霍乱毒素A基因内部翻译调控元件具有翻译起始功能

通过大肠杆菌体外转录－体外翻译系统,证明霍乱毒素Ａ基因内部的翻译调控元件具有翻译起始功能,且其翻译起始效率较ｃｔｘＡ基因高得多,当ｃｔｘＡ的起始密码突变时,从该元件起始的翻译效率下降,说明基因内翻译起以ｃｔｘＡ翻译起始的调控。结果进一步证实了霍乱毒素Ａ、Ｂ亚工比例表达调控的翻译弱化－翻译偶联机理。     

受强力霉素调节的自杀基因表达载体系统的构建和在乳腺癌细胞株(MCF-7)的表达

自杀基因治疗是恶性肿瘤基因治疗中最常用的途径之一,以递转录病毒载体-pRevTRE为基础进行载体构建,首先使用PCR技术对单纯疱疹病毒胸苷激酶基因（HSVtk)进行扩增,将HSVtk基因插入到pRe-vTRE,形成重组载体pRevTRE/HSVtk,用磷酸钙共沉淀法,经过两轮转染,分别将pRevTRE/HSVtk和pRevTet-On质粒导入乳腺癌细胞株（MCF-7）经过潮霉素B（HygromycinB)和G418筛选,建立了一株稳定的受四环素衍生物-强力霉素（Doxycycline,Dox)调控,表达HSVtk基因产物的人乳腺癌细胞株MCF/TRE/tk/Tet-On,HSVtk基因表达产物可以将无毒性的药物前体Ganciclovir(GCV)转变成一种有毒的代谢产物,从而杀死乳腺癌细胞株（MCF-7）,达到基因治疗的目的。     

与实验条件相关的基因功能模块聚类分析方法

针对细胞内基因功能模块化的现象,定义了“基因功能模块”和“特征功能模块”两个概念,并基于这两个概念提出一种“与实验条件相关的基因功能模块聚类算法”。该算法综合利用基因功能知识与基因表达谱信息,将基因聚类为与实验条件相关的基因功能模块。向基因表达谱中加入水平逐渐升高的数据噪音,根据基因功能模块对数据噪音的抵抗力,确定最稳定的基因功能模块,即特征功能模块。加噪音实验显示,在基因芯片技术可能发生的噪音范围内,该算法对噪音的稳健性优于层次聚类和模糊C均值聚类。将模块聚类算法应用在NCI60数据集上,发现了8个与实验条件高度相关的特征功能模块。     

籼稻蜡质基因5＇上游区与GUS基因编码区融合基因的构建和在水稻中的瞬间表达

为了鉴定水稻蜡质基因５＇上游区的顺式作用因子与研究它们在组织专一性表达中的作用,我们对籼稻品种２３２的蜡质基因５＇上游－１１５至－２１２０的区域进行了顺序测定,并将此基因的５＇上游区同报告基因ＧＵＳ构建成融合基因,用基因枪粒子轰击的方法将此融合基因导入水稻未成熟种子的幼胚和糊粉层细胞中,瞬间表达检测的结果表明,我们构建的融合基因中蜡质基因５＇上游区的长度已足以使ＧＵＳ基因在上述组织中表达。     

三芒山羊草中新型储藏蛋白基因Avenin-like的克隆及分析

目的:克隆三芒山羊草(Aegilops neglecta)中新型avenin-like基因,揭示avenin-like基因的表达模式,并对克隆的基因进行相关的分析.方法:利用RT-PCR的方法揭示avenin-like基因的表达模式,并用PCR方法从三芒山羊草中克隆新型的ave-nin-like基因.结果:avenin-like基因在胚乳中特异性表达;克隆得到新型的avenin-like基因;avenin-like基因属于醇溶蛋白超基因家族,含19个半胱氨酸残基、形成8对分子内二硫键和3对分子间二硫键.结论:新型avenin-like基因的克隆为小麦品质改良提供了很好的基因资源.     

Molecular analysis of the folC gene of Pseudomonas aeruginosa

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase-dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co-transcribed.     

基因枪介导真核表达质粒转染体外培养的COS-7细胞系的实验研究

目的：建立基因枪子弹制备及转染体外培养COS-7细胞系的方法。方法：以亚精氨、氯化钙沉淀法制备子弹（DNA＋金颗粒）,利用原子力显微镜观察子弹制备情况;采用基因枪方法分别将真核表达质粒pVax-Dsred-IRES-EGFP转染对照组和实验组COS-7细胞,转染后24h,利用激光扫描共聚焦显微镜观察细胞中红、绿荧光蛋白的表达。结果：制备了基因枪子弹,DNA紧密包裹在金颗粒周围;基因枪介导的pVax-Dsred-IRES-EGFP被转染入体外培养的COS-7细胞,转染后24h可检测到红、绿荧光,而对照组则没有荧光蛋白的表达。结论：国产新芝SJ-500型基因枪能够有效介导外源基因转移,基因枪转染的COS-7细胞能够有效表达报告基因。     

