应用PCR技术对在鸭粪中发现的疑似环孢子虫的18 S rDNA部分基因和ITS-1+基因进行了扩增,将扩增出的片段纯化后连接至pMD-18T载体上,选取阳性克隆进行序列测定,并利用NCBI在线BLAST程序和MEGA 4软件对测序结果进行了同源性比较和系统发...应用PCR技术对在鸭粪中发现的疑似环孢子虫的18 S rDNA部分基因和ITS-1+基因进行了扩增,将扩增出的片段纯化后连接至pMD-18T载体上,选取阳性克隆进行序列测定,并利用NCBI在线BLAST程序和MEGA 4软件对测序结果进行了同源性比较和系统发育树构建。结果显示,测得的18 SrDNA序列与环孢子虫的相似性最高(98%),且在系统树中位于同一分支上;测得的ITS-1序列高度特异,在GenBank中未发现同源性序列,可以确定其为环孢子虫的一个新种,暂命名为鸭源环孢子虫。展开更多
The rDNA internal transcribed spacer 1(ITS-1) regions of two Chinese fir provanences was amplified and cloned by PCR reaction.The PCR reaction was following:97℃ 5 minutes→95℃ 5 minhtes→Adding the Tag polymerase→9...The rDNA internal transcribed spacer 1(ITS-1) regions of two Chinese fir provanences was amplified and cloned by PCR reaction.The PCR reaction was following:97℃ 5 minutes→95℃ 5 minhtes→Adding the Tag polymerase→94℃ l?min 56℃ 1?min,72℃ 2?min;thirty\|six cycles→72 ℃ 10?min.High quality DNA template is necessary for the amplification of ITS-1 sequence,during the PCR reaction,ten minutes denaturation time and 56℃ annealing temperature are beneficial to amplification.The ITS-1 fragment was ligated to PUC19 plasmid,digested with Hind Ⅱ and transformed into competence cells of E.coli JM83 strain,the cloned strains harboring recombinant plasmid were obtained,those recombinant plasmids were used to sequence for ITS-1 fragment.Sequence analysis indicated that the sequence length is 273 bp,the using percentage of A\,T\,C\,G within ITS1 sequence of Chinese fir were 27.5%\,23%\,21.6%\,27.9% respectively and the G/C content of ITS1 sequence was 48.35%.Comparing with other plants,the G/C content of Chinese fir was less than other plants,whose ITS1 regions have been sequenced.As to ITSI sequence,there was no difference among two Chinese fir provenances,sequence analysis disclosed there were two repeat sequences [AAAG] n and [TTG] nappeared within ITS1 sequence of Chinese fir.展开更多
以采自云南地区的5个牛肝菌（Boletus sp.）样品为材料,根据样品的形态特征,并结合r DNA ITS和IGS1序列分析对5个样品进行分类鉴定,通过与Gen Bank上已登录的牛肝菌序列比对,采用邻接法（neighbor-joining,N-J）构建系统发育树。结果表...以采自云南地区的5个牛肝菌（Boletus sp.）样品为材料,根据样品的形态特征,并结合r DNA ITS和IGS1序列分析对5个样品进行分类鉴定,通过与Gen Bank上已登录的牛肝菌序列比对,采用邻接法（neighbor-joining,N-J）构建系统发育树。结果表明,所有供试材料的ITS和IGS1全长分别在739~787bp和537~583bp范围内,GC含量分别在48.31%~51.01%和48.42%~52.89%之间,遗传距离在0.017~0.248和0.002~0.225之间,上述数据表明云南5个牛肝菌样品之间存在一定的遗传差异。5个牛肝菌样品的ITS和IGS1序列聚类分析均表明,供试样品被分为两大类,其中样品YX1-2和QJ3-4聚为一个类群,样品CX2-10、QJ3-5和YX1-3聚为另一个大的类群,两种分析手段相互印证,且ITS序列聚类分析能将云南不同地点的牛肝菌鉴定到属甚至种的水平,上述分析结果表明ITS和IGS1序列分析相结合可作为牛肝菌亲缘关系鉴定的有效分子标记方法。展开更多
文摘应用PCR技术对在鸭粪中发现的疑似环孢子虫的18 S rDNA部分基因和ITS-1+基因进行了扩增,将扩增出的片段纯化后连接至pMD-18T载体上,选取阳性克隆进行序列测定,并利用NCBI在线BLAST程序和MEGA 4软件对测序结果进行了同源性比较和系统发育树构建。结果显示,测得的18 SrDNA序列与环孢子虫的相似性最高(98%),且在系统树中位于同一分支上;测得的ITS-1序列高度特异,在GenBank中未发现同源性序列,可以确定其为环孢子虫的一个新种,暂命名为鸭源环孢子虫。
文摘The rDNA internal transcribed spacer 1(ITS-1) regions of two Chinese fir provanences was amplified and cloned by PCR reaction.The PCR reaction was following:97℃ 5 minutes→95℃ 5 minhtes→Adding the Tag polymerase→94℃ l?min 56℃ 1?min,72℃ 2?min;thirty\|six cycles→72 ℃ 10?min.High quality DNA template is necessary for the amplification of ITS-1 sequence,during the PCR reaction,ten minutes denaturation time and 56℃ annealing temperature are beneficial to amplification.The ITS-1 fragment was ligated to PUC19 plasmid,digested with Hind Ⅱ and transformed into competence cells of E.coli JM83 strain,the cloned strains harboring recombinant plasmid were obtained,those recombinant plasmids were used to sequence for ITS-1 fragment.Sequence analysis indicated that the sequence length is 273 bp,the using percentage of A\,T\,C\,G within ITS1 sequence of Chinese fir were 27.5%\,23%\,21.6%\,27.9% respectively and the G/C content of ITS1 sequence was 48.35%.Comparing with other plants,the G/C content of Chinese fir was less than other plants,whose ITS1 regions have been sequenced.As to ITSI sequence,there was no difference among two Chinese fir provenances,sequence analysis disclosed there were two repeat sequences [AAAG] n and [TTG] nappeared within ITS1 sequence of Chinese fir.
文摘以采自云南地区的5个牛肝菌（Boletus sp.）样品为材料,根据样品的形态特征,并结合r DNA ITS和IGS1序列分析对5个样品进行分类鉴定,通过与Gen Bank上已登录的牛肝菌序列比对,采用邻接法（neighbor-joining,N-J）构建系统发育树。结果表明,所有供试材料的ITS和IGS1全长分别在739~787bp和537~583bp范围内,GC含量分别在48.31%~51.01%和48.42%~52.89%之间,遗传距离在0.017~0.248和0.002~0.225之间,上述数据表明云南5个牛肝菌样品之间存在一定的遗传差异。5个牛肝菌样品的ITS和IGS1序列聚类分析均表明,供试样品被分为两大类,其中样品YX1-2和QJ3-4聚为一个类群,样品CX2-10、QJ3-5和YX1-3聚为另一个大的类群,两种分析手段相互印证,且ITS序列聚类分析能将云南不同地点的牛肝菌鉴定到属甚至种的水平,上述分析结果表明ITS和IGS1序列分析相结合可作为牛肝菌亲缘关系鉴定的有效分子标记方法。
