乙肝病毒hbx基因诱导斑马鱼胚胎发育背部化畸形

为了研究HBX对胚胎发育的影响,从含有HBX基因的质粒上转录HBX mRNA,显微注射入斑马鱼早期胚胎,在bud期进行表型观察,并用全胚胎原位杂交法检测HBX对斑马鱼胚胎早期发育的影响,用脊索标记基因ntl和骨骼肌标记基因MyoD作为探针检测胚胎发育背部化的特征表型。首次发现HBX的表达引起斑马鱼胚胎早期发育背部化畸形,HBX过量表达的胚胎背部化表型与早期胚胎wnt缺失表达导致的胚胎发育背部化表型一致,HBX可能通过激活wnt/β-catenin信号通路的表达和调控,造成胚胎发育背部化的畸形。     

麻疯树干旱诱导差异表达基因片段的分离与功能分析

运用mRNA差异显示技术(DDRT-PCR),并结合反向Northern杂交筛选麻疯树(Jatropha.curcus)干旱胁迫下差异表达基因,最终从麻疯树叶片中分离出8条干旱胁迫诱导下表达上调的差异片段.经克隆、测序和基因信息分析后发现其中4个片段与GenBank中已知序列有较高同源性.对这4个差异表达片段的序列分析表明:JcDD-3和JcDD-13分别与1-磷酸-肌醇合成酶(MIPS)基因及甜菜碱醛脱氢酶(BADH)基因序列有较高同源性;JcDD-16与白杨中度抗旱叶片中提取的表达序列标签(CU230383)核酸序列同源;JcDD-25与植物富含亮氨酸重复序列型类受体蛋白激酶(LRR-RLK)基因序列同源.     

日本沼虾感光器Gqα基因的扩增及分析

采用分子生物学方法，提取日本沼虾感光器的总RNA，反转录成c DNA。根据Gq蛋白α亚基的保守序列设计简并引物，通过巢式PCR扩增出日本沼虾感光器Gqα基因的片段，并将该片段克隆到pMD 18-T载体上进行序列测定，结果显示此基因片段由369bp组成。翻译成氨基酸序列（123aa），Blast搜索结果比较该片段氨基酸序列与美洲鲎、美洲龙虾、冈比亚按蚊等6种动物的Gqα氨基酸保守序列具有高度同源性（83.74％～95％），其中与冈比亚按蚊的Gqα氨基酸序列同源性最高（95％）。     

血吸虫膜相关抗原基因的克隆

目的:筛选日本血吸虫(Schistosoma japonicum,S.japonicum)新的疫苗候选抗原基因.方法:以S.japonicum成虫DNA为模板,PCR扩增相关基因,然后将其克隆XpBS-T载体,通过菌落PCR对产物进行检测,对阳性克隆子测序,与GeneBank中的已知相关序列进行比对分析.结果:菌落PCR获得一条与PCR产物一致的DNA片段,序列测定结果表明PCR产物与S.japonicum 22.6 kDa抗原分子核苷酸序列具有高度同源性.其目的基因大小636 bp,具有一个189 bp的ORF,能编码63个氨基酸.ORF不是全长的阅读框,只获得部分编码区.表达产物可含有一个包含9个氨基酸残基的潜在螺旋跨膜片段(25～33位)和一个酪氨酸激酶磷酸化位点(38～39位).讨论:成功克隆出一个与S.japonicum 22.6kDa抗原编码基因有高度同源性的基因.预测该基因为膜相关蛋白基因,可编码血吸虫体表膜相关蛋白.在生理机能上,该膜相关蛋白可能是信号传递分子.     

家蚕浓核病毒BmDNV-1(伊那株)结构蛋白基因VP4的克隆、表达及抗体制备

为研发新的日本血吸虫(Schistosoma japonicum)疫苗候选抗原基因,为血吸虫诊断和疫苗研制提供依据,提取和纯化S.japonicum成虫DNA,PCR扩增相关基因,然后将其克隆入pBST载体,对菌落PCR阳性克隆子测序和分析.菌落PCR获得一条与PCR产物一致的DNA片段,PCR产物与S.japonicum 22 600抗原分子DNA序列具有高度同源性,636 bp大小,oRF189 bp,ORF小是全长的蒯读框,只获得部分编码区,能编码63个氨基酸,表达产物含有一个包含9个氨基酸残基的潜在螺旋跨膜片段(25～33位)和一个酪氨酸激酶磷酸化位点(38～39位).本研究所获取的阳性克隆是与S.japonicum 22 600抗原编码基因有高度同源性的基因.通过Blastn和BLASTP分析,预测该基因为体膜相关蛋白基因,可编码血吸虫体壁相关蛋白,可能是信号传递分子.     

