Isolation And Functional Analysis Of The RbcS Promoter From Glycine Max

Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco) in all higher plants is the key enzyme of photosynthesis and its content can account for 50% of total soluble protein in the chloroplast. It is structurally a hetero-16-mer, composed of eight small subunits and eight large subunits. The family rbcS genes can encode the small subunits of Rubisco and its expression is regulated by light, being leaf-specific. There are evidences that rbcS promoter is a high-performance expressive regulatory element. This study is about rbcS geen SRS4 promoter from Glycine max. On the condition that light-induced promoter is applied to genetic engineering and it will be very important to the culture of transgenic crop in the future.In this research, we cloned the rbcS gene SRS4 promoter and studied its function by Agrobacterium-mediated system. The main results were as follows:1.867bp of 5'flanking fragment from the rbcS gene SRS4 promoter of soybean were obtained by TAIL-PCR. Then, we gained 1540bp of rbcS gene SRS4 promoter by PCR. After analyzing the sequence, we found there were some basal promoter elements besides several light-induced items and other stress-induced ones.2. The plant expression vector with rbcS gene SRS4 promoter driving report gene gus was constructed successfully, named pCAMBIA-GrbcSP. pCAMBIA-GrbcSP and pCAMBIA1301 vectors were transformed into Agrobacterium tumefaciens LBA4404 by triparental hybrid method.3. Using tobacco as experimental material, we focused on the various factors that influence leaf-disc method by Agrobacterium mediation to setup a genetic transformation system, which is not only stabile, but also high-performance. Through the optimized system, we obtained several tobacco plants that have the propriety of Kan resistant.4. The tobacco resistant to Kan was analyzed by PCR and GUS staining, indicating that the special exogenous fragment had been integrated into the genome of tobacco and could drive gus gene to expression. From the result of GUS fluorescent quantitative analysis, rbcS gene SRS4 promoter from soybean can make the expression of gus gene in the transgenic tobacco being leaf-specific, besides, the quantity of the expression is consistent with 35S promoter.5. The pCAMBIA-GrbcSP transgenic plant was cultured 7d in treatment of dark, then raised under light, expression level of gus gene was increased with the length of illumination in the young leaves. So the promoter of soybean rbcS gene SRS4 was a light-induced promoter.