红斑丹毒丝菌C43065株spaA基因的克隆和表达

通过PCR从红斑丹毒丝菌C43065株基因组DNA中扩增出编码信号肽除外的成熟SpaA蛋白基因spaA,将其克隆到表达载体pET32a的BamHⅠ和Hind Ⅲ位点上,构建重组表达质粒pET-spaA,转化大肠杆菌BL21,在IPTG诱导下表达N端带有Trx标签的融合蛋白rSpaA,SDS-PAGE检测表达蛋白.DNA测序结果表明,spaA基因大小为1794 bp,编码由597个氨基酸残基组成的成熟SpaA蛋白,SDS-PAGE结果显示在大肠杆菌BL21中成功表达了分子量约为86 kDa的重组rSpaA,为进一步开展SpaA保护区域的研究奠定基础.     

鲈鱼cxcr4 cDNA克隆和序列分析

趋化因子受体(cxcr4)是一类重要的免疫调节因子受体,对原始生殖细胞的迁移和存活具有十分重要的作用。在对脊椎动物cxcr4进行初步生物信息学分析基础上设计引物,采用RT-PCR和RACE相结合的方法从鲈鱼卵巢克隆了cxcr4a全长和4b部分cDNAs序列,并预测其编码的氨基酸序列。结果表明,由于鱼类基因组经历一次特有的复制,cxcr4在鱼类存在两个复制基因。鲈鱼cxcr4a cDNA全长为1432 bp,编码311个氨基酸;cxcr4b部分cDNA片段长为905 bp,编码285个氨基酸。将鲈鱼cxcr4序列与所有已克隆的硬骨鱼类进行同源性比较,结果显示硬骨鱼类保守性较高,所有已经克隆的cxcr4按照进化地位的不同成簇聚类,表现出了较高的同源性。     

甲/戊型肝炎病毒重组抗原基因的克隆及表达

 将甲肝病毒vp1编码基因(aa 24~171)和戊型肝炎病毒ORF2基因(aa 431~615)通过一段编码柔性甘氨酸链(Gly4 Ser Gly4)的基因片断连接,获得编码HAV/HEV融合蛋白基因(AEAg342).将该融合基因插入质粒pBV220,构建表达质粒pBV220-AEAg342.将pBV220-AEAg342转化入大肠杆菌BL21中进行表达,表达量约占菌体总量的20%,经离子交换层析纯化,目的蛋白纯度达90%以上.Western blot证实该蛋白能与甲肝及戊肝患者阳性血清发生特异性反映,为进一步开发双价疫苗和联合诊断试剂奠定了一定基础.     

粘质沙雷氏菌抗铜基因的克隆及性质研究

 通过构建粘质沙雷氏菌KMR-3菌株的基因组DNA文库,克隆到了与该菌的铜抗性相关的基因,并对其部分特性进行了研究.结果表明克隆到的铜抗性基因所编码的蛋白属于CutF蛋白,由194个氨基酸编码,与莫氏耶尔森氏菌(Yersinia mollaretii)ATCC43969抗铜脂蛋白NlpE同源性最高,达到70%,并对该基因的调控序列(启动子、终止子、SD序列及转录起始位点)进行了分析.     

水稻几丁质酶基因(Oschi)cDNA的克隆及序列分析

 以成功克隆大蕉几丁质酶基因（MpChi-1）时设计的一对引物,采用RT-PCR技术从广亲和水稻品种（Oryza．satival L．Cpslo17）中扩增到一水稻几丁质酶的全长cDNA,长1 115 bp,命名为Oschi;此序列已在GenBank中注册,登录号为EU045451;将Oschi基因核苷酸测序结果在NCBI服务器上用BLAST搜索软件进行同源性基因检索;并将Oschi的cDNA序列及氨基酸序列与大蕉及其它单子叶植物的相应序列进行多重序列排列比较并构建系统树;结果显示此基因具有编码Ⅰ类几丁质酶的基本结构;与大蕉核苷酸序列相比同源性为99%,氨基酸序列同源性为99%,与预测登记的最相近的水稻［Oryza sativa(japonica cultivar-group)］核苷酸序列相比同源性为98%, 氨基酸序列同源性为98%;广亲和水稻Oschi基因与大蕉MpChi-1基因的序列及蛋白结构有较高的一致性。     

硬盘多播克隆技术与硬盘保护软件在机房维护中的应用

现在的计算机系统越来越大,如何快速安装与恢复系统就成了机房维护的关键。本文详细介绍了在网络环境下仅用U盘实现硬盘多播克隆技术方法和使用硬盘保护软件保护系统的方法,可以使机房的维护工作达到事半功倍的效果。     

Cloning and expression pro?les of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.)

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identi?cation, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame (ORF) encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except TaWRKY10 which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids. 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sar-coplasmic/endoplasmic reticulum.A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pead oyster.This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata,namely PCRT.PCRT encodes a deduced 414-amino acid protein,which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL.The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species.Semi-quantitative RT-PCR indicates that PCRT is ubiquitously ex-pressed in all tissues tested with the highest expression in the hemolymph and the mantle.In situ hybridiza-tion analysis of PCRT in the mantle showed strong signals in the inner fold,the inner side of middle fold,and the inner side of outer fold of the mantle epithelium.All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.     

Cloning and expression pro?les of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.)

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identi?cation, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame (ORF) encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except TaWRKY10 which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids. 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.