Functional Analysis Of IRES,a Regulatory Element For Translation Of HiPV Virus In Grey Planthopper,and Its Application In Bicistron Expression System In Insect Cells

Biosynthesis of most proteins in eukaryotic cells relies on the 5’cap structure to initiate translation.However,some RNA viruses lacking of the 5’cap structure can initiate downstream m RNA translation by using internal ribosome entry site（IRES）in the 5’untranslated region.Small brown planthopper（SBPH）as an important agricultural pest can transmit rice stripe virus（RSV）,which threats to rice production.Insect virus Himetobi P virus（HiPV）in SBPH contains IRES element.It was found that VP1 protein of HiPV could promote RSV proliferation in SBPH.So,it is important to study the mechanism of HiPV-IRES regulating protein synthesis for further revealing the interaction between HiPV and RSV.At the same time,this study can also provide new ideas for the exploitation of insect viruses to control SBPH.Therefore,in this study,HiPV-IRES was used as the object to identify the key functional region of its regulatory protein translation initiation and to clarify the function of its downstream asparagine repeat sequence.Further,a bicistronic expression system suitable for insect cells was established by using this element to achieve two proteins co-expressed by one promoter control.1.The secondary structure of HiPV-IRES was analyzed and the location of the pseudoknot structure was determined.The expression vector of p IE-IRES-YFP was constructed,in which the IRES located the downstream of promoter and the upstream of reporter gene yellow fluorescent protein（YFP）.After cell transfection,it was showed that IRES element could initiate protein synthesis of downstream genes in insect cells via fluorescence microscopy and Western blotting.Mutants of IRES with pseudoknot site-directed mutation were constructed and tested protein synthesis,and the results indicated that the pseudoknot structure formed by150-146 and 171-175 nucleotides in IRES was the key structure of initiation protein synthesis.The initiation protein synthesis efficiency of IRES from different HiPV isolates was analyzed,and the results showed that there was no significant difference between Japanese isolate IRES（standard type）and Jiangsu isolate IRES（variant type）.2.There was no homology in amino acid sequences of downstream viral proteins of IRES from different viruses,an aspartate repeat sequence（ANNNNNNNNTN）was located at downstream of HiPV-IRES.In order to explore the effect of repeat sequences on IRES initiating translation.IRES_NN containing repeat sequence and IRES_N’without repeat sequence were inserted into expression vector,respectively.After protein detection,it was found that aspartate repeat could improve the synthesis efficiency of downstream protein.Cr PV-IRES and DCV-IRES were inserted into the expression vector to compare the initiation protein synthesis efficiency with HiPV-IRES,and the results showed that the ability of Cr PV-IRES and HiPV-IRES in initiation protein synthesis was similar,and higher than DCV-IRES.3.Restriction enzyme sites were added at both ends of IRES,and the bicistronic expression original vector p IE-IRES was constructed.On this basis,VP1 gene was inserted into upstream of IRES,and YFP gene was inserted into downstream of IRES to get vector p IE-VP1-IRES-YFP.After protein detection,it was verified that the vector could express simultaneously two proteins,and the expression of two proteins did not affect each other.After transposing upstream and downstream genes,vector p IE-YFP-IRES-VP1 was obtained,and the two proteins are also co-expressed normally.These results suggested that a bicistronic expression system suitable for insect cells was established,in which one promoter leaded two proteins co-expression.It will provide an important reference for the improvement of protein expression system in insect cell.