RNAi And 5' Deletion Promoter Vector Construction Of OsWTF1 And OsWTF2 Genes And Their Rice Transfermation

AP2/ERF transcription factors are involved in the plant abiotic stress responses and plant epicuticular wax plays improtant role in conserve water, reduce UV damage, resist to pests and other abiotic stresses. OsWTF1 and OsWTF2 are transcription factor genes involved in wax accumulation and belong to the ERF subfamily of AP2/ERF family. For clarifying the role of rice OsWTF1 and OsWTF2 genes in wax metabolic regulation and the resistance to different stresses, the RNA interference expression vectors of OsWTF1 and OsWTF2 have been constructed and transformed into rice. In addition, different 5'end deletion promoter expression vectors of these two genes have been constructed successfully. The main results are as following:1. RNAi vectors of OsWTF1 and OsWTF2 genes were constructed successfully. A fragment of 607bp was amplified using the full length cDNA of OsWTFl as templates and is used as the sense and antisense fragment of RNA interference segment. Then a 128bp fragment of rice 3D gene was amplified from rice genomic DNA as stuffer sequence connecting sense and antisense fragments. The constructed RNA interference segment of OsWTF1 was then put into pCAMBA1301M to construct the OsWTF1 RNAi expression vector p1301m-OsWTFH. Likewise, a fragment of 499bp was amplified from the OsWTF2 full length cDNA as sense and antisense fragment of RNA interference segment. Then the OsWTF2 RNAi expression vector p1301m-OsWTF2I was constructed in the same way as OsWTF1.2. The interference expression vectors of OsWTF1 and OsWTF2 genes have been transformed into rice via Agrobacterium-mediated transformation. Five and four lines of OsWTF1 and OsWTF2 RNAi transgenic plants were obtained respectively. The inhibited expression of the OsWTF1 and OsWTF2 gene in these RNAi transgenic plants were confirmed at the RNA level. These transgenic will be useful materials for the further study of OsWTFl and OsWTF2 gene function.3. Different OsWTF1 5' end deletion promoter fragments (296,181bp) and OsWTF2 5'end deletion promoter fragments (544,385,256bp) were amplified, and then fused with GUS gene to construct five 5'end deletion promoter expression vectors:pOsWTF1-181, pOsWTF1-296, pOsWTF2-256, pOsWTF2-385, pOsWTF2-544 respectively. These vectors can be used to study the expression regulation function and the core sequence of the two gene promoters.