家蚕微孢子虫孢子DNA的提取、克隆及部分DNA序列分析

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。

EXTRACTION,CIONING AND SEQUENCING OF DNA FROM NOSEMA BONBYCIS

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.