大蒜花叶病毒外壳蛋白基因cDNA的克隆和序列分析

我们从自然发病的大蒜中分离得到了大蒜花叶病毒。以其基因组ＲＮＡ为模板合成了３＇末端部分ｃＤＮＡ。从中选出一批插入片段在２．０ｋｂ以上的重组克隆,经Ｎｏｒｔｈｅｒｎ点杂交分析证实了所选克隆与基因组ＲＮＡ同源。通过对若干个克隆的插入片段两端部分序列的测定,选出一个克隆ｐＧＭ４９５,其插入片段的长度约为２．４ｋｂ,３′末端存有一个Ｐｏｌｙ（Ａ）结构,它应包含了编码该病毒外壳蛋白全部序列。序列测定的结果表明,这个ｃＤＮＡ片段全长为２３７９ｂｐ,其中含有与酶切图谱分析结果相符的ＥｅｏＲＩ、ＰｓｔＩ及ＢａｍＨＩ酶切位点。第一个终止密码子ＴＡＡ与３′ｇ末端相距２６４ｂｐ,我们根据碱基序列推定的氨基酸序列与其它已发表的Ｐｏｔｙｖｉｒｕｓ的外壳蛋白氨基酸序列以及外壳蛋白翻译后加工的蛋白酶专一切点相比较后推测,编码该病毒外壳蛋白序列可能起始于３′末端上游的１１７０ｂｐ处,共编码３０２个氨基酸,其分子量为３６ｋＤ,略大于ＳＤＳ－ＰＡＧＥ所测定的３３ｋＤ,非编码区域长２６４ｂｐ,富含ＡＴ,并有多个终止密码子的存在。趾３′末端３２～２７ｂｐ处有一个ＡＡＴＡＡ序列。     

Cloning and Expression of Chlorobium vibrioforme Uroporphyrinogen Decarboxylase Gene in a Salmonella Heme Auxotroph

To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase (hemE) gene. A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain. The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant (hemA⁻ hemE⁻) by phage P22. Faster-growing colonies indicated complementation of the hemE mutation. Each clone was tested by backcross transduction of the mutant. Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella. HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone. This clone showed an active hemE gene within a 4-kb insert. Received: 21 February 2002 / Accepted: 8 May 2002     

拉布拉多犬嗅觉受体CfOLF4基因的克隆及序列分析

以常规PCR技术扩增警用拉布拉多犬(Canis familiaris)的嗅觉受体CfOLF4基因长879bp的目的片段,构建pMD-CfOLF4载体进行T-A克隆。重组质粒测序后与GenBank公布的犬CfOLF4基因进行序列分析比较,旨在了解拉布拉多犬嗅觉受体CfOLF4基因遗传的同源性和变异特性。     

Cloning and Nucleotide Sequence of the Endoinulinase-Encoding Gene, inu2, from Aspergillus ficuum

A 2.3 kb DNA fragment that contains a gene encoding endoinulinase, inu2, from Aspergillus ficuum ATCC 16882 was isolated and analyzed. It includes an open reading frame of 1,551 bp, coding for a polypeptide with calculated molecular weight of 55,790 Da, including a putative signal peptide of 22 amino acids. Alignment of amino acid sequences revealed 73.3% identity and 93.9% similarity between A. ficuum and Penicillium purpurogenum endoinulinase.     

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

Molecular Cloning and Nucleotide Sequence of the Superoxide Dismutase Gene and Characterization of Its Product from Bacillus subtilis

Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2,600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.     

马铃薯卷叶病毒外壳蛋白基因的合成,分子克隆和全序列分析

以分离自马铃薯主栽品种“紫花白”的马铃薯卷叶病毒(PLRV)分离物的RNA为模板,用人工合成的引物,用反转录和随后PCR扩增的方法合成了PLRV外壳蛋白(CP)基因的cDNA,并克隆于pUC19中。进一步用限制酶切和核苷酸序列分析表明,合成的cDNA由627个核苷酸组成(包括起始和终止密码),序列中有Hinc Ⅱ和BamH Ⅰ两个酶切位点,与国外报道一致。和国外的4个PLRV分离物CP基因序列对比结果,具有高度同源性,其同源率达99.0—99.7％。     

Molecular Cloning,Nucleotide Sequence,and Expression of the Structural Gene for Alkaline Serine Protease from Alkaliphilic Bacillus sp. 221

The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp. 221 was cloned in Escherichia coli and expressed in Bacillus suhtilis. An open reading frame of 1,140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp. 221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6—61.70/0). Also Bacillus sp. 221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.     

Cloning and expression of Chlorobium vibrioforme uroporphyrinogen decarboxylase gene in a Salmonella heme auxotroph

To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase ( hemE) gene. A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain. The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant ( hemA(-) hemE(-)) by phage P22. Faster-growing colonies indicated complementation of the hemE mutation. Each clone was tested by backcross transduction of the mutant. Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella. HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone. This clone showed an active hemE gene within a 4-kb insert.     

