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毛囊蠕形螨与皮脂蠕形螨基因组DNA的RAPD分析和序列比对
引用本文:赵亚娥,成慧.毛囊蠕形螨与皮脂蠕形螨基因组DNA的RAPD分析和序列比对[J].昆虫学报,2009,52(11):1273-1279.
作者姓名:赵亚娥  成慧
作者单位:西安交通大学医学院免疫学与病原生物学系,西安,710061
基金项目:国家自然科学基金,陕西省自然科学基金 
摘    要:【目的】分析毛囊蠕形螨Demodex folliculorumD.f.)和皮脂蠕形螨D. brevisD.b.)基因组DNA的多态性, 对相关条带进行测序分析。【方法】采用改良小昆虫DNA提取法提取两种人体蠕形螨基因组DNA, 选择RAPD技术对其进行多态性分析, 将相关条带分别与pMD18-T载体连接, 克隆、测序后进行酶切鉴定和分析。【结果】毛囊蠕形螨共扩增15条带, 皮脂蠕形螨共扩增12条带;两种蠕形螨既有共有条带, 又有特异性条带;根据条带差异计算得到两种间的遗传距离为0.5556. 毛囊蠕形螨约800 bp处特异性条带测序结果显示, 序列片段长度为855 bp(GenBank登录号为FI277970);特异性引物扩增和酶切鉴定均为毛囊蠕形螨所特有. 序列比对显示与阿糖胞苷DNA区域结合蛋白有46%的序列相似度。两种人体蠕形螨约300 bp处共有条带序列分析显示, 碱基序列均为341 bp(GenBank登录号分别为D.f. FI520176;D.b. FI520175), 在第84和第165位点有2个碱基不同, 分别是A/G和C/T互换, 同源性高达99.4%. 但未发现有开放阅读框和相似度高的序列。 【结论】序列片段为855 bp的特异性条带为毛囊蠕形螨所特有;341 bp碱基序列为毛囊蠕形螨和皮脂蠕形螨所共有, 同源性高达99.4%. RAPD技术可用于两种人体蠕形螨基因组DNA的多态性分析和物种鉴定。

关 键 词:毛囊蠕形螨  皮脂蠕形螨  RAPD分析  遗传距离  序列比对  物种鉴定  

RAPD analysis and sequence alignment of genomic DNA of hair follicle mites Demodex folliculorum and D.Brevis (Acari:Demodicidae)
ZHAO Ya-E,CHENG Hui.RAPD analysis and sequence alignment of genomic DNA of hair follicle mites Demodex folliculorum and D.Brevis (Acari:Demodicidae)[J].Acta Entomologica Sinica,2009,52(11):1273-1279.
Authors:ZHAO Ya-E  CHENG Hui
Abstract:Objective] Analysis of genomic DNA polymorphism and the related sequence of Demodex folliculorum and D. Brevis. Methods] The genomic DNA of the human Demodex was extracted by using improved DNA extraction method of mini-insects. Random amplified polymorphic DNA (RAPD) was applied to analyze the polymorphism. The related bands were connected with pMD18-T vector, and cloned, sequenced, and identified and analyzed after enzyme digestion. Results] There were 15 bands obtained in D. Folliculorum and 12 in D. Brevis. Some bands were shared by the two mites while others were species- specific. The genetic distance between the two Demodex species was 0. 5556. After recombination, the sequence of the specific band (about 800 bp) of D. Folliculorum was found to be 855 bp (GenBank accession no. FI277970) in size. The 855 bp fragment was confirmed to be characteristic for D. Folliculorum according to the PCR result with a specific primer and the result of enzyme digestion analysis. This sequence had 46% similarity to AraC-type DNA-binding domain-containing protein. The shared fragments (about 300 bp) by the two mites were both 341 bp in length (GenBank accession no. FI520176 and FI520175, respectively), and with two different bases at the 84th and 165th sites, which were A/G and C/T permutation, respectively. Their homology was 99. 4%, but there was no ORF and high similar sequence. Conclusions] The 855 bp fragment is specific to D. Folliculorum. The 341 bp fragment is shared by D. Folliculorum and D. Brevis with 99. 4% homology. RAPD can be used in genomic DNA polymorphism analysis and species identitfication of D. Folliculorum and D. Brevis.
Keywords:Demodex folliculorum  Demodex brevis  RAPD analysis  genetic distance  sequence alignment  species identification
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