Cloning And Functional Study Of D2-Type Cyclin Genes Pto CYCD2 In '741 Poplar'

Plant development is highly dependent on the precise regulation of cell division and proliferation,and the dynamic balance of cell division and proliferation is closely related to the regulation of the cell cycle.D-type cyclin(CYCD)is considered as the link between cell cycle regulation and plant growth development.CYCD regulates the G1/S phase transition of the cell cycle,which combines with Cyclin-Dependent Kinase(CDK)to form CYCD/CDK complex.The activated CYCD/CDK complex regulates the cell cycle through phosphorylation of downstream cell cycle response factors,which in turn affects plant growth and development.The current research on plant D2-type Cyclin genes mainly focus on herbaceous model plants,while no reports had been reported in woody plants.Taking the hybrid of(Populus alba L)× [(Populus davidiana Dode)+(Populus simonii Carr)] as the female parent and(Populus tomentosa Carr)as the male parent,‘ 741 Poplar ' is white poplar hybrid clones selected by two sexual crosses,which have the characteristics of fast growth,stable material,wide adaptability and beautiful crown shape.In this study,‘ 741 Poplar ' was used as experimental material,and two D2-type cyclin genes were identified: PtoCYCD2;1 and PtoCYCD2;2.using the methods of the search and align sequence,phylogenetic analysis,expression pattern analysis,protein localization analysis and genetic transformation,we hope to analyze on the biological function and studied the related signaling pathway of Cyclin regulate on cell division in poplar.Obtained results were as follows:(1)PtoCYCD2;1 and PtoCYCD2;2 contain an open reading frame of 1062 bp and 1041 bp,encoding proteins consist of 353 and 346 amino acids,respectively.They were considered as members of D2-type Cyclin gene superfamily by analysis of protein domain,system evolution analysis,multiple sequence alignment,PEST prediction and so on.Sequence analysis showed that PtoCYCD2;1 gene contains 6 exons and PtoCYCD2;2 gene contains 7 exons,and there are more single amino acid mutations in the Cyclin?C domain.Three-dimensional structural simulations show that the two proteins were different in the loop region.This indicated that PtoCYCD2;1 and PtoCYCD2;2 genes may have functional divergence.(2)Tissue expression patterns analysis showed that PtoCYCD2;1 and PtoCYCD2;2 genes were expressed in the root,stem,leaf,petiole,xylem,and bark.Among these six tissues,PtoCYCD2;1 and PtoCYCD2;2 genes showed the highest expression level in leaf tissue.The PtoCYCD2;1:GFP and PtoCYCD2;2:GFP fusion proteins were construct and transiently express in tobacco epidermal cells.GFP fluorescence was detected exclusively in the nucleus,indicating that PtoCYCD2;1 and PtoCYCD2;2 are nuclear-localized protein,performing biological functions in the nucleus.(3)Compared with the wild-type poplar(WT),‘ 741 Poplar ' overexpressing PtoCYCD2;1 and PtoCYCD2;2 genes showed reduced plant height,reduced stem diameter,and curled leaf in the direction of the far axis.Scanning Electron Microscope(SEM)analysis showed that,in the same surface area,the upper epidermal cells of transgenic poplar leaves became smaller in the average area,increased in number and irregular in shape compared with WT.Resin section showed that,compared with WT,the cells of the palisade tissue became shorter,the intercellular space of the sponge tissue was loose,and the dorsal and ventral development of the leaves changed in the transgenic poplar,and the xylem showed a different degree of reduction.Quantitative real time polymerase chain reaction(q RT-PCR)showed that,overexpressing PtoCYCD2;1 and PtoCYCD2;2 genes in ‘ 741 Poplar ',the expression levels of cyclin-dependent kinase genes CDKA;1,CDKB1;1 and CDKB2;1 were significantly up-regulated,while the expression levels of retinoblastoma-related protein1(RBR1)gene and cyclin-dependent kinase inhibitor(KRP)genes were significantly downregulated.(4)We determined the morphology of ‘ 741 Poplar ' overexpressing PtoCYCD2;1 and PtoCYCD2;2 genes.Taken together,PtoCYCD2;1 and PtoCYCD2;2 genes had evolved distinct difference in the regulation of plant height,stem diameter,xylem and some cell cycle regulatory protein genes(KRP5).This indicated that PtoCYCD2;1 and PtoCYCD2;2 genes have evolved functional differences in the growth and development of ‘ 741 Poplar '.Comprehensive the analysis of bioinformatics,tissue-specific expression,protein localization and the effect of overexpression on the growth and gene expression of poplar,the paper delved into the regulation process of PtoCYCD2;1 and PtoCYCD2;2 genes participate in the plant cell cycle,through promoting cell division affects the growth and development of poplar.Due to the difference in gene structure and genetic transformation characteristics,there existed functional differences between the two D2-type Cyclin genes.This study provided a theoretical basis for further understanding the function of CYCD2 gene in woody plants,and provided new data for revealing the cell cycle regulation mechanism of poplar.