Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses （enJSRV）

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia （enJSRV-NM）, we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame （orf-x） that overlaps the 3＇ end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain （Accession No.AF153615） and USA JSRV21 strain （Accession No. AF105220）. The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.     

绵羊肺腺瘤病毒NM株前病毒基因组的克隆与全序列分析

本研究参照GenBank中已发表的绵羊肺腺瘤病毒的基因组全序列,设计合成8对引物,从内蒙古某羊场自然感染绵羊肺腺瘤病的病肺肿瘤组织中提取总DNA为模板,对JSRV-NM株基因组分8段进行PCR扩增,产物分别为8个(531bp, 888bp, 949 bp, 944bp, 1428bp, 947bp, 1836bp, 538bp)基因片段,将其分别克隆入pMD-18 T载体中进行双向测序并拼接序列,获得完整的JSRV-NM株前病毒基因组全序列.结果表明,JSRV-NM株前病毒基因组全长7430bp,有相互重叠的4个较长的开放阅读框(ORF),分别代表gag、 pro、 pol 和 env基因.与绵羊肺腺瘤病毒Ⅰ型即南非代表株(NC-001494)和绵羊肺腺瘤病毒Ⅱ型即美国代表株(AF105220)的核苷酸同源性比较分别为90.4%和90%,推导出的氨基酸同源性分别为90%和89.1%.分析JSRV-NM株基因组结构,发现在LTR的上游和下游都具有外源性exJSRV特有的ScaⅠ酶切位点,在gag基因编码的NC区发现有2个较典型的"胱氨酸-组氨酸序列",可形成锌指结构.在env基因编码的TM区有特异性的"YXXM"基序.用地高辛标记外源性exJSRV特异的JSRV-2片段制成探针,原位杂交法检测自然感染绵羊肺腺瘤病(OPA)的病肺组织中JSRV-NM的 RNA及前病毒DNA,结果表明OPA患羊肺肿瘤细胞的胞浆和核内都有JSRV-2基因mRNA的表达, 说明JSRV-NM株是具有致瘤作用的外源性反转录病毒.这是我国首次报道的绵羊肺腺瘤病毒的基因组全序列.     

绵羊肺腺瘤病毒NM株前病毒基因组的克隆与全序列分析

本研究参照GenBank中已发表的绵羊肺腺瘤病毒的基因组全序列,设计合成8对引物,从内蒙古某羊场自然感染绵羊肺腺瘤病的病肺肿瘤组织中提取总DNA为模板,对JSRV-NM株基因组分8段进行PCR扩增,产物分别为8个(531bp,888bp,949bp,944bp,1428bp,947bp,1836bp,538bp)基因片段,将其分别克隆入pMD-18T载体中进行双向测序并拼接序列,获得完整的JSRV-NM株前病毒基因组全序列。结果表明,JSRV-NM株前病毒基因组全长7430bp,有相互重叠的4个较长的开放阅读框(ORF),分别代表gag、pro、pol和env基因。与绵羊肺腺瘤病毒Ⅰ型即南非代表株(NC-001494)和绵羊肺腺瘤病毒Ⅱ型即美国代表株(AF105220)的核苷酸同源性比较分别为90.4%和90%,推导出的氨基酸同源性分别为90%和89.1%。分析JSRV-NM株基因组结构,发现在LTR的上游和下游都具有外源性exJSRV特有的ScaⅠ酶切位点,在gag基因编码的NC区发现有2个较典型的“胱氨酸—组氨酸序列”,可形成锌指结构。在env基因编码的TM区有特异性的“YXXM”基序。用地高辛标记外源性exJSRV特异的JSRV-2片段制成探针,原位杂交法检测自然感染绵羊肺腺瘤病(OPA)的病肺组织中JSRV-NM的RNA及前病毒DNA,结果表明OPA患羊肺肿瘤细胞的胞浆和核内都有JSRV-2基因mRNA的表达,说明JSRV-NM株是具有致瘤作用的外源性反转录病毒。这是我国首次报道的绵羊肺腺瘤病毒的基因组全序列。     

Evolutionary dynamics of endogenous Jaagsiekte sheep retroviruses proliferation in the domestic sheep,mouflon and Pyrenean chamois

