[7-8]
parasites,aswellasgraftingbreeding.
Atpresent,theresistanceresearchesonCMVarefo?cusedonreplicaseproteinencodedbyRNA1genomeorCPproteinencodedbyRNA3subgenomic.Inthisstudy,basedonthepre?clonedCMVRNA2genomesequence,the2arepli?caseproteinwhichwasencodedbyRNA2genomewasusedtodesignRNAianti?viralvector,theconstructedRNAivectorwastransformedtotobaccothroughAgrobacterium?mediated
,andtheantiviralabilityoftransgenictobaccoplantsmethod
wasobtainedbychallengetesttodeterminewhethertheprocesscanbeappliedtotomatoproduction.
Received:May26,2010 Accepted:June29,2010SupportedbyInternationalScienceandTechnologyCooperationProgram(2008DFA30560);PreliminaryResearchSpecialFounda?tionof973Program(2008CB117018);ScientificResearchProjectforHighLevelofTalentsofShiheziUniversity(RCZX200732).
orrespondingauthor.E?mail:xbc@shzu.edu.cn?C
MaterialsandMethods
Materials
piRDG12andpBi35SG12weresavedbyKeyLaboratoryofAgricultrureBiotechnologyofShiheziUniversity/CollegeofLifeSciences,ShiheziUniversity;EscherichiacoliTOP10competentcellwaspurchasedfromShanghaiBioengineeringCompany;planttotalRNAextractionkitwaspurchasedfromTiangengBiotechnologyCompany;AMVreversetran?
,TNAligase,andPCRproductextractionkitscriptase4D
werepurchasedfromPromegaCorporation.Restrictionen?zymeswerepurchasedfromShanghaiBioengineeringCompa?ny.TomatocucumbermosaicvirusisolatesNS04wassavedbyKeyLaboratoryofAgricultrureBiotechnologyofShiheziUniversity/CollegeofLifeSciences,ShiheziUniversity.MethodsTotalRNAextractionoftobaccoplants TotalRNAwasextractedformNicotianaglutinosawhichhadsavedtomatoNS04isolates,andtheextractionmethodwasinaccordancewithRNAsimpleTotalRNAKitmanualoperationfromTian?gengBiotechnologyCompany.Primersdesignandsynthesis BasedonthesequencesinGenbank,theVectorNTIwasusedfortheprimersdesignandsynthesis,andtheprimersusedinthisstudyweresynthe?sizedbyShanghaiBiologicalEngineeringCompany.
Table1 PrimerandsequenceforPCRamplificationPrimernamePrimersequenceCMV2SP1CMV2ASP2CMV2SP3CMV2ASP4CR2?BgCR2?KpCR2?SB
GTTTATTTACAAGAGCGTACGGTTCAATCTCGAAGGCATCTCTGGAAGTATAACCTCCCAGTTCTCACC
GGATGGACAACCCGTTCACC
GGAGATCTGTTTGCTCACTTCATG BglIIGGGTACCGAATGACTCAGTCTT KpnIGGGAGCTCGGATCCCGTTCACCGTGAAAACGT
RNA2genomeamplificationandsequenceanalysisof
CMV TheextractedtotalRNAwasusedforthereversetran?
70
scriptionandsynthesisoffirststrand.Andthenthefirststrand
,theamplificationwascarriedoutbythewasusedasatemplate
useoftheCMV2SP1/CMV2ASP2,CMV2SP3/CMV2ASP4ofthedesignprimersCMVRNA2,afterthesequencingoftheamplifiedsequence,theDNAMAN(Version5.2.2,Lynnon