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1.
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。  相似文献   

2.
黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus, CGMMV)是葫芦科重要种传病毒之一.本文建立了免疫捕获RT-PCR(IC-RT-PCR)的分子检测方法直接从带毒种子中检测出该病毒,扩增获得其CP基因,并克隆到pMD18-T载体中,经核苷酸序列分析表明,该分离物CP基因全长为486个核苷酸,编码由161个氨基酸组成的17.3kDa蛋白,与国内外已报道的CGMMV的CP基因相比,其核苷酸序列的同源性为91.4%~99.4%,其推导的氨基酸同源性为98.8%~100%.IC-RT-PCR方法的建立,为检疫部门提供了从带毒种子中快速、准确检测该病毒的方法,很有应用前景.  相似文献   

3.
CMV甜瓜分离物外壳蛋白基因的克隆及其生物信息学分析   总被引：1，自引：0，他引：1  
从河南省临颖县采集的病毒感染的甜瓜样本经ELISA检测和接种分离获得黄瓜花叶病毒（Cucumber mosaic vi-rus,CMV）分离物。把该分离物接种于西葫芦,从发病的叶片中提取总RNA,并以此为模板经RT-PCR扩增获得CMV的外壳蛋白（cp）基因,将其克隆到pUCm-T质粒上。经序列测定和生物信息学分析,结果表明该cp基因由657个核苷酸组成,编码218个氨基酸。预测该蛋白的等电点为5。12,分子量约为24kD。其核苷酸序列与黄瓜花叶病毒亚组I的分离物有较高的同源性,达92.2%～93.9%,与亚组II的同源性仅为76.8%～77.8%,与我国报道的CMV分离物的cp基因序列比较,只有香蕉株系XB外核苷酸序列的同源性达91.8%～93.4%。根据这些分析,该CMV分离物属于亚组I。另外还对黄瓜花叶病毒外壳蛋白的高级结构作出了预测。  相似文献   

4.
用鸡源新城疫病毒凤阳分离株WF00C对9～10 日龄SPF鸡胚的尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF00C病毒的F基因,获得了1条1.7kb的特异性条带.用PCR产物直接测序.测序结果表明,扩增片段大小为1782bp,含有1个1662bp的开放性阅读框,编码554个氨基酸.核苷酸同源性分析表明:WF00C株与国内外其他NDV F基因的同源性为84.8%～97.5%,其中与国内标准强毒株F48E9的同源性为87.1%,说明WF00C与国内外的传统毒株有较大变异.与Taiwan95株和J株的同源性为94.1%和97.5%,说明WF00C与Taiwan95株和J株亲缘关系较近,具有较高的相似性.F蛋白裂解位点的氨基酸顺序为112Arg-Arg-Gln-Lys -Arg-Phe117, 表明为NDV的强毒株.蛋白疏水性和抗原性分析表明与标准强毒株相比没有太大的变异.  相似文献   

5.
用鸭源WF01D株新城疫病毒接种对9-10日龄SPF鸡胚尿囊腔,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF01D病毒的HN基因,获得了1条约1.8kb的特异性条带。PCR产物回收纯化后测序。测序结果表明,扩增片段大小为1844bp,含有1个1716bp的开放性阅读框,编码571个氨基酸。核苷酸同源性分析表明：WF01D与国内外其他NDV HN基因的同源性为81.2%-95.0%,其中与国内标准强毒株F48E9的同源性为84.3%,说明WF01D与国内外的传统毒株有较大变异。与Taiwan95株和NL/96株的同源性为93.8%和95.0%,说明WF01D与Taiwan95株和NL/96株亲缘关系较近,具有较高的相似性。  相似文献   

