家蚕微孢子虫孢子DNA的提取、克隆及部分DNA序列分析 |
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引用本文: | 章新愉 陈火胜. 家蚕微孢子虫孢子DNA的提取、克隆及部分DNA序列分析[J]. 蚕业科学, 1995, 21(2): 91-95 |
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作者姓名: | 章新愉 陈火胜 |
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摘 要: | 报道家蚕微孢子虫(Nosemabombycis简称N.b)孢子DNA的提取、克隆及部分DNA序列测定的结果。采用SDS-蛋白酶K将孢子破碎,用苯酚一氯仿法提取孢子的DNA。将N.b孢子DNA与pTZ18R质粒重组,转化于大肠杆菌DH5,获得4个阳性克隆株:pTZ18RN.b1,插入片段为1kb;pTZ18RN.b2,为1.2kb;pTZ18RN.b3为1.8kb及pTZ18RN.b4为1.9kb,经Southern印迹杂交,证实插入的DNA片段为家蚕N.b孢子所特有。与MG1(Nosemasp.),蓖麻蚕微孢子虫、蓝萤叶甲微孢子虫及蚕卵的DNA均无同源性。用双脱氧链终止法测定4个克隆株插入片段的部分DNA的序列,经检索尚未发现同源性的基因序列。讨论了PCR技术诊断家蚕微孢子虫孢子与近缘种的问题。
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关 键 词: | 家蚕,微孢子虫,核酸,克隆,核酸序列分析 |
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EXTRACTION,CIONING AND SEQUENCING OF DNA FROM NOSEMA BONBYCIS |
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Abstract: | This article reports the experimental results of extraction,cloning and partial se-quencing of Nosema bombycis DNA. The total nucleic acids were extracted from purifiedN. n. spores by SDS proteinase method. After digested with RNase and DNase,theethidium bromide stained pattern of agrose electrophoresis showed N. b. spore’s nucleicacids contain a high molecular weight of DNA and abundant of low molecular weight ofRNA. The N. b. spore DNA cleaved with restriction endonuclease EcoR Ⅰ and Hind Ⅲwere demonstrated smear pattern in AGE. Using plasmid pTZ18R as vector,Esherichiacoli DH5 as host cell,the EcoR Ⅰ cleaved DNA of N. b. were cloned by shot gunmethod. The 4 recombinant plasmids were screened and isolated. They were temporarilynamed as pTZ18RN. b. 1,pTZ18RN. b. 2,pTZ18RN. b. 3 and PTZ18RN. b. 4. The in-sert DNA size in the 4 recombinants were 1.0,1.2,1.8 and 1.9kb repectively. The DNAfragments were labelled with 32P-dCTP by random primer labelled method as a specificprobe. Hybridization of these probes with the DNA of N. b. spore,MG1,Nosema cynthiaand Nosema phyllobratica showed strong hybridization signals only in N. b. spore DNA.A Part of insert DNA fragments were sequenced with dideoxy chain terminationmethod. |
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Keywords: | Bombyx mori Nosema bombycis Nucieic acid Cloning Sequence |
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