抗黄瓜花叶病毒RNAi载体的构建及烟草的转化(2)

2021-09-24 20:08

[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

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