6.
构建马氏珠母贝腹足SMART cDNA质粒文库,测序和在GenBank中进行同源性查找,得到了核糖体40s亚基S13基因的全长cDNA序列。马氏珠母贝S13的cDNA全长554bp,推测的开放阅读框位于27-479,编码151个氨基酸,其中亮氨酸16个,所占比例为10.6%。马氏珠母贝S13同鲶鱼以及真菌纲的球毛壳的同源氨基酸序列相似性分别为90.05%和72.85%,很保守,可以用于种以上生物的进化关系研究。聚类分析显示,马氏珠母贝与脊椎动物和昆虫的亲缘关系较近,同线虫、水稻、啤酒酵母和球毛壳等动植物、真菌等相距较远。  相似文献   

7.
以牛的ANGPTL1基因为研究对象,利用生物信息学方法,对牛的ANGPTL1基因进行了电子克隆和序列分析,并对推导出的ANGPTL1蛋白结构与性质进行了初步分析。结果表明,牛的ANGPTL1基因序列长为2576 bp,该基因的编码序列长为1 476 bp,编码492个氨基酸,编码序列的两翼有135 bp的5’非编码区和785 bp的3’非编码区。DNA序列的G+C百分含量为44.31%,A+T百分含量为55.69%。该基因的核苷酸序列与人、黑猩猩、鼠和狗ANGPTL1基因的cDNA序列的相似性分别为91%、90%、82%和93%。在氨基酸序列上与人、黑猩猩、鼠和狗的相似性分别为95%、95%、92%和95%。用氨基酸序列构建的进化树显示,在人、牛、黑猩猩、狗、褐鼠、原鸡几种动物中,牛与狗的亲缘关系最近。  相似文献   

8.
为进一步研究克隆到的鹅、鸭、鹌鹑、斑鸠Mel 1c基因,对该序列进行特征分析,为Mel 1c基因的功能研究打下基础。参考NCBI其它物种的序列设计引物,克隆得到鹅、鸭、鹌鹑、斑鸠Mel 1c基因的部分c DNA序列,并对其编码的蛋白进行生物信息学分析。鹌鹑、鸭、鹅和斑鸠的Mel 1c部分片段,长度为850bp,编码283个氨基酸。通过分析鹌鹑、鸭、鹅和斑鸠Mel 1c的结构表明符合褪黑素受体的结构特征,含有一N端在胞内六个跨膜结构域,属于G蛋白偶联受体家族。该蛋白质为分泌蛋白,包含一个信号肽,其剪切位点位于26和27之间(VVA-VY)。同源性比对结果表明不同物种间Mel 1c基因序列及氨基酸序列的同源性较高均大于71%,Mel 1c在进化过程中比较保守。鹅、鹌鹑、斑鸠、鸭和原鸡的核酸和氨基酸的同源性最高分别大于92%和96%。Mel 1c符合进化规律,斑鸠、鹌鹑、鹅和鸭属于高等的非哺乳类脊椎动物,处于分子进化树的最顶层。  相似文献   

9.
珙桐rbcL基因的克隆与序列分析   总被引：1，自引：0，他引：1  
根据已知植物rbcL基因设计引物,以珙桐叶片总DNA为模板,PCR扩增目的DNA片段,克隆入pDM19-T载体.阳性克隆鉴定后进行测序,序列分析结果表明:该基因片断长为1266 bp,包括1031 bp的编码区序列,编码343个氨基酸;其编码区氨基酸与烟草、菠菜、玉米、甘薯、拟南芥、水稻、葡萄、地钱和葡萄柚等9个物种的同源性94.13%以上,并构建了它们间的亲缘关系树.该基因片断的预测晶体结构与菠菜的最相近,推测Lys86、Lys60、Lys179等残基对酶的活性影响较大.  相似文献   

