Conserved Domain Search分析它具有Lon蛋白酶的保守功能域。Northern杂交分析证实Oslon在叶中增强表达。分析还发现该基因在不育系珍汕97A的生殖器官（幼穗和花药）中存在可变剪接，RNA原位杂交初步显示该可变剪接可能发生在绒毡层中，可能与细胞质雄性不育有关。原核表达显示该基因可正常翻译出符合推测分子量的蛋白质。另外，基因组搜索表明它还存在两个同源基因。此外，我们通过筛选水稻幼苗cDNA文库分别克隆到细胞质和线粒体苹果酸脱氢酶基因。以Actin基因为内对照进行RT-PCR分析发现二者组织表达模式较为一致，在幼穗和未成熟胚中表达较高，而在叶和根中较低。原核表达证实二者都能编码与预期大小相符的蛋白质。进一步的检测表明OscMDH具有苹果酸脱氢酶活性。更多还原

【Abstract】 In order to study the leaf-specific expressed genes in Oryza sativa L., the subtracted library of rice leaf was established by suppression subtractive hybridization (SSH). After screening the library, 171 clones differentially expressed were obtained. All these inserts were put into GenBank separately. It was shown that 85 clones belonged to the cloned genes in rice, 45 clones belonged to the known rice gene that were not cloned and the other 41 clones belonged to unknown genes. The known genes were involved in the procedures of photosynthesis, metabolisms, membrane transduction, signal transduction and the resistance in the rice. Using the DNA fragments isolated from SSH, the full-length cDNAs of rice phosphoenolpyruvate carboxylase (Ospepc), invertase (Osinv) and Lon protease (Oslon) were cloned, separately. Their gene sequences and functions were also studied. After sequence alignment, it was found that Ospepc was highly homologous to those known PEPCs (84%-86%). Besides, some PEPC-specific conserved domains were detected in Ospepc. The expressing pattern of Ospepc was verified by quantitative PCR analysis that its signal was obviously stronger in leaf tissues than in roots. Meanwhile, it was shown that Osinv was homologous to the neutral/basic invertase in other plants (71%-80%). It was indicated from the RT-PCR results that the expression of Osinv was increased in leaves, and its expression level could be improved under salt and cold treatments. A putative Lon protease, which degraded the abnormal proteins in mitochondria, was translated from Oslon. It was shown from the alignment results that Oslon was homologous to other Lon proteases (77%-92%). From the conserved domain search, it was found that Oslon has all the important conserved domains found in other Lon proteases. It was convinced from Northern blot analysis that the expression of Oslon was increased in leaves. An alternative splicing of this gene was found in the reproductive organs (young panicles and anthers) in rice sterile line ZHENSHAN 97A. By RNA in situ hybridization analysis, it was shown that this alternative<WP=8>splicing was located probably in the tapetum, noting that it may be related to rice cytoplasmic male sterility. Oslon was also expressed as a fusion protein in prokaryotic system, which showed that the protein could be expressed as a normal protein with the correct size. At the same time, two homologous genes of Oslon were found in rice genome. Moreover, the cytoplasmic and mitochondrial malate dehydroxyase genes were cloned by screening the rice seedlings cDNA library. It was shown from the RT-PCR results that the expression patterns of them were according to each other. The highest expressions were detected in the young panicles and the premature embryos, while they were much lower in leaves and roots. Both of them could be expressed in prokaryotic system in the correct size. Furthermore, the MDH activity of OscMDH was detected.更多还原