缘管浒苔Rubisco酶大亚基基因编码序列rbcL克隆及分析

主要对缘管浒苔光合作用第一关键酶Rubisco大亚基基因(rbcL)进行了克隆分离.首先通过PCR特异性扩增叶绿体基因编码的缘管浒苔大亚基编码序列rbcL部分基因序列(1035 bp).依据基因步移原理,首次克隆得到缘管浒苔rbcL5'上游非翻译区序列(224 bp).据推测,rbcL 5'上游非翻译区序列存在类似原核生物的启动子元件-10区(TAAAAT)和-35区(TTGAAA).此外,依据3'-RACE(cDNA末端快速扩增技术)原理,克隆得到缘管浒苔rbcL3'末端cDNA序列(579 bp).     

缘管浒苔Rubisco酶大亚基基因编码序列rbcL克隆及分析

主要对缘管浒苔光合作用第一关键酶Rubisco大亚基基因(rbcL)进行了克隆分离.首先通过PCR特异性扩增叶绿体基因编码的缘管浒苔大亚基编码序列rbcL部分基因序列(1 035 bp).依据基因步移原理,首次克隆得到缘管浒苔rbcL 5′上游非翻译区序列(224 bp).据推测,rbcL 5′上游非翻译区序列存在类似原核生物的启动子元件-10区(TAAAAT)和-35区(TTGAAA).此外,依据3′-RACE(cDNA末端快速扩增技术)原理,克隆得到缘管浒苔rbcL 3′末端cDNA序列(579 bp).     

银合欢基因文库的构建和种子贮藏蛋白基因的分离

本文以λEMBL_3为载体,构建了银合欢(Leucaena leucocephala)的基因文库,所得重组子为3.5×10~6pfu。以大豆种子贮藏蛋白α′亚基基因为探针,从基因文库中分离得到了4个阳性克隆,并初步绘制了其中3个重组子的物理图谱。结果表明在这3个重组子的基因内部有一致的酶切位点。亚克隆的部分核苷酸序列与 GenBank 中的基因序列比较,表明与大豆种子贮藏蛋白α′亚基基因高度同源。     

药用植物灯盏花rbcL基因的克隆、生物信息学及适应性进化分析(英文)

目的:克隆药用植物灯盏花叶绿体rbcL基因,该基因编码二磷酸核酮糖羧化酶大亚基,并对该基因序列、蛋白质特性和适应性进化进行分析。方法:根据相关文献设计引物,利用PCR方法扩增并克隆完整rbcL基因序列,对rbcL蛋白进行结构建模和评价。结果:灯盏花rbcL基因长度为1 458bp,编码485氨基酸(GenBank登录号为KF482865)。通过与GenBank库中Erigeron tenuis氨基酸序列比较相似性超过95%。RbcL蛋白二级结构含有21个α-helices、7个β-sheets和一些卷曲。通过适应性进化分析在rbcL蛋白上有3个氨基酸正选择位点(76 E、131 Q和422 E)。结论:灯盏花rbcL基因的完整克隆有助于更深的研究灯盏花对特殊生境的适应,rbcL蛋白大亚基正选择位点的空间结构对于维持核酮糖结构具有重要的作用。     

蚕豆核酮糖1，5-磷酸羧化酶／加氧酶大亚基基因的分子克隆

核酮糖l,5－二磷酸羧化酶／加氧酶由大亚基(Ls)和小亚基组成。Ls由叶绿体DNA编码。蚕豆Ls的基因已被克隆到pBR322。应用几种限制性内切酶酶解以及Southern印迹法构建了该重组质粒的物理图谱。     

细菌人工染色体的研究和应用

细菌人工染色体 (Bacterialartificialchromosome ,BAC)是第二代大片段DNA的克隆载体系统。因其嵌合率低 ,遗传稳定性好 ,重组DNA容易分离和制备 ,转化效率高等 ,弥补了YAC的不足 ,很快在基因组研究中处于中心地位。近年来 ,已有多种BAC载体被构建出来 ,这些BAC载体在复杂基因组大片段文库的构建 ,基因的图位克隆 ,基因组物理图谱的构建 ,基因和基因组测序 ,基因组织结构分析 ,染色体组织和进化 ,以及基因的遗传转化和调控研究中得到了广泛的应用。     

