烟草脉带花叶病毒云南分离物全基因序列分析

应用电子显微镜负染色法在烟草叶片斑驳、脉间褪绿病样中观察到马铃薯Y病毒属病毒粒子,通过马铃薯Y病毒属兼并引物扩增获得目的片段,序列分析表明病样中病原物为TVBMV,命名为YN-YX-TVBMV。根据GenBank已知序列,分别合成引物,对YN-YX-TVBMV进行克隆及测序,得到一条6047 bp的不完整的病毒基因组核苷酸序列,将该序列与国内报道的TVBMV全基因序列进行对比分析发现该分离物与报道的TVBMV其他分离物具有明显的地域分布差异,即云南获得的分离物均独立聚为一簇。     

陕西烟区烟草脉带花叶病毒外壳蛋白基因的克隆和原核表达

根据烟草脉带花叶病毒(TVBMV)CP序列合成上下游引物,利用RT-PCR方法获得了(TVBMV)CP基因。序列分析表明,TVBMV CP基因全长813nt,编码271个氨基酸;与GenBank中已报道的9个分离CP基因相比,其核苷酸及氨基酸序列同源性分别为90.28%-98.15%和97.05%-100%。将TVBMV CP基因经BamHI/NotI双酶切定向插入到pET30a载体中,构建了原核表达载体pET30-TVBMV CP,重组质粒经BamHI和NotI双酶切鉴定及基因测序验证正确后,用IPTG进行诱导表达。结果表明:TVBMV CP在大肠杆菌中获得了高效表达,表达产物的分子量为37 kD。用表达产物为抗原免疫家兔制备了特异性抗血清,其效价为1/2048。用Western-blot检测田间样品,其结果表明,制备的抗血清可用于检测田间的发病植株。     

马铃薯Y病毒两个黑龙江烟草分离物的全基因组序列分析

为明确黑龙江烟区马铃薯Y病毒(Potato virus Y,PVY)分子株系和进化特征,从哈尔滨和牡丹江感病烟草采集到Harbin和MDJ两个样品,通过RT-PCR扩增了其全基因组序列,并进行了一致率、重组、系统发育和选择压力等分析。结果表明,2个分离物(GenBank登录号分别为MH933741和MH933742)基因组全长均为9699核苷酸。Harbin与MDJ和比利时分离物GBVC 26(JQ969039)一致率最高,为98.5%;MDJ与Harbin和GBVC 26一致率最高,为98.5%。Harbin是美国分离物Oz和瑞士分离物N-605的重组体,MDJ是美国分离物CW和N-605的重组体。利用全基因组序列构建的系统进化树显示,PVY分离物分为9个组,Harbin和MDJ均属PVYN-Wi组。PVY的11个基因均处于负选择,其中NIa蛋白基因的选择压力最大。      

烟草花叶病毒病的分子鉴定

为了建立烟草病毒病的分子鉴定体系,采用TMV,CMV和PVY3种病毒的检测引物对烟草花叶病样品进行了RT-PCR扩增,并从核酸水平上对病毒基因序列进行了分析。RT-PCR鉴定结果显示,该病害病原为TMV,测序结果显示扩增产物长923bp,序列比对后发现该序列与TMV3个中国分离物的同源性高达97%,序列分析结果表明该段序列位于TMV基因组3'末端,包含部分MP基因、完整CP基因和3'端非编码区。对CP基因进行了系统进化分析,且该序列已成功登陆GenBank。     

芝麻黄花叶病病原的分子鉴定

从芝麻黄花叶病株上分离得到花生条纹病毒2个分离物（Peanut stripe virus sesame isolate,PStV-sel和PStV-se2）。提取感病芝麻叶片的总RNA,RT-PCR扩增外壳蛋白基因（cp）片段。序列测定结果显示,印基因含有861个核苷酸,编码分子量为32．4kDa的蛋白。2个株系cp基因与PStV其它株系核苷酸序列一致性为94．4％～99．9％,氨基酸序列一致性为93．7％～100％。株系间CP氨基酸序列系统进化树结果表明,芝麻PStV分离物与中国其它寄主来源的PStV株系处于同一进化分支。     

烟草PVYN辽宁分离物全序列测定与分析

利用引起烟草脉坏死病的马铃薯Y病毒脉坏死株系辽宁分离物（Potato virus Y-vein necrosis strain Liaoning isolate, PVYN-LN）,以GenBank中已有的PVY全序列为基础,设计特异引物。通过总RNA提取、RT-PCR、3’RACE和5’RACE扩增及序列测定,测得PVYN-LN全长基因组序列（NCBI登录号为JQ971975）包括9714个碱基(含PolyA尾),整条序列仅包含一个由9186个碱基组成的开放读码框（ORF）,编码一条包含3061个氨基酸的多聚蛋白。对所得序列进行BLAST,利用MEGA软件对所测得的PVYN-LN全序列与GenBank中已有的PVY全序列及氨基酸进行系统进化分析,发现PVYN-LN分离物与已报道的PVYN株系具有较高的进化亲缘关系,该分离物属于PVYN株系。序列重组分析软件RDP4分析,该序列无重组。     

