麻疯树（Jatropha curcas L.）胚乳cDNA文库的筛选分析及Jc-Fbl1基因、Jc-TCTP1基因的克隆研究

【作者】 高帆；

【导师】 徐莺；

【作者基本信息】 四川大学 ， 植物学， 2007， 硕士

【摘要】 以本实验室构建的麻疯树胚乳cDNA文库为基础，随机测序并经前处理和聚类拼接后获得了47个EST序列；在GeneBank中利用Blastx、Blastn程序进行了同源性比对，用计算机批量处理结合手工分类的方法对EST序列进行了分类，按照功能分为10组：新陈代谢(10.64％)、细胞防御(8.51％)、蛋白合成(17.02％)、能量(6.38％)、运输(4.26％)、信号传导(8.51％)、细胞生长分裂(2.13％)、转录因子(4.26％)、结构蛋白(4.26％)和功能未知(34.04％)。在47条EST序列中，发现一条与多种植物、动物F-盒家族(F-box)相似的序列，随后从文库中获得了含有完整编码框的cDNA序列，并利用PCR方法获得基因组序列(Jc-Fbll)。得到的Jc-Fbll cDNA长1672bp，开放阅读框为1224bp，编码的蛋白质含有407个氨基酸残基(GenBank登录号为DQ900595)。Jc-Fbll推导的氨基酸序列与拟南芥、水稻中的同源蛋白相似性分别为52％和75％；与人、斑马鱼、线虫等动物的F-box蛋白的相似性达到48％～56％。应用Pfam Search寻找功能结构域，发现其除了含有F-盒家族类结构域外，还有富含亮氨酸重复单位(leucine-rich repeats，LRRs)。比对Jc-Fbll的基因组序列和cDNA序列，发现Jc-Fbll有两个长度分别为331bp和893bp的外显子和一个长362bp的插入序列，符合内含子的GT-AG法则，认为是Jc-Fbll的内含子。水稻、拟南芥基因组中的同源序列也出现了这种两个外显子、一个内含子的结构，且内含子位置较为保守。此外，为了进一步研究麻疯树F-box蛋白的功能，构建了同时含有Jc-Fbll完整开放阅读框序列和绿色荧光蛋白标签的真核表达载体pBI121-Jc-Fbl1-GFP，并完成酶切鉴定，以期为进一步研究此蛋白的功能及其在麻疯树胚乳发育中的作用奠定基础。同时，从此cDNA文库中还获得了一条与多种植物翻译控制肿瘤蛋白(translational controlled tumor protein，TCTP)序列相似的EST序列，随后从文库中获得了含有完整编码框的cDNA序列，并利用PCR方法获得基因组序列(Jc-TCTP1)(GenBank登录号为EF091818)。得到的Jc-TCTP1 cDNA长1410bp，含有一个由507个碱基组成的完整开放阅读框，5′非翻译区与3′非翻译区分别有526和377个核苷酸。Jc-TCTP1编码的推测蛋白产物由168个氨基酸残基组成，其氨基酸序列与橡胶树(Hevea brasiliensis)TCTP、油棕(Elaeis guineensis)TCTP、水稻[Oryza sativa(japonica cultivar-group)]TCTP及拟南芥(Arabidopsisthaliana)TCTP的同源性分别达到了93％、89％、84％和79％。用Pfam Searcah和Prosite search(Predict Protein)寻找功能结构域，发现其含有TCTP结构域，该结构域仅与人Mss4蛋白(一种缺乏鸟嘌呤的分子伴侣)有相似结构。比对Jc-TCTP1的基因组序列和cDNA序列，发现Jc-TCTP1有5个外显子和4个插入序列，均符合内含子的GT-AG法则，认为是Jc-TCTP1的内含子。为了进一步研究麻疯树Jc-TCTP1蛋白的功能，分别构建了仅含有Jc-TCTP1开放阅读框序列和带有5′非翻译区的开放阅读框序列的两种真核表达载体pBI121-Jc-TCTP1-1和pBIl21-Jc-TCTP1-2，已完成酶切鉴定，并获得了转基因烟草，以期为进一步研究此蛋白的功能及其在麻疯树胚乳发育中的作用奠定基础。更多还原

【Abstract】 50 randomly selected cDNA clones from Jatropha curcas(Linnaeus) endosperm cDNA library were sequenced and 47 expressed sequence tags(ESTs) information were obtained. With Blastn and Blastx analysis, these ESTs were further grouped into 10 functional categories including: metabolism(10.64％), cell defence(8.51％), protein synthesis(17.02％), energy(638％), transport facilitation(4.26％), signal transduction(8.51％), cell growth, division(2.13％), transcription(4.26％), cell structure(4.26％) and function unknown(34.04％).Among the 47 ESTs, a new gene was cloned and named as Jc-Fbll. The cloned cDNA encompasses a 1224bp open reading frame, which encodes a 407 amino acid peptide(GenBank ID: DQ900595). Alignments with F-box proteins in plants show 52％and 75％identity with Arabidopsis thaliana and Oryza sativa(japonica cultivar-group) respectively; while alignments with F-box proteins in animals show 48％～56％identity. Pfam search predicted some conserved domains or motifs in the deduced protein, including F-box and leucine-rich repeat domains. Genomic gene sequence analysis indicated two extrons and an intron with the length of 331bp, 893bp, and 362bp respectively. The similar genomic character was also found in the Arabidopsis thaliana and Oryza sativa homologues. Moreover, an eukaryotic expression vector, pBI121-Jc-Fbll-GFP, was constructed and confirmed for farther research work.Also from this cDNA library, another gene was cloned and named as Jc-TCTP1. Jc-TCTP1 cDNA encompasses an open reading frame with 507bp, which encodes a 168 amino acid peptide(GenBank ID: EF091818). Alignments with translational controlled tumor proteins(TCTP) in plants show 93％, 90％, 84％and 79％identity with Hevea brasiliensis, Elaeis guineensis, Oryza sativa(japonica cultivar-group) and Arabidopsis thaliana respectively. Pfam search and Prosite search predicted some conserved domains or motifs in the deduced protein, including a TCTP domain. Genomic gene sequence analysis indicated five extrons and four introns. Besides, two eukaryotic expression vectors, pBI121-Jc-TCTP1-1 and pBI121-Jc-TCTP1-2, were constructed and confirmed, the former one only contains Jc-TCTP1 ORF, while the later one contains Jc-TCTP1 ORF along with its 5’-UTR.更多还原