基于COⅠ与18S rRNA基因序列的4种肉食螨DNA条形码研究  

DNA barcoding of 4 species of cheyletid mites based on COⅠand 18S rRNA gene sequences

作　　者：詹雨娟 张兰香 孙梦涛 李心玫 王严 李梦珠 陶东东 孙恩涛[1] ZHAN Yu-Juan;ZHANG Lan-Xiang;SUN Meng-Tao;LI Xin-Mei;WANG Yan;LI Meng-Zhu;TAO Dong-Dong;SUN En-Tao(Department of Hygiene Inspection and Quarantine,Wannan Medical College,Wuhu 241002,China)

机构地区：[1]皖南医学院医学卫生检验与检疫学教研室,芜湖241002

出　　处：《中国血吸虫病防治杂志》2021年第1期66-70,78,共6页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(31870352);皖南医学院2019年青年卓越人才资助项目(Wyqnyx201902);皖南医学院美康大学生科技创新基金(MK201907);安徽省省级大学生创新创业项目(S201910368114)。

摘　　要：目的基于线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)与核糖体18S小亚基(18S rRNA)基因序列,分析确定4种肉食螨合适的DNA条形码,从而进一步完善肉食螨DNA条形码数据库。方法2018年5月—2019年7月于安徽省阜阳、芜湖、铜陵等3市小型面粉作坊采集分离肉食螨样本,经形态学鉴定后,提取单个螨基因组DNA,经PCR扩增、克隆和测序获得COⅠ与18S rRNA基因序列,所获序列进行BLAST比对。结合已报道肉食螨序列,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计算遗传距离,采用最大似然法构建系统发育树。结果马六甲肉食螨、转开肉食螨、鳞翅触足螨分子鉴定结果与形态学一致,而Gen Bank中缺少网真扇毛螨相关序列。4种肉食螨COI与18S r RNA基因序列(A+T)分别为69.6%和55.1%,碱基替换数分别为137对和46对。基于COⅠ与18S rRNA基因序列变异位点分析,发现4种肉食螨种间变异位点分别为154~321个和58~99个。4种肉食螨COⅠ与18S r RNA基因序列种内遗传距离均≤0.020,种间遗传距离分别为0.235~0.583和0.078~0.114。基于COⅠ与18S rRNA基因序列的系统发育树显示,4种肉食螨均能各自聚为一支,支持率均达100%,与形态学鉴定结果一致。结论线粒体COⅠ基因序列作为4种肉食螨DNA条形码优于18S rRNA基因序列,更适用于探索肉食螨的属、种低阶元亲缘关系。Objective To analyze the sequences of mitochondrial cytochrome C oxidase subunitⅠgene(COⅠ)and 18 S ribosomal RNA gene(18 S rRNA),so as to identify the feasible DNA barcodes for 4 species of cheyletid mites and improve the DNA barcoding database for cheyletid mites.Methods Cheyletid mite samples were collected from small-scale flour mills in Fuyang,Wuhu and Tongling cities of Anhui Province from May 2018 to July 2019,extracted and morphologically identified.Then,genomic DNA was extracted from a single cheyletid mite,and the COⅠand 18 S rRNA gene sequences"were obtained by PCR amplification,cloning and sequencing.The obtained sequences were aligned using the BLAST software.Multiple sequence alignment was done using the software ClustalX version 1.83 using the known gene sequences from cheyletid mites.The genetic distance was calculated using the software MEGA X,and the phylogenetic tree was created using the maximum likelihood method.Results The DNA barcoding results of Cheyletus malaccensis,C.carnifex and Cheletomorpha lepidopterorum were consistent with the morphological identification,while no sequences pertaining to Eucheyetia reticulate were retrieved in the GenBank database.The proportions of A+T were 69.6%and 55.1%in the COⅠand 18 S rRNA sequences of 4 cheyletid mites species,respectively,and the numbers of base substitutions were 137 and 46,respectively.There were 154 to 321 and 58 to 99 inter-species variation loci in the COI and 18 S rRNA gene sequences of 4 cheyletid mites species,respectively,and the intra-species genetic distance was all 0.020 or less in the COⅠand 18 S rRNA gene sequences of 4 cheyletid mites species,with inter-species genetic distance of 0.235 to 0.583 and 0.078 to 0.114,respectively.Phylogenetic analysis based on COⅠand 18 S rRNA genes showed that all four species of cheyletid mites were clustered into a branch with a 100%supportive rate,which was consistent with the morphological identification.Conclusion Mitochondrial COⅠgene is superior to 18 S rRNA gene as DNA barc

关 键 词：肉食螨 分子鉴定 COⅠ基因 18S rRNA基因 

分 类 号：R384.4[医药卫生—医学寄生虫学]