高致病性PRRSV ORF6和ORF7基因的遗传变异分析

从GenBank中选取8株国内报道的高致病性猪繁殖与呼吸综合征病毒株,应用DNAStar软件对其ORF6、ORF7基因序列进行分析,与VR-2332株、LV株、国内及周边国家分离株进行核苷酸和推导氨基酸同源性比较,并绘制系统进化树.结果表明,高致病性猪繁殖与呼吸综合征病毒株的ORF6、ORF7核苷酸与VR-2332株的同源性为95.1%-95.4%、93.8%-94.1%,与欧洲型（LV）的同源性为69.4%-69.6%、67.9%-68.5%;ORF6、ORF7推导氨基酸与VR-2332株的同源性为97.7%、94.4%-95.2%,与欧洲型（LV）的同源性为80.0%-80.6%、65.3%-66.1%.系统进化树表明,8株高致病性猪繁殖与呼吸综合征病毒株仍属于美洲型.说明猪繁殖与呼吸综合征病毒不断变异的现今,ORF6和ORF7基因依然高度保守.     

288株猪圆环病毒的序列比较及遗传进化分析

目的为进一步了解猪圆环病毒(Porcine circovirus,PCV)的遗传进化规律,为该病的预防和控制奠定基础。方法对来自世界各国的26株PCV1和262株PCV2的全序列用生物软件进行了核苷酸的同源性比较,并对其ORF1和ORF2进行了核苷酸和推导氨基酸的比较。结果从核苷酸水平来看,PCV1与PCV2明显分为两支,PCV1间分布没有规律,而PCV2间又分为以来自美国、加拿大、澳大利亚的序列为代表的亚型Ⅰ和以来自法国和新西兰的序列为代表的亚型Ⅱ;但从氨基酸水平来看,其分型并没有规律可循。从遗传进化树可以看出,PCV2的分布并没有地域性和时限性。数据统计显示,尽管相隔近十年之久,但其同源性并没有发生太大的改变,可见PCV2相当保守。另外对PCV2的碱基和氨基酸突变位点进行统计发现,PCV2序列间更倾向于碱基的颠换,但并无单个碱基和氨基酸的偏好性。结论PCV2相对保守,地域性和时限性分布不明显。     

啤酒花潜隐病毒新疆分离物的全基因组序列分析

对新疆啤酒花上获得的HpLV分离物HpLV-XJ进行了全长克隆和基因组序列分析。结果显示:HpLV-XJ的全基因组序列为8612个核苷酸(nt)(不包括poly A),含有6个开放阅读框(ORF),分别编码224 kDa(ORF1)、25kDa(ORF2)、11 kDa(ORF3)、7 kDa(ORF4)、34 kDa(ORF5)、和12 kDa(ORF6)蛋白。序列相似性分析结果表明,HpLV-XJ与HpLV(GenBank:AB032469)序列相似性达98.5%,6个开放阅读框的核苷酸序列相似性分别为98.3%、99.0%、97.6%、96.7%、99.5%和98.4%;由此推导的氨基酸序列相似性分别为98.6%、98.7%、97.2%、95.0%、99.4%和98.1%,各个基因的核苷酸序列和蛋白的氨基酸之间存在着一定的差异。     

鸡新城疫病毒ND-xx08株F基因的克隆及分析

新城疫病毒（NDV）ND-xx08毒株经10 d龄SPF鸡胚增殖后,提取其基因组RNA并反转录成cDNA,用NDV F基因特异性引物,经PCR扩增后获得与F基因预期大小一致的DNA片段。将NDV F基因片段克隆到pMD18-T载体上,并进行EcoR I和Hind III双酶切鉴定和测序鉴定。结果显示,ND-xx08毒株F基因片段的长度为1 662 bp,共编码554个氨基酸,F蛋白的裂解位点为112R-R-Q-K-R-F117,是典型强毒株氨基酸序列结构。将NDV ND-xx08株F基因的47 bp到420 bp序列与新城疫病毒基因型I至基因型Ⅸ毒株的相同序列绘制病毒基因进化树,显示ND-xx08分离株属于基因Ⅶe型。将NDV ND-xx08株F全基因与国内外发表的23株NDV F基因核苷酸序列和氨基酸序列的同源性比较分析,结果表明,其核苷酸序列的同源性在82.7%~97.8%之间,氨基酸同源性在87.5%~97.7%之间。     

