我国多斑按蚊复合体成员种的分子鉴别研究

目的建立我国多斑按蚊复合体5成员种的分子鉴别方法。方法测定并分析各成员种的rDNA-ITS2序列,并设计种特异引物,应用PCR法鉴别。结果多斑按蚊复合体5成员种的ITS2序列长度和GC含量分别为伪威氏按蚊328bp、58.54％、多斑按蚊330bp、57.85％、威氏按蚊337bp、59.05％、达罗毗按蚊334bp、58.68％和塞沃按蚊338bp、57.69％,各种内的ITS2序列保守,而种间的差异率范围为9.7％～18.9％。应用5.8S引物和5个种特异引物可以分别扩增出119、186、231、327和406bp等5条清晰不同的种特异条带,以鉴别各蚊种。结论基于ITS2序列差异建立的我国多斑按蚊复合体5成员种的PCR鉴别简便易行、可靠。     

应用rDNA-ITS2诊断性序列现场对3种重要蚊媒的鉴定

目的 建立利用核糖体DNA第二内转录间隔区(rDNA-ITS2)诊断性序列鉴别蚊媒的分子生物学方法. 方法 根据淡色库蚊、白纹伊蚊、嗜人按蚊rDNA-ITS2基因两侧的5.8S和28S保守区设计诊断性引物,进行3种蚊虫的rDNA-ITS2克隆鉴定,并利用其rDNA-ITS2特征进行现场蚊虫的鉴别. 结果 诊断性引物对于淡色库蚊、白纹伊蚊、嗜人按蚊DNA模板的扩增产物分别为440、514和558 bp,3种蚊虫阳性克隆的测序结果与GenBank-blast相应蚊种rDNA-ITS2序列的同源性均为99%.用Biosept、DNAstar等软件分别分析发现各蚊虫克隆的个体间存在一定程度的单核苷酸多态性(SNP).将现场采集的蚊虫诊断性引物扩增产物进行阳性克隆进行序列测定和分析,显示与淡色库蚊阳性对照的一致性为99%;与GenBank中淡色库蚊rDNA-ITS2基因的同源性为99%. 结论 通过对淡色库蚊、白纹伊蚊、嗜人按蚊核糖体DNA第二内转录间隔区(rDNA-ITS2)基因的序列分析,表明3种蚊媒的rDNA-ITS2基因具有可鉴别的种属特异性.利用同一体系的共享引物扩增,可以建立适用于3种蚊媒的现场鉴定方法.     

不同地区微小按蚊rDNA-ITS2序列差异

目的　比较我国云南、海南省、广西壮族自治区及泰国等不同地区微小按蚊核糖体 DNA第 2内转录间隔区 ITS2 )序列差异。　方法　取单只雌蚊蚊腿消化提取 DNA,PCR特异扩增 r DNA- ITS2片段 ,并对其扩增产物纯化、测序及分析。　结果　发现 2种不同的 ITS2序列 Gen Bank登录号 :AF4 16 783,AF4 16 784 ) ,分别与微小按蚊 A和 C同源 ,ITS2序列长度及 GC含量分别为 4 81bp,5 4 .0 4 %和 4 83bp,5 4 .2 3% ,两者无显著性差异 ;但 2 2个固定位点存在碱基置换和插入 /缺失 ,序列差异为 5 .8%。　结论　研究区内微小按蚊存在 A和 C两个不同的亲缘种     

基于核糖体 DNA 第二内转录间隔区基因的 9 种蚊虫分子鉴定

目的 分析常见 9 种蚊虫核糖体 DNA 第二内转录间隔区( rDNA-ITS2 )基因序列特征,为蚊虫种类分 子鉴定方法探讨提供依据。 方法 在山东省济宁市采集淡色库蚊、三带喙库蚊、二带喙库蚊、白纹伊蚊、刺扰伊蚊、中 华按蚊、骚扰阿蚊、常型曼蚊和黄色柯蚊成蚊,形态学种类鉴定后,提取上述 9 种蚊虫 DNA,PCR 扩增 rDNA-ITS2 基 因并测序;在 GenBank 中进行同源性比对,采用 Bioedit 7. 0 软件及 DNAMAN 软件对测序结果进行比对分析,通过 DNASTAR、ClustalX 1. 81 和 Mega6 软件分析列特征并构建系统进化树探讨系统发生关系。 结果 9 种蚊虫的 rDNA- ITS2 长度在 343 bp 与 577 bp 之间,共有 59 个保守位点、449 个变异位点、235 个简约信息位点和 191 个单态位点,9 种蚊虫种间序列同源性为 28. 21% ~ 53. 76%;所有蚊虫的 rDNA-ITS2 基因分子鉴定与形态学鉴定吻合率 100%,库 蚊属的淡色库蚊、三带喙库蚊和二带喙库蚊聚成一类,伊纹属的白纹伊蚊和刺扰伊蚊聚成一类,不同种蚊虫为独立 分支。 结论 核糖体 DNA 第二内转录间隔( rDNA-ITS2 )基因可用于蚊虫属和种的鉴定。     

