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)

• 1:贵阳医学院生物学教研室
• 2:重庆大学基因工程中心
• 3:贵阳医学院生物学教研室 贵州贵阳550004
• 4:重庆400030

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)

Email:

DOI: 10.19367/j.cnki.1000-2707.2005.01.002

参考文献(References)：

• [1]XiaoqingSu,FuzhenZou,QingGuo,etal.Areportona mosquito killingfunfus,Pythumcarolinianum[J].Fungal diversity,2001,129-133.
• [2]XiaoqingSu.Theeffectsoflagenidiumgiganteumonaedes albopictusandothermosquitoesinGuizhou,China[J].EntomologiaSinica,1994,1(3):283-288.
• [3]XinrongLiu,XiaoqingSu,FuzhenZou,etal.Construc tionofacDNAlibraryofpythumcarolinianum,amosqui to killingfungus[J].FungalDiversity,2001,53-59.
• [4]Jaronskist.SimplifiedproductionsysteuforthefungusLagenidiumgiganteunmforoperationalmosguitocontroe[J].Mogwtonews,1984,44(3):377-381.
• [5]郭庆,苏晓庆.大链壶菌卵孢子的研究Ⅲ.CA分离株四亚株产卵孢子能力的初步观察[J].贵阳医学院学报,1994,19(4):311-314.
• [6]萨姆布鲁克,D·W·拉塞尔.分子克隆实验指南[M].北京:科学出版社,2003.1595.
• [7]迪芬巴赫·CW,德维克斯勒·GS.PCR实验指南[M].北京:科学出版社,1998.288-295.
• [8]ZhuHeng,QuFeng,ZhuLihuang.ExtractionoffungalDNAapplicabletomolecularbiologicalanalysisusing benzylecholoride[J].ActaMycologicaSinica,1994,13(1):34-40.
• [9]GuY,NakamuraT,AlderH,etal.Thet(4;11)chro mosometranslocationofhumanacuteleukemiasfusestheALL 1gene,relatedtoDrosophilaTrithorax,totheAF 4 gene[J].Cell,1992,71:701.
• [10]JonesD.PanhandlePCR[J].PCRMethods,1995,4:195.
• [11]LokeshJoshi,RaymondJ,StLeger,etal.Cloningofacu ticle degradingproteasefromtheentomopathogenicfun gus,Beauveriabassianan[J].Microbiologyletters,1995,211-218.
• [12]BidochkaMK,KhachatouriansGG.IdentificationofBeauveriabassianaextracellularproteaseasavinlence factorinpathogenicitytowardthemigratorygrasshopper,Melanoplussanguinipes,J[J].InvertebrPathol,1990,56:362-370.
• [13]SHIXiao yun,SuXiao qing.IsolationandCloningofacD NAFragmentofPr1Genefrompythumcarolinianum[J].JournalofGuiyangMedicalCollege,2003,28(1):1-4.
• [14]TrigliaT,PetersonMG,KempDJ.Aprocedureforin vitroamplificationofDNAsegmentsthatlieoutsidethe boundariesofknownsequences[J].NucleicAcidsRes,1988,16:8186.
• [15]RosenthalA,JonesDSC.Genomicwalkingandsequen cingbyoligo cassettemediatedpolymerasechainreaction[J].NucleicAcidsRes,1990,18:3095-3096.
• [16]PfeiferGP,RiggsAD.Genomicsequencingbyligation mediatedPCR[J].MolBiotechnol,1996,5:281-288.
• [17]LohEY,ElliotJF,CwirlaS,etal.Polymerasechain reactionwithsingle sidedspecificity:analysisofTcell receptoredchain[J].Science,1989,243:217-220.
• [18]TerauchiR,KahlG.Rapidisolationofpromoterse quencesbyTAIL PCR:the5′ fankingregionsofPalandPgigenesfromyams(Dioscorea)[J].MolGenGenet,2000,263:554-560.
• [19]JonesDH,WinistorferSC.Sequencespecificgenera tionofaDNApanhandlepermitsPCRamplificationof unknownflankingDNA[J].NucleicAcidsResearch,1992,20(3):595-600.
• [20]MegonigalMD,RappaportEF,JonesDH,etal.Pan handlePCRstrategytoamplifyMLLgenomicbreakpoints intreatment relatedleukemias[J].ProcNatlAcadSciUSA,1997,94:11583-11588.
• [21]FelixCA,KimCS,MegonigalMD,etal.Panhandle polymerasechainreactionamplifiesMLLgenomictranslo cationbreakpointinvolvingunknownpartnergene[J].Blood,1997,90:4679-4686.
• [22]DHJones,SCWinistorfer.Genomewalkingwith2 to4 kbstepsusingpanhandlePCR[J].PCRMethodsAppl,1993,2:197.
• [23]JonesDH,WinistorferSC.Amethodfortheamplifi cationofunknownflankingDNA:Targetedinvertedre peatamplification[J].BioTechniques,1993,15:894-904.

文章评论(Comment)：