Establishment Of Protoplast Transient Expression System To Evaluate The Activities Of Rice Cis-regulatory Elements

Rice(Oryza sativa),one of the staple food crops,is a model crop system for studying monocotyledons genome.Recent advance in research on rice genome research include a high quality reference genome annotation,large-scale of transcriptome and epigenetics information,it provide great convenience to study gene regulation in rice.The gene expression and regulation in eukaryotes is controlled by orchestrated binding of regulatory proteins,including both activators and repressors,to promoters,enhancer and other cis-regulatory DNA elements(CREs).Transient expression system is a system which enables a specific DNA sequence to achieve short-term high expression in target cells within a short time.In this study,we took advantage of rice protoplast transient expression system to evaluate the activities of rice CREs.The study is mainly consisted of the following parts:1.Establishment of protoplast transient expression system to examine the activities of bidirectional promoters in rice.In terms of the signature of open chromatin and histone modifications profiles,several bidirectional promoters were identified in rice.Further two bidirectional promoters named BDP1 and BDP2 were selected according to the expression and histone modification of their target genes.A dual reporter vector,which employed bidirectional promoter to express GFP and RFP at the same time was constructed and transformed into rice protoplast.Then we used the protoplast transient expression system to test the activities of BDP1 and BDP2.Confocal microscopy was used to confirm their bidirectional expression activity.The experiment is simple to operate and efficient compared with stable transformation,which provides a useful method for identification and evaluation of genome-wide bidirectional promoters.2.Establishment of protoplast transient expression system to validate the activities of enhancers in rice.The vector used for enhancer verification was a GFP-based reporter system.In the vector used in the reporter system,the promoter has a very weak activity that unable to activate the GFP expression,thus the fluorescence could not be detected after the protoplast transformation.According to the feature that enhancers regulates gene expression at a distance,the enhancer sequence was inserted into the downstream of the promoter(about 4.5 kb),and fluorescence signals were observed after the transformation.If green fluorescence signals were distinctly observed,the enhancer was active.This experiment provided an efficient protoplast validation method for the identification of rice enhancers.3.Establishment of protoplast transient expression system of intron-derived enhancers in rice genome.In our previous work,we found an intron sequence with cis-regulatory activity in the 5’UTR region of LOC_Os04g57220 gene.we connected the promoter sequence of this gene and the corresponding 5’UTR sequence to the GFP protein,no fluorescence signal was observed after the protoplast transient transformation.Strong fluorescence signal was observed after the transformation,it suggested that the intron may have the enhancer activity.When the intron was present,the intensity of GFP expression increased about 2-fold.The establishment of this system provides an effective method for fast validation of intron in rice.