我国雷氏按蚊和嗜人按蚊的分子鉴别和分类地位的探讨

[目的 ]论证我国雷氏按蚊与嗜人按蚊的分类地位。 [方法 ]采用分子鉴别法 (鉴别PCR和rDNA ITS2序列 )检测辽宁及山东两省现场按蚊标本 ,并依据ITS2区序列建立分子系统树。 [结果 ]分子鉴别显示 ,辽宁和山东两省均存在雷氏按蚊和嗜人按蚊。我国雷氏按蚊 (辽宁和山东省 ,n =6 )和嗜人按蚊 (辽宁、云南、河南和四川省 ,n =10 )ITS2序列长度和GC含量分别为 45 1bp、 46 2 % ,44 8bp、 46 0 % ;各地雷氏按蚊和嗜人按蚊序列均无差异 ,但各地嗜人按蚊与对照组江苏的嗜人按蚊相比 ,种内序列差异为 0 88% ;雷氏按蚊与嗜人按蚊种间序列差异为 2 5 7%。分子系统树显示雷氏按蚊与中华按蚊、嗜人按蚊与凉山按蚊亲缘关系较近。 [结论 ]根据分子鉴别 ,确认我国雷氏按蚊与嗜人按蚊为同域分布的 2个独立种     

大劣按蚊和斯氏按蚊核糖体基因内转录第二间隔区序列分析

目的分析对约氏疟原虫不易感的大劣按蚊和易感的斯氏按蚊核糖体基因内转录第二间隔区（rDNAITS2）的基因特征。方法对大劣按蚊和斯氏按蚊ITS2基因进行PCR扩增，PCR产物进行限制性片断长度多态性分析（RFLP）和基因测序，并采用相关在线程序及软件进行序列分析。测序结果与GenBank数据库中其它传疟按蚊的ITS2序列进行多序列比对，构建分子系统树。结果大劣按蚊和斯氏按蚊的ITS2基因PCR产物经限制性内切酶Xho I消化，产生不同的酶切图谱；测序结果表明大劣按蚊和斯氏按蚊ITS2序列分别为715bp和466bp，且两种按蚊ITS2序列的GC含量和简单重复序列等特征存在明显差异，两者碱基差异水平为53．5％；分子进化树显示，大劣按蚊和斯氏按蚊形成单独的支系。结论大劣按蚊和斯氏按蚊ITS2基因具有不同的基因特征，其遗传多态性可能与产生对约氏疟原虫的易感性不同有关，为进一步研究按蚊与疟原虫之间的相互作用奠定了基础。     

山东省赫坎按蚊复合体成员种的分子鉴别研究

目的 进一步确认山东省赫坎按蚊复合体成员种。方法 采用鉴别PCR方法,对采自山东省8个地点经形态学初步鉴定的645只蚊虫进行检测,并对已检测的标本随机抽取数个样本,进行rDNA-ITS2序列测定。结果 645份样本中,中华按蚊占90.85％(586/645)、雷氏按蚊占5.27％(34/645),无扩增条带的标本占3.88％(25/645)。测定已鉴别的山东省中华按蚊、雷氏按蚊的ITS2序列长度分别为469bp和451hp,经Blast比对分析,显示与已公布的序列完全相同;3份无扩增条带标本的序列经核对为库蚊。结论 山东省分布的赫坎按蚊复合体成员种主要为中华按蚊、雷氏按蚊,其它成员种有待进一步证实。     

山东省赫坎按蚊复合体成员种的分子鉴别研究

目的 进一步确认山东省赫坎按蚊复合体成员种。方法 采用鉴别PCR方法，对采自山东省8个地点经形态学初步鉴定的645只蚊虫进行检测，并对已检测的标本随机抽取数个样本，进行rDNA-ITS2序列测定。结果 645份样本中，中华按蚊占90．85％(586／645)、雷氏按蚊占5．27％(34／645)，无扩增条带的标本占3．88％(25／645)。测定已鉴别的山东省中华按蚊、雷氏按蚊的ITS2序列长度分别为469bp和451bp，经Blast比对分析，显示与已公布的序列完全相同；3份无扩增条带标本的序列经核对为库蚊。结论 山东省分布的赫坎按蚊复合体成员种主要为中华按蚊、雷氏按蚊，其它成员种有待进一步证实。     

