Preliminary Study Of Transfer RNA Modification M~5C、m~1G And Gene Function In Arabidopsis Thaliana

Transfer RNA（t RNA）is adapter molecule connecting nuclear acids and proteins.In all domains of life,t RNA molecules contain modified nucleosides.These modifications are all derived from four basic nucleosides:adenosine,guanosine,uridine and cytosine.t RNA modificatons are important for the decoding process which converts message from m RNA into corresponding peptide sequence.Studies have shown that t RNA nucleoside modification can have wide influence depending on organisms,including pathogenic bacteria,human neural system disease,plant stress toleranc etc.Extensive study of t RNA modified nucleosides has been done in E.coli and S.cerevisiea,but much less in higher plants.In this work,we focused on functional study of t RNA nucleoside modification genes in model plant Arabidopsis thaliana,particularly the role of t RNA nucleoside modification genes on plant growth and development.According to sequence similarity with known modification genes in Sacccharomyces cerevisiae,we identified two Arabidopsis genes-At2g22400 for m⁵C,and At5g47680 for m¹G nucleoside modification,respectively.Both bioinformatic analysis and result from T-DNA insertion mutants suggested that,At5g47680 and At2g22400 indeed involved in m¹G9 and m⁵C49 modification.The main results of this work are described as following:1.Bioinformatics analysis shows that,At2g22400 is homologous with yeast Trm4p.At5g47680 contains m¹G-MT（methyl transferase）domain,the same as yeast Trm10p.Both protein is very similar to yeast homolog,shown with protein domain structure,motif composition and phylogeny relationship.2.Analysis of homozygous T-DNA mutant supports role of At2g22400 for m⁵C modification.Compared with wild type,At2g22400 T-DNA knock-out mutant showed≈40%decrease of m⁵C.At2g22400 mutant also had shorter petioles and delayed vegetative growth.3.We have completed vector construction for promoter-GUS analysis,and obtained positive transgenic plants with hygromycin selection.GUS staining will be performed in next generation.4.We constructed HIS-tag fusion protein of At2g22400 for protein in vitro expression and purification.The induction condition still need to optimized.We also in vitro transcribed t RNA_GUC^ASPas substrate for in vitro-methylation assay with purified His-tagged-At2g22400.This in vitro methylation assay will verify the methylation activity of Trm4p homolog At2g22400 for m⁵C modification.5.Previously we have shown that Arabidopsis Trm10 homolog is At5g47680,and it is responsible for m¹G modification.In this work,promoter-GUS assay showed that At5g47680 was mainly expressed in shoot apex and root tip.The expression pattern also suggests that At5g47680 might have some role on trichome development.6.Subcellular localization with transient tobacco transformation showed that At5g47680 protein was mainly located both in the nucleus and cytoplasm.7.Trm10p catalyzes the m¹G methylation on t RNA_GCC^Glyat position9.Using t RNA_GCC^Glyas substrate for in vitro methylation assay,we showed that At5g47680 could catalysis m¹G formation.Data also showed some unspecific methylated nucleosides such as Cm and m⁵C,the reaction might be optimized t reach the best specificity between t RNA substrate and purified protein.