目的基于核糖体18S r DNA基因对基氏蠊螨进行分子鉴定。方法采集和分离储藏物样本,进行螨类的形态学鉴定。提取单个螨的基因组DNA,经PCR扩增、克隆和测序获得COⅠ基因和18S r DNA基因,将所获序列进行Blast对比。检索Gen Bank数据库中蠊...目的基于核糖体18S r DNA基因对基氏蠊螨进行分子鉴定。方法采集和分离储藏物样本,进行螨类的形态学鉴定。提取单个螨的基因组DNA,经PCR扩增、克隆和测序获得COⅠ基因和18S r DNA基因,将所获序列进行Blast对比。检索Gen Bank数据库中蠊螨属18S r DNA基因序列,利用Clustal X 1.83软件进行多序列比对,基于MEGA X软件进行序列分析并以邻接法(Neighbor-Joining,NJ)构建系统发育树。结果采集的样本经形态和COⅠ基因双重鉴定为基氏蠊螨。同时,所选取的10个基氏蠊螨的18S r DNA基因序列完全一致,均表现出A/T碱基偏向性,与同属的Blattisocius tarsalis和Blattisocius everti分别有98.73%和98.94%的同源性。基于18S r DNA基因序列的系统进化树显示,基氏蠊螨与Blattisocius tarsalis和Blattisocius everti聚为一支。结论本研究建立了基氏蠊螨基于18S r DNA基因序列的分子鉴定方法,为基氏蠊螨的准确鉴定奠定基础。展开更多
The 18S ribosomal DNA gene (18S rDNA) sequences (approximately 1300 bp in length) were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in...The 18S ribosomal DNA gene (18S rDNA) sequences (approximately 1300 bp in length) were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode spe- cific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin6 I restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Al- though it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve se- quences were close to Pontonema vulgare and Adoncholaimus sp., four to Daptonema procerus and two (identical) to Enoplus brevis. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.展开更多
文摘目的基于核糖体18S r DNA基因对基氏蠊螨进行分子鉴定。方法采集和分离储藏物样本,进行螨类的形态学鉴定。提取单个螨的基因组DNA,经PCR扩增、克隆和测序获得COⅠ基因和18S r DNA基因,将所获序列进行Blast对比。检索Gen Bank数据库中蠊螨属18S r DNA基因序列,利用Clustal X 1.83软件进行多序列比对,基于MEGA X软件进行序列分析并以邻接法(Neighbor-Joining,NJ)构建系统发育树。结果采集的样本经形态和COⅠ基因双重鉴定为基氏蠊螨。同时,所选取的10个基氏蠊螨的18S r DNA基因序列完全一致,均表现出A/T碱基偏向性,与同属的Blattisocius tarsalis和Blattisocius everti分别有98.73%和98.94%的同源性。基于18S r DNA基因序列的系统进化树显示,基氏蠊螨与Blattisocius tarsalis和Blattisocius everti聚为一支。结论本研究建立了基氏蠊螨基于18S r DNA基因序列的分子鉴定方法,为基氏蠊螨的准确鉴定奠定基础。
基金This study was supported financially by the National Natural Science Foundation of China （No. 40176028）.
文摘The 18S ribosomal DNA gene (18S rDNA) sequences (approximately 1300 bp in length) were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode spe- cific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin6 I restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Al- though it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve se- quences were close to Pontonema vulgare and Adoncholaimus sp., four to Daptonema procerus and two (identical) to Enoplus brevis. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.
