A Preliminary Exploration Of The RNA M~5C Modification-related Proteins Functions In Petunia Hybrida

RNA m~5C methylation modification is a current research hotspot.With the increasingly advanced technology for detecting m~5C,people have detected m~5C methylation modification in various RNAs such as t RNA,r RNA,and mRNA.Studies have shown that Nop2 is the m~5C methyltransferase of r RNA,NSUN2 is the m~5C methyltransferase of t RNA and mRNA,DNMT2 is the m~5C methyltransferase of t RNA,and ALYREF is the m~5C-binding protein of mRNA.However,no m~5C demethylase for RNA has been found.Petunia(Petunia hybrida)is not only an important ornamental flower,but also a good material for studying the physiological mechanism of plants.Proteins related to m~5C methylation modification have not been reported in petunia.In this paper,seven protein homologs(PhNop2,PhNUSN2,PhDNMT2,PhALYREF1/2/3/4)related to m~5C modification were obtained by comparing amino acid sequences,and it was speculated that five Alk B family proteins(PhALKBH2/4/6/7/8)are RNA m~5C demethylases.VIGS technology was used to silence the 12 genes to observe the phenotype of the silenced plants.Dot hybridization was used to detect the m~5C methylation content of plants and subcellular localization was performed using fluorescent proteins.Genetic transformation of petunia was performed using CRISPR/Cas9 gene editing technology,plant expression vector p BI121-Ha,and tissue culture technology.This study preliminary explored the RNA m~5C methylation-related proteins in petunia,and obtained the following results:(1)PhNop2、PhNUSN2、PhDNMT2、PhALYREF1/2/3/4、PhALKBH2/4/6/7/8 gene fragments were successfully isolated to construct various recombinant vectors.(2)The leaves of PhNop2 silencing were irregular in shape and uneven and the color of the leaves became yellow.The leaves of PhALYREF1 and PhALYREF4 silencing became small and yellow,respectively.In addition,there were no significant changes in the phenotype of plants silenced by PhALKBH2/4/6/7/8,PhDNMT2,PhNSUN2 and PhALYREF2/3 genes.(3)In PhNop2-silenced plants,the upper epidermal cells of the leaves became larger and the cell edges were smoother.(4)The VIGS-mediated silencing of PhNop2 gene reduced the 5-methylcytosine content of mRNA and other RNAs.(5)PhNop2 was located in the nuclear.(6)We obtained some seedlings of overexpression and knockout of PhNop2 gene.