Cloning And Functional Characterization Of An Ethylene Response Factor ThERF1 Gene From Tamarix Hispida

The ERFs (ethylene response factors) are plant-specific transcription factors that play an important role in stress reponse. It can enhance plant resistance to biotic stress through interaction with GCC-box which presents in promoters of many pathogenesis-related (PR) genes. In addition, ERFs can response to abiotic stress through interaction with DRE cis elements and non GCC-box containing genes.An EST sequence encoding ThERF1 was obtained from cDNA library, and the full-length ORF (open read frame) sequence was cloned by TAIL-PCR method from Tamarix Androssowii. The ORF is 972 bp in length, encoding 323 amino acids, a predicted molecular mass of 34.1 kDa and a calculated pI of 8.95. Sequence analysis showed that the ERF contains a typical ERF domain in its N-terminus, with a conserved alanine (A) in the 14th position and aspartate acid (D) in the 19th position, both of which have been reported as conserved in the ERF subfamily. In addition to the ERF domain, ThERF1 also contains a conserved sequence (LDLNELP) similar to the ERF-associated amphiphilic repression (EAR) motif, which has been reported as a transcriptional repression domain. The promoter of ThERFl was cloned using TAIL-PCR. The promoter of 942 bp in length sequence was analyzed in detail. In addition to a number of basal promoter structural elements, it contains a wide range of stress-response cis-acting elements such as ABRE, TGACG-motif, MBS and CGTCA-motif.To examine whether ThERF1 could interact specifically with the GCC-box and/or DRE motif, yeast one-hybrid using a reporter gene containing three copies of the DRE, GCC-box and two GCC-box mutation sequences,3×DRE/GCC-box/M1/M2-pHIS2 was performed. The ORF of the ThERF1 was fused with the GAL4 DNA-activation domain as an effector plasmid. The results showed that the ThERF1 could interact with GCC-box and DRE-motif. However double substitution of C with T at positions 3 and 4, and G with T at positions 2 and 5 within the GCC-box eliminate the binding. This suggests that the bases of C3, C4, G2 and G5 winthin the GCC-box are necessary for the binding.To isolate proteins that interact with ThERF1, the entire ThERF1 protein fused with GAL4 binding domain was first used as bait and screened in yeast two-hybrid system. In the yeast strain Y2H, ThERF1 slightly but significantly activated the transcription of MEL1 reporter gene, although it did not activate the transcription of HIS3 reporter gene. Five clones designated elongation factor, aldehyde dehydrogenase, ATP synthase CF1 and unknown protein were isolated.To further characterize the function of ThERFl under abiotic stresses in plant, ThERFl was cloned into pROKⅡdriven by the CaMV35S promoter and transformed Arabidopsis by Arabidopsis mediated floral dip method.10 transgenic Arabidopsis were generated after screen and PCR confirmation.