贵州医科大学学报

2005, (01) 4-9

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用锅柄聚合酶链反应法克隆卡地腐霉Pr1基因的未知片段(英文)
Cloning of Unknown Partner Sequence of the Subtilisin-like Protease Gene of Pythium carolinianum with Panhandle PCR Technique

杨平,夏玉先,苏晓庆
YANG Ping 1, XIA Yu-xian 2, SU Xiao-qing 1* *(1.Department of Biology, Guiyang Medical College, Guiyang 550004, China; 2.Genetic Engineering Research Center, Chongqing University, Chongqing 400030, China)

摘要(Abstract):

目的:分离和克隆灭蚊真菌卡地腐霉类枯草杆菌蛋白酶基因已知片段的3′端未知序列。方法:采用 PANHANDLEPCR,以基因组DNA为模板,通过链内退火及延伸形成一个类似锅柄形状的结构,经嵌套式PCR 扩增得到未知目的片段。通过已知cDNA片段设计引物,PCR扩增得到该蛋白酶部分基因组序列,采用限制性 内切酶BamHⅠ消化卡地腐霉基因组DNA,经去磷酸化处理后,在消化片段3′端连接一段与卡地腐霉类枯草杆 菌蛋白酶基因5′端已知序列互补的5′端磷酸化寡核苷酸链NA,经链内退火及聚合酶延伸形成一锅柄状结构。 结果:获得一大小为876bp的PCR产物,经序列分析验证,该panhandlePCR产物包含了已知卡地腐霉类枯草杆 菌蛋白酶基因3′端未知的853bp核苷酸序列。结论:这种特殊的PCR方法可以高度有效地克隆和鉴定已知片 段两侧的未知基因片段。
Objective: To explore the DNA sequence of the subtilisin-like protease (Pr1) gene of Pythium carolinianum, a mosquito-killing fungus. Methods: A new approach called panhandle polymerase chain reaction (PPCR) was used to clone a 3′unknown sequence adjacent to a known sequence of Pr1 gene of P. carolinianum. The template was a segment of DNA with an intra-strand loop schematically shaped like a pan with a handle. Based on a partial Pr1 cDNA sequence of the fungus cloned previously, a 530 bp genomic sequence was cloned by designing a pair of specific primers. The genomic DNA was firstly digested to create a 5′overhang end with restriction enzyme BamH Ⅰ and then treated with calf intestinal alkaline phosphatase. Next, a 32-nucleotide single-stranded 5′phosphorylated oligonucleotide was ligated to the 3′ends of BamHⅠ-digested DNA. After denaturation, intra-strand annealing and polymerase extension, a panhandle was formed and then nested PCR was performed. Results: A 876 bp panhandle PCR product was subsequently subcloned and the sequence analysis indicated that it contains the 3′unknown partner sequence adjacent to the known sequence of Pr1 gene. Conclusion: Panhandle PCR is a quick and convenient new technique for cloning and identifying unknown partner genes.

关键词(KeyWords): 聚合酶链反应;克隆,分子;灭蚊;害虫防治,生物学;枯草杆菌蛋白酶;碱基序列;枯草菌素类蛋白酶;卡地腐霉
polymerase chain reaction; cloning,molecular; mosquito control; pest control,biological; subtilisin; base sequence; subtilisin-like protease; Pythium carolinianum

Abstract:

Keywords:

基金项目(Foundation): 贵州省省长基金资助项目;; 贵州省高层次人才特助经费资助项目

作者(Author): 杨平,夏玉先,苏晓庆
YANG Ping 1, XIA Yu-xian 2, SU Xiao-qing 1* *(1.Department of Biology, Guiyang Medical College, Guiyang 550004, China; 2.Genetic Engineering Research Center, Chongqing University, Chongqing 400030, China)

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DOI: 10.19367/j.cnki.1000-2707.2005.01.002

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