Beet Bvm14-madsbox Gene Promoter Of The Cloning And Transient Expression Analysis

Apomictic monosomic addition line M14 was obtained from the hybridization and intercross of Beta vulgaris L. and Beta corolliflora Zoss. M14 with the No.9 chromosome of Beta corolliflora Zoss.(VV+1C,2n=19)was found having a chromosome transmission frequency of 96.5%. It was proved that M14, having the characteristic of facultative apomixis served as ideal materials for cloning apomixes genes.In previous research, full-length cDNA sequences of BvM14-MADSbox gene was obtained by SSH and RACE. Bioinformatics analysis and constitutive expression in tobacco of BvM14-MADSbox gene indicated that it was from MADS-box genes family and it played an important role in differentiation and development of floral organ as well as in florescent regulation.As MADS-box gene played an important role in regulation during the course of growth, and in different developing phases, the gene had different space-time expressive patterns. If 35S was used as the promoter to research the function of BvM14-MADSbox gene, it may lead to some limitations so that 35S can't regulate gene expression properly. So in order to research the function of MADS-box gene further, cloning its specific promoter had a significant meaning. It would be beneficial to analysis the regulation mechanism of BvM14-MADSbox gene expression and utilize its specific promoter to regulate gene expression in transgenic plant so that it could break the limitation of constitutive expression.In the research, the 1 870bp sequence before initiative codon(ATG) for BvM14-MADSbox gene was obtained using Chromosome Walking from genome DNA of M14. The transcription start site, which localized at 152bp upstream of ATG, was predicted using http://www.fruitfly.org/seq_tools/promoter.html. The functional elements were analyzed by PlantCARE http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/. The result showed that the sequence contained the basic cis-regulatory elements: TATA-box and CAAT-box, besides, some other important cis-regulatory elements, such as ARE,ABRE,ERE,some relation to light elements and some cold-resistant dry-resistant elements are also found using the bioinformatics analysis.The BvM14-MADSbox 5'regulation sequence was deleted into four fragments by PCR, the fragments were called p1700, p1200, p700 and p300 individually, which replaced the CaMV35S promoter of pBI121 to construct the plant deletion expression vectors. The four vectors were pBI-p1700,pBI-p1200,pBI-p700 and pBI-p300 respectively.The deletion vectors were transferred into Agrobacterium tumefaciens EHA105 to infect leaf disc of tobacco. The result of transient expression of GUS gene indicted that three of the sequences had the function to drive the gene expression, the three sequences were p1700, p1200and p700. Besides, p1700 sequence had the strongest activity.The research result showed that the promoter sequence of BvM14-MADSbox gene was obtained by Chromosome Walking successfully.