Validation in vitro of miRNA Target Genes in Wheat
miRNAs are a class of endogenous non-coding small single-stranded RNA,which are widely found in the plants and animals.They are involved in various biological regulations such as cell differentiation,growth and development mainly by cutting target genes or inhibiting target gene expression.At present,it is short of a quick and simple method for verification of miRNA target genes.Based on the pre-screened wheat specific miRNA—Tae-miR9666a,this study was conducted to synthesize its precursor and precursor mutant,connect them to the expression vector,construct the target gene overexpression vector containing GFP(green fluorescent protein)label and co-transform it into tobacco,and verify the interaction between miRNA and the target gene by determining the cleavage of target gene through observing the fluorescence brightness of GFP.The results showed that the fluorescence brightness of the tobacco leaves with co-transmutation miRNA precur-sors was lower,indicating that the target gene was cut by miRNA,and the subsequent GFP could not be ex-pressed normally.The fluorescence brightness of tobacco leaves of co-transmutation miRNA precursor mutants was comparable to that of single transmutation target genes,indicating that miRNA precursor mutants did not cut the target genes,resulting in normal GFP expression.This experiment effectively demonstrated the cleavage of miRNA target genes,and also indicated that it was feasible to verify the interaction between miRNA and tar-get genes by using Nicotiana benthamiana.
万方数据
维普
