Optimization Of Tombusvirus-based Plant RNA Virus-based Expression System

Plant RN A virus-based vector expression system has been developed in recent years,which is a new genetic expression potential platform.Tomato bushy stunt virus(TBSV)is ideal material to construct the new plant expression vector of RNA virus.Tomato bushy stunt virus(TBSV)is used as experimental material,previous studies have shown that Tombusvirus-based plant RNA vector by agrobacterium into exogenous gene GFP expressed in host plants.But its expression efficiency is still in a relatively low leveL Regarding the issue above,based on the TBSV plant RNA virus vector,we expected that by probing into the exogenous gene GFP TBSV vector in sectional locus,and downstream related sequence effects on viral vector express efficiency,which need to optimize TBSV viral vector structure in order to get efficient TBSV virus expression vector.First of all,in the process of optimization in theTBSV virus expression system,in order to explore the translation initiation site AUG position influence on viral carrier expression efficiency,I designed and built the fusion model expression vector pTBSV-WY2.1 on the basis of the vector pTBSV-WY2.0.Then using the method of agrobacterium infiltration inoculation in host tobacco varieties(N.excelsiana),looply through the entire comparison,the level of molecules such as proteins,nucleic acids research have shown that,resulting that translation initiation site AUG location efficiency significantly influences the expression of viral vector and translation level of change is the main reason of the expression difference between the two vector.In addition,TBSV viral genome RNA degraded rapidly in 8 days.It's speculation that virus induced gene silencing(VIGS).So it is for the recovery of P19 genes laid a certain foundation.Secondly,In order to explore the pl9 genes in host plants N.excelsiana will cause a host of water and can further improve the virus expression vector of continuity and efficiency of foreign gene expression,pTBSV-WY2.1 vector recovery on the basis of the inhibition of gene silencing P19.The results show that P19 gene product to replace the host plants after N.excelsiana have no obvious adverse reactions.P19 genes expressed in expressing duration and efficiency have remarkable enhancement effect in Virus Vectors,and cis of p19 genes effect more apparently.Thirdly,in order to further clarify the start codon AUG upstream flanking sequences and their locations on the efficiency of the viral vector expression.On the basis results above,we built four different virus expression system of TBS V The experimental results showed that initiation codon AUG upstream flanking sequences and their length have no significant influence for the efficiency of expression vector.It's speculation that the expression of vector sequence location efficiency may play a key role.Fourthly,in order to explore the local hairpin instead of RNA-RNA feasibility of long-range interaction transcription to sg mRNA2 and sg mRNA2 transcription affect viral vector expression efficiency.We designed three different virus vectors that choose the appropriate local structure(such as structure of G4,Hairpin stem-loop structure)to replace the complex structure.The results show that sg mRNA2 transcription has directly influenced the viral vector expression efficiency.It's speculation that the effect has directly related to the two vial gene product encoded by sg mRNA2.The fifth,in order to explore the exogenous gene GFP downstream of CP gene sequences the influence of different length of viral carrier expression efficiency,we have designed and constructed three kinds of viral vectors successfully.From the perspective of the quantitative results of ELISA,3 'end of CP gene sequences of different length and negatively correlated with the expression of exogenous gene GFP quantity relationship.The sixth,the mfold software to predict the secondary structure of oriented,two plant virus expression vectors was designed that named dATGdC and mATGdC,Which is used to explore the sg mRNAl secondary structure that effects on viral vector expression efficiency.The results showed that sg mRNAl secondary structure on the efficiency of the viral carrier expression does have a certain degree of influence.It's speculation that there are the other secondary structures which playing an important role,excepting outside Y-shaped domain.In the end,In order to improve the ease of fusion vector pTBS V-WY2.1-P 19 in the later application,exogenous gene insertion sites in vector before joining the enzyme loci Fsp I build new viral vector pTBSV-WY2.1-FSP.The comparative analysis show that we have built two TBSV viral vectors(pTBSV-WY2.1-FSP and dATGdC),which have achieved satisfactory result,in terms of the expression of exogenous gene GFP quantity.Preliminary explicate that building the virus expression vectors need the foundation of further application.The expression quantity is about 5.0 mg in per gram fresh leaves on 10th day in integration vector pTBSV-WY2.1-FSP.The expression quantity is about 3.0 mg in per gram fresh leaves in non-fusion vector dATGdC.This article attemps to improve or solve the problem,which preliminary laboratory construction of TBSV retroviral expression vector of expression efficiency is relatively low,which lays a foundation for its further application.And through the study of related mechanism,in-depth understanding of plant RNA virus expression System and provide reference for other plant virus RNA-based expression systems.