进化速率不同的分子标记对微小按蚊新亲缘种的研究

目的选择进化速率不同的核糖体rDNA-ITS2、rDNA-28S-D3基因和线粒体DNA（mtDNA）COⅡ基因作为分子标记，研究我国微小按蚊变异及新的亲缘种。方法用蛋白酶K消化单蚊蚊腿，提取基因组DNA，PCR特异扩增rDNA-ITS2、rDNA-28S-D3和mtDNA-COⅡ基因片段，对PCR产物进行纯化、测序和序列分析，并基于mtDNA-COⅡ基因采用最大似然法构建种系发生树。分析微小按蚊变异地位和进化关系。结果1）云南元江微小按蚊rDNA-ITS2扩增片段约700bp，其他微小按蚊为480bp左右；2）ITS2和D3序列分析显示3种单倍型，其中元江微小按蚊基因长度与碱基组成显著不同；3）mtDNA-COⅡ种系发生树显示元江微小按蚊与其他微小按蚊的亲缘关系较远。结论我国存在微小按蚊A和C之外的新的微小按蚊亲缘种。     

赫坎按蚊种群近缘种核糖体基因内转录第二间隔区序列分析

目的　分析赫坎按蚊种群内4个近缘种核糖体基因内转录第二间隔区(rDNAITS2)特征。　方法　采用特异性ITS2引物对中华按蚊和嗜人按蚊江苏实验株、辽宁省沈阳市和朝鲜开城现场捕获的嗜人按蚊、八代按蚊和雷氏按蚊rDNAITS2进行PCR扩增、基因测序和限制性内切酶酶切位点图谱分析。　结果与结论　赫坎按蚊种群近缘种中华按蚊、嗜人按蚊、八代按蚊和雷氏按蚊rDNAITS2分别为472、452、456和456bp,各基因序列存在明显差异并存在不同的限制性内切酶酶切位点     

Multilocus molecular and phylogenetic analysis of phlebotomine sand flies (Diptera: Psychodidae) from southern Italy

This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of phlebotomine sand flies (Diptera: Psychodidae) from the Mediterranean region. A ～900 bp long fragment of the mitochondrial DNA encompassing regions within cytb and nd1 gene and the complete ITS2 ribosomal region (～500 bp) were sequenced and characterized for Phlebotomus perniciosus, Phlebotomus perfiliewi, Phlebotomus neglectus, Phlebotomus papatasi, and Sergentomyia minuta, captured in two sites of southern Italy. From one to eight mitochondrial haplotypes and from one to three ITS2 sequence types were found for the examined specimens according to the different sand fly species. The mean interspecific difference in the mitochondrial sequences was of 16.1%, with an overall intraspecific nucleotide variation from 0.1 to 2.8%. A higher interspecific difference (mean 25.1%) was recorded for the ITS2 sequence, with an overall intraspecific nucleotide variation up to 4.9%. The sequence types alignment of ITS2 region showed that all phlebotomine specimens possessed a split 5.8S rRNA, consisting of a mature 5.8S rRNA and a 2S rRNA separated by a short transcribed spacer. Phylogenetic analysis of the Phlebotomus spp. sequences, herein determined and of those available in GenBank? were concordant in clustering P. neglectus, P. perfiliewi and P. papatasi with the same species collected from different geographic areas of the Mediterranean basin in four main clades for mtDNA and ITS2, respectively. This study demonstrates the utility of multilocus sequencing, provides a dataset for the molecular identification of the most prevalent phlebotomine sand flies in southern Europe and defines the phylogenetic relationships among species examined.     

Molecular characterization and species identification of the Anopheles dirus and An. minimus complexes in north-east India using r-DNA ITS-2

The sibling species composition of the Anopheles minimus and Anopheles dirus complexes is poorly known in the highly malarious north-eastern region of India where these two vector taxa are accountable for most of the malaria transmission among 30.7 million inhabitants. Prevalent members of these two complexes in this part of India were identified using sequences for the second internal transcribed spacer (ITS2) of ribosomal DNA. Anopheles baimaii (species D) of the An. dirus complex and An. minimus s.s. (species A) of the An. minimus complex were detected in Arunachal Pradesh, Assam, Meghalaya and Nagaland states. No intraspecific variation was observed in the ITS2 sequence (479bp) of An. baimaii whereas a single substitution was detected in the ITS2 sequence (372bp) of An. minimus from Nagaland state.     