石河子一串红花叶病病原的分子鉴定

为了防治石河子一串红花叶病,采用提取dsRNA/RNA、RT-PCR、克隆和序列分析对病原进行了检测鉴定。结果表明:从表现花叶的S4分离物中提取dsRNA,得到大小约4700和6300 bp的片段,以此dsRNA为模板,用蚕豆病毒属(Fabavirus)的兼并引物进行RT-PCR,扩增得到1条约390 bp片段特异性条带,序列测定该片段大小为391nt,blast比对分析与蚕豆萎蔫病毒2号(BBWV2)的同源性最高,表明S4分离物为BBWV2。RNA1基因组全序列测定及分析结果表明,基因组大小为5957nt(不含poly A),仅编码1个开放阅读框(ORF),与已报道BBWV2的RNA1序列同源性为78.4%-100.0%。     

PRRSV福建流行毒株结构蛋白基因之克隆分析

对福建省流行的PRRS病毒FJ-1的结构蛋白基因进行了克隆、测序.FJ-1结构蛋白基因序列长3 188个核苷酸,包含7个开放阅读框(ORF).将FJ-1结构蛋白基因与国内外已发表的18个报道全长结构蛋白基因的PRRSV毒株进行核苷酸序列和推定的氨基酸序列比较,发现:其与17个美洲型毒株核苷酸同源性达到89.7%～92.4%,推定各个ORF编码氨基酸的同源性在85.6%～98.6%之间;而与欧洲型毒株Lelystad核苷酸同源性为54.9%,推定各个ORF编码氨基酸同源性为53.2%～78.2%.遗传进化树分析表明FJ-1与美洲型毒株进化距离近,而与欧洲型毒株进化距离远.从分子水平上证明了福建省流行的PRRS病毒属于美洲型毒株.     

鹅细小病毒C日v强毒株VP3基因克隆及遗传进化

根据Genbank中的鹅细小病毒(GPV)B株全基因序列,设计合成一对引物,应用PCR技术扩增了GPV强毒株CHv的VP3基因片段,将扩增后的VP3基因重组到pMD18-T质粒载体上,并对擂人片段进行序列测定,将测序结果及由该结果推导的氨基酸序列与国内外分离的GPV,M DPV,PPV和CPV等不同宿主的细小病毒的VP3进行比对分析.结果表明:中国四川分离的GPV CHv株VP3基因长1 605 bp,编码 534个氨基酸,与国内外10株GPV的VP3基因进行比较,核苷酸同源性为93.4%-99.8%,氨基酸同源性为96.5%99.3%,其变异较小,是GPV保持一个血清型的分子基础.与番鸭细小病毒的核苷酸和氨基酸同源性分别为79.6%和89.9%,而与其他种属的细小病毒同源性均在30%以下,表明它们与GPV CHv株亲缘关系较远.     

水稻黑条矮缩病毒基因组第九组分cDNA的克隆及序列分析

从我国发病的玉米材料中提取水稻黑条矮缩病毒,抽提病毒RNA,利用RT-PCR等手段,获得了病毒基因组第九组分（S9）cDNA克隆。序列分析结果表明：S9全长1900bp,含有2个不重叠的ORF,编码蛋白的分子质量分别为39.9,24.2ku,与日本株S9核苷酸序列同源性为89%,与意大利株MRDV S8同源性为86%。     