金鱼蛋白磷酸酶2A—B＂家族Alpha和Gamma调节基因的cDNA克隆及mRNA表达

以金鱼卵巢组织作为材料,运用RTPCR和cDNA末端快速扩增（RACE）技术克隆得到蛋白磷酸酶2A（PP2A）调节亚基B＂家族中γ基因（GSPR）cDNA全序列和α基因（PR130）cDNA部分序列．结果显示γ基因cDNA全长1885bp,编码一个含457个氨基酸的蛋白质．而α基因cDNA长1781bp,编码的多肽共含426个氨基酸,系v亚基的部分蛋白序列．它们与已知其他物种对应的B＂家族蛋白质均有着很高的同源性．结构预测分析发现,α和γ两条多肽均存在PP2A调节亚基B＂家族成员共有的两个EFHAND结构域．用RT—PCR的方法检测了这两个基因在金鱼不同组织和胚胎发育不同时期的mRNA表达水平．结果表明,γ亚基在金鱼多种组织和各个胚胎发育时期中均有较高表达且表达量比较平均,仅在卵巢和精巢组织中最为丰富．与γ亚基表达模式相比,α亚基表达呈现明显的组织和胚胎发育阶段差异性,在卵巢和肌肉组织中最高,在肾脏和鳃中最低,在原肠胚、神经胚、脑泡分化和体色素等胚胎发育时期的表达较弱,而在其他时期中的表达较强．据此,推测α、γ两调节亚基可能在金鱼不同组织和胚胎发育过程中起着特定的作用．此外,进一步的分析发现,α基因除参与PP2A全酶功能外还可能具有独立的功能．     

鸡新城疫病毒ND-xx08株F基因的克隆及分析

新城疫病毒（NDV）ND-xx08毒株经10 d龄SPF鸡胚增殖后,提取其基因组RNA并反转录成cDNA,用NDV F基因特异性引物,经PCR扩增后获得与F基因预期大小一致的DNA片段。将NDV F基因片段克隆到pMD18-T载体上,并进行EcoR I和Hind III双酶切鉴定和测序鉴定。结果显示,ND-xx08毒株F基因片段的长度为1 662 bp,共编码554个氨基酸,F蛋白的裂解位点为112R-R-Q-K-R-F117,是典型强毒株氨基酸序列结构。将NDV ND-xx08株F基因的47 bp到420 bp序列与新城疫病毒基因型I至基因型Ⅸ毒株的相同序列绘制病毒基因进化树,显示ND-xx08分离株属于基因Ⅶe型。将NDV ND-xx08株F全基因与国内外发表的23株NDV F基因核苷酸序列和氨基酸序列的同源性比较分析,结果表明,其核苷酸序列的同源性在82.7%~97.8%之间,氨基酸同源性在87.5%~97.7%之间。     

花椰菜（Brassica oleracea vat．botrytis）抗黑腐病差异表达cDNA片段的克隆及功能的初步研究

采用花椰菜抗黑腐病近等基因系C731（感病系）和C712（抗病系）作为材料,利用cDNA—AFLP技术,研究了花椰菜黑腐病抗性在黑腐病菌侵染和非侵染条件下基因表达的情况．获得了两个阳性克隆M2和M6．Northern杂交和点杂交进一步证明M2是组成型表达的cDNA片段,只是在病菌侵染与非侵染条件下表达丰度不同．而M6是差异表达的cDNA片段．Southern杂交表明M6 cDNA片段在花椰菜基因组中是单拷贝序列．随后的序列分析发现M6cDNA片段与拟南芥1号染色体的BACF19P19中66665～66813bp有84％同源性,该片段编码拟南芥的2A6蛋白的部分序列．推测的氨基酸序列与拟南芥的2A6蛋白部分序列有91％同源性．用H2O2作为外源分子胁迫处理的初步功能分析发现：M6cDNA片段受H2O2诱导,在诱导早期16～24h高度表达,其在叶片中的积累呈逐渐上升趋势．根据序列分析和初步的功能分析的结果推测：M6cDNA片段可能就是编码花椰菜中类2A6蛋白的部分基因片段．是参与花椰菜抗病反应信号途径的相关调控基因片段．     

水稻花粉特异性启动子的克隆与表达

利用PCR技术,从水稻幼苗总DNA中扩增并克隆了一段DNA片段。将该片段与报告基因GUS基因融合,构成植物表达载体。经农杆菌转化烟草萌发花粉,共培养约24h后,作瞬时表达检测。结果表明,该克隆片段具有启动子功能。DNA序列分析表明,该克隆片段长度为526bp,含有TATA盒及CAAT盒。与原基因（PSI）启动子序列比较,同源性为52％。部分顺式调控元件相同。     

斑马鱼Ngly1的生物学特性及克隆表达研究

为研究斑马鱼ngly1基因的功能,我们利用序列比对、同源建模和系统发育分析,发现斑马鱼Ngly1蛋白与其哺乳动物同源物在进化上高度保守,并且其三级结构与小鼠Ngly1十分相似。通过基因克隆获得了斑马鱼ngly1基因的全长cDNA,转染HEK293T细胞后观察到分子量约98kD的GFP-Ngly1融合蛋白。通过荧光定量PCR技术对斑马鱼胚胎发育和不同组织中ngly1的表达量进行分析,结果显示斑马鱼ngly1 mRNA在胚胎发育的全过程中都可以检测到,包括二细胞期,24 h,48 h和72 h,并且在胚胎发育第5天时达到高峰。在成鱼中,相比于其他组织,ngly1在卵巢和睾丸中高表达,在肝脏中低表达。     

