凌云牛心李DNA条形码筛选及其亲缘关系分析

本研究通过对9份凌云牛心李进行ITS、matK及rbcL序列PCR扩增和序列比对分析,旨在筛选适合于凌云牛心李开展遗传多样性研究的DNA条形码。通过序列比对分析,9份李种质ITS序列长度在713 bp~717 bp之间,G+C含量59.33%~60.95%之间,存在27个变异位点;matK序列长度为935 bp~937 bp之间,G+C含量33.33%~33.55%之间,存在5个变异位点;rbcL序列长度为743 bp,G+C含量42.20%~43.07%,存在3个变异位点。相对于matK及rbcL序列,ITS序列进化速率较快,存在更为丰富的遗传变异位点,更适宜用来开展凌云牛心李的分子鉴定及亲缘关系分析研究。基于ITS序列构建了9份凌云牛心李、22份其他地区李种质以及5份杏、5份梅种质的NJ系统进化树,凌云牛心李与其他李种质亲缘关系较近,其中N2、N3、N8和N9中国宁波的茄皮李和日本的大石早生李聚为一个类群,而N1、N4、N5、N6和N7与中国嘉兴的槜李、中国山东的玉皇李、中国贵州的九阡李聚为一个分支,表明凌云牛心李作为地方优质李品种,在长期种植过程中种内产生了较丰富的遗传变异。     

细口杯环线虫形态学和分子特征分析

为探讨细口杯环线虫(Cylicocyclus leptostomum)的分类地位和系统发育关系,本试验利用数码显微镜对细口杯环线虫进行了形态观察,运用PCR扩增其核糖体DNA内部转录间隔区(rDNA-ITS)序列,并从GenBank中下载12种圆线虫的ITS序列,以马圆形线虫(Strongylus equinus)为外群,运用最大似然法(maximum likelihood,ML)构建系统发育树。结果显示,细口杯环线虫中等大小,口囊呈圆柱形,宽度大于深度,口囊底部有小齿;口囊壁前端薄,后端基部有明显的环箍形增厚;外叶冠由20～24个小叶组成,内叶冠由50～60个小叶组成;食道漏斗较小;雄虫生殖锥较长,呈圆锥形;雌虫尾部直,尾尖呈指形;所测ITS序列总长度为837bp,其中ITS1长366bp,5.8S长153bp,ITS2长318bp;ITS1的GC含量(46.0%)明显高于ITS2(39.8%);经BLAST同源性比对分析,本研究线虫ITS序列与GenBank上登录的同种线虫序列(登录号:AJ004849、Y08587)同源性达99.85%,与阿氏杯环线虫的同源性达99.0%,与杯环属内其他线虫的同源性仅为93.33%～98.45%;ITS序列种间差异远大于种内差异;系统进化分析显示,细口杯环线虫与阿氏杯环线虫的亲缘关系较近,而与杯环属内其他线虫的亲缘关系相对较远。综上所述,ITS序列可作为鉴定寄生线虫的分子遗传标记,证实所采标本是细口杯环线虫,并首次在国内报道了细口杯环线虫的ITS序列,为该线虫的进一步研究奠定基础。     

桑属植物ITS序列研究与系统发育分析

用PCR产物直接测序法对桑属的 9个种 3个变种共 13份桑种质和构属构树的ITS序列进行了测定。结果表明 :桑属植物ITS1长度平均约为 189bp;桑属 5 8SrRNA为 15 2bp ;ITS2长度平均为 2 12bp ;桑属植物ITS序列G +C含量为 6 0 %左右。用DNASTAR软件构建了桑属植物ITS序列的系统发育树 ,并探讨了参试桑种质的亲缘关系。     

