Expression And Functional Analysis Of PtROP And PtRab Gene Family In Poyulus

Small GTP-binding proteins as molecular switches regulate various biological processes in eukaryotic cells.All small GTP-bing proteins compose the Ras superfamily.This superfamily can be divided into five distinct families: Ras,Rab,Rho,Arf,and Ran,based on the structural and functional similarities.Compared to mammals and yeast,plants have evolved obviously different Ras superfamily: plant Ras superfamily doesn’t contain Ras family;the remarkable expansion of RabA subgroup is one of the most striking features of the plant Rab family;plant Rho family forms the plant-unique ROP family.Many reports suggest that ROP and Rab proteins are involved in plant growth and development,stress tolerance,hormone response,and secondary cell wall formation.But there is little research on roles of ROP and Rab proteins in woody plant,characterized as perennials with secondary growth.In this study,we analyzed the phylogenetic relationship,gene structure,conserved motifs,duplication of paralogous pairs,expression patterns in various tissues and different stresses conditions of poplar ROP and Rab gene families using bioinformatics methods.In the study,we identified 13 ROP genes in the Populus trichocarpa genome.Their gene structures were similar and functional motifs were conserved.Based on amino acid sequence similarity and hypervariable C-terminal domain,poplar PtROP were divided into four classes: PtROP10;PtROP8,PtROP11,and PtROP12;PtROP7,PtROP9,and PtROP13;PtROP1-6.To explore the duplication among PtROP genes,we found 4 paralogous pairs(PtROP1/PtROP4,PtROP2/PtROP6,PtROP8/PtROP11,and PtROP9/Pt ROP13)in PtROP gene family,and the PtROP paralogous pairs were estimated to occur between 8.74 to 17.30 million years ago,suggesting all the pairs were generated by whole genome duplication and underwent pure selection.We found that members of PtROP gene family had different expression patterns in various tissues and under different stress conditions.Based on the functional metwork analysis,PtROP may involve in signal transduction,tRNA modification,and cytoskeleton formation.We found 67 Rab genes in the Populus trichocarpa genome,and All of them were distributed unevenly on 18 of 19 Populus chromosomes,and no Rab gene on Chr 17.After analysis the structure of PtRab genes,we found gene structures(exon/intron)were evidently different among the eight subfamilies,but similar among members in the same subfamily.RabA subfamily had the simplest structure with only one exon.The same as mammalian and yeast,Poplar Rab proteins had four conserved motifs related to GTP binding and hydrolysis.Although a little sequence difference was existed in the four motifs among different Rab subfamilies,the basic arrangement of the four motifs was relatively conserved in all PtRab proteins.The promoters of PtRab genes were searched in the PlantCARE database and many cis-elements involved in hormone and stress response were found.This implied PtRab might participated in biological processes in response to hormones and stresses.We found 27 paralogous pairs in PtRab gene family and these pairs were estimated to occurr between 9.96 to 28.14 million years ago,thus all the pairs were also generated by whole genome duplication and underwent pure selection.We analyzed the expression patterns of PtRab genes in different tissues and different conditions using microarray data.In various tissues,most of the members in PtRab family were highly expressed in phloem and xylem,especially the members in RabC subfamily.Comparatively,most PtRabs were relatively low expressed in reproductive organs including male and female catkins.To validate the expression patterns of PtRab genes and the divergence between paralogous pairs,the expression of 27 PtRab paralogous pairs were analyzed based on our RNA-seq data.Only four pairs(PtRabA1i/PtRabA1 j,PtRabA2a/PtRabA2 b,PtRabC2a/PtRabC2 d,and PtRabF2b/PtRabF2c)showed similar expression patterns across 6 different tissues.There were six paralogous pairs(PtRabA1d/PtRabA1 h,PtRabA2d/PtRabA2 e,PtRabA3a/PtRabA3 b,PtRabA4b/PtRabA4 d,PtRabE1a/PtRabE1 c,and PtRabG3e/PtRabG3g)showed significant differences in their mRNA abundance between two genes in each pair.PtRab genes were not sensitive to drought stress based on the results of gene chip data and semi-quantitative PCR.However,RabE1 b was highly expressed in response to salt stress both at early(2 h)and late stage(12 h)of treatment.Over-expression PtRabE1 bQL enhanced the salt stress tolerance and adventitious roots formation of transgenic poplars under stress.