Regulation Mechanism Of BpERF11 Gene From Betula Platyphylla Response To Severe Salt And Drought Stress

AP2/ERF transcription factors are specific in plant, they could regulate plant response to different biotic and abiotic stress. In this study,7 ERF genes were cloned and their response to salt and drought was analyzed by RT-PCR. The result showed that BpERF genes could be induced by NaCl and PEG stress, we selected BpERF11 gene which could be significantly induced by salt and drought treatment to study it’s functions.Firstly, pROKⅡ-BpERF11-GFP fusion expression vector was constructed, and was transformed to onion epidermal cells by gene gun instantaneous conversion technology. The results showed that BpERF11 gene was located in nucleus. Secondly, overexpression and RNAi-silence vectors of BpERF11 gene were constructed and were transformed to WT birch by Agrobacterium-mediated method. The overexpression and RNAi-silence transgene lines were obtained and the functions of BpERF11under abiotic stress were studied.We analyzed the phenotype difference between transgenic lines and WT plant. The result showed that, plant height, fresh weight and dry weight of overexpression lines were much lower than WT, but higher than WT in RNAi-silence lines.To analysis stress resistance of different transgenic lines and WT plant under NaCl and mannitol stress, we performed DAB、NBT and Evans blue stain test and measured SOD, POD activity, MDA content, chlorophyll content, electrolytic leakage, H2O2content, proline content, water loss rate and stomatal aperture. Result showed that, SOD, POD activity, chlorophyll content and proline content were much lower than WT in overexpression lines, but they are higher than WT in RNAi-silence lines. MDA content, electrolytic leakage, H2O2content, water loss rate and stomatal aperture are higher than WT in overexpression lines, but they were much lower in RNAi-silence lines. These results indicated that the RNAi-silence birch enhanced stress tolerance by increase SOD and POD activity, chlorophyll and proline content, decrease MDA content, electrolytic leakage, H2O2content, water loss rate and stomatal aperture.The expression level of stress resistance genes, such as SOD, POD, P5CS and LEA genes under salt and drought stress were analyzed by real time RT-PCR. Result showed that the expression level of SOD, POD, LEA and P5CS genes were lower in overexpression lines than WT, however, they were higher in RNAi-silence lines. In RNAi-silence lines, expression level of PRODH and P5CDH are downregulated, but they were upregulated in overexpression lines. These results indicated that overexpression of BpERF11 gene decreased plant stress resistance by reducing the expression level of SOD, POD, proline synthesis gene and LEA genes.Yeast one-hybrid (Y1H) was performed to determine the binding of BpERF11 to GCC-box, DRE motif and other unknown motifs. Result showed that BpERFll could bind to GCC-box and DRE motif, but they could not bind to their mutant motifs. We obtained an unknown motif from random component library which could interact with BpERF11, and identified the core sequence of this motif as "GGCTTA".Different expression profile between overexpression and RNAi-silence lines was analyzed by expression profile microarray technology. We found peroxidase genes, proline rich protein genes, adenosylmethionine decarboxylase genes, leucine repeated receptor protein kinase, serine/threonine protein kinase genes, aquaporins genes, aldehyde dehydrogenase genes, glutathione S-transferase genes. These results indicated that BpERF11 could regulate expression of these stress response genes.