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2007,38(5):425431ActaVeterinariaetZootechnicaSinicamiR2206,,,3(,,430070):miRNAs,miR2206(Precursor),miRNART2PCR,miRNA,miR2206:;miR2206;;:S828.2:A:036626964(2007)0520425207:2007201211:(A06056);(30672238);(2006CB102105);(2006ABC008):(19852),,,,E2mail:jianhaichen@yahoo.com.cn3:,,,,,E2mail:shzhao@mail.hzau.edu.cnCloning,ExpressionandBioinformaticsAnalysisofmiR2206PrecursorinthePigCHENJian2hai,WEIWen2juan,XIAOXiao,ZHAOShu2hong3(KeyLaboratoryofAgriculturalAnimalGenetics,BreedingandReproductionofMinistryofEducation,KeyLaboratoryofSwineGeneticsandBreeding,MinistryofAgriculture,HuazhongAgriculturalUniversity,Wuhan430070,China)Abstract:MiRNAsplayimportantrolesinskeletalmuscledevelopment.Inthisstudy,thepor2cinemiR2206precursorisclonedandsequencedfromskeletalmusclethroughcomparativegenom2icsapproach.Thesecondarystructurewaspredicted.RT2PCRresultsindicatedthatthemiR2206precursorisexpressedinmostofthetissuesandexhibitedthehighestexpressionlevelinskeletalmuscle.Homologyanalysisrevealedthatpre2miR2206ishighlyconservedamongdifferentspe2cies.Keywords:pig;miR2206precursor;cloning;expressionMicroRNAs(miRNAs)22RNA,[1]miR2NARNA(pol),(pri2miRNA)(7MGpppG)(Poly(A))[2]pri2miRNADrosha(DGCR8Pasha)70ntmiRNA(pre2miR2NA)[2,3]Ran2GTPExportin5[4,5],,Dicer22ntmiRNA:miRNA3[6]miRNA3[7],miRNARNA(RISC)miRISC,miRNAmRNA3UTR[810]mRNA(38)mRNA()[1,11,12]62%miRNA(Inter2gentic);34%(Intronic);4%(Exonic)[13]miRNAlin24let27,(pre2miR2NA),Pol2IIPol2IIIRNA,miRNAmRNA(pre2mRNA)Pol2II,miRNApre2mRNA[2,14]miRNAs,,mir17292[15,16]50%miR2NAs,[17],miRNA,miR2134miR2154miRNAsDlk12Gtl2,(Im2printing)[18]miRNAs,,miRNAsmiRNAs,miRNAs[1925],miRNAmiR21,miR2133miR2206[26]miRNAs,miRNAs,,,miRNA,miRNA,,11.1TripureRNAsafe;RNATURBODNA2freeTMAmbion;M2MLVPromege;RandomPrimerTOYOBO;TaqDNAdNTPspMD182TDL2000marker;PCR90d1.21.2.1miRNAmiRBase()miRNAMap()miRNA,Blast,NCBI,(ht2tp://=9823)miR2206(83bp),has2mir2206(MI0000490),95%(emb|CT842532.2|),,primerpremier5.0:PL(52GGCCACATGCTTCTTTATATCC23)PR(52CACACACTTCCTTACATTCCAT2AG23),63bp,miR2206(Homologoussequence)11hsa2mir2206Fig.1Alignmentofhsa2mir2206andhomologoussequenceinthepig1.2.2RNA90d;90d7()Tripure6245:miR2206RNA,RNA(TURBODNA2freeTM)RNADNA,DNA2%RNA,Du640(Beckman)RNAOD1.82.0RNA1.2.3RT2PCRRNA,PromegeM2MLVcDNAcDNA2(50L):RNA1g/L,2LRNA0.15mL,2LRandomPrimer(20pmol/L),DEPC15L,705min,RNA,12