硕士论文-桑粒肩天牛幼虫肠道优势菌群研究及Cry3A重组质粒

重庆大学硕士学位论文桑粒肩天牛幼虫肠道优势菌群研究及Cry3A重组质粒载体构建姓名：冯霞申请学位级别：硕士专业：生物医学工程指导教师：殷幼平20050501I20-90%16SrRNAGFPCry3AGFPCry3ACry3A1289Staphylococcus7.84±0.61100Enterobacter7.42±0.23100KlebsiellaStreptococcusProteusSerratiaEscherichiaBacillusMicrococcus29PCR16SrRNA9StaphylococcusEnterobacterKlebsiellaStreptococcusProteusSerratiaEscherichiaBacillusMicrococcusBLAST16SrRNA98.7899.0298.8697.8999.24%98.99%98.44%86.67%99.18%99.23%99.34%99.64%98.35%99.87%99.08%99.16%99.32%99.29%316SrRNAStaphylococcusEnterobacter16SIIrRNA16SrRNA98.1299.044GFPCry3AStaphylococcusAmpErm16SrRNAGFPCry3AIIIABSTRACTLonghornBeetleCerambycidaeisabiggroupofinsectsinColeopteranandmostofthelarvaeboringintreetrunkorbranchandusingwoodfiberasmeat.About20%to90%forestsaredamagedbythesepestsandmillionsyuanofmoneyislostedeveryyearinChina.However,thechemicalcontrolsoftenresµLtedingrievouspollutionstoenvironmentandkilledthenaturalenemies,andtraditionalbiocontrolstrategytooktime,hadlowefficacyandcostalot.Therefore,newbiocontrolmethodsandtechniquesareeagerlydemandedinpractice.Anewbiocontroltheoriesandapproacharealwaysbasedontheresearchofinsectphysiology.Recently,akindofnewapproachforpestcontrol,basedontheresearchofmicro-communityandmicro-ecosystemofinsectintestines,isbecomingoneofthehottopicsallovertheworld.BytraditionalcµLtureand16SrRNAsequenceanalysis,theintestinalmicrobeflorainAprionagermari(Hope)larvawasanalyzedandtheirpredominantflorawasfoundout.Meanwhile,thisstudyalsoconstructedaclonevector,whichwascontainingfluorescentproteingeneGFPandBtnocuousproteingeneCry3A,andtransformedtheclonevectorintothepredominantflora.Bythatway,thesettleofthepredominancefloraandBtgeneCry3AexpressioninA.germari’gutcoµLdbemonitored.Theaimwastoprovideatheoreticalandtechnologicalsupportfortheresearchofnovelgenicengineeringbio-insecticide.ThemainresµLtswereshowedasfollows:(1)Twentyeightdifferentbacterialstrains,whichobtainedfromA.germarilarvaegut,belongedto9differentgenus,bytraditionalcµLturemethods.Staphylococcuswasthepredominantflorawithcountof7.84±0.61andtheisolationrate(Thenumberoflarvaewhichhadthebacteria/thetotalnumberofthelarvaehadbeendetected)100.ThesecondpredominantflorawasEnterobacterwithcountof7.42±0.23andtheisolationrate100also.Theisolationofotherbacteriawascomparativelylow.TheycoµLdberankedorderlyasKlebsiella,Streptococcus,Proteus,Serratia,Escherichia,BacillusandMicrococcusaccordingtotheirisolationrates.(2)Theninedifferentbacteriastrains,whichwereabtainedthroµghtraditionalmeansfromA.germarilarvae’gut,wereanalyzedby16SrRNAsequenceanalysis.ItwasshowedthattheywereStaphylococcus,Enterobacter,Klebsiella,Streptococcus,Proteus,Serratia,Escherichia,BacillusandMicrococcusrespectivelyandtheywereIVconsistentwiththeresµLtsofcµLture.Their16SrRNAsequencessimilarityratesinBLASTwererespectively98.78,99.02,98.86,97.89,99.24%,98.99%,98.44%,86.67%and99.18%(byfirstpairpimers);99.23%,99.34%,99.64%,98.35%,99.87%,99.08%,99.16%,99.32%and99.29%(bysecondpairpimers).(3)16SrRNAsequenceanalysisrevealedthatStaphylococcusandEnterobacterweretherealpredominantflorainA.germarilarvae’gutalso.(4)Therecombinedclonevector,whichcontainedgeneGFPandCry3A,hadbeenconstructedsuccessfµLlyandverifiedbymolecµLarweightdetecting.ThentheclonevectorwastransformedintotheintestinalpredominantfloraStaphylococcusofA.germarilarvaegut.However,thetransformantbacteriacoµLdn'texpressgreenfluorescentproteinasexpected.Thereasonsneedtobefurtherinvestigated.Keywords:Aprionagermari(Hope),intestinalpredominantflora,16SrDNA,geneGFP,geneCry3A,cloneplasmidvector1111.1[1]19921998199920020.67200250111.7300“”41075002004596.8120-90%Scriveneretal1997[2][3-7][8]12[9][10]IsopteraBlataira,(Orthopteroa)(Lepidoptera)[11](microcommunity)microecosystemautochonousfloraautochthonyallochthonousfloraallochtonyindigenousfloraresidentfloratransientflora1.21.2.1VPMR2000[12]PenaeuschinensisVibriosAeromonasPseudomonasPhotobacteriumAcinetobacterFlavobacteriumFlexibacterChromobacteriumVibriosPhotobacterium1399.5-99.9%[13]1.2.21983PCR1986PaceWoeserRNA[14][15]53005600[16][17][18]16SrRNA16S-23SrDNARNARAPDGeorginaLhold200210[18]KristianDaly20010.9PH7.0[19]12314AyyamperumalJeyaprakash2003ApismelliferacapensisApismelliferascutellata[20]A.F.Reeson2003VespµLagermanica[21]YuichiHongoh2003ReticµLitermessperatus[22]rRNARNARNADNADNArRNA()DNA[23-27]DNA5-10DNADNADNAStahlSDS3[28]DNADNAShortProtocolsinMolecµLarBiology[29]116SrRNAa.16SrRNArRNA(Conserveddomain)(Variabledomain)rRNA(Phylogenesis)16SrRNA1540bp16SrRNA1516SrRNA16SrRNA16SrRNA16SrRNADNA16SrRNA16SrRNA3b.16SrRNAPCR16SrRNA[30]1.116SrRNAPCR94-962-5min94-9530-60s45-5560s68-722-4min68-72610min[31-33]PCRDNARNA16SrRNAPCR16SrRNA(Chimericproducut)16SrRNA1.116SrRNAPCRTable1.1Somestandarduniversalprimersfor16SrRNAPCRatpresentPrimers*Sequences(5-3)Ranges*frD:PfEcoR1Sal1rHindBamHXmalrRNArP1rP2rP3317**5-3,DNADNA16PCR16SrRNA“”PCR[34]c.16SrRNA16SrRNA41)PCR16SrRNA2002AI-3AI-3Halomonadaceae[35]BettinaA.Moser16SrRNAMicrosporidiathelohaniaVairimorpha99.2[36]KristianDaly200116SrDNA[19]NguyetThuPhung200416SrDNA,[37]GeorginaLhold200216S