解淀粉芽孢杆菌pyrR基因敲除载体的构建

【目的】构建用于解淀粉芽孢杆菌（Bacillus amyloliquefaciens）pyrR基因敲除的温敏型敲除载体,为研究pyrR蛋白缺失对胞苷合成的影响奠定基础。【方法】设计并合成含有限制性内切酶酶切位点的引物,以解淀粉芽孢杆菌基因组DNA为模板,利用PCR分别扩增pyrR基因的上游同源序列和下游同源序列,再定向克隆至含卡那抗性基因的p KS1温敏型载体上,构建敲除载体p KS1-pyrR。【结果】经特异性引物对pyrR-S1/pyrR-A1和pyrR-S2/pyrR-A2扩增,获得解淀粉芽孢杆菌目的DNA片段pyrR-up和pyrR-down,大小约500 bp;目的片段经克隆载体p...

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Published in南方农业学报 Vol. 46; no. 8; pp. 1339 - 1344
Main Author 吴庆 刘慧燕 方海田 何建国 贺晓光 王梦娇 于丽男
Format Journal Article
LanguageChinese
Published 宁夏大学农学院,银川,750021 2015
Subjects
pyrR基因
基因敲除
温敏型敲除载体
胞苷
解淀粉芽孢杆菌
pyrR基因
基因敲除
cytidine
胞苷
B.amyloliquefaciens
pyrR gene
temperature sensitive knock-out vector
解淀粉芽孢杆菌
gene knock-out
温敏型敲除载体
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Summary:【目的】构建用于解淀粉芽孢杆菌（Bacillus amyloliquefaciens）pyrR基因敲除的温敏型敲除载体,为研究pyrR蛋白缺失对胞苷合成的影响奠定基础。【方法】设计并合成含有限制性内切酶酶切位点的引物,以解淀粉芽孢杆菌基因组DNA为模板,利用PCR分别扩增pyrR基因的上游同源序列和下游同源序列,再定向克隆至含卡那抗性基因的p KS1温敏型载体上,构建敲除载体p KS1-pyrR。【结果】经特异性引物对pyrR-S1/pyrR-A1和pyrR-S2/pyrR-A2扩增,获得解淀粉芽孢杆菌目的DNA片段pyrR-up和pyrR-down,大小约500 bp;目的片段经克隆载体p MD19-T连接、Trans1-T1转化,获得的重组质粒分别以Kpn I/Hind III、Pst I/Not I双酶切鉴定和测序后,可获得大小约500 bp的上、下游克隆载体T-pyrR-up和T-pyrR-down。上游克隆载体T-pyrR-up经Kpn I与Hind III双酶切及T4 DNA连接、Trans1-T1转化后,进行重组载体检测和质粒双酶切鉴定,得到大小分别为433、5064 bp的片段,与连接长度一致,构建获得带标记基因的重组载体p KS1-pyrR-up。采用Pst I和Not I对T-pyrR-down和p KS1-pyrR-up同时进行双酶切,经连接、转化、双酶切鉴定后,得到两条长度分别为446和5497 bp的片段,与连接前长度一致,证明pyrR的上、下游同源臂已正确插入到p KS1载体中,获得敲除载体p KS1-pyrR。【结论】构建的解淀粉芽孢杆菌pyrR基因敲除载体p KS1-pyrR可用于研究敲除pyrR基因及pyrR蛋白缺失对胞苷过量合成的影响。
Bibliography:Objective】The present study was conducted to construct a temperature-sensitive knock-out vector for pyrR gene in Bacillus amyloliquefaciens in order to lay the foundation for studying the effects of pyrR protein deficiency on cytidine synthesis. 【Method】The primers with restricted endonucleases locus were designed firstly, followed by amplifying the upstream and downstream arms＇ sequences of pyrR gene in B. amyloliquefaciens by using genome DNA as template and PCR method. Then, the amplified fragments were cloned into the temperature sensitive vector pKS1 to construct temperature sensitive knock-out vector pKS1-pyrR containing upstream and downstream homologous sequences of pyrR gene,and kanamycin gene. 【 Result 】 The obtained target fragments of pyrR-up and pyrR-down in B. amyloliquefaciens were about 500 bp in length after amplified with specific primer pair pyrR-S1/pyrR-A1 and pyrR-S2/pyrR-A2. The target fragments were linked to p MD19-T and transformed into Trans1-T1 to get recombinant plasmid, and about
ISSN:2095-1191
DOI:10.3969/j:issn.2095-1191.2015.08.1339