侵染新疆辣椒CMV的外壳蛋白基因的克隆和序列分析

从表现黄化的辣椒病株上获得分离物XJ-P,用1对CMV基因引物进行RT-PCR,扩增得到了774bp的特异性核苷酸片段。将PCR产物克隆到pMD18-T载体中再转化到大肠杆菌TOP10。经限制性内切酶酶切鉴定,重组克隆中含有与PCR产物大小相同的774bp的插入片段。cDNA全序列分析表明,所扩增出的774bpCMV基因,含1个657bp开放阅读框架（ORF）,编码218个氨基酸组成的蛋白,所克隆的基因包含完整的CMVCP基因。所测得的XJ-P分离物其CP基因与CMV亚组Ⅰ、亚组Ⅱ各株系之间的核苷酸同源性分别为90.84%-94.22%和74.37%-76.52%,氨基酸同源性分别为94.04%-98.17%和80.37%-82.19%,因此将该分离物鉴定为黄瓜花叶病毒亚组Ⅰ。     

PRV广东株gE基因去信号肽片段的克隆与序列测定

根据伪狂犬病病毒（PRV）Rice株gE基因的序列设计并合成了1对引物,以我国PRV地方毒株广东株的基因组DNA为模板,通过PCR方法获得了一大小约1.6kb的DNA片段,并将其克隆到pMD18-T载体上进行测序,序列测定结果显示,该片段长1665bp,编码555个氨基酸,与PRV Rice株gE基因的核苷酸序列同源性为97.7%,氨基酸序列同源性为95.9%。     

两株产人参皂苷糖苷酶酵母菌株的18S rDNA和ITS序列分析

对两株产人参皂苷糖苷酶的酵母菌株进行核糖体18S rDNA和ITS序列克隆测定,获得了长度分别为1 477和1 478 bp的18S rDNA序列和长度分别为791和727 bp的ITS序列,对获得的基因序列进行比对及同源性分析,结果显示,两株酵母菌的18S rDNA序列的相似性达100%,而ITS序列的相似性则为66%,均与NCBI数据库中登录的啤酒酵母的相应序列同源性最高.从GenBank中选取部分不同种属的酵母菌ITS序列,以ITS为对象构建系统发育树,从分子生物学角度确定了两株酵母菌为酵母菌属的不同种类.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

黑曲霉β-葡萄糖苷酶基因的分离

以黑曲霉基因组DNA、mRNA为模板,利用种属相似性设计了一对引物,采用PCR与RT-PCR技术得到β-葡萄糖苷酶基因的DNA和cDNA全序列.测序表明:克隆得到的DNA序列全长2 924 bp,cDNA序列全长2 583 bp,编码860个氨基酸.编码序列推导的蛋白质序列相似性分析显示:完整蛋白质序列同源性高达97%～99%.该研究为β-葡萄糖苷酶基因的遗传转化及应用 奠定了基础.     

Genetic diversity of rhizobial populations recovered from three Lotus species cultivated in the infra-arid Tunisian soils

Eighty-three bacterial strains isolated from root nodules of Lotus creticus, L. pusillus, and L. arabicus grown in infra-arid Tunisian soils were characterized using a polyphasic approach including phenotypic analysis, rep-PCR and PCR-RFLP analyses of the 16S rRNA gene. Phenotypically, all isolates are fast growers the majority of which grow at a pH of between 5.5 and 9. Most of the tested isolates tolerate NaCl concentrations from 1.39% to 3.48%. By rep-PCR fingerprinting, the genomic similarity varied from 30% to 98%. All tested isolates were clustered into 32 rep-PCR clusters at the similarity level of 80%. The genomic divergence of strains revealed by rep/PCR analysis appeared to be very important since a molecular polymorphism delimiting symbionts for each species of Lotus was identified. With the high-resolution of rep-PCR profiles of the isolates obtained using Pearson’s/UPGMA analysis, the isolates were resolved into 60 different profiling groups to undergo 16S ARDRA analyses. The analysis of all restriction fragments from each strain based on the UPGMA algorithm from the combined patterns showed that Lotus isolates are very diverse and that they were affiliated to Sinorhizobium, Rhizobium, and Mesorhizobium genera.     

