鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

NF-κB决定小鼠胎肝激酶-1基因启动子的基本活性

为克隆小鼠胎肝激酶-1（fetal liver kinase-1,FLK-1）基因上游启动子序列,并观察其不同截短片段在小鼠血管内皮细胞中的启动子活性,以小鼠全基因组为模板,通过PCR扩增方法获得-258～+299 bp、-96～+299 bp、-71～+299 bp、-36～+299 bp大小的FLK-1启动子片段,将其定向克隆入pGL3 Basic,构建荧光素酶报告基因载体,并制备NF -κB结合位点的突变或缺失体.在阳离子脂质体介导下,报告基因载体瞬时转染小鼠血管内皮细胞株SVEC 4-10.结果发现,在小鼠血管内皮细胞中,各FLK-1启动子片段均有活性;－71～－36 bp区存在FLK-1启动子的核心调控元件.针对该区域NF-κB结合位点进行突变或缺失,能导致启动子活性显著降低;凝胶电泳迁移率实验表明该区段能结合转录因子NF-κB.结果提示,成功克隆了在血管内皮细胞中具有活性的FLK-1上游启动子序列,NF-κB是决定其基本活性的重要转录因子,为进一步研究FLK-1基因的转录调控机制奠定了基础.     

构建重组系统分析回文结构对基因表达的影响

回文结构序列是最常见的一种DNA序列,它是许多基因表达调控因子的顺式作用元件。本文设计了不同长度的回文结构序列,构建了Cre\|loxP重组系统,通过共转化含有cre基因的质粒和含有loxp位点的质粒,观察共转化菌株质粒图谱的变化,研究了重组系统在细菌中对基因表达的作用,同时研究不同长度回文结构对基因表达的影响,结果发现长度在20bp以下回文序列对下游基因的表达没有影响,而长于34bp的回文结构序列则会抑制下游基因的表达。     

水稻minE基因的克隆及分析

 植物叶绿体与原核生物分裂机制相似,其中MinE蛋白在细菌分裂过程中具有重要作用. 为了研究植物MinE蛋白在叶绿体分裂过程中的功能及其进化,利用RT PCR技术克隆了水稻叶绿体分裂相关基因OsMinE,并在GenBank登录（No. AY496951）.OsMinE基因cDNA全长1 035 bp,其ORF为711 bp,编码236个氨基酸.与原核生物MinE蛋白相比,水稻OsMinE具有明显延伸的N端与C端.其N端102个氨基酸残基为预测的叶绿体导肽序列,C端延伸保守,推测赋予植物MinE蛋白新的功能.植物minE基因结构分析显示,水稻、拟南芥、杨树都仅含有1个内含子,且插入位置及相位相同.这表明,该内含子可能在单子叶、双子叶植物分化前产生.水稻OsMinE基因在大肠杆菌细胞中的表达严重影响了细胞的分裂,初步证明了水稻MinE蛋白与原核细胞MinE蛋白功能类似.水稻OsMinE基因的克隆为进一步研究叶绿体的分裂机制奠定了基础.     

康氏木霉纤维素酶转录因子ACEII与外切纤维二糖水解酶基因 I（cbh1）启动子的结合分析

ACEI、ACEII和Xyr1是康氏木霉中调控纤维素酶基因表达的转录因子。体外实验已证实ACEI和Xyr1可与cbh1启动子上的287bp序列（-304bp~-18bp）结合从而调控cbh1基因转录,但ACEII是否可与此序列结合仍未清楚。为进一步研究ACEII调控纤维素酶基因表达的机制,利用PCR技术扩增康氏木霉ACEII DNA结合区的基因序列,并使其在大肠杆菌中表达。凝胶迁移率移动试验表明ACEII DNA结合区不能与cbh1启动子的287bp序列结合。提示了康氏木霉cbh1基因在诱导表达时起调控作用的主要是Xyr1,而不是ACEII。这对阐明真菌纤维素酶基因表达调控的分子机制具有重要的意义。     

人HAS3启动子的结构与功能初步分析

透明质酸合酶3（hyaluronan synthase 3,HAS3）,是一个参与透明质酸合成的酶分子,在上皮形成和肿瘤转移等过程中起重要作用.为了进一步研究HAS3基因的转录调控机制,本研究克隆鉴定了HAS3基因的启动子.首先应用5′RACE（rapid amplification of cDNA ends,cDNA末端快速扩增）技术鉴定了HAS3基因的转录起始位点,发现了丰度和转录起始位点不同的两种新的HAS3基因剪接变异体.通过PCR定向克隆策略,构建了覆盖HAS3基因5′端侧翼区起始密码子ATG上游约4.3 kb区域的一系列HAS3基因启动子荧光素酶报告基因重组体.启动子活性分析表明,HAS3基因启动子定位于转录起始位点附近约450 bp的区域内.转录因子结合位点分析表明,HAS3基因启动子缺乏典型的TATA盒,但含有典型的GC盒以及C/EBP等其它潜在的转录因子结合位点. 结果提示,Sp1和C/EBP等转录因子可能参与HAS3基因的转录调控.     

