油桐尺蠖核型多角体病毒DNA聚合酶基因的克隆和序列分析

用PCR方法从油桐尺蠖核型多角体病毒(BusuNPV)中扩增出DNA聚合酶基因片段,经pGEM-T载体克隆到大肠杆菌DH5α菌株中。经自动序列分析仪测出DNA聚合酶基因2379bp长的核苷酸序列,推导出793的氨基酸序列。氨基酸同源性比较显示,BusuNPV与HzSNPV的同源性最高,达57%;与OpMNPV的同源性最低,为39.6%。     

吸血诱导的埃及伊蚊海口株后期胰蛋白酶基因测序及与美国株同源性比较

应用逆转录-聚合酶链式反应（RT-PCR）技术从吸血后24 h埃及伊蚊海口株总RNA中扩增出了后期胰蛋白酶编码区cDNA序列。采用自动DNA分析仪进行序列分析,并与已知埃及伊蚊美国株后期胰蛋白酶基因及推导的氨基酸序列进行了同源性比较。结果表明：埃及伊蚊海口株后期胰蛋白酶基因序列与美国株同源性达98%,有11个碱基发生变异;氨基酸同源性达99%,仅有3个氨基酸发生变异,但与催化位点密切相关的氨基酸及N末端氨基酸序列完全一致。以上结果显示,埃及伊蚊胰蛋白酶不同地理株间存在微小的差异。     

变色栓菌漆酶基因的克隆与序列分析

本研究以PCR扩增的方法,从变色栓(菌Trametes versicolor)的基因组DNA中扩增出了一预期大小的DNA片断,并将其克隆到了pUCM-T载体上。经筛选、酶切、PCR鉴定,序列分析,证明该片断为变色栓菌漆酶基因的克隆。该基因的开放阅读框由1560个核苷酸组成,编码一个由519个氨基酸组成的多肽。与GeneBank中发表的Laccase基因(AY081188)序列比较发现,编码的氨基酸序列同源性为96%,而核苷酸序列的同源性为92%。     

不同致病型马立克氏病病毒DNA聚合酶基因序列比较

为了分析马立克氏病病毒(MDV)致病型与其DNA聚合酶基因的关系,本研究比较了9个不同致病型的该基因的同源性关系,这包括四种不同致病型的国际参考株即弱毒疫苗株CVI988/Ripens株、强毒株GA、超强毒株Md5和特超强毒株648A;中国疫苗株814、中国强毒参考株.Jing-1及3个中国野毒株.结果表明MDV的DNA聚合酶基因非常保守,在比较的9个毒株间,该基因上游约369个碱基的调控序列的同源性在96.7%～100%之间,该基因编码的1220个氨基酸序列的同源性在99.2%～100%之间.尽管不同毒株在一些位点上出现了氨基酸的变异,但这些变异与病毒的致病型或地域分布没有明显的关系.     

枳壳CBF转录激活因子基因片段的克隆

根据不同植物CBF同源基因的保守区设计合成简并引物,采用PCR技术首次从枳壳基因组中分离出一个DNA片段并克隆到pMD18-T载体中.序列测定和分析表明,该片段长464bp,与拟南芥3个CBF基因的核酸序列及其推导的氨基酸序列分别具有79%和67%～69%的同源性,而且推导的氨基酸序列含有同源性更高的AP2DNA结合域和CBF蛋白的两段特征序列PKK/RPAGRxKFxETRHP和DSAWR.结果 表明,本研究克隆的片段为枳壳CBF基因片段.     

蓝舌病毒血清5型毒株S7基因编码区的分子克隆与序列分析

目的:对蓝舌病毒(BTV)血清5型毒株(BTV-5)的S7基因编码区(ORF)进行克隆和序列分析。方法:用TRIzol LS试剂提取病毒总RNA,经反转录-聚合酶链反应(RT-PCR)扩增BTV-5型毒株S7基因的编码区,将扩增片段克隆到pGEM-T Easy载体上,对阳性克隆进行核苷酸序列测定;采用DNAStar和DNASIS v2.5软件对环状病毒属不同种群的S7基因ORF序列及其推导的氨基酸序列进行同源性及系统进化树分析。结果:克隆的基因片段长1050bp,为S7基因开放性读码框的全长序列,编码349个氨基酸残基;与环状病毒属不同血清型毒株比较,核酸序列同源性范围为42.2%～96.6%,推导的氨基酸序列同源性范围为40.4%～99.7%。结论:蓝舌病毒与非洲马瘟病毒、鹿流行性出血热病毒分属于不同种群,群内不同血清型的S7基因ORF序列及其推导的氨基酸序列显示出很高的同源性,而不同种群之间的同源性很低。     

