结球甘蓝抗TuMV相关基因的克隆

以结球甘蓝高抗TuMV自交不亲和系84075为材料,构建了cDNA文库。根据抗病基因保守序列（NBS－LRR）设计一对简并引物,以84075的基因组DNA和cDNA为模板,进行PCR扩增,分别扩增出一条513bp片段,扩增片段进行克隆测序。选取两个与抗病基因同源性较高的克隆片段作探针（命名Borl,Bor2）,对构建的cDNA文库进行筛选,得到一个阳性克隆（命名TuR2）,测序及序列分析表明,该基因总长为762bp,编码226个氨基酸、包含681bp的开放阅读框。与已克隆的抗病基因有不同程度的同源性。利用TuR2作探针,进行了Southern杂交、Northern杂交以及抗病性的共分离检测分析。结果表明,TuR2可能吧单拷贝形式存在,其表达是组成成型的,且无组织特异性;初步确定是一个与结球甘蓝抗TuMV相关的基因。     

牛β-酪蛋白5′端上游调控序列的克隆和序列分析

该文用PCR扩增了牛β－酪蛋白基因5′－端上游调控序列,并对其进行了克隆和序列分析。采集成年母牛肝,提取DNA。在牛β－酪蛋白基因外显子1和上游调控区内设计引物,扩增其上游调控序列。两条引物长均为19个核苷酸,引物间跨度为635bp。以牛肝DNA为模板,进行PCR扩增,扩增产物在2％琼脂糖凝胶上电泳,可见特异的目的条带。从凝胶中回收目的片段,克隆到pGEM－T载体中。重组质粒提取DNA,进行序列分析。测序结果与文献发表的类似序列相比,仅有4个碱基不同,同源性达99．4％。表明获得了牛β－酪蛋白基因5′－端上游调控序列的克隆。     

嗜热菌Aquifex pyrophilus中mbh2基因簇的鉴定和部分基因的克隆及测序

基于嗜热菌Aquifex aeolicus的mbhS2基因,设计合成特异性引物,以Aquifex pyrophilus的染色体DNA为模板,应用PCR技术扩增出目的片段。序列测定结果显示,其推导出的氨基酸序列与A.aeolicus的相应序列具有85％的同源性。以此PCR片段为探针,从A.pyrophilus的Nco Ⅰ部分基因组文加（4－6kb)中筛选出含5kb大小插入片段的阳性克隆,进而对其进行了亚克隆及测序。结果表明,该插入了包含A.pyrophilus mbh2基因簇的小亚基基因mbhS2,orf1基因的部分orf2基因。所推导出的氨基酸序列与A.aeolicus的MbhS2和Orf963相比较的同源性分别为81％和60％。     

雄牛特异的SRY同源序列的扩增和分析

利用人、兔、鼠SRY序列设计引物,应用PCR扩增牛的SRY序列,获得200bp的雄牛特异的扩增片段。克隆该扩增片段,获得重组质粒pCH21,进行序列分析,并与人、兔和鼠SRY的对应区域比较,具有高度同源性。用pCH21 DNA作探针与牛的基因组DNA酶切图谱杂交,显示了雄牛特异的I．7kb的杂交带。分析200bp的PCR扩增片段是牛的SRY基因片段。用同一对引物扩增人和山羊的DNA样品,也获得雄性特异的200bp的扩增片段。     

利用聚合酶链反应扩增基因片段

聚合酶链反应(PCR)是一种模拟天然DNA复制过程的核酸扩增法,具有敏感特异、产率高、操作简单、容易自动化等特点。由于它有成指数倍(2~n)扩增靶序列的能力,又被称之为“无细胞分子克隆”或“试管内分子克隆”。我们以化学合成的寡核苷酸的粗提物为引物,含目的基因片段的重组质粒DNA为模板,利用PCR技术成功地扩增出数百至3544bp的特异性     

