Cloning and characterization of two ERF transcription factor genes in Brazil banana

HOU Xiaowan; HU Wei; XU Biyu; ZHANG Lubin; JIN Zhiqiang

doi:10.13925/j.cnki.gsxb.20160436

Abstract：[Objective] Banana (Musaceae,Musa) is one of the most important food plants,widely distributing in tropical and subtropical countries.Banana is particularly sensitive to most of the abiotic stresses,such as freezing,drought and salt,which would cause heave decrease of the yield and quality.Therefore,investigation the molecular mechanism of the response of banana to abiotic stresses is of prime importance for improving stress-resistant ability.Moreover,over the last decades APETALA2/Ethylene Responsive Factor (AP2/ERF) proteins have been found to be involved in a variety of biological processes.Based on the number of the structure domain,AP2/ERF superfamily was divided into four families as AP2/ERF,ERF,RAV and soloist.The proteins of ERF family only contained an AP2/ERF structure domain and were studied more deeply than others in all transcription factors family.The genes of ERF family played an important role in regulating plant response to abiotic stresses.The purpose of this study is to clone the key ERF transcription factors of banana and to identify their functions in response to abiotic stresses in order to provide a basis for improving stress-resistant ability.[Methods] The relative transcriptome results of AP2/ERF superfamily genes of Brazilian banana involved in responding to abiotic stresses of leaves were obtained.The expression profile analysis on AP2/ERF super family genes of banana were conducted by using MeV software.In view of the sensitivity to low temperature stress,MaERF25 and MaERF27 were isolated from banana using RNA reverse transcription cDNA mixture from various tissues and organs of banana as template.By using ExPASy,DNAMAN and some other related biological analysis softwares and on the basis of protein sequence homology,the sequences homologue and the basic characteristics of protein were analyzed.In addition,the transient expression vectors containing MaERF25 and MaERF 27gene were constructed on the basis of plant expression vector pCAMBIA1302.PCAMBIA1302-MaERF25-GFP,pCAMBIA 1302-MaERF27-GFP and empty vector (pCAMBIA 1302-GFP) were respectively introduced into onion epidermal cells by Agrobacterium tumefaciens-mediated transient transformation and the fluorescent signals were observed by a FluoViewTM FV1000 laser scanning confocal microscope to analyze the effecting position.The transcriptional activating activity of MaERF25 and MaERF27 proteins was detected in yeast strain AH 109.The yeast expression vectors including full length and deletion fragment of those two genes,MaERF25,MaERF25-N/MaERF25-C and MaERF27,MaERF27-N/MaERF27C,were respectively constructed on the basis of the vector pBKGT7 (pBD) carrying the His and LacZ reporter genes.Then,the expression vectors were transformed into yeast competent cells to detect its transcriptional activating activity and to confirm its transcriptional activating region.Finally,the expression level of MaERF25 and MaERF27 under different abiotic stress and ABA treatment were verified by Mx3005P quantitative real-time PCR.[Results]The study acquired 110 gene expression information from the transcriptome results.The result of heatmap analysis showed that AP2/ERF super family get the higher gene expression level (90.91％) under cold stress treatment compared with that under other aboitic stress treatments,the up-regulated genes accounted for 73.64％ under cold treatment.In addition,under high-salt treatment,the differential expression genes accounted for 82.72％,and the up-regulated genes possessed 45.45％.Under mannitol treatment,the differential expression genes occounted for 79.54％,and the up-regulated genes had the proportion of 42.27％.MaERF25 (GSMUA_Achr2T20280) and MaERF27 (GSMUA_Achr10T15510) were chosen as the research objects acorrding to the results of heatmap analysis,and their CDS sequences were cloned.Sequence analysis confirmed that their CDS sequences had higher consistence with Elaeis,Phoenix dactylifera,Nelumbo nucifera and Vitis vinifera.The conserved domain analysis by NCBI showed that the amino acid sequence of MaERF25 and MaERF27 were respectively located in the 52-110 and 68-126 amino acids both harboring a single AP2 domain,which were consistent with the ERF families' features and indicated that MaERF25 and MaERF27 were AP2/ERF transcription factors.MaERF25 and MaERF27 full-length sequences respectively inserted into the plant expression vector of 35S:GFP (pCAMBIA1302-GFP).Subcellular localization analysis revealed that MaERF25 and MaERF27 were located in the nucleus.MaERF25,MaERF25-N,MaERF25-C,MaERF27,MaERF27-N and MaERF27-C full-length sequences inserted into pBKGT7 vector,and then transformed into AH109 yeast strains,respectively.Yeast one-hybrid assays demonstrated the presence of transcriptional activity in the MaERF25 and MaERF27 proteins and their C-terminal domains.The transcription response of the two TFs in response to mannitol,salinity,low temperature and the treatment of abscisic acid (ABA),showed MaERF25 and MaERF27 were involved in the response to freezing,drought,salinity and abscisic acid,and were induced in different degrees.The highest level of induction of the two genes was found when treated with low temperature.[Conclusion]Two ERF transcription factor family genes,MaERF25 and MaERF27,were isolated from banana.Their proteins were all located in nuclear and had transcriptional activating activity,being involved in the responses to abiotic stresses.