Functional Identification Of Transcription Factor PtrERF36 From Poncirus Trifoliata In Cold Tolerance

Trifoliate orange(Poncirus trifoliata(L.)Raf.)has excellent tolerance to cold stress which is widely used as a rootstock in citrus industry.It has been frequently used in cold resistance research.AP2/ERF proteins are a large family of transcription factors in plants.Many studies have shown that some members in this family are involved in various physiological process,such as plant development,wounding,pathogen defense and abiotic stress.But the functions of most ERF members remain unknown.In current studies,functions of PtrERF36 in cold tolerance was examined.The main results are as follows:1.The vector of PtrERF36-RNAi was constructed and transformed into Poncirus trifoliata with Agrobacterium-mediated transformation.A total of 202 transgenic plants were examined by PCR and 29 were positive plants.The positive rate is 14.36%.The expression levels of PtrERF36 in 6 transgenic plants were measured;all transgenic plants showed a decreased PtrERF36 expression level than wild type(WT),especially #47 and #56.2.Cold resistance analysis showed that the electrolyte leakage and the content of MDA and ROS(H2O2 and O2-)in the two transgenic lines(#47 and #56)were higher than that of WT after 0 ℃ treatment.In addition,POD and SOD activities were lower in the transgenic plant compared with WT.These results demonstrated that knock-down of PtrERF36 caused a decreased cold tolerance in lemon,thus,we investigate that PtrERF36 may play a positive role in cold tolerance.3.RT-PCR analysis of several cold-related genes in PtrERF36-RNAi transgenic lines demonstrated that POD,CAT,SOD,COR15,HOS1 and ICE1 were down-regulated in the transgenic lines.Although all these genes expression level were incresed after cold treatment but their transcript levels were still lower in the RNAi lines compared with the WT.The greatest suppression was observed in POD.In addition,there was a GCC-box in the promoter of POD,suggesting that the POD may be a target of PtrERF36.4.The promoter of PtrERF36 was isolated from genomic DNA of Poncirus trifoliata by PCR amplification.Promoter analysis revealed that some stress-related motifs exited in the promoter of PtrERF36,such as ABRE and DRE/CRT.