【作者】 张兵；

【导师】 于军； 胡松年；

【作者基本信息】 浙江大学 ， 生物学， 2006， 博士

【摘要】 本文详尽报道了五步蛇毒腺细胞功能基因表达序列标签和毒素基因进化研究、家猪脑组织基因表达分析以及海洋弧菌噬菌体EH3-23P1全基因组测序及序列分析结果。ESTs是对cDNA克隆5’端或3’端测序得到的长度在500bp左右的序列，是研究基因表达和差异表达以及功能的重要手段。作为非常特化的器官，蛇毒腺合成和分泌大量复杂的毒素蛋白。由于蛇毒素独特的药用价值，毒腺基因表达已经被广泛的研究，但毒素分泌的分子机制、毒腺的细胞结构和代谢还知之甚少，由此引发一个基本问题就是毒腺除了表达大量的毒素蛋白以外还有哪些基因表达?为了阐明这些问题，我们选取五步蛇毒腺作为基因表达研究模型，构建毒腺非标准化cDNA文库，5’末端测序得到8，696条高质量的表达序列标签序列，聚类处理后产生3，416个类，其中40.16％的ESTs编码毒素相关蛋白，39.85％的ESTs属于细胞功能基因，另外20.00％的ESTs在现有的数据库中找不到同源序列。分析细胞功能转录本，我们发现一些毒素分泌相关基因和腺体特异基因的高表达，如钙离子结合的EF-hand蛋白、蛋白质二硫键异构酶等；与能量代谢相关的基因如肌酸激酶和NADH脱氢酶也以高水平的表达；另外，毒腺细胞结构蛋白与哺乳动物肌肉组织相似。同时，和其它蛇毒腺基因表达比较分析，发现五步蛇毒液成分没有神经生长因子类毒素基因的表达。分析两个毒素蛋白家族groupⅢmetalloproteinase和serine protease在Colubroidea亚目的进化关系表明了早期的单次基因获得事件。五步蛇毒腺ESTs数据为蛇类功能基因组和毒腺的基因表达研究提供了重要的资源，也为进一步阐明蛇毒分泌机制和毒素进化提供了重要的序列信息。为理解家猪脑组织发育过程中基因表达的变化，构建了丹麦长白猪7个脑组织不同发育时期和不同部位的非标准化cDNA文库，随机测序处理后得到43，122条高质量的5’末端ESTs序列，聚类后产生16，101个类，其中30.6％代表已知功能的基因，75.1％与已知核酸序列有显著相似性，73.3％和已有的家猪ESTs序列有显著相似性，另外21.5％找不到任何同源序列，代表新基因。通过出生前后脑组织基因表达谱的比较，发现多个基因在这两个发育阶段表达有显著差异，预示着家猪脑组织的发育和功能的分化。基因组学的发展能够将全基因组序列应用到噬菌体鉴定以及进化研究和生物学特性的理解中。我们从中国珠江入海口海水中分离出一株弧菌EH3-23和它的裂解性噬菌体EH3-23P1，电镜图片显示EH3-23P1属于有尾噬菌体目，通过鸟枪法全基因组测序，得到一个113，861bp闭合环状的双链DNA序列，平均GC含量为40.4％。基因组注释后发现，EH3-23P1编码195个蛋白质基因，3个tRNA基因，12个不依赖于rho因子的转录终止子。在195个推定的蛋白编码基因中，30个和已知功能基因有显著相似性，30个比对上已有的未知功能基因，135个是EH3-23P1特异基因。在EH3-23P1基因组中识别出一个重要的磷酸代谢补偿基因phoH；另外，EH3-23P1基因组并没有末端冗余的重复序列，表明非序列变换的基因组复制模式。更多还原

【Abstract】 In this report, we analyzed the expressed sequence tags involved in cellular function from the venomous gland of Deinagkistrodon acutus and the evolution of toxin related genes and gene expression of porcine brain and a complete genome sequence of vibriophage EH3-23P1.EST represents a segmental DNA sequence from cDNA 5’/3’ end sequencing and has been an important tool for studying the gene expression, difference expression and function. The snake venomous gland is a specialized organ, which synthesizes and secretes the complex and abundant toxin proteins. Though gene expression in the snake venomous glands has been extensively studied, the focus has been on the components of the venom. As far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. Therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components? To examine extensively the transcripts expressed in the venom gland of Deinagkistrodon acutus and unveil the potential of its products on cellular structure and functional aspects, we generated 8,696 expressed sequence tags (ESTs) from a non-normalized cDNA library. All ESTs were clustered into 3,416 clusters, of which 40.16% of total ESTs belong to recognized toxin-coding sequences; 39.85% are similar to cellular transcripts; and 20.00% have no significant similarity to any known sequences. By analyzing cellular functional transcripts, we found high expression of some venom related genes and gland-specific genes, such as calglandulin EF-hand protein gene and protein disulfide Isomerase gene. The transcripts of creatine kinase and NADH dehydrogenase were also identified at high level. Moreover, abundant cellular structural proteins similar to mammalian muscle tissues were also identified. The comparative analysis with gene expression of other snake venomous glands, the toxin protein of never growth factor is absent of the venom of Deinagkistrodon acutus. The phylogenetic analysis of two snake venom toxin families of group III metalloproteinase and serine protease in suborder Colubroidea showed an early single recruitment event in the viperids evolutionary process. Gene cataloguing and profiling of the venom gland of Deinagkistrodon acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissue. So this study provides a first global view of the genetic programs for the venom gland of Deinagkistrodon acutus described so far and an insight into molecular mechanism of toxin secreting.To understand gene expression variation of porcine brain among several developmental stages, seven cDNA libraries from Landrace brain were constructed. We obtained 43,122 high quality expressed sequence tags from 5’ end cDNA clone sequencing. After clustering, 16,101 clusters were produced. Of these clusters, 30.6% showed similarity with known functional sequences; 75.1% had homology with nucleic acid database; 73.3% matched porcine ESTs from database; 21.5% represented novel genes. Comparison of gene expression indicated that the level of many genes expression showed significant difference between prenatal and postnatal porcine brain, which implied the development of porcine and functional differentiation.Based on complete genome sequences, we can identify bacteriophages and understand their evolution and biological character. We isolated a vibrio strain named EH3-23 in estuarine seawater of Southern China Pearl River and a lytic EH3-23P1. The electron micrograph showed that EH3-23P1 belong to Caudovirales. The 113,861 bp closed circular genome of EH3-23P1 was determined by whole genome shotgun sequencing, which has 40.4% of an average G+C content. EH3-23P1 encodes 195 putative protein-encoding open reading frames (CDSs), 3 tRNAs, 12 rho-independent transcription terminators. Of putative 195 CDSs, 30 match known function genes, 30 match known hypothetic genes, 135 are unique to phage EH3-23P1. We identified a phosphate starvation gene phoH in EH3-23P1. No terminal redundant repeat sequences were identified in EH3-23P1 genome, implying non-permute DNA replication mode.更多还原