RNA聚合酶σ38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3.1kb EcoRⅠ_XhoⅠ片段。通过抗性基因（抗庆大霉素基因）的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪_1_羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10.2μg/mL分别减少到20.4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0.5μg/mL和20.5μg/mL分别提高到75.4μg/mL和185.6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β_半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果: σ38亚基正调控吩嗪_1_羧酸的表达,而负调控藤黄绿脓菌素的表达。     

球孢白僵菌羧基转运蛋白基因BbJEN1及其上游序列的克隆与分析

在分析一株球孢白僵菌TDNA插入突变体T12的Tagging序列的基础上,根据与其具有高度同源性的一条金龟子绿僵菌EST序列（编号为AJ273226）设计简并引物,用YADE法从球孢白僵菌中扩增出该EST的同源序列及其延伸序列。序列分析表明,该片段与粗糙脉孢霉的羧基转运蛋白JEN1具有高度同源性,由此确定该序列为球孢白僵菌羧基转运蛋白JEN1基因的部分序列。然后利用YADE法延伸扩增该序列的上、下游序列,获得球孢白僵菌羧基转运蛋白JEN1的全长DNA序列,命名为GBbJEN1。利用3′RACE扩增出球孢白僵菌羧基转运蛋白JEN1的cDNA序列,命名为BbJEN1。BbJEN1全长1656bp,编码514个氨基酸的蛋白。推测蛋白分子量为55975.37Da,等电点9.32。氨基酸序列与金龟子绿僵菌、粗糙脉孢霉和酿酒酵母羧基转运蛋白JEN1的同源性分别为77%、66%和30%。序列分析表明,GBbJEN1含有2个内含子。Southern杂交表明,GBbJEN1基因在球孢白僵菌基因组中为单拷贝。利用RTPCR法对BbJEN1的表达特性进行了分析,结果发现BbJEN1基因的转录受蟑螂壳、蝉蜕等昆虫体壁的诱导,受葡萄糖的抑制。进一步利用YADE法获得了长为977bp的GBbJEN1上游序列,其中含有可能的葡萄糖抑制调控序列和压力反应元件。     

水母雪莲Myb转录调控因子SmP基因的克隆及序列分析

采用TDPCR(Touch down PCR)法从水母雪莲(Saussurea medusa Maxim)红色系愈伤组织cDNA文库中筛选并克隆了雪莲Myb转录调控因子SmP (S.medusa Maxim Mybrelated P gene)基因。序列分析表明该基因全长969bp,包括一个771bp的完整开放阅读框架（ORF）,编码一个256氨基酸残基的蛋白质。氨基酸序列的同源性分析表明在N-端具有两个典型的R2R3-Myb-DNA结合结构域。C-端富含亲水的丝氨酸S(18.38%),且以寡聚体的形式存在,具有转录调控因子激活结构域常见的特征。     

Expression of the human cancer/testis antigen NY-SAR-35 is activated by CpG island hypomethylation

The novel cancer/testis antigen gene, NY-SAR-35, is expressed exclusively in normal testis and in various histological types of tumor. However, the NY-SAR-35 gene expression is observed to be aberrant in several cancer cell lines and tissues. The analysis of methylation status of the NY-SAR-35 gene promoter in various cancer cell lines showed that its expression was related to methylation of the promoter region. Treatment of human cancer cell lines with the demethylating agent 5-aza-2′-deoxycytidine activated the expression of the NY-SAR-35 gene. In addition, transfection experiments on various fragments of the CpG-rich gene promoter indicate that in vitro methylation of the NY-SAR-35 gene promoter results in the loss of promoter activity. The expression of NY-SAR-35 is therefore activated by hypomethylation of the CpG island in the gene promoter.     

The gene S promoter of hepatitis B virus confers constitutive gene expression.

The properties of the promoter of the hepatitis B surface antigen (HBsAg) were studied using recombinants containing either this promoter or the SV40 early promoter. Mouse L cells were transfected with these recombinants and the levels of gene expression obtained with the two promoters were compared. The level of expression of a cellular gene, the human fibroblast interferon gene, obtained with the HBsAg promoter was comparable to that obtained with the SV40 early promoter. Similarly when the HBsAg gene was controlled by the SV40 early promoter the level of HBsAg synthesis is in the same range as that observed with its own promoter. Together these results suggest that although the HBsAg gene codes for a structural viral protein, its expression is constitutive as for an early gene. The implications of these observations on the synthesis of HBV particles in vivo are discussed.     