山东烟区首次发现番茄斑萎病毒侵染

为确定番茄斑萎病毒（TSWV）在山东烟草上的侵染,基于GenBank中已有的TSWV基因组序列设计该病毒特异性引物,通过总RNA提取,RT-PCR扩增,全基因组序列测定和拼接分析等,结果表明,山东临沂疑似病样中含有TSWV,该TSWV分离物具有3条RNA链,大小分别为8911、4773和2971 bp,与国内已报道的其他TSWV分离物核酸序列同源性均在99.0%以上,蛋白氨基酸序列同源性均在98.0%以上。系统进化分析结果显示,该分离物与已报道的TSWV云南分离物聚类到同一分支中,推测山东烟区中的TSWV可能由云南烟区通过带毒介体的迁入而传入。该研究为山东烟区TSWV的发生流行溯源和该危险性病毒病的精准测报及防控提供科学依据。     

迟钝爱德华氏菌FimA基因的克隆及序列分析

以迟钝爱德华氏菌Et、XA、EA分离株的DNA为模板,采用PCR技术,扩增菌毛亚基(FimA)基因的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:Et、XA、EAFimA基因由534个核苷酸组成,编码177个氨基酸。Et、XA、EA之间FimA基因核苷酸同源性为99%,与其它分离物核苷酸同源性为76%～77%,氨基酸同源性为81%～82%。     

福寿螺(Ampullaria crossean)mfc 基因的分子克隆和序列分析

目的:从福寿螺胃组织细胞中扩增多功能纤维素酶基因,与pGEM T-easy载体连接,转化大肠杆菌删DH5 a.结果:该基因全长为1 225 bp,上游5'端非翻译区19 bp,3'端非编码区21 bp,开放阅读框(0pen reading frame,ORF)1 185bp,编码395个氨基酸,推测等电点pI为6.57,分子质量45.8k Da.推测的多功能纤维素酶具有糖苷水解酶保守的氨基酸序列,属于糖苷水解酶第10家族.同源性比对结果显示,该基因及推测的氨基酸序列与egx(GenBank Accession No.AAP 31 839)的同源性分别为99.3%和99.8%,在8个位点出现核苷酸差异.在1个位点出现氨基酸差异.通过软件分析并预测,该基因编码的蛋白共存在14个丝氨酸、苏氨酸和酪氨酸磷酸化位点.没有信号肽序列.结论:成功克隆出福寿螺多功能纤维素酶基因.     

烟草黄瓜花叶病毒湖北分离物全基因组序列分析及亚组鉴定

为明确我国湖北烟区烟草黄瓜花叶病毒(Cucumber mosaic virus,CMV)的亚组类型,对湖北地区侵染烟草的CMV HB24株系进行基因组全长克隆和序列分析。结果表明,HB24株系RNA1(GenBank序列号:KC019299)全长为3363个核苷酸(nt),编码993个氨基酸的1a蛋白;RNA2(GenBank序列号:KC019300)全长为3049nt,编码859个氨基酸的2a蛋白和111个氨基酸的2b蛋白;RNA3(GenBank序列号:KC019301)全长为2216nt,编码279个氨基酸的3a蛋白和218个氨基酸的CP蛋白。核苷酸序列同源性分析表明,HB24与Fny,SD和Q株系RNA1同源性分别为91.6%,91.1%和75.9%,RNA2同源性分别为91.8%,97.8%和69.5%,RNA3同源性分别为92.1%,93.4%和73.2%;与同属花生矮化病毒(Peanut stunt virus,PSV)ER株系RNA1,RNA2和RNA3的同源性分别为65.5%,56.7%和54.1%,与同属番茄不孕病毒(Tomato aspermy virus,TAV)V株系同源性分别为66.9%,57.7%和51.5%。HB24 RNA3 5′NTR(Nontranslated region)的结构分析以及RNA3 5′NTR和CP基因核苷酸序列系统进化树的分析表明,HB24属于CMV亚组IB。     

PVY、CMV双价抗性RNA沉默表达载体的构建

采用RT-PCR方法克隆了PVYCP基因、PVYHC-Pro基因、CMVCP基因和CMV2b基因的保守片段,分别将PVYCP片段和CMV2b片段、PVYHC-Pro片段和CMV2b片段、PVYHC-Pro片段和CMVCP片段连接起来,以此为靶标构建并得到3个RNA沉默表达载体pCAMPb、pCAMHb和pCAMHP。获得的载体可以应用于植物转基因抗病毒育种工程中。     

