Identification And Functional Analysis Of VQ Gene Family In Chinese Cabbage(Brassica Rapa L.ssp.pekinensis)

Chinese cabbage(Brassica rapa L.ssp.pekinensis),an important vegetable crop known for its high nutritional value,is widely cultivated in China.The leafy head is the main part that is eaten,whose growth and development is co-regulated by genes and environment.VQ protein family is a plant-specific transcription regulation cofactor,which plays an important role in regulation of plant growth,development and responses to various external environment stresses and it is named after the invariant valine-glutamine(VQ)dipeptide.Over the past several years,researches have found that not only VQ protein family participated in regulating the growth and development of seed,hypocotyl,flower and leaf,but also played an important role in response to the stresses of drought,salt,temperature and pathogen.At present,VQ multi-gene families had been identified in Arabidopsis thaliana,rice,soybean,grape,maize and so on,however,to our knowledge,the VQ gene family from Chinese cabbage has not been characterized.In this study,Chinese cabbage VQ motif–containing proteins were analyzed in detail and some progresses were as follows.1.The bioinformatics analysis of Chinese cabbage VQ motif–containing proteinsOn the basis of the published amino acid sequence of VQ domain,57 Chinese cabbage VQ motif–containing proteins were identified.The analyses of gene structure displayed,these 57 BrVQ genes showed that their CDS ranged from 282 bp to 1707 bp and more than 90% VQ genes had no intron.The analyses of protein characteristic showed the predicted protein lengths varied in size from 93 to 568 amino acids,the molecular weight of the BrVQ proteins ranged from 10.4 to 63.0 k Da and the theoretical isoelectric point(p I)from 4.67 to 10.53.VQ proteins only contained a highly conserved VQ domain “Fxxx VQx L/F/VTG”(where x is any residue);however,the amino acid sequences of other regions have diversity.Basing on the difference of L and G residues,these BrVQ proteins were divided into six types(LTG?FTG?VTG?LTS?LTV and YTG);Furthermore,56 BrVQs were dispersedly distributed on 10 chromosomes except for 1 BrVQ gene.Syntenic Analysis showed a total of 53 BrVQs derived from 13 blocks of seven t PCK chromosomes of the ancestor,respectively,and 41 BrVQs were detected to have counterparts on segmental duplication whereas 2 BrVQs on tandemly duplicated.Phylogenetic Tree indicated there was similar evolution process between BrVQs and AtVQs but different from Os VQs.2.The expression pattern analysis of Chinese cabbage VQ genesWe performed q RT-PCR expression analysis of 57 BrVQs in six tissues,including root,dwarf stem,old leaf,young leaf,flower and flower bud.The result are as follows,26 BrVQs showed higher expression levels in the root than other tissues,13 BrVQs were expressed more in the old leaf than other tissues,7 BrVQs were expressed mainly in the flower bud,4 BrVQs were expressed mainly in the young leaf;3 BrVQs were expressed mainly in the dwarf stem and only 1 BrVQ was expressed in the flower.Besides,some BrVQ paralogs showed similar expression patterns in different tissues,however,some exhibited different expression patterns.The expression patterns of BrVQs under different abiotic stresses and hormones were analyzed,the result displayed that 15?11?29?11 BrVQs were induced by PEG6000?Na Cl?35? and 4?,respectively,and 18?25?23 BrVQs were induced by GA3?ABA and SA,respectively.Comparison of the expression patterns of BrVQ genes and their orthologs in Arabidopsis showed that some orthologs had similar expression patterns,however,some not.The above results indicated BrVQs possibly participate in regulating pant growth,development,and resistance to different stimuli.3.The cloning and function analysis of Arabidopsis VQ3 geneArabidopsis and Chinese cabbage are Cruciferae plants,the research on AtVQs function are beneficial to better understand BrVQs function.At present,VQ3 gene from Arabidopsis and Chinese cabbage has not been reported,so we firstly studied on Arabidopsis thaliana VQ3/At1G21326 gene.The result as follows,AtVQ3 gene sequence length was 720 bp which encode a proten of 239 aa and it had no intron.The expression pattern of AtVQ3 in different tissues and under various stimuli were analyzed,AtVQ3 was expressed in root more higher than others which was similar to BrVQ3-1,however,except for ABA,AtVQ3 was induced by PEG6000?Na Cl?35??4 ? and GA3,especially under Na Cl,which was different to BrVQ3-1.The phenotypic analysis of AtVQ3 transgenetic plant showed that it inhibited plant growth and development including root and hypocotyl length,leaf size,flowering time;besides,AtVQ3 over-expression plants exhibited lower seed germination rate and worse seedling growth situation than AtVQ3 interference plants.The above results indicated AtVQ3 had negative regulation on Arabidopsis thaliana growth,development and resisetance to high salt.4.The transcriptome analysis of wide type,35S:AtVQ3-8 and AtVQ3-RNAi-3 transgenetic plantsTo more comprehensive understand the function of AtVQ3 gene on Arabidopsis growth,development and salt sensitivity,the RNA-Seq of wide type,35S:AtVQ3-8 and AtVQ3-RNAi-3 transgenetic plants were analyzed in detail.The result as follows,in the comparison of wide type and 35S:AtVQ3-8,94 differentially expressed genes(DEGs)were up-regulated and 50 DEGs were down-regulated;in the comparison of wide type and AtVQ3-RNAi-3,57 DEGs were up-regulated and 48 DEGs were down-regulated;in the comparison of AtVQ3-RNAi-3 and 35S:AtVQ3-8,134 DEGs were up-regulated and 137 DEGs were down-regulated.Futher analysis showed,in 35S:AtVQ3-8 transgenetic plant,the obviously up-regulted genes included four auxin early response SAUR genes,brassinosteroid signal pathway related genes BZS1 and RALF23,plant growth related transcription factor MIF3 and LBD38,however,some cell wall synthesis related genes EXPs and EXTs,one SAUR gene,plant flowering related genes ELF4,SOC1 and JAC1,calmodulin coding genes,drought and salt response genes GOLS2,ATNCED3 and ATCIPK6,had a notably down-regulation trend.On the contrary,above-mentioned genes displayed opposite expression trend in AtVQ3-RNAi-3 transgenetic plants.The above results indicated VQ3 gene could co-regulate plant growth,development,and salt sensitivity with multi-genes via multi-pathways.5.The cloning and function analysis of Chinese cabbage VQ3-1 geneTo understand the biology function of BrVQ3-1 gene,we constructed BrVQ3-1 over-expression vector and obtained Arabidopsis 35S: BrVQ3-1 transgenetic plants.The result as follows,BrVQ3-1 gene had similar function with AtVQ3 gene on Arabidopsis growth and developmet,however,35S:BrVQ3-1 transgenetic plants have insensitive to salt treatment.Moreover,we selected 9 genes from transcriptome data of AtVQ3 transgenetic plant,then these genes expression levels were detected by q RT-PCR in 35S:BrVQ3-1 plant,the result showed 5 genes(EXPA8,EXT3,SAUR31,BZS1 and ELF4)displayed abvious down-regulation trend in 35S:BrVQ3-1 plant,which was similar to that in 35S:AtVQ3-8 plant,and further illustrated AtVQ3 and BrVQ3-1 had similar function in regulating plant growth and development.