基于mtDNA?COⅠ基因的山东省蚊种群遗传多样性分析

目的　探讨山东省不同地理株及实验室不同抗性品系淡色库蚊及5种常见蚊种的线粒体DNA细胞色素C氧化酶亚基Ⅰ（mtDNA?COⅠ）基因序列特征,分析其遗传多样性。方法　采用山东省济南、济宁、青岛等地现场采集及实验室选育的淡色库蚊成蚊,以济宁市现场采集的5种蚊（淡色库蚊、三带喙库蚊、中华按蚊、白纹伊蚊、骚扰阿蚊）作为样本,PCR扩增mtDNA?COⅠ基因并测序,分析序列特征并构建系统进化树。结果　PCR特异扩增8个不同地理株及4个实验室抗性品系的淡色库蚊mtDNA?COⅠ区域,获得长度均为528 bp的扩增片段,A+T含量为67.4%,存在两个变异位点。不同地理株淡色库蚊mtDNA?COⅠ基因同源性为99.95%,不同抗性品系淡色库蚊基因序列相同,所有蚊虫COⅠ基因具有高度保守性。5种常见蚊虫的mtDNA?COⅠ基因片段长度均为528 bp,有408个保守位点,120个变异位点,42个简约信息位点,78个单态位点,A+T含量为65.7%～68.0%,同源性分析发现蚊种间基因同源性为86.17%～92.05%,所有蚊虫的COⅠ基因分子鉴定与其形态学相吻合。系统进化关系显示,同种和同属之间呈明显的聚集,但阿蚊属成单独的分支,与其他蚊种亲缘关系较远。结论　线粒体COⅠ作为不同地理株及实验室不同品系淡色库蚊的遗传进化分子标记不够理想,但该基因具有种间和属间特异性,可用于蚊虫属和种的区分。     

广州市白纹伊蚊细胞色素C氧化酶亚基Ι基因遗传多态性分析

目的 探索广州市白纹伊蚊线粒体细胞色素C氧化酶亚基I基因（COI）序列的特征,分析同一城市不同环境下白纹伊蚊的遗传变异。方法 单蚊抽提广州市不同点采集的白纹伊蚊基因组DNA,扩增细胞色素C氧化酶亚基I基因片段,联合单链构象多态性PCR（PCR-SSCP）技术分析COI基因片段多态性并筛选部分样品进行克隆测序,用软件比对分析序列的差异。结果 广州市不同采样点白纹伊蚊PCR扩增后获得709bp的COI基因部分序列,序列分析出现12个变异位点,变异率为1.69%。聚类分析显示广州株白纹伊蚊不同采样点的群体间出现部分分化。结论 广州市不同采样点的白纹伊蚊线粒体COI基因出现部分遗传多态性,可能与城市化发展下白纹伊蚊孳生环境的变化有一定关系。     

ITS2 characterization and Anopheles species identification of the subgenus Cellia

In Mizoram, the origin and molecular nature of Anopheles species is poorly understood, despite the region having high malarial incidence and rich biodiversity. A diagnostic PCR assay for distinguishing the Cellia subgenera members of Anopheles species was developed based on the interspecific ITS2 variation. No intraspecific variation was found and the size (362–604 bp) and GC content (48.8–58.9%) of the ITS2 were highly variable among Anophelines. The ITS2 of A. vagus is significantly longer than those of other Anopheles species. Significant relationship was observed among repeats, minimum free energy and RNA secondary structures. Different types of microsatellites were identified and among them dinucleotide, pentanucleotide and polynucleotide microsatellites were predominant. Variation in the length of the ITS2 between species was due to indels in simple repeats. Four domain types of RNA secondary structures were identified and the lowest free energy values were predicted using the computer software, RNAfold. Types I and II were observed only in Neocellia and Myzomyia series and Types III and IV were common in Neocellia and Pyretophorus series. ITS2-based PCR protocol provides a means for vector ecologists, malaria epidemiologists and control personnel to accurately identify members of the subgenera Cellia and a better understanding of their genomic status in Mizoram.     