min,M2MLV1.5L(200U/L),5Buffer10L,dNTPs2.5L(10mmol/L),RNAsafe1L(40U/L),DEPC20L,35L,371h,955min,cDNA(1.2g/L)-20cDNAPCR,PCR:7.2L,1L10Buffer,0.6L(25nmol/L),0.2LdNTPs(10mmol/L),PLPR0.2L(10pmol/L),0.1LTaqDNA(2U/L),0.5LcDNA(1.2g/L),10L,PCR:955min,9430s,5830s,7220s,30;725min,4GAPDH,GAPDH2F(52CCT2TCATTGACCTCCACTAC23)GAPDH2R(52GTTGTCATACTTCTCATGGTTC23),321bpGAPDH2FGAPDH2R0.3L(10pmol/L),6030s,7225s,RT2PCRmiRNA,31.2.4RNAcDNA,PLPRPCR,PCR3%,PCRpMD182T,(DH5),,PCR,1.2.5miRNA3.2mfold()miR2206miR22061.2.6miRNAmiRBasemiRNAMapmiR2206,NCBIBLAST()(DNAstar)MegAlign,ClustalWmiR2206,22.1cDNA90dcDNA,PLPRRT2PCR3%,2RT2PCR,PCRpMD182T,NCBIBlast,miRNAMapmiR22066,miR2NA7,2(Correspondencesbetweenhu2manandpigchromosomalsegments)(),76,miR22062.2miRNAmfoldmiR2206,(86bp)2(3),3,72438M.DL2000marker;12.pre2miR2206M.DL2000marker;1,2.Porcinepre2miR22062pre2miR2206Fig.2Porcinepre2miR2206amplifiedbyRT2PCR,AG,3miR22062,,,miR220652UGGAAUGUAAGGAAGUGUGUGA232.3miR2206miRNA,BLASTRNAmiR22063pre2miR22062Fig.3Homosapiensandsusscrofapre2miR2206stem2loop(MI0000490),(MI0000249),(MI0000948),(MI0002619),(MI0002620),(MI0004863),(MI0001207),(MI0002045),(MI0002046),(MI0005317)95%85%87%95%95%86%89%100%81%87%,(MI0002045)23bp,miRNAmiR2206,G,22nt,3AClustalW4,,,,,miRNAs,(100)Thelengthbetweeneachpairofbranchesrepresentthedistancebetweeneachpairofsequences,thescaleinthebottomrepresentthesumofthenucleotidesubstitutions(100)4miR2206Fig.4Thephylogeneticstreeofpre2miR2206indifferentspecies8245:miR22062.4miR2206RT2PCR,pre2miR220690d7,5303540,30,,35,,,40PCR,35GAPDH28,miR2206,,,,,,,GeneToolsmiRNA,35PCRGAP2DH,,6miR2206,,M.DL2000marker;1.;2.;3.;4.;5.;6.;7.;GAPDH.M.DL2000marker;1.Heart;2.Liver;3.Spleen;4.Lung;5.Kidney;6.Skeletalmuscle;7.Placenta;GAPDH.Internalcontrol.Thesameasbelow5pre2miR2206Fig.5Expressionpatternofporcinepre2miR22066pre2miR2206Fig.6Tissuedistributionofporcinepre2miR22063miRNA:,cD2NANorthern[27]miR2NAmiR22067,619,7Andersond[28]6miR22063.8kbmiR2133b,miR2133b,miR2133b(MI0000822)(=9823),7,miR22063.8kbmiR2206/1217,,[28]miRNA,Andersond[28]NorthernmiR2206,RT2PCRpre2miR2206,Schmittgen[29]RT2PCR6miRNAs,miRNAmiR2NASawera[30]mir17292,NorthernmiR2NAsmiRNAs,miRNAsmiRNAsmiRNAsRosenberg[31]MyoDmiR2206follistatin2like1utrophinKim[32]miR2206p180DNA392438Sweetman[33](Somites)FGF24miR2206Rao[26]miR21,miR2133miR2206miRNAAnderson[28]miR2206connex2in43mRNA,miR22068ntseedCx4332UTR,