柞蚕核型多角体病毒泛素类似基因的克隆与序列分析

从感病的柞蚕Antheraea pernyi蛹中分离纯化柞蚕核型多角体病毒 (ApNPV),提取基因组DNA,分别构建ApNPV DNA的HindⅢ和SalⅠ酶切片段文库。对基因文库中1个克隆进行序列分析,得到1个长度为321 bp的序列,其中包含一个编码76个氨基酸的开放阅读框,预测的分子量为8.46 kD,系泛素类似基因。在读码框的上游调控序列中,具有典型的晚期基因启动子序列ataag。氨基酸序列同源性分析结果表明,ApNPV与黄杉毒蛾Orgyia pseudotsugata核型多角体病毒 (OpNPV)的同源性最高 (96.1%),与苜蓿尺蠖Autographa californica核型多角体病毒 (AcNPV) 的同源性为86.8%,与棉褐带卷蛾Adoxophyes orana 颗粒体病毒 (AoGV) 的同源性最低（71.1%）,但与人类、线虫和酵母的泛素同源性分别为77.6%、76.3%和76.3%。一些氨基酸残基在真核生物中保守,在杆状病毒中不保守,个别氨基酸残基是杆状病毒所特有的,这些氨基酸序列的改变对杆状病毒泛素基因的作用有待进一步研究。     

甘蔗乙烯合成酶基因家族三个成员的克隆与序列分析

ACC（1-aminocyclopropane-1-carboxylic acid）合成酶是高等植物乙烯生物合成途径中的限速酶.根据已克隆的植物ACS（1-aminocyclopropane-1-carboxylic acid synthase）基因同源序列,设计简并引物,以甘蔗叶片总DNA为模板,通过PCR扩增,得到3条特异性强的扩增片段：Sc-ACS1为1 041 bp、Sc-ACS2为1 345 bp和Sc-ACS3为1 707 bp.将序列在GenBank核酸数据库进行同源性搜索,结果表明,3个片段均为ACS基因,推导编码的蛋白质序列分别包含326、242和310个氨基酸.其中,Sc-A CS1和Sc-ACS3同源性最高,核苷酸序列和蛋白质氨基酸序列分别有98%和96%同源,与禾本科植物玉米Zm ACS6、水稻OS-ACS2、毛竹等ACS基因家族也有很高的同源性,核苷酸序列同源性为88%-98%,蛋白质氨基酸序列同源性为73%-81%.甘蔗Sc-ACS2与水稻OS-ACS5在核苷酸和氨基酸序列上分别有91%和79%同源性,但与甘蔗Sc-ACS1和Sc-ACS3基因成员之间,氨基酸同源性分别只有45%和49%.系统进化分析表明,Sc-ACS1和Sc-ACS3基因与玉米Zm ACS6基因亲缘关系最近,而Sc-ACS2基因与水稻OS-ACS5基因亲缘关系最近.Southern杂交表明三基因在基因组中确实存在而且是多拷贝基因.三个片段已在GenBank数据库中注册,注册号分别为AY620985、AY620986和AY788919.     

杨树叶绿体3′rps12基因,rps7基因和ndhB基因片段的克隆及序列分析

通过烟草叶绿体相关序列设计引物 ,从杨树的叶绿体基因组中克隆出 2个相邻的DNA片段 .经分析发现 ,这 2个DNA片段包含核糖体蛋白 3′rps12、rps7基因和NADH脱氢酶第二亚基ndhB基因片段 .利用DNAMAN等软件 ,将扩增到的杨树叶绿体DNA片段与烟草、拟南芥、玉米和黑松的相关序列进行比较 ,证实所扩增的片段具有较高的保守性 ,尤其是在这 3个基因的编码区 ,同源性均在 90 %以上 .插入或缺失常发生在基因间隔区 ,同源性在 80 %左右 ;对其编码区所推导的氨基酸序列进行了比较 ,同源性均在 92 %以上 .首次克隆了杨树叶绿体的部分DNA序列 ,详细报道并分析了杨树 3′rps12、rps7基因和ndhB基因片段及其边界序列信息 .所报告的基因序列均已登录GenBank .     

猪水肿病大肠杆菌志贺样毒素Ⅱ型突变体新基因的分子特征

从仔猪水肿病样品中分离的一株大肠杆菌菌株NP9621,抽提染色体DNA,构建NP9621的总DNA文库,经菌落原位杂交筛选、限制酶酶切和Southern印迹等分析,获得含有志贺样毒素基因的重组质粒,核苷酸序列分析表明, 该基因的 A亚基与SLTIIes的同源性为97.6%～98.1%,与SLTIIc、SLTIId及EHEC O157:H7产生的SLTII 的A亚基之间的同源性分别为93.1%、93.0%和92.9%;B亚基与SLTIIes的同源性为99.62%～100%,与SLTIic、SLTIid及SLTII的同源性分别为81.5%、81.1%和81.5%;与29种已知序列的SLTII 、SLTI及志贺毒素STX进行聚类分析,结果证实大肠杆菌NP9621菌株中的志贺样毒素基因属于一种新的SLTIIe亚型。SLTIie/NP9621的A亚基与其它SLTIIe之间存在7～9个氨基酸的差异,B亚基的氨基酸序列完全相同,与A亚基毒力活性密切相关的氨基酸分别为Thr4、Glu167、Arg170和Arg176。     

