Phylogenetic Analysis Of RBCS Gene Family And Funcational Studies Of Promoter Of TaRBCS11 In Wheat

Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)is a key enzyme in eukaryote photosynthesis,and its small subunits are encoded by a multigene family,which highly expressed in most plant species and involved in stress responses.In this study,hexaploid wheat Triticum aestivum cv.Chinese Spring were slected as research materials.The members of RBCS genes were identified in the current sequenced Triticum aestivum genome and were clustered into four subgroups in phylogenetic tree.From the perspective of comparative genomics we explored the origin,evolution,classification and the possible ways of regulation of RBCS genes in wheat.RBCS genes from different subfamilies were also cloned and analyzed.Combining with q RT-PCR and chip-technology,induced expression profiles and tissues expression profiles of Ta RBCS genes were analysised.Finally,the funcation of promoter of TaRBCS11 gene were studied.Main results are as follows:1.20 RBCS family genes were identified in the current sequenced Triticum aestivum genome,and 31 RBCSs were identified from other 2 kinds of monocots and 4 kinds of dicots.Phylogenetic results showed that Ta RBCS genes are clustered into four subgroups(subgroups A,B1,B2 and B3).Homology analysis suggested that wheat has a close genetic relationship with Aegilops tauschii and Triticum urartu.The upstream regions of one subfamily genes have some similarity.Many light-responsive motifs were observed at the relatively the same position in the proximal region of these promoters.The stress-regulated elements were also discovered in these promoters.These results demonstrated that the expression of Ta RBCSs could be induced by regulating stress-regulated elements of their promoters.2.Ta RBCS2(The members of Sub B1),TaRBCS3(The members of Sub B3),Ta RBCS7(The members of Sub B2)and TaRBCS11(The members of Sub A)were cloned and compared.All cloned sequences had more than 99% similarity when compared with the predicted sequences in the wheat genome.The 4 Ta RBCSs were 90.36~94.49% similar to each other,which suggested that wheat RBCS gene family were highly conserved during evolution.Moreover,three-dimensional structure comparison showed that all RBCS families share a similar structure with variations.This ensures the correct assembly of Rubisco holoenzymes and effect of catalytic activity.3.Induced expression profiles of Ta RBCS genes showed that Ta RBCSs were obviously response to drought,salt and dark stresses in wheat by decreasing their expression levels.Subgroups A and B2 were significantly down-regulated after 48 h of drought stress,while expression of subgroups A,B1,B2 and B3 exhibited up-regulated to some extent after ABA treatment.These results indicated that wheat RBCS gene families participated in abiotic stress response.Besides,tissues expression profiles indicated that Ta RBCSs transcripts were constitutively expressed in leaves,flowers,and roots of wheat and the transcription level of Ta RBCSs in leaves and flowers were higher than that of roots.Notably,expression of Ta RBCSs increased in early and then decreased in inflorescence.4.A 1839 bp upstream sequence of TaRBCS11 gene was isolated from the genomic DNA of wheat.The sequence was submitted to the Plant CARE database and the analysis result revealed that the sequence contained large numbers of light-related cis-elements,and dehydration,salt and ABA response-related cis-elements.Meanwhile,quantitative analysis of GUS activity showed that GUS activities decreased gradually as the length of Ta RBCS promoters decreased.Assay of tobacco leaves transformed the complete promoter in the fusion construct indicated that P1656 was negatively regulated by salt,ABA,dark,and especially under drought.The 5‘ deletion analysis of Ta RBCS promoter confirmed that a region between-804 and-474 positions included the drought and salt-response region,and that ABA-response elements might be located in the region between-1597 and-1198.