3种冠环线虫rDNA-ITS的PCR扩增及序列分析

本试验利用PCR扩增3种冠环线虫5个样品的核糖体DNA内转录间隔区(ITS)及5.8S片段,将PCR扩增产物纯化后直接进行序列测定和分析。序列比对和分析结果显示,所测样品ITS1-5.8S-ITS2的长度范围为748~843 bp,总变异位点(包括gaps)119个,简约信息位点18个;其中ITS1和ITS2的长度范围分别为367~370 bp和228~320 bp,变异位点分别为14个和105个,简约信息位点均为9个。所有测试样品的5.8S片段完全相同,长度为153 bp。5条序列ITS1区的G+C含量(48.0%~48.5%)明显高于ITS2区(37.7%~40.3%)。通过序列两两比对,3种冠环线虫ITS1和ITS2的种间差异性分别为1.9%~3.5%和5.6%~31.8%;而种内差异性分别为0~0.5%和0~0.9%。并且所测序列与GenBank中已知序列的同源性为99.07%~99.41%。本研究认为,ITS序列可以作为冠环线虫种类鉴定的分子标记。     

黄鳝胃瘤线虫ITS及5.8S rDNA的克隆及序列分析

本研究旨在阐明黄鳝胃瘤线虫湖南分离株的核糖体DNA(rDNA)内转录间隔区(ITS)及5.8 S rDNA序列的遗传变异情况,并用ITS序列重构胃瘤线虫与其它线虫的种群遗传关系.利用聚合酶链反应(PCR)扩增胃瘤线虫rDNA的ITS-1、5.8S及ITS-2片段,将PCR扩增出的片段纯化后克隆至pGEM-T Easy载体,重组质粒通过菌落PCR鉴定后,对阳性菌落进行序列测定并进行序列分析.结果显示所获得的胃瘤线虫ITS及5.8 S rDNA序列总长存在一定差异(922～927 bp),其中包含部分的18S、28 S及全部的ITS-1 (350～351 bp)、5.8S(102 bp)及ITS-2 (340～344 bp)序列.本研究系国内首次报道胃瘤线虫的ITS序列,其结果为黄鳝胃瘤线虫的分类鉴定以及进一步的分子流行病学调查和群体遗传研究奠定了基础.     

Culicoides arakawae (Diptera: Ceratopogonidae) efficiently blood-fed and infected with Leucocytozoon caulleryi through a natural membrane

Culicoides arakawae, the most common Culicoides sp. on chicken farms in East Asia, is an important blood-sucking insect and Leucocytozoon caulleryi vector. How parasites, in an ingested blood bolus, enter the midgut of insects and deal with this complex and biochemically hostile environment is poorly understood. However, successful blood-feeding through a membrane in C. arakawae is beneficial for studying this phenomenon. Therefore, a membrane-feeding method for C. arakawae was developed in. The blood-feeding success rates of C. arakawae fed through five different membranes were: turkey egg at 43.7+/-11.7%, chicken egg at 45.2+/-12.1%, duck egg at 38.8+/-12.0%, pig gut at 0% and chick skin at 0%. In fertility measurements, the average number of eggs produced for C. arakawae fed through egg-shell membrane, at 77.7+/-15.1 per female, was significantly higher (P<0.01) than the 46.7+/-10.6 found in C. arakawae fed on the breast skin of a live chicken. Meanwhile, in parasite infectivity tests, C. arakawae could be infected by L. caulleryi when the vector was blood-fed with infective blood cells reconstituted with specific pathogen-free (SPF) sera through an egg-shell membrane. The sporozoite average and infection rates of inoculated chicks were 166.8+/-12.5 and 100%, respectively. In conclusion, feeding C. arakawae blood through fowl-egg-shell membranes should be an efficient method for in vitro infection of midges as the engorged midges are infected by parasites and display reproductive potential. Furthermore, the method is practical for feeding a large number of midges.     

Molecular detection of Culicoides spp. and Culicoides imicola, the principal vector of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe

Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap network for the collection of Culicoides, was implemented in 2002 in southern mainland France. The morphological identification of Culicoides can be both tedious and time-consuming because its size ranges from 1.5 to 3 mm. Therefore, an ITS1 rDNA polymerase chain reaction (PCR)-based diagnostic assay was developed to rapidly and reliably identify Culicoides spp. and C. imicola. The aim of this work was to set up a rapid test for the detection of C. imicola amongst a pool of insects collected in areas at risk for BT. The sequence similarity of the rDNA (nuclear ribosomal DNA), which is greater within species than between species, is the foundation of its utilisation in species-diagnostic assays. The alignment of the 11 ITS1 sequences of Culicoides obtained from Genbank and EMBL databases helped us to identify one region in the 5' end and one in the 3' end that appear highly conserved. PCR primers were designed within these regions to amplify genus-specific fragments. In order to set up a C. imicola-specific PCR, another forward primer was designed and used in combination with the previously designed reverse primer. These primers proved to be highly specific and sensitive and permitted a rapid diagnostic separation of C. imicola from Culicoides spp.     

