Study On The Functions Of Tomato OPA3-like Gene

Helper component proteinase(HC-Pro)encoded by PVY(Potato virus Y)is a multifunctional protein involving in aphid transmission of the virus,genome amplification,symptom expression,the suppression of RNA silencing and other processes.Using yeast two hybrid system,we screened out a gene from the tomato c DNA library which can interact with HC-Pro.The putative amino acid sequence of this gene is homologous to OPA3,which we call OPA3-like.Objective: Bioinformatics analysis of OPA3-like protein function,then obtained the tomato OPA3-like fusion protein.These experiments prepared for the preparation of OPA3-like antibodies and follow-up experiments;determine the subcellular localization of OPA3-like protein;obtain OPA3-like overexpression and gene silencing plants through transgenic technology,in order to establishment the foundation of studying on the relationship between OPA3-like and PVY infection.Methods: The basic physical and chemical properties,signal peptide,transmembrane region and subcellular localization of OPA3-like protein were analyzed by the Vector NTI and online software such as Ex PASy,Signal P,Plant�m PLoc,et al.Three prokaryotic expression vectors,p ET22b-OPA3-like,p ET28a-OPA3-like and p ET32a-OPA3-like,had been constructed,and then transformed each of them into E.coli BL21(DE3)and Rosetta(DE3)respectively to analyze the prokaryotic expression of OPA3-like gene;constructed the subcellular localization vectors,p SP-OPA3-like-GFP and p CBGFP-OPA3,which the C terminal or N terminal of OPA3-like protein fused with GFP respectively.Transformed tobacco with the fusion vectors and the localization of target protein was observed by confocal laser scanning microscope;constructed OPA3-like overexpression vector p BP-OPA3-like and obtained transgenic tobacco and tomato.RNAi expression vector p BRi-OPA3-like was also transformed into tomato to obtain transgenic tomato.Results and conclusions:(1)The bioinformatics analysis results showed that tomato OPA3-like protein contained rare codons,transmembrane region,the N terminal chloroplast localization signal sequence,so the tomato OPA3-like protein may be a membrane protein localized in chloroplast.(2)Three prokaryotic expression vectors were transformed to BL21(DE3)and Rosetta(DE3)bacteria which can provide rare codons.The results indicated that for the same recombinant vector,the expression of the fusion protein had no obvious differences in two strains.This meant that the important factor affecting protein expression isn't rare codons;there's not expression of fusion protein after transformed p ET22b-OPA3-like vector.But when transformed p ET28a-OPA3-like vector,the two strains both can express a 20 k Da fusion protein.Also,after transformed p ET32a-OPA3-like vector,we could obtain a 37 k Da fusion protein and the expression was the highest.All the results indicated that different expression vectors with different protein labels and promoters affect protein expression.Thioredoxin A(Trx A),a fusion protein contained in p ET-32a(+),wasthe reason that the OPA3-like fusion protein can highly expressed;soluble analysis showed that the fusion protein mainly exists as inclusion bodies.(3)The GFP-OPA3-like and OPA3-like-GFP transgenic tobacco were obtained,the fluorescence observation shows that OPA3-like protein of tomato was mainly located in the chloroplast,the green fluorescence mainly accumulated in chloroplasts,while the fusion protein of OPA3-like N-terminal and GFP sequence was partially localized on the protective cell membrane,which may be that the GFP affects the chloroplast localization signal of N-terminus of OPA3-like protein.(4)Constructed the OPA3-like overexpression vector and RNAi expression vector,then we obtained tomato OPA3-like gene silencing transgenic tomato;Obtained OPA3-like overexpression transgenic tomato and tobacco plants.However,we found many other differences between the transgenic tobacco plants and the wild type,the young leaf color was deeper to form a clear vein;mature leaf had uneven color,petal color was deeper,but stigma color was lighter than wild type.These results speculated that OPA3-like may be involved in the synthesis of pigment.