| Calcineurin B-like protein（CBL）is a kind of calcium binding protein,which plays an important role in plant stress signal transduction.CBL protein and its intera-cting protein kinase CIPK（CBL interacting protein kinase）are the basic elements of CBL/CIPK signal system.This signal system plays a key role in abiotic stress such as high salt,drought and low temperature.As an essential second messenger in plants,calcium ion transmits stress signals through the spatiotemporal changes of its concent-ration in plants under stress.CBL protein can sense stress-induced Ca2+signals and bind to Ca2+,thereby activating its downstream CBL-interacting protein kinases（CIPKs）and forming CBL-CIPK complexes,which regulate transcription factors,stress response genes as well as the resistance of different cellular levels and tissue parts,so as to help plants respond to the impact of stress.Promoters driving gene expression are important elements in the regulation of gene expression at the transcriptional level.In-depth study on the structure and function of the promoters will help to understand the mechanism of genes in plant growth and development as well as plant defense under stress,thus providing a basis for targeted modification of plant resistance.In this study,full-length GmCBL4 gene promoter（CblP1）and its 5’deletion fragments with different lengths were cloned from soybean,and plant expression vectors were constructed to transform tobacco,which laid a foundation for the analysis of promoter function.Additionally,the GmCBL10 gene of soybean was cloned and tobacco was transformed,which laid a foundation for the functional analysis of this gene and the cultivation of resistant soybean varieties using transgenic technology.The main results of this study are as follows.1.The primers were designed according to the upstream sequence of the GmCBL4gene of soybean in the plant genome database Phytozome,and the promoter sequence of the GmCBL4 gene was cloned from soybean,with the sequence length of 1,876 bp,named CblP1.Online promoter analysis software PLACE and PlantCARE promoter prediction tools revealed that this sequence contained conservative domains TATA-box and CAAT-box,some abiotic stress-responsive cis-acting elements（drought-induction related element MBS,low temperature induction-related element LTR）,light-induction related signal transduction elements,hormone-responsive cis-acting elements,pollen development-specific activating elements,etc.The prediction results demonstrated that this promoter had the characteristics of stress induction.pCAMBIA1301-CblP1,a plant binary expression vector driven by this promoter,was constructed and transformed into tobacco plants by the Agrobacterium-mediated method.Finally,two positive transgenic tobacco plants with promoter CblP1 fused with the gus reporter gene were obtained.2.In order to explore the core sequence functional area of this promoter and further analyze the 5’-end deletion of this promoter,four 5’-end deletion fragments with different lengths were cloned,with sequence length of 1,399 bp（Cbl P2）,932 bp（CblP3）,550 bp（CblP4）and 248 bp（CblP5）,respectively.Additionally,plant binary expression vectors driven by the 5’-end deletion fragments were constructed,pCAMBIA1301-CblP2,pCAMBIA1301-CblP3,pCAMBIA1301-CblP4 and pCAMBIA1301-CblP5,respectively.Through Agrobacterium-mediated transform-ation of tobacco,two positive transgenic tobacco plants with gus reporter gene fused with different promoter deletion fragments were obtained.3.Transform the above 5 strains containing different recombinant plant expression vectors into tobacco transiently,and after GUS histochemical staining analysis,it was found that the full-length sequence Cbl P1（1876bp）and the 5’deleted sequence CblP2（1399bp）,CblP3（932bp）had the initiating activity,and Cbl P3 has the strongest initiating activity;CblP4（550bp）and CblP5（248bp）have no initiating activity.It is speculated that the core functional area of the promoter may be in the region of-932～-550.4.With cDNA for reverse transcription of soybean RNA as template,the coding region sequence of soybean GmCBL10 gene was cloned,with a total length of 1094bp,including 792bp ORF region,encoding 263 amino acids.Based on the prediction analysis of the physicochemical properties secondary structure,signal peptide,transmembrane domain and subcellular localization of the protein encoded by this gene,it was found that the protein was an acidic and hydrophilic protein,with no signal peptide but a typical EF-hand domain and a transmembrane domain,and was mainly located in cytoplasm and mitochondria.Phylogenetic analysis showed that GmCBL10had the closest relationship with the homologous protein in phaseolus vulgaris.The sequence of soybean GmCBL10 gene with homologous recombination joint was cloned.The plant expression vector pCPB-GmCBL10 was constructed by homologous recombination.The Agrobacterium EHA105 was transformed using the freeze-thaw method,and tobacco was transformed by the leaf disk method.Finally,3 positive transgenic plants were identified by PCR. |