10.
灰斑古毒蛾核型多角体病毒几丁质酶基因及其分子进化   总被引：5，自引：0，他引：5  
通过对构建的灰斑古毒蛾Orgyia ericae单粒包埋型核型多角体病毒(OrerSNPV)基因组DNA的EcoRI酶切片段质粒文库的测序分析,在EcoRI—J片段(6．4kb)中鉴定出了编码几丁质酶(ChiA)基因开放阅读框(ORF),chiA基因编码区由1695bp组成,编码564个氨基酸,预计蛋白质分子量为62kD。这也是首次在基因序列水平上研究OrerSNPV。将OrerSNPV ChiA氨基酸序列与其它已知的18种杆状病毒ChiA氨基酸序列联配比较,结果表明OrerSNPV与其它杆状病毒ChiA氨基酸有60．3—69．5％同源性,其中与BmNPV、CfDEFNPV和LdMNPV的同源性最高。根据氨基酸序列绘制的分子进化树表明杆状病毒chiA基因至少可以分为SlMNPV、GV和其它NPV三个分支,OrerSNPV与LdMNPV在进化树上关系最为接近。  相似文献   

11.
Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV is known to have four segmented, single-stranded RNA molecules and causes rice stripe disease in the rice fields of China, Japan, and Korea. Based on the complete genomic sequences of the determined 6 RSV isolates (from Yunnan, Jiangsu, Zhejiang, and Liaoning Provinces, China) and 27 other RSV isolates (from Yunnan, Jiangsu, Anhui, Henan, and Shandong Provinces of China, also Japan and Korea) downloaded from GenBank, we provided a genotyping profile of RSV field isolates and described the population structure of RSV. All RSV isolates, except isolate CX, could be divided into two subtypes, one including 6 isolates from Yunnan Province, and the other including 26 isolates from different parts of China, Japan, and Korea, which were referred to as subtype II and subtype I, respectively. The amino acid distances between subtypes range from 0.053 to 0.085. RSV isolates in Yunnan Province were genetically differentiated from other parts of China, Japan, and Korea and showed infrequent gene flow. The RSV populations collected from other parts of China, Japan, and Korea were only composed of subtype I and showed very low genetic diversity. We speculated that isolate CX may be the result of recombination of isolates from two subtypes. Two potential recombination events were detected in RNA4 of isolate CX.  相似文献   

12.
To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi’an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi’an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%–99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites.  相似文献   

13.
Genome segments S8 of two Chinese isolates of rice black-streaked dwarf virus (RBSDV), one from Zhejiang Province and another from Hebei Province, were amplified by RT-PCR and sequenced. Both segments consisted of 1936 nts in full length (EMBL accession numbers were AJ297431 and AJ297432, respectively) and contained only one big open reading frame which encoded a polypeptide with molecular weight of 68kD. The two Chinese isolates shared 94.0% and 96.5% identity at nucleotide and amino acid level, respectively. They shared 94.5-94.9% and 92.5-92.9% homology with S8 of RBSDV Japanese isolate at nucleotide and amino acid level, respectively; shared 85.1-87.6% and 91.7-91.9% homology with S7 of Italian MRDV (maize rough dwarf virus).  相似文献   

14.
For a long time, classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics. A new subspecies of Demodex folliculorum has been proposed, but not confirmed. Here, cox1 partial sequences of nine isolates of three Demodex species from two geographical sources (China and Spain) were studied to conduct molecular identification of D. folliculorum. Sequencing showed that the mitochondrial cox1 fragments of five D. folliculorum isolates from the facial skin of Chinese individuals were 429 bp long and that their sequence identity was 97.4%. The average sequence divergence was 1.24% among the five Chinese isolates, 0.94% between the two geographical isolate groups (China (5) and Spain (1)), and 2.15% between the two facial tissue sources (facial skin (6) and eyelids (1)). The genetic distance and rate of third-position nucleotide transition/transversion were 0.0125, 2.7 (3/1) among the five Chinese isolates, 0.0094, 3.1 (3/1) between the two geographical isolate groups, and 0.0217, 4.4 (3/1) between the two facial tissue sources. Phylogenetic trees showed that D. folliculorum from the two geographical isolate groups did not form sister clades, while those from different facial tissue sources did. According to the molecular characteristics, it appears that subspecies differentiation might not have occurred and that D. folliculorum isolates from the two geographical sources are of the same population. However, population differentiation might be occurring between isolates from facial skin and eyelids.  相似文献   

