Cloning And Functional Analysis Of BpAP2 Gene And Its Promoter

The APETALA2(AP2)family is one of the subfamilies of APETALA2/ethylene-responsive element binding protein(AP2/EREBP)family of transcription factors and is ubiquitous in plants.It is widely involved in plant growth and development,stress response and various physiological and biochemical reactions.The AP2 gene is a member of this family and is expressed in various tissues and organs of plants.The promoter regulates gene expression at the transcriptional level,and the gene-specific promoter directly regulates the expression time,expression site,and expression intensity of the downstream gene through its cis-acting element.This study cloned AP2 gene and its series of promoter fragments from Betula platyphylla Suk.The specific results are as follows:(1)Cloning of AP2 gene promoter of B.platyphylla and analysis of its transcriptional activity.The promoter sequence proBpAP2 of AP2 gene with a length of 2308 bp and the promoter fragment Pn(n = 1,2,...5,6)deleted from the 5' end of AP2 gene were cloned from the genome of Betula platyphylla Suk..The proBpAP2 and six 5' end deletion fragments of the promoter were cloned into the pBI121 plant expression vector by restriction enzyme digestion.Diffusion of B.platyphylla and Arabidopsis thaliana by Agrobacterium-mediated method,and then they were analyzed by GUS histochemical staining.The results showed that proBpAP2 and the six deletions had promoter activity in the entire A.thaliana and B.platyphylla leaves,and the activity of proBpAP2 in vegetative organs and female wings and samara of B.platyphylla,indicating that they may participate in the development of corresponding tissues and organs.(2)Sequence analysis and hormone response of proBpAP2.Bioinformatics analysis revealed that the proBpAP2 sequence has a large number of light-responsive elements and hormone-responsive elements,such as gibberellin and abscisic acid,in addition to the conserved elements commonly found in eukaryotic promoters such as TATA-box and CAAT box.According to the results of element analysis,A.thaliana transformed with proBpAP2::GUS was treated with gibberellin(GA3)and methyl jasmonate(MeJA),respectively,and the results showed that promoter activity decreased at the initial stage of MeJA treatment,and activity increased with the increase of treatment time.At the initial stage of GA3 treatment,promoter activity first increased,and gradually decreased with time.This indicates that the promoter responds to the regulation of GA3 and MeJA.(3)Validation of BpAP2 gene in A.thaliana.The BpAP2 gene was cloned and the 35S::BpAP2 overexpression vector was constructed by restriction enzyme digestion.The wild type A.thaliana(Col)was transformed with Agrobacterium-mediated floral dip method.After observation and analysis of transgenic plants,it was found that the number of rosette leaves of A.thaliana overexpressing the BpAP2 gene was greater than that of the wild type and the rosette leaf was larger than that of the wild type;the whole plant height did not change significantly compared with the wild type;it has early twitching,flowering and pod setting in transgenic plants;in addition,the main roots of the transgenic plants were longer than the wild type,the root hairs were longer than the wild type and the number was larger,and the trichomes on the surface of the leaves were different from those of the wild type.The trichomes on leaf surface were different from the tridentate branches of Col and showed a binary branching state.These results indicate that the BpAP2 gene affects the number and size of leaves,the elongation of primary root and root hairs and the number of root hairs while participates in the regulation of flowering and morphogenesis of trichomes.