含蚕豆叶绿体rRNA基因重组质粒物理图谱的构建

以 pBR322 DNA 为载体,Escherichia coli HB101为受体菌,克隆了含蚕豆叶绿体 rRNA基因的二个 BamHI 片段。应用几种限制性内切酶酶切以及 Southern 印迹法构建了这二个特异片段的物理图谱。重组质粒 pVFB16含有一个4.70kb 的 BamHI 片段,其上含有完整的16S rRNA 基因;重组质粒 pVFB32含有一个5.65kb 的 BamHI 片段,其上含有23S rRNA基因,23S—4.5S/5S rRNA 基因的间隔区及4.5S/5S rRNA 基因。     

小麦及其相关物种rbcL基因序列的比较研究

用4个抗白粉病小麦品系构建了一个混合cDNA文库,从该文库中分离到一个新的小麦rbcL基因全长cDNA克隆。至此,已经有3个不同的小麦rbcL基因序列被报道,新rbcL基因cDNA与以前的2个序列分别有1个和3个碱基的差异。新的cDNA长1519bp(基因库查询号：AY328025)。同源性比较发现,新的小麦rbcL基因的cDNA序列与以前报告的小麦rbcL基因的cDNA序列(gi：344052)有一个碱基的差异,从而导致所编码的多肽在144位由Tyr变为Cys。进一步比较分析了在相关物种中rbcL基因的cDNA序列,结果显示,rbcL基因的编码区在不同属的物种间高度保守,并从分子水平表明小麦与大麦、新麦草、赖草、披碱草属等的亲源关系比与水稻、玉米等的近。     

基因组细菌人工染色体文库(BAC)的构建及应用

细菌人工染色体 (BAC)是一种承载DNA大片段的克隆载体系统 ,用于人、动物和植物基因组文库构建。BAC具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点。BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础 ,可用于真核生物重要基因及全基因组物理作图、重要性状基因的图位克隆、基因结构及功能分析。本文主要综述了细菌人工染色体的构建与其鉴定 ,及其在物理图谱构建、图位克隆、转基因技术等研究上的应用。     

高粱叶绿体psbD基因在重组质粒pSD_2Ⅱ中的定位及psbD基因的亚克隆

利用pUC19我们构建了高粱叶绿体基因文库,从库中我们筛选到4个含psbD基因的阳性克隆。Southern分析证明重组质粒由pUC19和高梁叶绿体DNA的Pst10片段组成。通过8种限制性内切酶酶切分析和Southern分析测出了重组质粒pSD_2Ⅱ的物理图谱,并确定了高梁psbD基因在重组质粒中的精确位置。通过构建pSAC_2质粒,可以将Pst10片段中非psbD部分去除,使psbD可以直接用EcoRⅠ从pSAC_2上切下,为研究高粱psbD基因的表达和序列分析奠定了基础。     

单纯疱疹病毒2型特异性DNA片段的克隆和鉴定

用核酸限制性内切酶BamHI对单纯疱疹病毒2型(HSV—2)的DNA进行酶解,回收位于基因组中的反向重复序列区的Bam HIG片段,然后将其克隆在载体质粒PUC 8的Bam HI切点上,进一步用核酸限制性内切酶Eco RI和KPNI对这一重组质粒联合酶解,移去EcoRI—KPNI小片段,经末端修饰后,将其连接得到新的重组质粒pRC102,它含有一小段HSV—2的DNA序列。以此质粒为探针,分别与HSV—1、HSV—2及细胞DNA进行斑点杂交;与HSV—1和HSV—2酶解后的DNA片段进行Southern转印系交。两组实验结果显示,pRC102质粒DNA只与HSV—2 DNA特异性杂交,其HSV—2的型特异性良好。     

Cloning of soybean leghemoglobin structural gene sequences synthesized in vitro.

Double-stranded soybean leghemoglobin DNA was synthesized from leghemoglobin mRNA isolated from soybean nodules. The dsDNA was inserted into the Bam H1 site of plasmid pBR322 using the poly-dAT-joiner method. A cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The restriction cleavage map and the DNA sequence of a selected part of the inserted DNA are in complete accordance with the amino-acid sequence of soybean leghemoglobin.     