葡萄球菌肠毒素A全长基因的克隆和序列测定

分析和克隆超抗原（SAg）葡萄球菌肠毒素A（SEA）全长基因,为进行SAg基因应用于肿瘤导向治疗和基因治疗的研究奠定基础。设计并合成一对针对SEA全长基因的特异性引物,用PCR反应从产SEA的标准葡萄球菌菌株的基因组中扩增出SEA全长基因。PCR产物与克隆载体pGEM-T连接后进行基因序列测定。成功地从标准葡萄球菌菌株的基因组中扩增出一条约770bp的条带。基因序列测定表明,与巳发表的SEA全长基国序列完全一致。     

利用套叠PCR技术进行基因突变和拼接

利用套叠PCR技术（又称重叠区扩增基因拼接法）对hGM-CSF基因内第28位氨基酸处的糖基化位点进行突变和进行人促性腺激素基因,腺苷酸激酶短肽与胰岛素样生长因子－基因三者之间的拼接,结果表明采用该技术能在体外实行有效的基因重组和定点突变,其成功率为100％,这一技术不需要内切酶消化和连接酶处理,技术操作员简单易行,在基因拼接,基因内部突变方面具有良好的应用价值。     

Role of gene order in developmental control of human gamma- and beta-globin gene expression.

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.     

人组织激肽释放酶基因在哺乳动物细胞中的表达

克隆人胰腺组织激肽释放酶基因 (hKK) ,构建融合荧光蛋白基因的真核表达载体 ,在CHO细胞中表达 ,为开发激肽释放酶基因工程产品以及开展基因治疗高血压研究奠定了基础。提取人胰腺组织总RNA后 ,RT PCR扩增KK ,构建中间载体KSKK。从KSKK中切出激肽释放酶基因 ,插入真核表达载体pEGFP C2 ,构建出激肽释放酶带有荧光蛋白报告基因的表达载体pEGC KK ,测序分析后转染CHO细胞 ,荧光显微镜观察激肽释放酶基因表达。并进行SDS PAGE及Westernblot分析。成功克隆激肽释放酶基因 ,并在CHO细胞获得表达 ,克隆的人组织激肽释放酶基因可用于激肽释放酶基因工程产品开发以及基因治疗研究。     

金鱼草AmPDS基因克隆及调节类胡萝卜素合成功能分析

八氢番茄红素脱氢酶(phytoene desaturase,PDS)是类胡萝卜素进行生物合成途径中的关键酶。为了深入探究金鱼草PDS基因的功能,该研究以‘马里兰’金鱼草(Antirrhinum majus‘Maryland True Pink’)为材料,对其PDS基因(AmPDS)全长序列及蛋白结构进行分析,并克隆了AmPDS基因片段;采用qRT-PCR技术检测AmPDS基因在不同时期及部位的相对表达水平,利用VIGS技术验证AmPDS基因功能,用紫外分光法测定叶片中各类色素含量。结果显示:(1)成功克隆AmPDS基因片段(500 bp);AmPDS基因cDNA全长1743 bp,编码580个氨基酸;其蛋白分子量为64.75 kD,理论等电点6.66;同源比对分析显示AmPDS基因与芝麻(Sesamum indicum)的序列相似性最高。(2)qRT-PCR分析表明,AmPDS基因在全株均有表达,且在全盛期花朵的上瓣和叶片中表达量最高。(3)构建pTRV2-AmPDS载体,建立了金鱼草的VIGS沉默体系,AmPDS基因沉默效率约为53%,与阴性对照相比叶片中各类色素含量均显著降低。研究认为,AmPDS基因是金鱼草类胡萝卜素生物合成途径中的关键基因,可作为金鱼草VIGS沉默体系的指示基因,为后续研究金鱼草其他基因功能奠定基础。     

Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG

The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.     

Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151.

The gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.     

利用Red重组系统敲除大肠杆菌 O157:H7的waaL 基因

目的:利用λ噬菌体Red重组系统敲除大肠杆菌O157:H7的waaL基因。方法:以pKD4为模板扩增出与waaL基因上下游同源的、含有卡那霉素抗性基因的PCR产物。然后电击转化到大肠杆菌 O157:H7 中,利用Red重组系统,通过卡那霉素抗性基因两侧的waaL基因序列在体内与waaL基因发生同源重组,置换了 O157:H7 基因组中的waaL基因。并进一步利用卡那霉素抗性基因两侧的FRT位点,通过FLP位点专一性重组将卡那霉素抗性基因敲除。结果:成功构建了敲除waaL基因且不带卡那霉素抗性基因的菌株。