Comparison of promoter region constructs for in vivo intramuscular expression

BACKGROUND: High transgene expression is generally expected after gene transfer. However, different level, kinetics and localization of expression might be needed for relevant therapeutic applications. Former studies have compared various promoter regions driving gene expression leading to conflicting results. In the present work, two promoter families have been compared using the efficient in vivo intramuscular electrotransfer technique. METHODS: Three promoter regions were constructed by associating the strong ubiquitous cytomegalovirus (CMV) enhancer-promoter to its homologous intron A or to a heterologous intron, or to a hybrid intron. Promoter regions derived from the muscle creatine kinase (MCK) promoter were also studied. The expression of the same transgene (SeAP or neurotrophin-3) under control of these different promoters was compared after plasmid electrotransfer in mouse tibialis-cranialis skeletal muscle. RESULTS: Heterologous intron association to the CMV promoter did not modify gene expression kinetics nor increase gene expression level. Usefulness of intron A or hybrid intron association to the CMV promoter depended on the gene. The various MCK promoters drove efficient gene expression but lower than that obtained with the CMV promoter. Furthermore, peak value was reached earlier with MCK promoter regions (14 days). CONCLUSION: For applications of gene transfer restricted to skeletal muscle, the MCK promoter or a MCK promoter variant would be a promising alternative to the CMV promoter. Indeed, it has been demonstrated that the use of MCK promoter limits humoral and cell-mediated immune responses. Furthermore, the MCK promoter decreases the initial expression peak that may be detrimental, drives a sustained gene expression, and improves gene transfer safety.     

Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells

Recombinant baculoviruses have emerged as a new gene delivery vehicle for mammalian cells. Thus, a shuttle promoter that mediates gene expression in both insect and mammalian cells will facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. This study described the generation of three recombinant baculoviruses with an EGFP reporter gene under the control of the white spot syndrome virus (WSSV) ie1 promoter, or either of two control promoters, the baculovirus early-to-late (ETL) promoter and polyhedrin promoter. The resulting recombinant baculoviruses were used to infect insect Sf9 cells and transduce several mammalian cell lines to test the expression of EGFP. We found that the WSSV ie1 promoter displayed a strong promoter activity in both insect and mammalian cells, and showed a stronger promoter activity than the ETL promoter in some mammalian cell lines. The activity of the WSSV ie1 promoter, but not the ETL promoter, can be enhanced by sodium butyrate, a histone deacetylase inhibitor. A transient plasmid transfection assay indicated that the WSSV ie1 promoter activity in mammalian cells is independent of baculovirus gene expression, differing from the ETL promoter, which was shown to be baculovirus-dependent. This study demonstrates, for the first time, that the WSSV ie1 promoter can function as a baculovirus-independent shuttle promoter between insect cells and mammalian cells. This novel shuttle promoter will facilitate the application of baculovirus-based vectors in gene expression, gene therapy, and non-replicative vector vaccines.     

Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.     

斑马鱼心肌特异性启动子的克隆及其功能鉴定*

目的:探讨心室肌球蛋白重链(vmhc)基因启动子的心肌组织特异性.方法:利用PCR技术从斑马鱼基因组中克隆了vmhc编码区5’上游大小为1952bp的调控区域,应用酶切连接方法将vmhc启动子插入pGEFP-N1质粒,成功构建pEGFP-vmhc重组载体.再应用高保真DNA聚合酶PCR扩增包含vmhc启动子序列,增强型绿色荧光蛋白(EGFP)基因序列及3'UTR序列的基因片段,经过纯化后通过显微注射将vmhc-EGFP基因片段导入斑马鱼受精卵中.结果:注射后的斑马鱼心脏中出现绿色荧光,而其他部位无荧光出现.结论:vmhc启动子能够正确有效地驱动外源基因在斑马鱼心脏中特异表达,适合应用于心血管疾病的基因功能研究,基因靶向治疗等.     

家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants

The rice Ubiquitin1 (Ubi1) promoter was tested to evaluate its capacity to express the heterologous gusA gene encoding β-glucuronidase in transgenic rice tissue relative to the commonly used Ubi1 corn promoter and the rice gibberellic acid insensitive (GAI) gene promoter element. Experimental results showed increased expression of gusA gene in rice tissue when driven by the native Ubi1 promoter when compared to the use of corn Ubi1 promoter. Results further indicated that the cis-regulatory elements present in the native promoter element might have been responsible for high expression. However, the gusA gene expression level when driven by the rice GAI promoter was notably lower than both Ubi1 promoters. The present study, thus, for the first time helped to demonstrate that the native Ubi1 promoter is a promising genetic element in transgenic approaches for constitutive expression of any gene in rice tissue.