The phylogenetic history of immunodeficiency viruses

Knowledge of the phylogenetic history of the human immunodeficiency viruses (HIV-1 and HIV-2) is important for our understanding of the epidemiology of AIDS, the disease caused by these viruses. Reconstruction of the evolutionary tree is hampered, however, by two problems. One is the high variation in nucleotide sequence between the known HIV isolates which can create formidable difficulties in identifying homologous genomic sites that may be used in a molecular phylogenetic reconstruction. Another impediment has been the lack of unequivocal time calibration points: there is only a sparse 'fossil record' for HIV and limited historical epidemiological data. We have largely overcome these difficulties by: (1) a thorough optimal-sequence alignment analysis; (2) the inclusion of sequences of an early (1976) HIV-1 isolate, a recent (1986) HIV-2 isolate and two simian immunodeficiency viruses (SIV) along with five other HIV-1 isolates; and (3) the reconstruction of a minimum-length evolutionary tree based on the envelope-gene variable positions. We conclude that HIV-1 may have evolved from its common ancestor with HIV-2 as recently as 40 years ago.     

菊花‘千手观音’LEAFY基因转录区基因组克隆和结构分析

LEAFY(LFY)基因在植物花发育过程中具有重要作用,不仅控制着花序分生组织向花分生组织的转变,而且控制着开花时间.通过基因组PCR扩增,获得了菊花‘千手观音’LFY同源基因序列.序列分析表明该基因包括2个内含子和3个外显子.其内含子1的2个序列长短不同,差异明显.2个内含子与甘菊的LFY同源基因DFL相比都表现出了丰富的变异性.其外显子推测的氨基酸序列与甘菊DFL的氨基酸序列相似性达99%.系统进化分析表明‘千手观音’的LFY同源基因与所有的菊属植物的LFY基因在树的同一枝上,且距双子叶植物的距离近于单子叶或裸子植物.Southern杂交表明,‘千手观音’基因组中LFY同源基因以两个拷贝形式存在.     

海甘蓝硫甙合成关键酶S—GT基因克隆及种子特异性hpRNAi载体构建

根据甘蓝型油菜S-GT（thiohydroximate S-glucosyltransferase）基因cDNA序列设计引物,以海甘蓝总DNA为模板进行PCR扩增,获得S-GT基因全长。克隆的海甘蓝S—GT序列与甘蓝型油菜序列相比,除74bp的内含予部分外有92个碱基的差别,相似性高达93．4％。分析显示该序列均有完整的开放阅读框,并表明所克隆的海甘蓝S-GT序列编码465个氨基酸,在第10个位点上比甘蓝型油菜序列少一个丙氨酸（A）,总共有23个氨基酸不同,相似性为95．06％。根据获得的基因序列设计引物扩增出同一基因序列相同但是带有不同酶切位点的两个片段,将两个片段反向插入到已构建的带有种子特异表达载体内含子的两端,成功构建了海甘蓝S-GT基因的种子特异性hpRNAi载体,为特异性降低海甘蓝的种子硫甙奠定了基础。     

胜利油田纯梁采油厂某区块油藏中微生物菌群分析

构建细菌16S rDNA基因文库以及硫酸盐还原菌的dsrAB基因文库,研究了胜利油田纯梁采油厂35区块某油井采出水中微生物的多样性以及SRB的菌群结构组成.结果表明,样品中微生物的多样性指数不高,样品中的微生物主要来自β变形菌纲以及γ变形菌纲,SRB主要由Thermodes-ulfobacterium、Desulfomicrobium、Thermodesulforhabdus以及Archaeoglobus fulgidus等属的微生物组成.将序列相似性≥97%作为一个OTU的划分原则,16S rDNA文库中有2个优势OTU,分别与Arco-bacter以及Pseudomonas stutzeri相近,分别占总克隆数的44%和56%;dsrAB基因文库有3个优势OTU,分别与Archaeoglobus fulgidus、Archaeoglobus infectus、Desulfotomaculum geothermicum相近,分别占总克隆数的46.6%、16.6%以及13.3%.16S rDNA基因序列与已知序列相似性较高,优势菌种在国内油田中较为常见,但不同油藏之间菌群结构存在较大差异.dsrAB序列与已知序列的相似性较小,可能存在新的SRB.     

Molecular characterization of H1N1 influenza A viruses from human cases in North America

Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A （H1N1） virus in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites （IPSIQSR↓GL）, and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug （i.e. oseltamivir and zanamivir） because no mutations have been observed. Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A （H1N1） virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.