The oncogenic exogenous Jaagsiekte sheep retrovirus (JSRV), responsible for ovine pulmonary adenocarcinoma, has several endogenous counterparts termed enJSRVs. Although many of these elements have been inactivated over time by the accumulation of deleterious mutations or internal recombination leading to solo long terminal repeat (LTR) formation, several members of enJSRVs have been identified as nearly intact and probably represent recent integration events. To determine the level of enJSRV polymorphism in the sheep population and related species, we have undertaken a study by characterizing enJSRVs copies and independent integration sites in six domestic sheep and two wild species of the sheep lineage. enJSRVs copies were detected by amplifying the env-LTR region by PCR, and for the detection of the insertion sites, we used two approaches: (1) an in silico approach based on the recently published Sheep Reference Genome Assembly (OARv3.0) and (2) an experimental approach based on PCR suppression and inverse PCR techniques. In total, 103 enJSRV sequences were generated across 10 individuals and enJSRV integrations were found on 11 of the 28 sheep chromosomes. These findings suggest that there are still uncharacterized enJSRVs, and that some of the integration sites are variable among the different species, breeds of the same species, subspecies and geographic locations.     

家蝇防御素基因的cDNA克隆及序列分析

Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.     

Complete sequence of the chloroplast genome from pear (Pyrus pyrifolia): genome structure and comparative analysis

The chloroplast genome of Pyrus was found to be 159,922?bp in length which included a pair of inverted repeats (IRs) of 26,392?bp, separated by a small single-copy region of 19,237?bp and a large single-copy region (LSC) of 87,901?bp. A total of 130 predicted genes (113 unique genes and 17 genes, which were duplicated in the IR) including 79 protein-coding genes, four ribosomal RNA genes and 30 tRNA genes were identified based on similarity to homologs from the chloroplast genome of Nicotiana tabacum. Genome organization was very similar to the inferred ancestral angiosperm chloroplast genome. Comparisons between Pyrus, Malus, and Prunus in Rosaceae revealed 220 indels (??10?bp). Excluding ycf1 and ycf2, which contained deletions in the coding region, all of these were detected in the spacer or intron regions. Three insertions and 13 deletions were detected in Pyrus compared to the same loci in Malus and Prunus. After comparing 89 noncoding chloroplast DNA regions in Pyrus and Malus, highly variable regions such as ndhC-trnV and trnR-atpA were identified. In Pyrus and Malus, the IR/LSC borders were 62?bp shorter than those of Prunus. In addition, there were length mutations at the IRa/LSC junction and in trnH. A total of 67 simple sequence repeats (more than 10 repeated motifs) were identified in the Pyrus chloroplast genome. The indels and simple sequence repeats will be useful evolutionary tools at both intra- and interspecific levels. Phylogenetic analysis demonstrated a close relationship between Pyrus and Prunus in the Rosaceae.     

Visual verification of close disposition between a rice A genome-specific DNA sequence (TrsA) and the telomere sequence

A rice A genome-specific tandem repeat sequence (TrsA) and telomeric nucleotide sequences, (TTTAGGG)_n, were simultaneously detected by multicolor fluorescence in situ hybridization (McFISH) using rice prometaphase chromosomes. Six pairs of TrsA sites visualized by fluorescence signals were all localized on the long arms close to the telomeric regions. Differences in the copy number of TrsA at the different sites were visualized both by the size of the telomeric condensation block stained with Giemsa solution and the signal intensity after FISH with TrsA. McFISH analyses using interphase nuclei could resolve close disposition of TrsA and telomere and also gave rough estimation of the distance between them. The functional significance of the close disposition of TrsA and telomere is discussed.     

棉铃虫P450基因HarmCYP9A33的克隆、 序列分析及原核表达

夜间活动昆虫如夜蛾类主要通过嗅觉来寻找配偶、 寄主植物和产卵场所, 是研究昆虫嗅觉分子机制的理想材料。P450为多功能单加氧酶, 在昆虫对各种内源与外源物质的代谢中起重要作用。为研究P450在昆虫嗅觉中的作用, 本研究采用RT-PCR和RACE技术, 从夜蛾科昆虫棉铃虫Helicoverpa armigera (Hübner)雄蛾触角中扩增得到一条全长1 772 bp的P450基因, 命名为HarmCYP9A33 (GenBank登录号为JX486677)。序列分析表明, HarmCYP9A33开放阅读框全长1 590 bp, 编码529个氨基酸残基, 预测蛋白质分子量和等电点分别为61. 62 kD和7. 97; HarmCYP9A33与甘蓝夜蛾Mamestra brassicae触角毛形感器中高表达的MbraCYP9A13蛋白的氨基酸序列一致性最高, 达75%, 蛋白二级结构相似, 6个底物识别位点(substrate recognition sites, SRSs)序列一致性达61%, 其中底物与酶结合通道开关Ⅰ螺旋中SRS4序列完全相同, 与棉铃虫CYP9A亚家族蛋白有一定的结构相似性。Real-time PCR检测表明, HarmCYP9A33在雌、 雄蛾各组织中均有表达, 以腹部表达量最高, 其次为头部; 在卵至成虫各个时期也均表达, 以蛹中表达量最高; 在触角中的表达量随羽化时间而变化, 且多高于卵和幼虫中的表达量。SDS-PAGE检测和Western blot鉴定表明HarmCYP9A33体外融合表达成功。本研究为深入探讨该基因在棉铃虫触角感器细胞中的定位及其生物学功能奠定了基础。     

Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.     

版纳微型猪近交系AQP3克隆、序列分析及组织表达

目的 克隆版纳微型猪近交系aquaporin 3(AQP3)基因,并利用生物信息学方法分析其序列特征,研究其在猪各组织中的表达情况.方法 从版纳微型猪近交系脾脏中提取总RNA,利用RT-PCR方法扩增猪AQP3编码区序列,将纯化的片段与pMD18-T载体连接,转化宿主菌DH 5α,筛选阳性克隆进行测序.并采用半定量RT...     

白鱀豚BDNF基因的扩增和序列分析

利用聚合酶链式反应,首次从白鱀豚基因组DNA 中扩增和克隆到脑源神经营养因子的编码区。在该段序列中含有一个长为747 bp 的开放阅读框,无内含子,编码一个由248 个氨基酸组成的蛋白质,预计分子量为27 953.7道尔顿。其中包括由18 个氨基酸残基组成的信号肽区,111 个氨基酸残基组成的前肽区及119 个氨基酸残基组成的成熟区。序列分析表明,白鱀豚脑源神经营养因子基因编码区的核苷酸序列与其它哺乳动物相似性超过90%,而与猪牛相似性相对较高（分别为95% 和94.7%）。氨基酸序列比较发现,白鱀豚BDNF 前体蛋白的氨基酸序列与其它哺乳动物具有94.5% ～99.5%的相似性,显示了极高的保守性。通过邻接法进行的系统发生分析中,鲸目和食肉目的物种分别聚为单系;与其它哺乳动物相比,鲸类与有蹄类的牛和猪的亲缘关系相对较近,这与鲸类和有蹄类之间具有相对较近的亲缘关系相符。
     

甜菜夜蛾过氧化氢酶cDNA序列克隆、 序列分析和表达特征

过氧化氢酶 (catalase, CAT)作为生物体内的重要物质, 其主要功能是参与活性氧代谢过程, 在清除H₂O₂、 超氧自由基和过氧化物以及阻止羟基自由基形成等方面发挥着重要作用。本研究利用RT-PCR技术和RACE方法首次克隆和分析了甜菜夜蛾Spodoptera exigua （Hübner）CAT基因, 命名为SexiCAT, GenBank登录号为JN051294, 其cNDA序列全长为1 755 bp, 开放阅读框长1 524 bp, 推测编码507个氨基酸。经氨基酸序列比对, 此多肽序列具有高度保守性, 与其他昆虫CAT的序列一致性分别为： 家蚕Bombyx mori （87%）、 黑腹果蝇Drosophila melanogaster （73%）、 埃及伊蚊Aedes aegypti （71%）和赤拟谷盗Tribolium castaneum （70%）。对该基因在甜菜夜蛾各个发育时期以及不同组织表达量的荧光定量PCR分析表明, SexiCAT基因在甜菜夜蛾各个发育阶段的表达水平存在显著差异, 其中成虫期的表达量最高, 是卵期表达量的7倍, 幼虫期次之, 卵期最低; SexiCAT基因在5龄幼虫体壁、 中肠、 脂肪体和马氏管组织中都有表达, 但在脂肪体中表达量最高。甜菜夜蛾SexiCAT基因的成功克隆及同源建模将为今后对其功能研究以及作为靶标设计新型氧化酶抑制剂提供了基础。     