Cloning and roles of goldfish maternal factor β-Catenin cDNA in embryonic development

Interaction between nucleus and cytoplasm has been focused in the field of animal embryonic development, in which study of maternal factors is required positively. β-Catenin, an important maternal factor in early embryogenesis, has been analyzed in its expression pattern and functions in this paper. We have cloned goldfish β-Catenin cDNA gene and compared it with zebrafish β-Catenin cDNA. High homology was found in cDNA and in amino acid sequences between them, 93% (2227/2384 bp) and 98.5% (768/780 aa) respectively. The expression pattern of β-Catenin by in situ hybridization and the roles of β-Catenin on embryonic development by co-injection of anti-sense RNA and reporter gene, EGFP have been investigated in the whole process of goldfish embryonic development. The results suggest that β-Catenin presents dynamic distribution, mainly locates at body axis, dorsal tissues, head and tail structures after being fertilized. The loss of β-Catenin activity would cause serious destruction of embryo in dorsal tissues and in anteroposterior axes, and leads embryos to die before larva get hatched.     

Expression of genes of the T-cell antigen receptor complex in precursor thymocytes

The antigen receptor on T lymphocytes has recently been characterized as a heterodimeric, transmembrane glycoprotein consisting of disulphide-linked alpha (acidic) and beta (basic) subunits of relative molecular mass (Mr) 40,000-45,000 each. The genes encoding these proteins have been cloned and shown to resemble immunoglobulin genes in both overall structure and the requirement for DNA rearrangement before expression. In humans, three additional proteins, termed the T3 complex, are found associated with the clonotypic receptor, and a role for T3 in receptor expression has been proposed. Despite these recent advances in characterizing the antigen receptor complex, there is as yet little understanding of T-cell maturation, particularly the stage of T-cell ontogeny at which the genes encoding the antigen receptor and its associated structures are expressed and assembled. In the adult, stem cells destined to differentiate into T cells arise in the bone marrow and migrate to the thymus, where T-cell precursors proliferate, develop a preference for recognizing antigens in the context of self MHC molecules and are released to the periphery. Recently, cells that have the properties of immature murine thymocytes have been isolated and described. We have now analysed these cells with a series of molecular probes and we describe three distinct patterns of T-cell antigen receptor gene rearrangements in developing thymocytes.     

Functional and molecular organisation of an antigen-specific suppressor factor from a T-cell hybridoma

Thymus-dependent (T) lymphocytes have been shown to have antigen specificity. The antigen receptor on T lymphocytes, in contrast to that on B lymphocytes, does not appear to be of the conventional immunoglobulin (Ig) type. Studies on the antigen-specific factors derived from helper and suppressor T cells (Ts) demonstrated that they possess determinants with antigen binding affinity and products of genes in the H-2 complex (MHC). Furthermore, antibodies against the variable region of Ig heavy chains or idiotypes have been shown to react with T-cell antigen receptors as well as antigen-specific helper and suppressor T-cell factors (TsF). It is, therefore, conceivable that at least two gene products are involved in the structural entity of these receptors: one each coded for by genes in either. To establish the molecular nature of the recognition component of T cells we have used homogeneous TsF from a T-cell hybridoma with a specific function. We report here that the antigen binding and I-J coded molecules on TsF are independently synthesised in the cytoplasm, and are secreted as an associated form of the two molecules; this association is required for antigen-specific suppression of antibody response.     

Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication

Dissection and reconstitution of the adenovirus DNA replication machinery has led to the discovery of two HeLa nuclear proteins which are required in conjunction with three viral proteins. One of these, nuclear factor I (NF-I), recognizes an internal region of the origin between nucleotides 25 and 40 and by binding to one side of the helix stimulates the initiation reaction up to 30-fold. NFI-binding sites have been observed upstream of several cellular genes, such as chicken lysozyme, human IgM and human c-myc, and coincide in most cases with DNase I hypersensitive regions. Here we report the identification of a novel DNA-binding protein from HeLa nuclei, designated NF-III, that recognizes a sequence in the adenovirus origin very close to the NFI-binding site, between nucleotides 36 and 54. This sequence includes the partially conserved nucleotides TATGATAATGAG. NF-III stimulates DNA replication four- to sixfold by increasing the initiation efficiency. Potential cellular binding sites include promoter elements of the histone H2B gene, the human interferon beta gene, the human and mouse immunoglobulin VK and VH genes and the mammal/chicken/Xenopus laevis U1 and U2 small nuclear RNA genes. Furthermore, a subset of the herpes simplex virus immediate early promoter specific TAATGARAT elements is homologous with the adenovirus 2 (Ad-2) NFIII-binding site.