| Grain size and grain weight are important yield components in common wheat(Triticum aestivum).Identification of key genes involved in plant regeneration is of great significance for establishment of efficient regeneration and genetic transformation system in wheat.The BABAY BOOM(BBM)transcription factor has been proven as a key regulator of plant cell totipotency.In this study,we cloned the BBM homologs from wheat,and studied the biological function of wheat.The main results are as follows:We use the amino acid sequence of Arabisopsis thaliana BBM as the query sequence to search the wheat database in the Ensembl Plants database.The TaBBM genes were found located on chromosome 3,respectively named TaBBM-3A 、TaBBM-3B and TaBBM-3D.Using Cultivar Chinese spring wheat embryos as material,specific primers were designed and three TaBBM genes cloned by RT-PCR technology.Their CDS sequences contain 2109 bp,2250 bp and 2247 bp nucleotide residues,respectively.Phylogenetic tree analysis indicated that TaBBMs and Os BBM3 were in the same branch.The TaBBM protein was located in the nucleus showed by subcellular localization experiments.The result of q RT-PCR analysis revealed that TaBBM genes were expressed in the young grain with highest level in the embryo of Chinese spring wheat.To investigate the biological function of TaBBM genes in wheat,a series expression vectors including TaBBM-3A-OX,TaBBM-3B-OX,TaBBM-3B-DOWN,p ER8-TaBBM-3A and ami R-TaBBMs were constructed.Using young embryos of Fielder as receptor materials,Agrobacterium tumefaciens mediated genetic transformation was carried out.So far,T4 generation positive plants of TaBBM-3A-OX,TaBBM-3B-OX,TaBBM-3B-DOWN,p ER8-TaBBM-3A and ami R-TaBBMs have been obtained.The expression level of TaBBMs was up-regulated in overexpression plants,and down-regulated in TaBBM-3B-DOWN and ami R-TaBBMs transgenic plants examined by q RT-PCR results.Plant regeneration assay indicated that overexpression of TaBBM-3A and TaBBM-3B significantly enhanced the plant regeneration frequency and shoot number of wheat immature embryo explants in comparison to that of wild type.Morphological observation showed that TaBBM-3A overexpression promoted embryonic cell formation and maintenance.It was demonstrated that overexpression of BBM genes of winter rape(Brassica napus)and A.thaliana induced somatic embryogenesis.Our results revealed that the function of TaBBMs is different from that of Bn BBM and At BBM in plant regenration.Compared with the Fielder plant,TaBBM-3B-DOWN and ami R-TaBBMs transgenic plants showed larger grain size(GS)with significantly increased grain width,grain length and grain thickness as well as grain weight(GW).However,both the GS and GW of TaBBM-3A-OX and TaBBM-3B-OX transgenic plants were decreased.The results suggested that TaBBMs are negative regulation factors of GS and GW in wheat.Morphological and histological observation revealed that decreased TaBBMs expression level facilitated cell expansion of the grain.In order to address the mechanism of TaBBMs in GS regulation,young grains of12 days after flowering of both the Fielder and ami R-TaBBMs transgenic plants were sampled for RNA-seq.The result showed that the expression level of 1435 genes was up-regulated and 8507 genes down-regulated in the transgenic plants.The reliability of transcriptome sequencing data was verified by q RT-PCR method.The GO analysis of the differentially expressed genes showed that the molecular function of the differentially expressed genes in transgenic plants was significantly enriched in metabolic process and cellular process.KEGG analysis showed that the differentially expressed genes in the transgenic plants were highly enriched in signal transduction,biosynthesis of secondary metabolites,amino acid metabolism,lipid metabolism and carbon metabolism functions.Functional classification of down-regulated genes in transgenic plants revealed that the differentially expressed genes were highly enriched in biosynthesis and metabolism.The up-regulated genes in transgenic plants were significantly enriched in pathways including cellular process and signal transduction.The q RT-PCR verification results showed that the expression level of two Expansin(EXP)genes was up-regulated in ami R-TaBBMs plants,suggesting that TaBBM genes might indirectly regulate Ta EXPs to mediate GS and GW in wheat.The results provided important information for understanding the mechanisms of TaBBMs in regulation of wheat GS and GW.Taken together,suppression of TaBBMs expression caused increased GS and GW,indicating that TaBBMs are negative regulation factors of these important traits.In addition,TaBBMs overexpression can promote plant regeneration.The results provided important ideas and clues for wheat high yield breeding and molecular design breeding as well as efficient plant generation by use of gene editing technology to artificially manipulate the expression level of TaBBMs in wheat. |
