Cloning Of Arabidopsis CBF4Promoter And Its Activity Detection

Drought is restricting the development of world agriculture, is the world’s common agricultural environmental problems.Now, the world of arid and half arid area covers an area of approximately1/3of the total area of the world. Our country is agricultural big country, but the arid and semi-arid area has reached or even exceeded the area of our country land more than half.Drought severely limits crop normal growth and development and yield of crop.In our country agriculture output statistics found that year after year, drought on crop yield losses sustained in abiotic stress in the first, second only to abiotic stress in plant diseases and insect pests of crop loss caused by.The drought caused crop yield, fruit quality drops, more serious harm to the agricultural production of our country.So for the drought stress research has more and more get of broad agriculture scientists attention.With the technology of gene engineering in agriculture in the widely cited, drought stress genes in plant stress resistance breeding research on deepening, for stress inducible promoter research has gradually become a plant molecular genetics orientation improve crop quality research. A variety of different types, different uses of the promoter to find, for the use of gene engineering method of directional regulation of expression of gene with a wide selection of space expansion. Selection of appropriate promoter for transferred target genes expression and regulation is very important, now commonly used promoter as a constitutive promoter, the regulation of gene expression has no time and space, resulting in a plant resource waste, and inducible promoter from a molecular perspective makes up for this shortcoming, so that the target gene only in specific induction of factors began to express.In this study, Arabidopsis thaliana drought stress inducible transcription factor CBF4promoter sequence is studied, compare the whole sequence and deletion sequences under drought stress and drought stress conditions on the downstream reporter gene expression intensity and expression characteristics, analysis of the functional element, discuss its under drought conditions on reporter gene expression regulation mechanism.From gene engineering considerations, to drought stress type promoter element analysis and function identification, study stress promoter of components and component interactions, and confirmed the promoter sequences in the core region, for the use of genetic engineering technology in improving the quality of crop, increase crop yield, improve crop resistance, looking for new high promoter laid the foundation.The main results of this study are as follows: 1.In the Arabidopsis thaliana genome as a template, were cloned from CBF4transcription factor of the upstream sequence of1260bp, named4A, and this sequence as the basis, through the5’series of deletion method to obtain4A sequence deletion sequences of4B565bp and4C470bp fragment, and4A,4B,4C three fragments of PMD18-T respectively with the carrier connection was constructed, containing4A,4B,4C three cloning vector pMD-4A, pMD-4B, pMD-4C.2.By sequencing the4A,4B,4C, the results showed that3sequences are containing inducible promoter functional components.3. The vectors of p1301-4A, p1301-4B, p1301-4C, p1301-4A-GFP, p1301-4B-GFP and p1301-4C-GFP were transformed by Agrobacterium EHA105, comprising these vectors Agrobacterium engineering bacteria, by Agrobacterium mediated transformation of tobacco leaf disc transformation method, get the transgenic plants.4The transgenic tobacco with4A,4B,4C for the purpose of sequence, as detected by PCR and Southern hybridization for molecular biological identification, to determine the integration of exogenous DNA copy number, the results show that, in a single copy gene integrated into the genome of tobacco.5.The transgenic tobacco GUS staining and GFP fluorescence observation, determine the4A,4B,4C sequence with start function, the results showed that4A,4B,4C sequence can start to follow GUS and GFP gene, with the promoter function.6.4A,4B,4C start-up sequence of GUS and GFP gene protein in quantitative analysis, in which GUS protein by spectrophotometry, GFP protein by using the ELISA analysis, the results show that,4C sequence has the strongest start capability.