小花棘豆(Oxytripis glabra DC)毒素基因的cDNA克隆及序列测定

在完成小花棘豆毒素 95 %氨基酸序列的基础上 ,根椐已知的氨基酸序列 ,设计合成了特异简并引物 .以小花棘豆总RNA为模板 ,逆转录合成cDNA第一链 ,用置换法合成双链cDNA .用特异引物对此双链cDNA进行PCR扩增 ,将扩增后的目的基因与用SmaⅠ酶切的质粒pUC 18连接 ,转化大肠杆菌JM10 7.筛选阳性克隆进行序列分析 ,获得了OXY基因的全部序列 .经测序后测得基因序列与原氨基酸序列对照完全一致 .GenBnak数据检索说明 ,OXY基因编码序列确定是一个从未报道的序列 .此研究结果对该毒素的应用研究奠定了基础 .     

豌豆外源凝集素基因的克隆及序列分析

从豌豆幼叶分离基因组ＤＮＡ,设计特异引物,用聚合酶链式反应方法扩增出豌豆外源凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＥｃｏＲＶ位点。进一步亚克隆至ｐＵＣ１９。序列分析表明,克隆到的片段大小为８３２ｂｐ,包含了豌豆外源凝集素基因完整的编码序列。该基因无内含子,同报道的已知序列相比,其核苷酸序列及推测的氨基酸序列的同源率分别为９９．６％和９８．９％。     

A novel ochre mutation in the beta-thalassemia gene of a Thai. Identification by direct cloning of the entire beta-globin gene amplified using polymerase chain reactions

The beta-globin genes from a Thai patient compound heterozygous for beta-thalassemia and HbE disease were investigated. The 3.0-kilobase fragment containing the entire beta-globin gene was amplified by polymerase chain reaction, using Taq DNA polymerase followed by direct cloning of the amplified product into plasmid DNA. Sequence analysis of the thalassemia gene revealed only one base change, a C-A transversion within codon for an amino acid 35. This new mutation creates a premature terminator, TAA, an ochre codon, and results in a beta 0-thalassemia phenotype. The same result was obtained when this mutation was analyzed using a conventional cloning technique, direct sequencing of the amplified product, and hybridization with allele-specific oligonucleotide probes. No misincorporation was detected in the sequence analysis of the 3.0-kilobase insert of five clones of the amplified products obtained from genomic DNA of a normal individual. This approach is a rapid and accurate method for molecular cloning of the beta-globin gene and also other genes, the partial nucleotide sequences of which are known.     

Insertional inactivation of an Escherichia coli urease gene by IS3411.

Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.     

淋巴细胞存在有δ阿片肽受体的分子证据

以小鼠的成神经细胞瘤和大鼠神经胶质瘤的杂交细胞δ阿片肽受体的基因外显子Ⅲ序列为依据合成引物,应用ＲＴ－ＰＣＲ方法扩增人外周血淋巴细胞ｍＲＮＡ的一片段ｃＤＮＡ,扩增产物经纯化后进行核苷酸序列测定．测序结果表明该片段与杂交瘤细胞的δ阿片肽受体基因序列相比有５个同源区,其中碱基的同源性达６３％．实验结果从分子水平表明了人淋巴细胞表面存在有阿片肽受体的可能性．     

中国牛朊病毒基因的克隆和序列分析

从中国牛外周血中分离淋巴细胞,提取基因组DNA.用所设计引物以聚合酶链式反应扩增出不致病的朊病毒蛋白(PrP~c)基因,并克隆到pGEM-Teasy Vector.序列分析表明所克隆的牛PrP~c的片段大小为795bp,包含了牛朊病毒基因的完整编码区序列.该基因无内含子,同国外报道的已知序列完全相同.     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.     

Complete sequence of the coding region of human elongation factor 2 (EF-2) by enzymatic amplification of cDNA from human ovarian granulosa cells

The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starting with total double-stranded cDNA from human ovarian granulosa cells. The amplified DNA fragment with a length of 1765 bp was restricted and sequenced by the shot gun approach. From the sequences obtained from the amplified fragment and the cDNA insert of pHGR81 [Rapp et al. (1988) Biol. Chem. Hoppe-Seyler 369, 247-250] respectively, the DNA sequence containing the complete coding as well as the 3'-untranslated region was assembled.     

Monoacylglycerol lipase from moderately thermophilic Bacillus sp. strain H-257: molecular cloning, sequencing, and expression in Escherichia coli of the gene

Monoacylglycerol lipase [MGLP, EC 3.1.1.23] is produced intracellularly by the moderately thermophilic Bacillus sp. strain H-257. The gene encoding MGLP was cloned, sequenced, and expressed in Escherichia coli. A genomic library of Bacillus sp. strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino acid sequence of a purified MGLP. The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp. strain H-257 DNA. Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa. The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved among lipases, esterases, and serine proteases. The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology). When pMGLP31 was introduced into E. coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greater extent than Bacillus sp. strain H-257. The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.