Sequence heterogeneity of the merozoite surface protein-1 gene (MSP-1) of Plasmodium vivax wild isolates in southeastern Iran

The merozoite surface protein of Plasmodium vivax (PvMSP-1) has been considered as a vaccine candidate, which exhibits antigenic diversity among isolates. We investigated the extent of sequence variation in the polymorphic region 5 of PvMSP-1 in order to characterize the genetic structure and composition of P. vivax in clinical isolates from Iranshahr and Chahbahar districts of Sistan and Baluchistan province, Iran. The PvMSP-1 gene amplification revealed size variation among the isolates, ranging from 430 to 550 bp. Sequences were obtained for 15 Iranian and one Pakistani isolates and 14 different alleles were detected. Results also showed three distinct sequence types of the polymorphic region. Sequence analysis has shown several single nucleotide polymorphisms to occur in this block of PvMSP-1, creating different alleles in the progeny and also microheterogeneity in the region. Thus, this study provides preliminary evidence of sequence heterogeneity in the Iranian P. vivax population.     

基于cDNA末端快速扩增的刚地弓形虫核仁G蛋白-1的克隆

目的　克隆弓形虫核仁G蛋白- 1(NOG1)基因的全长cDNA序列。方法　根据NCBIGeneBank中登录的弓形虫NOG1基因的唯一EST序列设计引物,利用cDNA 5’-末端快速扩增法(5’-RACE)和cDNA 3’-末端快速扩增法(3’-RACE)分别对已知序列进行延伸,将扩增获得的3’-端和5’-端未知序列与位于中间位置的已知序列进行拼接,然后经Blast检验全长序列的正确性。结果　5’-RACE扩增获得3个不同长度的片断,最长片断全长12 87bp ,去除引物和夹杂的已知序列后,通过5’-RACE在5’端扩增获得未知序列为1196bp。3’-RACE扩增后得到一条特异性条带,位于大约1.7kb左右,测序全长为16 34bp ,去除引物和已知序列,经3’-RACE扩增获得的3’-端未知序列为12 0 4bp。Blast全长为316 7bp的3段拼接序列,发现其覆盖NOG1基因cDNA的完整ORF或者CDS序列(6 5 0bp 2 80 9bp)。推导翻译出的蛋白质一级结构包含719个氨基酸(aa) ,在所有已发现物种的NOG1中,弓形虫NOG1基因的cDNA和相应的aa序列都是最长的。该序列已登录NCBIGeneBank ,核酸序列登录号AY6 86 734,对应蛋白质的aa序列登录号为AAT94 2 90。结论　本研究首次克隆获得了弓形虫NOG1基因的全长cDNA序列,并对相应的aa序列进行了推导翻译和简单分析。     

手足口病患儿感染肠道病毒71型全基因组基因特征分析

目的从手足口病患儿粪便中分离肠道病毒71型(EV71),对其进行全基因组测序,了解秦皇岛市EV71全基因组基因特征,为手足口病的综合防治提供依据。方法从秦皇岛市各医院收集手足口病患儿粪便标本,进行EV71检测,阳性标本进行病毒分离及核酸提取,通过RT-PCR进行全基因组序列扩增,通过电泳、测序及序列拼接一系列操作分析其基因特征。结果用提取的核酸扩增出7 406bp的目的基因片段测序后与已记录的EV71相应序列进行比对完全一致,且测序峰图较好,可信度也较高;各基因片段间通过核苷酸序列拼接后得出,秦皇岛市医院EV71全基因组序列长7 406bp,编码区各基因片段的长度分别为:2A450bp、2B297bp、2C987bp、3A258bp、3B66bp、3C549bp、3D1 386bp、VPl 891bp、VP2 762bp、VP3 726bp、VP4 207bp。结论秦皇岛市EV71分离株为C4a型全基因组序列长7 406bp,可为当地手足口病的防控提供参考。     

Extensive polymorphism in the mitochondrial DNA of apes.

Ape species are 2-10 times more variable than the human species with respect to the nucleotide sequence of mtDNA, even though ape populations have been smaller than the human population for at least 10,000 years. This finding was made by comparing purified mtDNAs from 27 individuals with the aid of 25 restriction endonucleases; for an additional 59 individuals, comparisons were made with fewer enzymes by using the blot hybridization method. The amount of intraspecific sequence divergence was greatest between orangutans of Borneo and Sumatra. Among common chimpanzees, a large component of the variation is due to two highly distinct forms of mtDNA that may reflect a major geographic subdivision. The least amount of sequence variation occurred among lowland gorillas, which exhibit only twice as much sequence variation as humans. The large intraspecific differences among apes, together with the geological and protein evidence, leads us to propose that each ape species is the remnant of an ancient and widespread population that became subdivided geographically and reduced in size and range, perhaps by hominid competition. The low variation among human mtDNAs is consistent with geological evidence that the human species is young. The distribution of site changes within the mitochondrial genome was also examined. Comparison of closely related mtDNAs shows that the ribosomal RNA genes have diverged more slowly than the rest of the genome.