羊驼线粒体12srRNA/tRNA-Val/16rRNA 基因序列测定及分子进化研究

对GaneBank中登录的部分骆驼科动物线粒体12 srRNA/tRNA-Val/16 rRNA基因序列进行同源性比较,并借助DNAstar软件设计引物,扩增并进行序列分析,旨在揭示其遗传变异的规律。PCR条件优化后成功扩增出长度为1 014 bp的DNA片段,b lastn分析显示,该片断与骆驼科动物的同源性高于95%,所获得片断包括30bp的12 srRNA基因部分序列,71 bp的tRNA-Val基因全序列和913 bp的16 srRNA基因部分序列。利用RNA-structure 4.2 RNA分析软件绘制了部分骆驼科动物的线粒体tRNA-Val推导性二级结构图,比较结果显示,羊驼线粒体tRNA-Val氨基酸臂和反密码子环呈属间特异性遗传。     

猪FGL2基因cDNA末端序列检测及结构分析

检测猪FGL2基因cDNA末端序列并对该基因结构初步分析。α-32P dCTP放射性同位素标记cDNA探针筛选猪基因组DNA文库;cDNA末端快速扩增(rapid amplification of cDNA end,RACE)。以猪正常小肠及心脏组织提取新鲜总RNA,反转录后作为模板,设计基因特异性引物,采用Advantage 2 聚合酶混合物进行PCR扩增;依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物,以人FGL2基因3′末端序列设计下游引物,以猪基因组DNA为模板采用Advantage 2 聚合酶混合物进行PCR反应;PCR载体重组质粒DNA亚克隆扩增。同位素探针未能筛选到特异阳性克隆,RACE反应检测到特异性转录起始位置及第一个转录终止位置,但仍未检测到第二个转录终止位置。猪基因组DNA行PCR扩增成功检测到猪FGL2基因3′末端未知序列及第二个转录终止位置。     

猴血中SFV病毒基因PCR检测方法的建立及应用

针对猴泡沫病毒SFV（Simian Foany Virus）多聚酶区（pol区）设计两对引物,以猴血DNA为模板者嵌套式PCR扩增,得到500bp左右基因片段,克隆进入pUC-19载体,经测序鉴定为SFV465bp的pol区基因片段,将此段序列与SFV各型425bp的pol区基因片段进行同源性比较,它与SFV－1型的同源性最高,为92.00%。在此基础上,用这两对引物对158例猴血DNA进行检测,阳性54例,阳性率为34.2%,发现猴群中有较高的SFV病毒的感染。     

猪附红细胞体快速诊断方法的研究

浙江大学动物预防医学研究所侯玉存慧和浙江出入境检疫局蔡渭明等5位科研工作者根据GenBank上已报道的附红细胞体16S rRNA的序列设计一对特异性引物,进行PCR扩增,并将产物克隆到T-vector后测序,发现扩增到的目的基因片段为404bp,它与GenBank上MycoplasmaSuis序列同源性为97％。试验建立的PCR诊断方法特异性强,     

苦荞中查尔酮合成酶全长基因的克隆及其序列分析

选用乌克兰伊琳娜苦荞为试验材料,以从叶片中提取的RNA为模板,应用RACE技术结合CODEHOP引物设计方法成功克隆出苦荞中查尔酮合成酶cDNA序列后,将其序列电子合并其全长后设计基因全长特异性引物.以DNA为模板进行PCR扩增出基因序列.通过生物信息学分析表明,该基因金长为1 906 bp,具有一个463 bp的内含子序列,编码区长度为1188 bp,编码395个氨基酸.Blastn序列比对发现本试验所获得的CHS基因序列与相近物种Rheum palmatum(登录号:DQ205352.1)的CHS基因同源性达86％.将得到的序列命名为RaCHS并提交GenBank,登录号为HQ434624.应用Clustalxl.81和MEGA4软件构建系统进化树,并进行了同源性分析.     