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板,采用PCR技术,扩增气溶素基因(aerA)的DNA片段,将其克隆到pMDl8-T载体上.通过序列测定,分析结果表明:所克隆的1393 bp片段为aerA部分序列,编码产生464个氨基酸.BZ与NK之间aerA核苷酸同源性为97.6%.氨基酸同源性为98.3%,与其它分离物核苷酸同源性为71.6%～97.5%,氨基酸同源性为68.0%～98.9%.利用邻接法构建了aerA分子树状图,树状图分析表明:气单胞菌属各分离物聚为三支,其中嗜水气单胞菌各菌株之间关系密切,被聚类为同一支.     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板, 采用PCR技术, 扩增气溶素基因(aerA)的DNA片段, 将其克隆到pMD18-T载体上。通过序列测定, 分析结果表明：所克隆的1393 bp片段为aerA部分序列, 编码产生464个氨基酸。BZ与NK之间aerA核苷酸同源性为97.6%, 氨基酸同源性为98.3%, 与其它分离物核苷酸同源性为71.6%~97.5%, 氨基酸同源性为68.0%~98.9%。利用邻接法构建了aerA分子树状图, 树状图分析表明：气单胞菌属各分离物聚为三支, 其中嗜水气单胞菌各菌株之间关系密切, 被聚类为同一支。     

小麦返白系胆色素原脱氨酶纯化及编码cDNA序列分析

以小麦(Triticum aestivum)返白系F772为材料,通过粗提、加热处理、硫酸铵分级沉淀和凝胶柱层析等方法,对胆色素原脱氨酶（PBGD）进行了提取纯化,纯化倍数为1092,得率为15%.纯化的PBGD约为37 kD.对其部分生化性质的研究表明：高浓度的NH+4对酶活性有强烈的抑制,光照处理可以使酶活性降低.对纯化后的PBGD N末端氨基酸序列进行了测定.根据N端序列设计简并引物,获得了PBGD的cDNA全部编码区序列.它编码一个351个氨基酸的前体蛋白,有一个叶绿体导肽的序列.通过比较小麦PBGD与来自与其他物种的同源蛋白表明,有些氨基酸残基非常保守.     

Cloning and expression of a structural gene from Chlorobium vibrioforme that complements the hemA mutation in Escherichia coli.

Escherichia coli SASX41B carries the hemA mutation and requires delta-aminolevulinic acid for growth. Strain SASX41B was transformed to prototrophy with pYA1, a plasmid vector carrying a 5.8-kilobase insert of genomic DNA from the green sulfur bacterium Chlorobium vibrioforme. Cell extracts prepared from transformed cells are able to catalyze transfer of label from [1-14C]glutamate or [3,4-3H]glutamyl-tRNA to delta-aminolevullinic acid at rates much higher than extracts of wild-type cells can, whereas extracts prepared from untransformed strain SASX41B cells lack both activities. By comparing the relative abilities of glutamyl-tRNAs derived from several heterologous cell types to function as substrates for the dehydrogenase reaction in extracts of HB101 and SASX41B cells transformed by pYA1, it was determined that the expressed dehydrogenase in the transformed cells resembled that of C. vibrioforme and not that of E. coli. Thus it can be concluded that plasmid pYA1 contains inserted DNA that codes for a structural component of C. vibrioforme glutamyl-tRNA dehydrogenase which confers glutamyl-tRNA substrate specificity.     

耐辐射奇球菌超氧化物歧化酶基因的克隆与序列分析

By using a 453 bp length gene fragment of superoxide dismutase(SOD)as a probe,which was firstly amplified from Deinococcus radiodurans genomic DNA by PCR with degenerate oligonucleotide primers corresponding to the conservative regions of known SODs,a putative SOD gene was identified from the database of D.radiodurans whole genome.Its 636 bp length open reading frame and 5′ and 3′ flanking sequence was determined.The conventional E.coli ribosomal and RNA polymerase binding sites were found upstream from SOD encoding region and an inverted repeat sequence downstream of the termination codon.The deduced 211 amino acid sequence of the structural gene showed a high similarity to other manganese and iron containing SODs in normally conserve regions.     

大豆11S球蛋白Gy5(A3B4)的基因克隆和序列分析

大豆11Ｓ球蛋白(Ｇｌｙｃｉｎｉｎ)是大豆种子的主要贮藏蛋白,分子量为360ｋＤ,由6对相同的蛋白亚基(每对亚基的分子量约60ｋＤ)构成。每对亚基又是由一个酸性Ａ肽(35～45ｋＤ)和一个碱性Ｂ肽(22ｋＤ)通过二硫键连接而成。Ａ肽和Ｂ肽源自同一个基因,即首先由一个大的ｍＲ?..     

Cloning and Nucleotide Sequence of the Mycodextranase Gene from Streptomyces sp. J-13-3

Mycodextranase (EC 3.2.1.61) is an α-glucanase that cleaves α-1,4-bonds of alternating α-1,3- and α-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.