Molecular cloning of the genome of human spumaretrovirus

DNA of human spumaretrovirus (HSRV) was cloned from both cDNA and from viral DNA into phage lambda and bacterial plasmid vectors. The recombinant plasmids harboring viral DNA were characterized by Southern blot hybridization and restriction mapping. Physical maps were constructed from cDNA and found to be colinear with the restriction maps obtained from viral DNA. The recombinant clones isolated contained viral DNA inserts which range in size from 2.2 kb to 15.4 kb. The recombinant clones allowed to construct a physical map of the complete HSRV provirus of 12.2 kb.     

Introduction of new DNA sequences into previously selected regions of a plasmid genome by means of the formation of heteroduplexes

A new method for obtaining the recombinant DNA based on heteroduplex-initiated site-directed insertion of alien nucleotide sequences is proposed. To generate a single-stranded region, plasmid DNA was nicked with restriction endonuclease in the presence of ethidium bromide with subsequent exonuclease III controlled digestion. The inserted DNA sequences flanked by nucleotide sequences complementary to single-stranded region were annealed with plasmid DNA and E. coli cells were transformed by the resulting heteroduplex molecules. The presented data show the possibility to insert as many as 200 nucleotides. The yield of recombinant DNA varied from 16 to 0.7% as the number of nucleotides inserted correspondingly varied from 15 to 200. The site of insertion does not depend crucially on the localization of the restriction site used.     

HSV-tk基因逆转录病毒重组体的构建与DNA序列分析

目的　构建含有单纯疱疹病毒Ⅰ型胸苷激酶 (HSV1 tk)基因的逆转录病毒重组载体pLXSN TK。方法设计一对寡核苷酸引物 ,用PCR方法从质粒pHSV10 6中特异扩增HSV tk基因片段 ( 1168bp) ,分别用BamHI和Eco RI酶切后 ,定向连接到质粒pLXSN中 ,转化宿主菌TG1,分别用上述内切酶 ,PCR和DNA测序鉴定重组质粒。结果　酶切鉴定所切下的片段和PCR扩增的片段大小均与预计相符 ,测序结果与文献报道序列及预计结果一致 ,证实符合表达框架。结论　成功构建了HSV tk嵌合重组质粒pLXSN TK。     

Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus.

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.     

Adenovirus types 2 and 5 functions elicit replication and late expression of adenovirus type 12 DNA in hamster cells.

A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging.     

A library of genes of plasmid PSS120 of Shigella sonnei

The gene library of S. sonnei plasmid pSS120 was constructed with the use of plasmid pSL5 as vector. The complete restriction of the vector DNA and the partial restriction of the DNA of plasmid pSS120 were carried out by means of the enzyme EcoRI. The restricted DNA was ligated and packed in vitro into the capsid of phage lambda. The titer of negative colonies obtained after packing was 0.8 X 10(3) clones per 1 microgram of S. sonnei DNA. The total number of detected clones was 250. On the basis of the results, obtained in the analysis of the inserts of the DNA of plasmid pSS120 into the DNA of recombinant clones, the theoretical volume of the library, equal to 92 clones, was calculated. The collection of clones thus obtained will be used for checking the presence of the determinants of invasiveness and phase I antigen, localized in the DNA of plasmid pSS120.     

Cloning of chicken lysozyme structural gene sequences synthesized in vitro.

Double-stranded chicken lysozyme cDNA was synthesized from an oviduct mRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.     

Cloning of Herpes simplex virus 2 DNA fragments in a plasmid vector

DNA isolated from virions of Herpes simplex type 2 (HSV-2) strain 333 was digested with various restriction enzymes and joined to the EK2 plasmid vector pBR322. The viral DNA sequences present in the hybrids were analyzed by restriction enzyme mapping and hybridization to fragments of HSV-2 DNA. The collection of recombinant molecules represents approx. 75% of the HSV-2-genome. In most cases, the structure of the recombinants seemed identical to the organization of authentic fragments of HSV-2 DNA, however, a few hybrids contained rearrangements of viral and plasmid sequences.