棕熊蛔虫ITS rDNA的PCR扩增与序列分析

目的以核糖体DNA（rDNA）的第一与第二内转录间隔区序列（ITS-1及ITS-2）鉴定从广州动物园棕熊分离的蛔虫种类。方法应用PCR方法以保守引物NC5和NC2扩增蛔虫样本的ITS及5．8SrDNA序列,将PCR扩增出的片段纯化后克隆至pGEM-TEsay载体,用菌落PCR及酶切鉴定阳性菌落,对阳性菌落进行测序,并与Gen．Bank“公布的人蛔虫（Ascaris lumbricoides）、猪蛔虫（Ascariis suum）、浣熊贝利斯蛔虫（Baylisascaris procyonh）及狮弓蛔虫（Toxascaris leonina）ITS序列比较。结果从棕熊分离的2个蛔虫样本的ITS及5．8SrDNA总长为859—861bp,种内相似性为99．7％,与GenBank^TM公布的人蛔虫、猪蛔虫、浣熊贝利斯蛔虫及狮弓蛔虫的相似性分别为84．6％、84．5％、89．3％及72．3％,序列差异明显。结论棕熊蛔虫不同于上述种类蛔虫,可能为Baylisacaris transfuga.     

Characterization of Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle by internal transcribed spacers of nuclear ribosomal DNA

In the present study, samples representing Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle in Heilongjiang Province, China, were characterized and grouped genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The rDNA region including the ITS-1, 5.8S, ITS-2, and flanking 18S and 28S rDNA sequences was amplified by polymerase chain reaction (PCR), then sequenced and compared with that of other members of the hookworms available in GenBank?, and phylogenetic relationships between them were reconstructed using the Maximum-Parsimony method. The ITS-1, 5.8S, and ITS-2 sequences of the sheep hookworm were 381, 153, and 231 bp in length, respectively, and the corresponding sequences of the cattle hookworm were 392, 153, and 240 bp in length. The identity of ITS sequences of B. trigonocephalum and B. phlebotomum from sheep and cattle was 87.4%. A PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay using restriction endonuclease Nde I was established for the unequivocal differentiation of the two hookworm species. Phylogenetic analyses based on the ITS sequences revealed that B. trigonocephalum and B. phlebotomum were closely related, but they represent two different species.     

大熊猫蛔虫ITS及5.8SrDNA序列的克隆与分析

基于核糖体DNA第一与第二转录间隔序列以及5.8S序列,以分离自广州动物园大熊猫体内的蛔虫为研究对象,用保守引物NC_5和NC_2对核糖体DNA（rDNA）的内转录间隔区ITS-1,ITS-2及5.8S序列进行PCR扩增,扩增后的片段纯化后克隆至pGEM-Teasy载体,重组质粒通过菌液PCR鉴定后,对阳性菌落进行序列测定及分析,鉴定大熊猫蛔虫的种类。结果显示,目的片段总长为910 bp,2个不同样品之间的ITS及5.8S序列没有差异,与GenBank~（TM）中的拜林蛔线虫（Baylisascaris transfuga）、猪蛔虫（Ascaris suum）和人蛔虫（Ascaris lumbricoides）的ITS序列相似性分别为96.6%、82.9%和82.7%。结果表明,此次分离的大熊猫蛔线虫可能为拜林蛔线虫。     