小地老虎几丁质酶基因的克隆与序列分析

利用昆虫几丁质酶对几丁质的调控作用破坏几丁质新陈代谢的平衡来防治害虫, 在生物防治策略中具有很大的发展潜力。从处于预蛹期的小地老虎Agrotls ipsilon (Hufnagel)体中肠内提取总的RNA, 经反转录, 利用cDNA末端快速扩增技术（RACE）获得了几丁质酶基因的cDNA序列。该基因序列已经登录GenBank并获得登录号为EU035316。该序列长度为2 823个碱基, 含有一个1 674个碱基的开放读码框。开放读码框编码558个氨基酸残基, 预测的分子量为62.5 kDa, 等电点5.12。推导得到的氨基酸序列含有2个N-位糖基化位点,20个O-位糖基化位点, 含有2个几丁质酶所具有的保守序列：N-端的催化区和C-端的几丁质结合区。氨基酸序列与其他昆虫, 特别是鳞翅目昆虫的几丁质酶高度同源。     

飞蝗谷胱甘肽S-转移酶基因克隆、 序列分析及表达特征

谷胱甘肽S-转移酶（glutathione S-transferase, GST）是一类广泛分布的多功能超家族酶系, 其中Omega家族GST在昆虫体内担负重要生理功能。为探讨飞蝗Locusta migratoria Omega家族GST功能, 利用RT-PCR技术克隆得到1条飞蝗谷胱甘肽S-转移酶Omega家族基因全长cDNA, 命名为LmGSTo1 （GenBank登录号： JQ750592）。该基因开放阅读框长738 bp, 编码245个氨基酸。该酶含有N-端和C-端2个结构域, N-端结构域由5个β-折叠和3个α螺旋组成, 包括4个GSH结合位点; C-端结构域由8个α螺旋组成, 含有5个底物结合位点。Real-time PCR结果表明, LmGSTo1在飞蝗不同龄期均有表达, 在胃盲囊和中肠表达量较低, 在前肠、马氏管、肌肉和脂肪体表达量较高; 溴氰菊酯处理可导致LmGSTo1表达水平显著下降。这些结果为进一步研究LmGSTo1基因功能提供了依据。     

中华鳖HMG1基因的克隆与序列分析

为了解中华鳖(Pelodiscus sinensis)HMG1(High mobility group 1)的基因结构,利用RT-PCR,从中华鳖肝脏组织的总RNA中,克隆并测序了中华鳖HMG1cDNA片段,结果表明,中华鳖HMG1基因的开放读码框(Open reading frame,ORF)长度为606 bp,编码202个氨基酸。中华鳖HMG1多肽链主要包含三个保守的区域:位于多肽链N端的HMG盒区1(第9—80个氨基酸之间);位于多肽链中心的HMG盒区2(第89—162个氨基酸之间);位于多肽链C端的富含酸性氨基酸区域(第163—202个氨基酸之间)。在2个HMG盒区范围内,中华鳖HMG1多肽链与红原鸡、人、虹鳟等物种的HMG1多肽链相比,氨基酸同源性依次为96.5%、74%和67%。排序比较显示,不同物种HMG1多肽链之间的富含酸性氨基酸区域的长度是不同的,暗示了HMG1多肽链富含酸性氨基酸区域的长度可能受到选择压力的影响,但这种选择压力没有使谷氨酸和天冬氨酸这两种酸性氨基酸之间区分开来。系统发生分析表明,脊椎动物HMG1基因的HMG盒区1和盒区2分别形成了2个亚族。本研究首次报道爬行动物的HMG1基因。       

北极狐GHR基因cDNA的克隆及序列分析

本文根据狗(AF133835)的GHR基因cDNA编码全序列设计了三对引物,利用RT-PCR方法克隆出北极狐GHR基因编码区全长cDNA序列(GenBank accession No.EU304325)。结果表明,北极狐GHR的ORF为1917bp,编码638个氨基酸的前体蛋白,由18个氨基酸的信号肽和620个氨基酸的成熟肽组成。通过同源性比较发现北极狐与狗的同源性最高,达到98%。另外,利用邻接法(NJ法)构建的分子系统进化树聚类结果表明,北极狐与狗先聚为一类,该聚类结果与传统的物种进化关系基本一致。另外,通过氨基酸对位序列比较发现,北极狐GHR在氨基酸序列上存在明显的特异性,如45和451位分别为A和E,而其它物种均分别为T(大鼠为K)和A(牛羊为V,鼠为T)。     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant,Polyrhachis vicina

Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full‐length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599‐amino acid protein that exhibits a high degree of homology with other mAchRs. Real‐time quantitative RT‐PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains. © 2011 Wiley Periodicals, Inc.