Mitochondrial genes are found on minicircle DNA molecules in the mesozoan animal Dicyema

Animal mitochondrial DNA genomes are generally single circular molecules, 14-20 kb in size, containing a number of functional RNAs and 13 protein-coding genes. Among these, the COI, COII and COIII genes encode three subunits of cytochrome c oxidase. We have isolated and characterized these three mitochondrial genes from the mesozoan Dicyema, a primitive multicellular animal. Surprisingly, the COI, COII and COIII genes are encoded on three small, separate circular DNA molecules (minicircles) of length 1700, 1599 and 1697 bp, respectively. We estimated the copy number of each minicircle at 100 to 1000 per cell, and have shown a mitochondrial localization of the minicircles by in situ hybridization. Furthermore, we could not detect a putative "maxicircle" DNA molecule containing any combination of the COI, COII and COIII genes using either PCR or genomic Southern hybridization. Thus, our results show a novel mitochondrial genome organization in the mesozoan animal Dicyema.     

家鸭细胞色素C氧化酶Ⅲ（COⅢ）基因的克隆及序列分析

线粒体是存在于绝大多数真核细胞内的一种基本的、重要的细胞器,是细胞进行氧化磷酸化的场所。不同脊椎动物同源基因序列的比较显示,细胞色素b(Cytb)、细胞色素C氧化酶Ⅰ（COⅠ）、细胞色素C氧化酶Ⅱ（COⅡ）、细胞色素C氧化酶Ⅲ（COⅢ） 基因最保守,同源性最高,ATPase6、ATPase8基因、ND基因变异比较大。本试验以家鸭（家鸭起源于绿头鸭：Anas platyrhynchos）肝脏的线粒体DNA为模板,按照GenBank已经公布的潜鸭族（AF090337）的全序列及其绿头鸭的mtDNA部分序列（L22476、L16770、L22477）设计合成特异引物进行PCR扩增,克隆并测定了线粒体细胞色素C氧化酶Ⅲ亚基（COⅢ）的全序列784bp以及ATPase6基因的3'端和tRNA-Gly基因的5'端序列共934bp。 用DNAStar分析软件对家鸭与GenBank中7种禽类的COⅢ序列进行比较分析,显示家鸭与这些动物的COⅢ基因具有较高的同源性,与同科潜鸭属中的Aythya Americana的相似性 最高为90.6 %,与同目不同科的加拿大雁、小天鹅、白额雁的相似性分别为89%、88.6%、88.6%。根据家鸭与其他7种禽类的COⅢ基因序列相似性所建立的进化树,与传统的分类地位基本吻合。     

Detection of cariogenic bacterial genes by microchip electrophoresis

Allele-specific PCR primers were designed, based on the dextranase (dex) gene, to identify Streptococcus mutans and Streptococcus sobrinus in dental plaque; subsequently, PCR products were detected via microchip electrophoresis (ME). In order to amplify the dex gene fragment of S. mutans and S. sobrinus, the following two PCR methods were established. Duplex allele-specific PCR primers were designed on a region of low DNA homology; furthermore, 211 and 126-bp fragments were amplified for S. mutans and S. sobrinus, respectively. Common PCR primer for single allele-specific PCR was designed so as to sandwich a region exhibiting high homology and amplify PCR product of different DNA size due to deletion of small DNA fragment in two dex genes. S. mutans and S. sobrinus were amplified, leading to the generation of 202 and 226-bp products, respectively. Analysis of DNA base size by ME in order to achieve efficient separation employed a polymer mixture consisting of hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO). In the presence of a polymer mixture of 0.125% PEO/0.6% HPMC, two PCR products were obtained, displaying degree of separation of 226 bp/202 bp of 2.67 (Rs). Reproducibility (CV%, n = 7) was 0.3%; additionally, separation time was approximately 85 s. This method was applied to the detection of S. mutans and S. sobrinus in dental plaque. Detection of the dex genes of S. mutans and S. sobrinus characterized by quickness, precision and high sensitivity was possible.     