卡拉干斯齿线虫的形态及ITS和COⅠ序列分析

利用光学显微镜和PCR扩增技术,对采自河南省马肠道内的卡拉干斯齿线虫(Skrjabinodentus caragandicus)进行形态学观察和分子序列测定。结果表明,卡拉干斯齿线虫中等大小,口囊壁呈"S"状,前端极度膨大,厚度10μm~12μm,后端稍薄,约4μm~6μm,外叶冠由8个长而宽的小叶组成,内叶冠由16个~18个短而宽的小叶组成,食道漏斗发达;雄虫生殖锥长337μm,引器长245μm;所测核糖体DNA(rDNA)内转录间隔区(ITS)及5.8S片段长度为853bp,其中ITS1长369bp,ITS2长331bp,ITS1区的GC含量(47.2%)明显高于ITS2区(40.7%);线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因长度为393bp,A、T、G、C碱基含量分别为25.2%、42.7%、21.4%、10.7%。卡拉干斯齿线虫在河南省属首次报道,为河南省新记录种。     

PCR-SSCP对我国鲁道夫对盲囊线虫的分子鉴定

对来自青海湖的鲁道夫对盲囊线虫（Contracaecum rudolphii）核糖体DNA第一、第二内转录间隔区（ITS-1、ITS-2）进行PCR扩增、DNA单链构象多态性（SSCP）分析及序列分析。并与来自欧洲的2个姊妹种鲁道夫对盲囊线虫进行了比较。结果显示，我国青海湖的鲁道夫对盲囊线虫与来自意大利的（.rudolphii B具有一样的SSCP带型及ITS序列，但不同于C．rudolphiiA．因此属于C.rudolphii B。本试验证实了ITS片段可作为遗传标记用于鉴别鲁道夫对盲囊线虫的姊妹种，从而为鲁道夫对盲囊线虫的进一步研究奠定了基础。     

斯氏艾美耳球虫ITS1-5.8S rRNA-ITS2序列的克隆及PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18SrRNA和28SrRNA进行序列比对分析,在18SrRNA 3′端和28SrRNA 5′端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8SrRNA-ITS2序列,其大小为1 178bp,其中ITS1序列长度为423bp,5.8SrRNA为155bp,ITS2为600bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列相似性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

基于ITS1-5.8S rRNA-ITS2序列兔肝球虫PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18S rRNA和28S rRNA进行序列比对分析,在18S rRNA 3’端和28S rRNA 5’端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8S rRNA-ITS2序列,其大小为1178bp,其中ITS1序列长度为423bp,5.8S rRNA为155 bp,ITS2为600 bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列同源性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.     

狼尾草属牧草rDNA的ITS序列分析

对来自福建、江苏、海南等15份狼尾草属牧草的5.8SrDNA、ITS1及ITS2片段进行克隆及序列分析,并登录于GenBank数据库,采用DNAMAN、CLUSTALX、MEGA等软件分析其遗传关系聚类图。结果显示,克隆的目的片段长度为573～586bp,聚类结果总体能较好地反应狼尾草属牧草之间的遗传距离,其中杂交狼尾草和细茎杂交狼尾草可能存在着同种异名的现象。     

白鹈鹕体内对盲囊线虫种类的鉴定及其ITS序列分析

为了鉴定从广东某地圈养的白鹈鹕体内的线虫种类,2个样本通过形态学观察初步鉴定为对盲囊线虫后,对虫体核糖体DNA（rDNA）内转录间隔区ITS-1、ITS-2及5.8SrDNA序列进行扩增和测序,并与GenBankTM公布的序列进行比对,构建系统发育树分析,确定其种类.结果显示,白鹈鹕体内线虫符合对盲囊线虫的形态学特征,其ITS-1序列长度为463 bp,与Contracaecum sp.（GenBankTM登录号：KF990496.1）ITS-1的序列相似性分别为98.9％、99.8％;ITS-2序列长度分别为288-289 bp,与C.bancrofii（GenBankTM登录号：FM177887.1）的ITS-2序列的相似性分别为96％、95％;5.8SrDNA序列长度为157 bp.通过以ITS-1序列为分子标记的系统发育树分析,白鹈鹕体内线虫与C.bancrofti属同一分支,应为C.bancrofii.形态学鉴定方法结合ITS序列分析可以准确鉴定白鹈鹕体内线虫的种类,为其他线虫的快速准确鉴定提供参考.     

