Construction Of The Vector Containing IPT Gene Which Directed By GmSARK Promoter And Genetic Transformation Into Plants

The plant can not be a normal physiological and biochemical processes with Stress conditions. Plants died will reduce the productions.As a stress condition,drought has been extensive research and discussion by many researchers. With drought conditions, plants will die in the early age,it will result the reducing of the productions. So if we delay the senescence of the plants and make sure it will grow normally with stress condition through biotechnological methods.It’s not only increase the resistance capacity of the plants,but also increase the crop yields and land utilization.Therefore we can use the means through delaying senescence of the plants. Generally, under stress conditions, the content and activity of the plants’hormone often changed, and these changes will affect the physiological processes of the plants. As a regulatory factor, Cytokinin (Cytokinin, CTK) plays a very important role in growth and development process of plants.It can promote the expansion of cells, induction the differentiation of the shoots、roots and chloroplasts, remove the apical dominance, dealy leaf senescence, stimulate the development of seeds and fruits, and enhance the resistance of plants.The isopentenyl transferase gene(IPT gene)is an enzyme that catalyzes the rate-limiting step in cytokinin synthesis. It will stimulate the synthesis of cytokinin, regulat plant growth and development, plants with IPT gene will delay senescence, increase the yields. Senescence associated receptor protein Kinase(SARK) which is a gene encoding a maturation/senescence-dependent receptor protein kinase whose promoter plays an important role in senescence signaling pathway of plant development, and it can control the IPT expression modestly. In this study,we isolated the IPT gene and GmSARK promoter based on the previous research. The recombinant expression vector was transformed into soybean by means of transfering with Agrobacterium tumefaciens.1. The fragments of IPT gene had been amplified through PCR using the designed primers and the plasmid which contain IPT gene.2. The fragments of GmSARK promoter had been amplified through PCR using the designed primers and the total DNA from williams82soybean.3. The plant binary vector p3301-GmSARK-IPT was constructed by substitution of GmSARK promoter and IPT gene for35S promoter. 4. Soybean species named "JiYu72" was used in this study. The IPT genes were transferred into it by Agrobacterium-mediated method. Finally, we got32transgenic plantlets separately. The result of PCR analysis showed that10transgenic plants were positive lines.5. The Southern blot of transgenic soybean plants indicate that the target gene was transcribed into4transgenic lines.6. The plant expression vector of p3301-GmSARK-IPT was constructed and transformed into Arabidopsis by means of Floral dip with Agrobacterium tumefaciens.Transgenic plants were obtained by PPT herbicides selection and8positive transformants were screened followed by a further PCR identification. The T1transgenic plant lines were treated by drought and dark treatments and the transcription of foreign genes in transformants was confirmed by semi-quantitive RT-PCR.DNA PCR results prove that the target gene is integrated into the genome of Arabidopsis.RT-PCR results indicate that the target gene is transcribed in the transgenic line.Heterologous expressing the IPT gene directed by GmSARK promoter in transgenic plants was first used in this study,and we integrated the the target gene into the genome of transgenic plants successfully.Thus, this research paved a strong foundation for the research of the stress resistance of GmSARK::IPT fusion gene.