Screening Of The Host Gene Interaction With Apple Chlorotic Leaf Spot Virus

Apple chlorotic leaf spot virus (ACLSV) is one of most economic important latentviruses on apple, which distributes worldwide. The virus infection alone or in combinationwith other viruses can cause tree weak, chronic damage and yield dropping significantly. Inrecent years, the extensive study of ACLSV on biology characteristics have been made,including the symptoms, serological relationship, and molecular variation and so on, butmost of them stand by the aspect of virus itself. The interaction between host and viruslacks of efforts. The function of virus proteins, viral pathogenesis and host susceptibilitymechanisms are not clear. In this study, the host proteins interacting with ACLSV werescreened by yeast two-hybrid system on the basis of construction of cDNA library ofMalus sylvestris cv. R12740-7A, the woody indicator and host simultaneously for ACLSV.The results will contribute to clarify the functions of the host proteins, the pathogenicmechanism of the virus, the regulation of virus on the host growth, and the response of hostresistance to virus. It will provide important information for antiviral germplasm resourcesof apple. The main results are as follows:1. The pGADT7-based eukaryotic expression vector of yeast two-hybrid cDNAlibrary was constructed using Malus sylvestris cv. R12740-7A tissue culture as materials.Total RNA of M. sylvestris was extracted and purified. The dscDNA was synthesized usingof SMART method, enzyme digested and constructed into the library vector pGADT7,which was electronic transformed into the E. coli DH10B.46clones were randomly pickedfrom the library,75%library inserts were larger than1kb and recombination rate was100%, which indicated the library meet the slibrary construction standards. It could be usedfor the the subsequent study of apple molecular biology and virus-host interaction laid thefoundation.2The yeast two-hybrid bait vectors of ACLSV coat protein (CP) and movementprotein (MP) on the basis of pGBKT7vector were constructed. CP and MP genes ofACLSV were amplified by reverse transcription PCR (RT-PCR) and constructed into theyeast two-hybrid bait vector pGBKT7. The recombinant pGBKT7-CP and pGBKT7-MPvectors were obtained, which were transformed into yeast Y2H gold cells. The toxicity andself-activating activity was test. The results showed bait vector did not showed toxic andself-activation and could be used for yeast two-hybrid system.3Seventy-nine prey genes were obtained from the cDNA library using ACLSV CP and MP as bait proteins. Analysis by bioinformatics,13of them may be related to theinteraction of virus. Sequential transformation was made by PEG/LiAc method. The baitplasmid pGBKT7-CP and pGBKT7-MP were transformed into yeast Y2H gold. Then thelibrary plasmid was transformed into yeast Y2H Gold that containing the bait plasmidpGBKT7-CP, pGBKT7-MP respectively, the transformation products was cultured withdifferent (DO) dropout medium (Two-DO, Three-DO and Four-DO). The positive cloneswas picked from Four-DO medium and then propagated and extracted. The size of cDNAinserts were identified using universal primers of pGADT7by PCR. The yeast plasmidswith cDNA fragments larger than500bp were transformed into E. coli DH5α and selectedby Amp resistance. The positive clones identified by PCR once more, were sequenced. Theresults showed that79target genes were obtained, the cDNA insert size, sequence integrity,correctness of reading frame of which were analyzed by bioinformatics. The sequenceswere blasted in Genbank database. According to the information note of the homologoussequences,13of them may be related to interaction of virus, which was selectedas target genes for further study.