Functional Analysis Of Cotton PsbP Promoter

Gene expression and regulation is an important part of plant genetic engineering, moreover, the promoter play an central role of transcriptional regulation, which largely determines gene expression in time, space and intensity. Currently, constitutive promoter is widely used in commercial transgenic crops, and foreign gene driven by them can express constantly and stably throughout all tissues of plant at every developmental stage, at a high metabolic expense to host plant. Meanwhile, excessively expression of foreign gene may also have some potential harmful effects. Thus, utilization of specific promoter, replacing the constitutive promoter, in order to drive foreign gene expression in certain time, place and intensity, is a significant part of plant genetic engineering.PsbP is a peripheral protein of oxygen-evolving complex in the chloroplast photosystem II , which is a indispensable component to ensure the functional integrity of PS II,and whose essential function is maintaining Ca2 + concentration in the water splitting process. The full length cDNA of GhPsbP gene and its promoter were cloned form leaves of cotton, meanwhile, six promoter deletion mutants fused with GUS gene were designed and seven plant expression vectors were constructed and transformed into tobacco NC89. The results of histochemical localization and expression analysis showed that the promoter of PsbP gene specifically express in green tissue of plant, and its structure and function were determined. The core promoter of PsbP gene was localized between -1 and -97bp, which can drive GUS gene specific expression in leaves of tobacco. Sequence of -677 to -1084bp control gene specifically expression in stem and mature leaves, including a high frequency of 5'-TTTTA-3 'and 5'-TATAAAT-3' motif which is a potential positive regulatory elements and may be related to its function. -1055~-2077bp segment distributed enhancer elements, whose efficiency is five times higher than the core promoter. The expression of GUS drived by full-length promoter is higher than all of mutants.A binary plant expression vector, pBI121-PE, carrying the CP4 5-enolpyruvylshikimate- -3-phosphate synthase (EPSPS) drived by cotton PsbP promoter has been constructed. Tobacco leaves were transformed with Agrobacterium tumefaciens LBA4404 harboring the pBI-PE121 plasmid, The results of PCR detection showed that 32 transformed tobacco plants were positive. Semi-quantitative and real-time PCR analysis of EPSP synthase expression showed that,the CP4 epsps drivened by PsbP promoter expressspecificly in green tissue of transgenic tobacco which showed higher resistant to the glyphosate of working concentration. it provide the theoretical basis and experimental evidence for resisting glyphosate of tansgenic engineering in choosing proper gene and expression elements,but also for the application of green tissue specific promoter.