Transformation Of Osmotic Stress Correlative Genes Into Tobacco And Study The Function Of The Genes

Drought,high salt and low temperature are three common stress conditions that adversely affect plant growth and crop production.And they are the important problems which affect the agriculture yield of our country even the world.Therefore how to enhance the crops resist drought,high salt and low temperature ability is the question which one urgently awaits to solve.With the rapid development of the gene engineering technique,some functional genes have been transferred to plants.But the improving is limited.At present the regulatory genes has been the research emphases.If the regulatory genes can be transferred to plants,we maybe gain the plants which had excellence characters.It is the important precondition condition to cloning and identification the function of the regulatory genes. In this study,we used tobacco to analysis the function of DREB2A,OsCDPK7 and OsMAPK4 genes.We constructed several plant expression vectors.And the tobacco was transformed by Agrobacterium Tumefaciens.Transgene plants were obtained,and the generation of T1 was inspected by resistance identification.The function of the targeted genes was primarily analyzed.The main results was summarized as follows: 1. Construction of plant expression vectors There were four plant expression vectors were constructed,they were pBM29A,pBME12,pBC729A and pBC7E12.The plant expression vector pBM29A contained the OsMAPK4 gene,which was expressed in dicotyledonous plant and induced by osmotic stress conduction.The plant expression vector pBME12 contained the OsMAPK4 gene,which was constitutively expressed in dicotyledonous plant.The plant expression vector pBC729A contained the OsCDPK7 gene,which was expressed in dicotyledonous plant and induced by osmotic stress conduction.The plant expression vector pBC7E12 contained the OsCDPK7 gene,which was constitutively expressed in dicotyledonous plant. 2. Optimization of tobacco genetic transformation system 1 Identified the concentration of Km on stage of adventitious buds differentiation:Km concentration is 20 mg/L.2 The optimization of antibiotics is Amoxicillin and Clavulanate Potassiium(2:1)andthe concentration is 100 mg/L. 3. Transformation mediated by Agrobacterium Tumefaciens DREB2A gene,OsMAPK4 gene and OsCDPK7 gene,which were respectively regulated by promoter E12 and rd29A,were transferred into tobacco "Longjiang 911". And each combination all obtained the massive resistant seedlings. 4. PCR identification of resistent seedlings There were 66 PCR positive plants be obtained,which were transformed with LBA4404(pB2A29A);there were 17 PCR positive plants be obtained,which were transformed with LBA4404(pB2AE12);there were 13 PCR positive plants be obtained,which were transformed with LBA4404(pBM29A);there were 45 PCR positive plants be obtained,which were transformed with LBA4404(pBME12);there were 11 PCR positive plants be obtained,which were transformed with LBA4404(pBC729A);there were 36 PCR positive plants be obtained,which were transformed with LBA4404(pBC7E12). 5. Southern blot identification of PCR positive plants 15 PCR positive plants that were transformed with OsCDPK7 gene were inspected by Southern bolt,and 9 plants were Southern blot positive plants.There was 1 Southern blot positive plant with pBC729A,and 8 Southern blot positive plants with pBC7E12. 18 PCR positive plants that were transformed with OsMAPK4 gene were inspected by Southern bolt,and 6 plants were Southern blot positive plants.There was 2 Southern blot positive plants with pBM29A,and 4 Southern blot positive plants with pBME12. 6. Analysis of genes function The generation of T1 was inspected by resistance identification.The function of the target genes was primarily analyzed.