Cloning And Functional Identification Of The GPAT Gene Promoter From Lilium Regale Wilson

Promoters are of great value for gene function studies and research into the regulation of plant development. Constitutive expression promoter such as CaMV35S promoter, which is likely to promote the expression of exogenous gene in transgenic plants, is now being widely used in the function of plant genes. While it can drive the foreign gene is highly expressed in a host, but because of its constitutive expression characteristics, it is also in the host plant throughout the growing highly expressed during development of various periods and various organizations, thus overexpression of the gene of interest, for plant growth impediment. Tissue-specific expression promoter and inducible promoters can overcome these shortcomings, they can be expressed in a particular tissue or special induction conditions, foreign plant gene expression can be precisely controlled to avoid the target gene overexpression caused the plant adverse effects. Lily has an important value in terms of ornamental and edible medicine, but it is vulnerable to adverse environment (such as cold, drought, disease, etc.) influence. So the study Lily cold-related gene glycerol-3-phosphate acyltransferase (GPAT) gene promoter has an important value for the stress resistance molecular breeding of lily and other plants.In this study, cold variety regale as experimental material, the application of chromosome walking techniques to regale GPAT gene promoter was cloned, and its function were studied. The main results are as follows:1. According to regale GPAT gene coding sequence to design primers, to the genomic DNA of regale as a template, chromosome walking technique using two methods adapter PCR and FPNI-PCR PCR amplification, and ultimately was cloned 644 bp start sequences, and named it GPATp. To two methods of adapter PCR and FPNI-PCR were compared and found FPNI-PCR cloning of promoter sequences better than adapter PCR. Cis-acting elements and predicted sites PlantCARE and NewPLACE to 644 bp promoter sequences were analyzed by the promoter, the promoter sequence was found except TATABOX, CAATBOX core element comprising, there are many other important cis-acting elements, and speculated that the promoter is not only a tissue-specific promoter but also an inducible promoter.2. The three deletion construct promoter vector:PCR amplified through three GPATp promoter deletion fragments were GPATp1 (644 bp), GPATp2 (275 bp) and GPATp3 (163 bp), and replacing each plant expression vector pCAMBIA 1304 (including GFP/GUS fusion reporter gene) 35S promoter to construct three recombinant plant expression vector GPATp1-GFP/GUS, GPATp2-GFP/GUS and GPATp3-GFP/GUS.3. The use of Agrobacterium-mediated method GPATpl-GFP/GUS, GPATp2-GFP/GUS, GPATp3-GFP/GUS and 35S-GFP/GUS vector into tobacco leaves, were GUS gene transient expression analysis, the results showed that:163 bp the GPAT promoter can drive the expression of GUS gene, its promoter does not have activity; minimal promoter 275 bp promoter weaker activity and predicted TATABOX at-222/-217 bp region, thus having activity at 230 bp about; 644 bp of promoter activity is high, so the cis-acting elements upstream of a positive regulatory role greatly increases the activity of the promoter; normal and low temperature (4℃) treatment, the promoter activity can not see significant changes, whether in response to cold-inducible promoter should be further studied stable expression.4. Transformation of Tobacco with Agrobacterium mediated leaf disc method, after screening, PCR validation has been successfully transferred to GPATpl-GFP/GUS, GPATp2-GFP/GUS and 35S-GFP/GUS vectors transformed tobacco plants. Late in-depth study to regale GPAT gene promoter function provides a good foundation.