Function Analysis Of TRNA-m1G37 Nucleoside Modification Gene AtTrm5a And Primary Cell Wall Related Transcription Factors In Arabidopsis Thaliana

The thesis focus on function of tRNA-m~1G37 nucleoside modification gene At Trm5a and primary cell wall related transcription factors in Arabidopsis thaliana.In the first chapter,we studed the function of tRNA-m~1G37 nucleoside modification gene At Trm5a.m~1G modification at position 37 of tRNA is one of the oldest tRNA nucleoside modifications.The enzyme that catalyzes m~1G37 modification was originally found in bacteria which is catalyzeed by Trm D,whereas the enzyme responsible for this modification in eukaryotes and archaea is Trm5.The absence of tRNA-m~1G37modification in bacteria and yeast can affect the normal growth of cells,resulting in temperature-sensitive phenotypes.However,the study on this modification in higher plants has not been reported.In this study,two candidate genes,At Trm5a and At Trm5b,were found in Arabidopsis thaliana by sequence alignment using the reported Saccharomyces cerevisiae(S.cerevisiae)Trm5p sequence.The functional domain of methyltransferase was found in the two candidate genes.By studying the function of At Trm5a,we found that:1)In vitro,At Trm5a can catalyze the formation of m~1G at the 37position of tRNA,and absence of the N-terminal(D1 domain)can still catalyze the formation of m~1G,but the activity is reduced.Only the C-terminal(D3)cannot maintain the structure of the protein.2)Heterologous expression of At Trm5a in the yeast trm5mutant could restore its growth defect phenotype,and its m~1G content returned to normal level.3)At Trm5a located in the nucleus,and its localization was not affected by the truncation of the N-terminal(D1 domain).4)At Trm5a was mainly expressed in fast growing tissues.5)In the attrm5a mutant:the contents of m~1G and m~1I were significantly decreased;the plant growth slowly,root length become shorter,flowering delayed,growth period was longer,filaments beaome shorter and seed setting rate were lower;the contents of IAA and JA hormone changed;the 80S ribosome was decreased,while40S and 60S ribosome were increased.6)After using attrm5a genetic complementarity,the mutant's phenotype was restored and the contents of m~1G and m~1I were increased to the same level of the wild type.7)Proteomic data indicated that in attrm5a mutant proteins which involved in photosynthesis,ribosome function and cell signaling pathway were decreased.According to the research on At Trm5b,we known that At Trm5b had a long N terminal and the deletion mutant was homozygously lethal.However,in vitro,At Trm5b could not catalyze the formation of m~1G at the 37 position of tRNA.In the second chapter,we introduced the function of primary cell wall related transcription factors in Arabidopsis thaliana.The synthesis of plany cell walls requires coordination on deposition of pectin,cellulose and hemicellulose polymers,this complicated process demands transcriptional regulation of gene expression by various transcription factors(TFs).Studies for NAC and MYB TFs involved in second cell wall formation are well documented,but reports for primary cell wall TFs are scarce.In this study,we used Arabidopsis thaliana as a model system to study the function of primary cell wall TFs.We selected 19 candidate TFs by Y1H.Further functional analysis was conducted on the 19 candidate genes and got the main results are as followings:1)We constructed OE vectors for 6 candidate TFs,each includes TF,TF-EAR(dominant negative),TF-VP16(dominant positive)versions,we transformed those plasmids into Col.0 and obtained transgenic plants.Upon these we found At2g44730-EAR and At MYB70-EAR might be detrimental for plant growth,why we did not obtain any positive transgenic lines.2)We ordered T-DNA mutants for 16 candidate TFs,isolated homozygous mutant out of 14 TFs,analyzed gene expression from 11 mutants.We found9 mutants where corresponding TFs were dramatically down-regulated,except for S1 and S7 mutants.We also investigated primary cell wall genes that were coexpressed with Ces As,the result suggested At DDF1?At E2F2?At DF1?At2g44730?At ARF9?At Rap2.6L?At HB23 and At ILR3 act as activators,whereas At1g61730 and At GBF2 act as repressors for primary cell wall genes.3)Compared to WT,S3,S7,S8,S15,S17,S22 and S23mutants showed shorter root length on plate under light conditions,S1,S3,S7 and S15showed shorter hypocotyl length under dark conditions.4)S8 mutants had less mucilage adherence on seed coat,radial fibers were shorter than WT,pectin staining suggested less pectin esterification,suggesting mutation in At DF1(S8)resulted in structural change of pectin and cellulose,which also affected seed germination under low water potential conditions.