Functional Study Of PpcERF Gene Isolated From Chinese Cherry

ERF are transcription factors and are important members AP2/EREBP super gene family, and play activation or inhibition capabilities of gene expression in plants. The amino acid sequences contain a highly conserved AP2 binding domain that includes 60 amino acids. ERF are widely involved in plant growth and development, and often act on regulating the expression of a variety of functional genes, and involved in plant defense responses, thereby improving the ability of plant to stress resilience adversity.An encoding sequence of ERF was identified from transcriptome library of Chinese cherry (Prunus pseudocerasus), named PpcERF. The structure PpcERF be analyzed, and in order to analyze the expression and functional characteristics of PpcERF using real-time quantitative PCR and genetic transformation tools. The main results are as follows:1. The ORF of PpcERF was 1059 bp, which encoded 352 amino acids (aa). The hydrophilic PpcERF contained one conservative AP2/ERF domain. The amino acid sequences were similar to Prunus plants. The cluster analysis revealed that PpcERF have closer genetic relationship to Prunus persica. PpcERF and ERF5/6 have high similarity after aligning with Arabidopsis gene database, suggested that PpcERF might have similar functions with ERF5/6 of Arabidopsis.2. In order to be able to accurately analyze gene expression of PpcERF using Real-time PCR, the stability of seven housekeeping genesincluding18SrRNA, ACTB, TUB, UBCE, TIF5A, EF1B and GAPDH was analyzed. Analysis via Genorm, Bestkeeper, NormFinder and △Ct programs showed that GAPDH was morereliable reference gene under 4℃ and NaCl treatment, and ACTB and UBCE were more stabilized gene under ABA treatment and in the process of flower bud dormancy release respectively.3. The expression characteristion of PpcERF under different processing conditions was analyzed as indicated by qRT-PCR analysis, the expression level of PpcERF displayed significantly up-regulated during H2O2 and PEG2000 treatment, and dropped off again. The results showed that was a trend that decline after rising first under treatment. The expression level of PpcERF was suppressed by 4℃. During transition of flower bud dormancy, the expression of PpcERF peaked at 236 C.U while the change of expression level of PpcERF was not obviously under ABA and NaCl treatment. The results shown above indicated that PpcERF might response to drought stress, oxidative stress and low temperature stress.4. In order to further reveal functions of PpcERF, a plant expression vector was constructed driven under the 35S promoter. The transgenic lines of Arabidopsis were obtained showed that the transgenic Arabidopsis is small as a whole, leaf curl, and low degree of thick leaves. Arabidopsis seed germination were influenced under different concentration of H2O2, and showed that transgenic Arabidopsis seeds maintained at a high level about vigor and germination rates in a 4 mmol/L H2O2. The analysis of downstream genes by real-time PCR showed that the expression level of GA2-OX6 and PRX33 in transgenic Arabidopsis were higher than wild-type Arabidopsis. PpcERF might affect leaf the normal growth by regulating the activity of the GA, and increase the antioxidant capacity by regulating the expression of antioxidant gene.In conclusion, the structure, expression characteristion and function of PpcERF were analyzed. The results oprovide theoretical support for plant stress resistance.