15.
Papaya leaf curl China virus (PaLCuCNV) was previously reported as a distinct begomovirus infecting papaya in southern China. Based on molecular diagnostic survey, 13 PaLCuCNV isolates were obtained from tomato plants showing leaf curl symptoms in Henan and Guangxi Provinces of China. Complete nucleotide sequences of 5 representative isolates (AJ558116, AJ558117, AJ704604, FN256260, and FN297834) were determined to be 2738–2751 nucleotides, which share 91.7%–97.9% sequence identities with PaLCuCNV isolate G2 (AJ558123). DNA-β was not found to be associated with PaLCuCNV isolates. To investigate the infectivity of PaLCuCNV, an in-fectious clone of PaLCuCNV-[CN:HeNZM1] was constructed and agro-inoculated into Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants, which induced severe leaf curling and crinkling symptoms in these plants. Southern blot analysis and polymerase chain reaction (PCR) indicated a systemic infection of test plants by the agro-infectious clone.  相似文献   

16.
The molecular cloning of the G1 genome segment of the Z10 strain of Hantaan virus and analysis of the first gene data on the currently widely used Hantavirus vaccine strain in China are presented. The G1 genome segment of Z10 virus was amplified by the method of RT-PCR, and the products were cloned into the pGEM-T vector after identification and purification. The clone was sequenced by Sanger’s dideoxy chain termination method. The 1449 bases of the Z10 G1 genome segment, and the coding for 483 amino acids were determined. The base compositions for the G1 segment RNA determined from cDNA sequence information, were 20.6% A, 21.6% G, 18.8% C and 30.0% U. These values are similar to those of the 76/118, Lee and SR virus. As compared to the Z10 G1 segment, the sequence homology at the nucleotide level is 87% (76/118, type I), 86% (Lee, type I), 86% (Hojo, type I), 67% (R22, type II), and 59% (K22, type III). The Z10 virus G1 protein amino acid sequence identity with other Hantaan viruses (94–95% homology) was higher than that with Seoul type viruses (77–80%). More amino acid sequence heterogeneity between the Z10 and 76/118 was observed in the N terminal, especially the diverging cluster of ten amino acids at position 84 to 93 of the Z10 virus G1 protein. Conclusion: (1) The Z10 strain is one of the Hantaan viruses. (2) The important region of the Z10 G1 segment was conservative. (3) Although substantial divergence of nucleotide sequences of the G1 genome segment was found between the Z10 strain and other type I Hantaviruses, relatively high amino acid sequence homology was shown among them. Thus, good immune protection could be obtained with the inactivated Meriones unguiculatus kidney cell vaccine against other strains of Hantaviruses.  相似文献   

17.
Using degenerate primers and RT-PCR,RACE techniques,a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase(SAD)is cloned from developing seeds of Jatropha curcas L.The segment contains a 1191 bp of complete open reading frame(ORF).Analysis in the BLAST on NCB! shows that Jatropha curcas SAD(JSAD)gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids.The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs.The nucleotide and peptide identity of JSAD to Ricinus communis SAD(RSAD)is up to 89% and 96.2% respectively.Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.  相似文献   

18.
利用西方蜜蜂雄蜂头部5’LongSAGE文库中的一组标签序列,在蜜蜂基因组序列第9号连锁群上定位一个新的西方蜜蜂表皮蛋白基因转录起始位点,并克隆了该基因的eDNA序列,继而利用此cDNA序列分析了该基因的内含子和外显子结构．分析结果显示：该基因的DNA序列长1253bp,含有4个外显子和3个内含子,其cDNA长598bp,编码一个169AA的富含Val和Pro残基（23．67％,20．12％）多肽序列．BLAST分析发现,该蛋白与已知的4个昆虫表皮蛋白序列的相似性为47．54％,且其C末端有一个共有基序,说明这5个蛋白可能属于同一个表皮蛋白家族．该研究结果提供了一个新的蜜蜂表皮蛋白基因,且该基因在雄蜂头部表达水平较高．  相似文献   

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