3个黑龙江烟区烟草花叶病毒分离物的全基因组序列测定与分析

为明确黑龙江烟区烟草花叶病毒（Tobacco mosaic virus,TMV）分子株系和进化特征,从哈尔滨和牡丹江感病烟草采集到HEB1、HEB2和MDJ三个样品,通过RT-PCR扩增了其全基因组序列,并进行了一致率、重组、系统发育和选择压力等分析。结果表明,3个分离物全长均为6395核苷酸（GenBank登录号分别为MH595919、MH595920和MH595921）。HEB1和MDJ与辽宁分离物Beipiao（HE818412）一致率最高,分别为98.9%和99.6%;HEB2与云南分离物Chuxiong1（HE818417）一致率最高,为97.1%。HEB1是MDJ和HEB2的重组体。在根据全基因组序列构建的系统进化树中,TMV分为3个组,其中HEB1和MDJ均属I（U1）组,HEB2属Ⅱ（Rakkyo）组。TMV的4个基因均处于负选择,其中衣壳蛋白基因的选择压力最大。      

Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1)

The yeast Kluyveromyces marxianus strain BKM Y-719 produces an efficient pectin-degrading endopolygalacturonase (EPG) that cleaves the internal alpha-1,4-D-glycosidic linkages to yield oligomers of varying sizes. The EPG1 gene encoding this industrially important EPG was cloned by using the polymerase chain reaction (PCR) technique and degenerate primers to generate a 135 bp DNA fragment with which a genomic library was screened. The cloned fragment contained an open reading frame (ORF) of 1083 bp, encoding a 361 amino acid polypeptide. The predicted amino acid (aa) sequence of EPG showed similarity with polygalacturonases (PGs) of fungi. Analysis of the aa sequence indicated that the first 25 aa constitute a signal sequence and a motif (C218XGGHGXSIGSVG230) that is usually associated with a PG active site. Pulsed-field gel electrophoresis resolved chromosomal bands for K. marxianus BKM Y-719 and using chromoblotting it seems that EPG1 is present as only a single copy in the genome.     

DNA sequence of the mat2,3 region of Schizosaccharomyces kambucha shares high homology with the corresponding sequence from Sz. pombe

To define conserved sequences for mat1 imprinting and silencing of the mat2,3 region of Schizosaccharomyces pombe, we determined the DNA sequence of the cognate region (mat2,3 region) of another fission yeast, Sz. kambucha, a yeast species isolated from Kambucha tea mix. The entire mat2,3 region shows more than 98% identity between the two species. Sequence similarity is even higher (99.3%) for mating-type cassettes; deduced amino acid sequences of three of the four Mat peptides (Pi, Pc and Mi) are identical between the two species, while the fourth (Mc) has a single amino acid polymorphism. Comparison of the sequence motif of the imprint site essential for mat1 switching shows that mat-P of Sz. kambucha has a sequence identical to the conserved motif present in Sz. pombe. However, this sequence motif of nine bases differs by one base for mat-M of Sz. kambucha. The sequence of the K region shows about 98% identity between the two species, with the cenH region showing 98.3% homology. Thus, the arrangement of the mat2,3 region in both yeasts is conserved and shows 1-2% nucleotide sequence variation throughout the region. The DNA sequence of the mat2,3 region from Sz. kambucha has been submitted to GenBank under Accession No. AY271822.     

基于内转录间隔区序列对云南12株鹅膏菌的分子分析

目的 基于内转录间隔区(internal transcribed spacer, ITS)序列对云南省12株鹅膏菌进行分类鉴定。方法 采用十六烷基三甲基溴化铵法(cetyltrimethylammonium Ammonium Bromide, CTAB)法提取12株鹅膏菌DNA, 以ITS4和ITS5为引物进行PCR扩增, 完成DNA序列测序, 对序列进行分析并对发育树进行构建。 结果 12个样品的ITS序列长度574~755 bp, GC含量39%~47%, 平均遗传距离为0.329, YN05、YN03与其他10株鹅膏亲缘关系较远。结论 ITS序列高度保守, 在真菌的科属种上能够初步实现对物种的鉴定和系统 发育分析, 为建立云南省鹅膏属分子数据库提供基础数据。     

An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme

The lipase-encoding Arxula adeninivorans ALIP1 gene was isolated using fragments of lipase isolates obtained by trypsin digestion for the definition of oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1347 bp encoding a 420 amino acid protein of some 50 kDa preceded by an N-terminal 28 prepro-secretion sequence. The deduced amino acid sequence was found to be similar to the lipases from Candida albicans and C. parapsilosis (34-38% identity) and more distantly related to other lipases. The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases. The expression of the gene is regulated by carbon source. In media supplemented with Tween 20, induction of the ALIP1 gene and accumulation of the encoded lipase in the medium is observed, thus demonstrating gene regulation by lipophilic compounds. The enzyme characteristics are analysed from isolates of native strains as well as from those of recombinant strains expressing the ALIP1 gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins a molecular mass of 100 kDa was determined, indicating a dimeric structure, a pH optimum at pH 7.5 and a temperature optimum at 30 degrees C. The enzyme hydrolyses all ester bonds in all triglyceride substrates tested. Middle-sized chain fatty acids are more efficiently hydrolysed than short- and long-chain fatty acids, with the highest activity on C8/C10 fatty acid esters pNP-caprylate, pNP-caprate and tricaprylin.