Identification,cloning And Functional Analysis Of BBM Gene In Betula Luminifera

BBM（BABY BOOM）,as an important member of plant AP2/ERF transcription factor famlily,plays critical roles in cell proliferation,somatic embryogenesis and other biological processes in plant.Betula luminifera is a deciduous tree of the genus Betula of the Betula family.It has become an ideal material for forest tree genetic research due to its excellent quality and short juvenile phase.In this study,based on the whole genome and transcriptome data of B.luminifera,the AP2/ERF gene family was first identified,and Bl BBM was then cloned.In order to further explore the function of Bl BBM,its expression pattern during seed development and germination,and under 2,4-D treatment was analyzed.Through Agrobacterium-mediated method,genetic transformation was performed for Bl BBM by using hybrid poplar 84K and B.luminifera..All these works provide important basis for further reveal the roles of Bl BBM in callus induction and adventious root formation.The main findings are as follows:1.In the B.luminifera genome,51 AP2/ERF genes were identified,which can be classified into four subfamilies including AP2,ERF,DREB and RAV subfamily.And,17 genes belong to AP2 subfamily,2 genes belong to RAV subfamily,DREB and ERF subfamily contains 21 and 11genes.Expression analysis in different tissue/organs showed that expressions of most B.luminifera AP2/ERF genes have strong tissue-specific patterns,five of them are only expressed in a single tissue or organ.For example,Bl BBM is only strongly expressed in roots.It means that the function of these genes may be related to the development of specific tissues or organs.2.Based on the identification results of the AP2/ERF gene family,our study obtained the BBM homologous gene Bl BBM from B.luminifera through the analysis of gene structure and conservative motifs.Bl BBM gene has similar gene structure and motif distribution to the BBM homologous genes of other species,suggesting that it may have similar functions to its homologs genes in other species.Expression pattern analysis showed that the expression level of Bl BBM gene was higher in the early and the middle stages of seed development of Betula luminifera,and gradually decreased in the middle and late stages of development,showing a trend of first rising and then falling,which was similar to the expression patterns of other BBM genes.This suggests that Bl BBM gene may be involved in the early development of plant embryos.In the process of seed germination,the expression of Bl BBM gene also showed a pattern of rising first and then falling,which may be related to its regulatory function on root development.In the process of2,4-D inducing callus formation,the expression of Bl BBM was up-regulated,and showed a dosage dependent pattern,among which 4 mg·L^-1 2,4-D has a best induction effect on the formation of callus and the expression of Bl BBM.These results indicate that auxin may be an important mediator for Bl BBM to perform the biological functions.3.Using hybrid poplar 84K and Betula luminifera as materials,the biological function of Bl BBM was studied by Agrobacterium-mediated transformation.The results showed that:（1）Transient expression of Bl BBM gene in poplar can induce adventitious roots in leaves without hormones.Compared with the control,poplar leaves that transiently expressed Bl BBM have a higher frequency,a larger number in adventitious roots.Further expression analysis also confirmed that the occurrence of adventitious roots is related to the high level of Bl BBM gene expression.Overexpression of Bl BBM gene on culture medium containing phytohormones could promote the formation of callus in poplar leaves.Compared with the control,the callus formation on the leaves of poplar overexpressing Bl BBM takes place earlier,the callus induction rate is higher,and the average number of calluses formed per leaf is larger.The callus is larger in size,more densely distributed,and better in vigor;（3）Bl BBM gene can also promote the production of more adventitious roots in the stems of the seedlings of B.luminifera.In the two different treatment methods of puncture and injection,Bl BBM gene can both promote formation of adventitious roots.The phenotype of adventitious roots is also directly related to the expression level of Bl BBM gene.（4）Overexpression of Bl BBM gene can also promote the formation of callus on the leaves of B.luminifera,which has similar results to that of poplar leaves.The above results indicate that the Bl BBM gene of B.luminifera has the dual function of regulating callus formation and adventitious root formation,and may interplay with auxin pathway.