一种简便、快捷的胰蛋白酶抑制剂基因的分离与克隆方法

从 3个豇豆品种幼嫩叶片中分离出核基因组 DNA,参照已知的几种 Bowman-Birk型胰蛋白酶抑制剂基因序列 ,设计合成了 2 7bp,且含有 Bam H I位点的寡核苷酸引物 ,分别以 3种豇豆核基因组 DNA为模板 ,PCR扩增 ,均得到长度约为 3 40 bp的 DNA片段。产物 DNA片段经 DNA序列分析 ,结果表明三者的碱基序列相同 ,与报道的胰蛋白酶抑制剂基因相比 ,同源性为 1 0 0 %和 99.7%。     

人端粒酶RNA基因的克隆与鉴定

以人血基因组ＤＮＡ为模板,合成两段２０个寡聚核苷酸为引物,经过ＰＣＲ扩增,得到４８０ｂｐ的片段,克隆到ｐＭＤ１８－Ｔ载体中,经电泳、酶切、ＰＣＲ鉴定后测定序列。序列分析表明氙克隆的人端粒酶ＲＮＡ（ｈｕｍａｎ ｔｅｌｏｍｅａｓｅ ＲＮＡ,ｈＴＲ）基因含有４８０ｂｐ,包括约４５０ｂｐ的编码模板区主序列和约３０ｂｐ的上游调控区序列,其中模板区的１１个核苷酸（５’－ＣＵＡＡＣＣＣＵＡＡＣ－３’）合成端粒亚     

Molecular detection of Cotesia vestalis (Hymenoptera: Braconidae) in the beet webworm Loxostege sticticalis L. (Lepidoptera: Crambidae)

Genomic DNA samples from larvae of the beet webworm Loxostege sticticalis collected in the south‐western Russia were used to amplify mitochondrial cytochrome oxidase unit I (COI) gene. In a small proportion of samples, the sequenced product showed considerable heterogeneity due to admixture of a minor sequence. A preliminary BLAST analysis of a 100‐bp‐long fragment of this minor sequence showed its maximal similarity to the COI gene region of Cotesia, a genus of braconid larval endoparasitoids of Lepidoptera. An additional primer was designed to specifically amplify ca 300 bp of the COI gene region from Braconidae. As many as seven of 25 samples were positive by PCR. Sequencing of the amplified products in all these samples showed nucleotide sequence identity to the COI region of Cotesia vestalis (Cotesia plutellae) and the presence of two molecular haplotypes among individual parasitoid samples.     

小麦双引物RAPD分析方法的研究

ＲＡＰＤ标记是近几年迅速发展的一种新型分子标记,标准的ＲＡＰＤ的反应是以１０个寡聚核苷酸作引物,通过ＰＣＲ反应扩增出基因组的部分片段,我们在研究外源ＤＮＡ导入小麦后外源遗传物质的追踪时,对这个方法进行了改进,采取了双引物进行扩增,结果双引物反应能够比单引物反应扩增出更多的多态性片段。分子杂交结果表明,双引物扩增出的新片段与单引物扩增片段无同源性,并对双引物扩增出的多态性片段产生的可能原因进行讨论。     

PCR primers and amplification methods for 12S ribosomal DNA, the control region, cytochrome oxidase I, and cytochrome b in bufonids and other frogs, and an overview of PCR primers which have amplified DNA in amphibians successfully

New primers (N = 24) for the amplification and sequencing of the complete or near complete 12S ribosomal DNA, about 1000 bp of the control region, 390 bp of cytochrome oxidase I, and the near complete cytochrome b are described. The 12S ribosomal DNA primers successfully amplify DNA in tetrapods; other primers successfully amplify DNA in bufonoids and other anurans. An overview of published literature and sequence data banks identified 170 mitochondrial and 96 nuclear DNA primers that have been used or are highly likely to be useful in amphibians. Primer sequences, their locations within genes, and sequence location and identity in Xenopus and human and/or mouse are presented for each primer. The utility of each primer was estimated by identifying the smallest, yet most inclusive, taxonomic category within which each primer has been successful. Primers from all published sources are mapped together. We hope that these new primers, as well as the list of primers that have been useful in amphibians, will encourage further systematic and population genetic studies of amphibians.     

Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies

The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the other enzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from the occlusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second set of primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeus monodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV was obtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA. This sensitive method could detect MBV even in tissue homogenates.