Culicoides arakawae (Diptera: Ceratopogonidae) population succession in relation to leucocytozoonosis prevalence on a chicken farm in Taiwan

Leucocytozoonosis caused by Leucocytozoon caulleryi is a significant disease prevalent in open chicken houses of southern and eastern Asia. L. caulleryi is transmitted by Culicoides arakawae, a blood-sucking vector. Leucocytozoonosis prevalence is influenced by vector population succession. Thus this examination was performed on a farm to investigate vector population succession and leucocytozoonosis prevalence in experimental chicks and to obtain the ecology data for assessing the prevalence. The findings were as follows: (1) C. arakawae adults might be highly host specific because they were rarely discovered on cattle or pig farms, and none of the experimental chickens were infected by L. caulleryi on those farms. (2) Identifying and counting gorged and gravid C. arakawae to assess leucocytozoonosis prevalence is a practical strategy. The critical vector index should be 5.0 calculated by dividing the smallest vector mean from the prevalent period by the largest vector mean from the population not causing leucotocytozoonosis. (3) Taking vector means from three or more collections each month, should be the best assessment of leucocytozoonosis prevalence because C. arakawae succession appears to have a 3-week periodicity. Hopefully, these findings will contribute to assessing leucocytozoonosis prevalence.     

小熊猫蛔虫ITS及5.8S rDNA序列的克隆与分析

以广州动物园小熊猫体内分离出的蛔虫为研究对象,运用PCR方法,以保守引物NC5和NC2扩增其核糖体DNA（rDNA）的内转录间隔区（ITS）和5.8S序列,并对扩增后的片段进行纯化、克隆至pGEM-Teasy载体、转化、测序和序列分析,以鉴定小熊猫蛔虫的种类。结果显示2条蛔虫样品的ITS及5.8S rDNA序列基本一致,总长为913 bp,样品间序列相似性为99.7%。将序列与GenBank^TM公布的相关序列进行比较分析,结果显示2条蛔虫的ITS及5.8S序列与黑熊横走贝蛔虫（Baylisascaris transfuga注册号AB571304）相似性分别为98.1%、98.4%,与大熊猫西氏贝蛔虫（Baylisascaris schroederi注册号JN210912）相似性分别为96.9%、97.1%,与猪蛔虫（Ascaris suum注册号AB571302）相似性分别为89.9%、90.1%,与人蛔虫（Ascaris lumbricoides注册号AB571296）相似性分别为89.8%、90.1%,ITS-1序列与浣熊贝蛔虫（Baylisascaris procyonis注册号AB053230）相似性分别为92.0%、92.3%。研究结果表明小熊猫体内分离的蛔虫可能为贝蛔属蛔虫,从而为蛔虫的进一步分类、鉴定和遗传变异研究奠定了基础。     

Amplification of ribosomal DNA of Anoplocephalidae: Anoplocephala perfoliata diagnosis by PCR as a possible alternative to coprological methods

The diagnosis of tapeworm infections in horses relies on copro-diagnostic methods, which are time-consuming and of limited sensitivity for determination of the exact prevalence. The development of serological tests has slightly improved the detection of tapeworm infections, but more sensitive methods are still required. A polymerase chain reaction (PCR)-based approach may constitute a valuable tool to improve tapeworm diagnosis. Nuclear ribosomal DNA (rDNA) is a useful target for species and/or strain markers. Partial 18S, the internal transcribed spacer 1 (ITS-1), the 5.8S, the internal transcribed spacer 2 (ITS-2), and partial 28S rDNA of the equine tapeworms Anoplocephala perfoliata and Anoplocephaloides mamillana were amplified and sequenced. The lengths and GC contents of the regions sequenced were 2087-2091bp and 49.35-49.69% for A. perfoliata, and 2110-2119bp and 49.15-49.32% for A. mamillana, respectively. Sequence alignment and comparison of both taxa showed 79.3-80.2% identity. The lowest identities were found in the ITS regions with 39.9-43.5% for the ITS-1 and 59.5-61.2% for the ITS-2. No matches of the ITS-2 of A. perfoliata and A. mamillana were found with other species by BLAST search. For this reason, ITS-2 sequences seemed appropriate as accurate species markers and A. perfoliata ITS-2 primers were developed. The ITS-2 PCR enabled the detection of genomic DNA as low as 0.5 pgs. First efforts on the practical application of the PCR-based approach were made. A 6-mg fragment of a tapeworm proglottid was detected in 0.5 and 1g of faeces.