The Effects Of RNA Polymerase ? Promoter And Its Terminator On Plant Viral Expression System

The plant RNA viral vector expression system is a promising new foreign gene expression platform.Biosafety issues are bottlenecks in the current research field of plant viral vector expression systems.The type I promoter has interspecies specificity and can indirectly increase the biosafety of the viral vector expression system by reducing the host range of the recombinant virus.At present,the type I promoter-mediated transcription of viral expression vectors has been widely used in animal virus research,but has rarely been reported in plant systems.Whether a plant type I promoter can be as efficient as a mammalian type 1 promoter to maintain high efficiency and improve the biosafety of viral vectors remains to be verified.In view of this,the tomato bushy stunt virus(TBSV)was used as the experimental material,and the green fluorescent protein(GFP)was used as the reporter gene to construct a TBSV virus expression vector mediated by N.benthamiana type I promoter.Inoculation of recipient plant N.benthamiana leaves by agrobacterium diafiltration to analysis of viral vector-mediated expression of recombinant GFP in inoculated leaves.Exploring the effect of cis-regulatory sequence,transcriptional properties and type I terminator of N.benthamiana type I promoter on viral vectors.The validity and main features of the plant viral vector expression system mediated by type I promoters were initially identified.The study found that,first of all,the length of the N.benthamiana type I promoter has no significant effect on the promoter transcription frequency and viral vector expression efficiency.A promoter of only 77 nt in length can initiate transcription of viral genomic RNA and there is no upstream control element(UCE)similar to the animal type I promoter in the promoter.Second,the upstream T-string sequence of the transcription initiation site of the promoter and the nucleotide A/G exchange at the transcription initiation site also have no significant effect on the promoter transcription frequency and the efficiency of expression of the viral vector.Third,the downstream extension of the transcription initiation site(extending to+42)has a significant positive regulation of the transcriptional strength of the promoter.The effect of increasing the transcription frequency of the promoter and effectively increasing the expression level of the viral vector is about 2-fold.Fourth,the Enhancer-like sequence(ELS sequence)derived from the 35S promoter can enhance the transcription frequency of the promoter and increase the expression level of the viral vector by 3 to 5 times.The modified N.benthamiana type I promoter has similar transcriptional intensity to the enhanced 35S promoter.Fifth,the N.benthamiana type I promoter has transcriptional non-specificity.This transcriptional feature is independent of the downstream extension sequence of the transcription initiation site and the ELS sequence and is its inherent characteristic.Non-specific recognition of type I promoter by RNA polymerase II in the receptor N.benthamiana cells is a possible cause of non-specific transcription of type I promoters.Sixth,the single base mutation in the TATA box of the core sequence of the N.benthamiana type I promoter can improve the transcription specificity to some extent without affecting the transcription efficiency of the type I promoter.However,the non-specific recognition and transcription of the type I promoter by the receptor cells RNA polymerase II is still not completely eliminated.Seventh,the type I terminator neither increases the expression efficiency of the viral vector nor enhances the transcriptional specificity of the N.benthamiana type I promoter and the presence or absence of the terminating element does not affect the efficiency of expression of the viral vector.The high level of self-healing ability of the TBSV virus at its 3' end is a possible cause of this result.Related studies have initially established an RNA polymerase I-mediated transcription of plant viral vector expression system,which lays a foundation for further exploring the potential of the type I promoter-mediated plant virus expression system in biosafety and its potential and application prospects in practical applications.