Cloning, sequencing and expression analysis of auxin response factors (MdARF) in apple

Abstract：【Objective】Auxin response factors (ARFs) are a class of transcription factors that can activate or repress gene expression via binding with auxin response elements (AuxREs, TGTCTC/TGTCCC/TGTCGG) in the promoters of down-stream target genes including Gretchen Hagen 3 (GH3) , Auxin/Indole-3-acetic acid (Aux/IAA) and Small Auxin Up RNA (SAUR) . Most ARF transcription factors contain three characteristic domains: the N-terminal B3-like DNA-binding domain (DBD) , the Cterminal dimerization domain (CTD) containing a region called motif III/IV, which allows the dimerization of ARF or ARFs and Aux/IAA proteins, and the variable middle region (MR) , which is responsible for gene activation or repression. The evolutionary analysis showed that the Arabidopsis ARF gene family was divided into five groups: Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ. A large number of studies indicated that ARF transcription factors played multiple roles in the regulation of plant developmental and physiological processes, including apical dominance, embryogenesis, vascular elongation, lateral root initiation, flow-ering and fruit development. However, little information was available on the involvement of MdARF transcription factors in IAA signal transduction pathway and in response to various abiotic stresses.Therefore, the aim of this study was to characterize novel apple (Malus domestica‘Zihong Fuji') auxin response factor genes (MdARFs) involved in regulation of growth, development and abiotic stress responses. The transcriptional level of MdARFs in different tissues and under IAA, salt and mannitol treatments were determined to provide the foundation for studying the biological function of MdARFs gene.【Methods】Total RNA was extracted from‘Zihong Fuji'leaves using the CTAB method and the first strand cDNA was synthesized by PrimeScriptTM 1 STStrand cDNA Synthesis Kit. The full-length cDNA sequences of MdARFs were isolated by homologous comparison and RT-PCR methods, the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics software, such as BLAST P, Conserved Domains of NCBI (https://www.ncbi.nlm.nih.gov/cdd) , Pfam 26.0 (http://pfam.sanger.ac.uk/) , and DNAMAN 6.0.3. Phylogenetic tree was constructed using the MEGA 6.0 software to investigate the evolutional relationship between MdARFs and other ARF proteins from Arabidopsis, rice, maize, grape and tomato. The subcellular localization prediction programs of SoftBerry ProtComp9.0 (http://linux1.softberry.com/berry.phtml) , CELLO v.2.5 (http://cello.life.nctu.edu.tw/) and PSORT (http://psort. hgc. jp/form. html) were used to determine the possible subcellular localization of MdARFs. The expression levels of MdARFs were detected in 16 different tissues using the array from NCBI GEO database. The expression levels of MdARFs were detected under 40 mg · L^-1 IAA, 150 mmol· L^-1 NaCl and 300 mmol· L^-1 mannitol treatments using qRT-PCR method with BIO-RAD IQ5 Real-time PCR Detection Systems (USA) .【Results】Ten MdARF genes (designated as MdARF4-13; GenBank Accession No. MG021615-MG021624) were isolated from‘Zihong Fuji'leaves using RT-PCR method. The cDNAs of MdARFs contained open reading frame (ORF) of 2 136, 3 345, 2 052, 2 400, 2 532, 2 691, 1 782, 2 532, 3 342 and 2 034 bp in length which encoded proteins of 711, 1 114, 683, 799, 843, 896, 593, 843, 1 113 and 677 amino acid residues with calculated molecular weight (MW) of 78.28, 123.44, 75.66, 88.50, 93.93, 98.05, 65.37, 93.93, 123.96 and 75.28 ku and predicted isoeletric point (pI) of 7.74, 6.90, 6.39, 6.63, 6.14, 5.31, 6.04, 6.74, 6.53 and 6.22, respectively. The results of phylogenetic analysis revealed that ARF proteins were divided into five classes: Class Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ.MdARF3 and MdARF7 belonged to Class Ⅰ (AtARF3/4-like) ; MdARF4 and MdARF10 constituted Class Ⅱ (AtARF10/16/17-like) ; MdARF1, 6, 11 and MdARF13 belonged to Class Ⅲ (AtARF1/2-like) ;MdARF5, 9 and MdARF12 constituted Class Ⅳ (AtARF5/7/19-like) ; MdARF2 and MdARF8 belonged to Class Ⅴ (AtARF6/8-like) . The results of subcellular localization prediction showed that all MdARFs were located in the nucleus. The ARF could be determined as transcriptional activator or transcriptional repressor by the amino acid compositions in the MR domains. Analysis of amino acid compositions in the MR domains of MdARFs showed that MdARF2, MdARF5, MdARF8, MdARF9 and MdARF12 were probable transcriptional activators; while MdARF1, MdARF3, MdARF4, MdARF6, MdARF7, MdARF10, MdARF11 and MdARF13 were probable transcriptional repressors. The expression profiles of 16 different tissues of apple (GSE42873) were downloaded when the NCBI GEO database was used to detect the expression of MdARFs in different tissues. The arrays results indicated that MdARF genes (except MdARF4) were relatively highly expressed in the flowers (M64 and M74) and the fruits (M74-100 DAM, M20-100 DAM, M74-harvest and M20-harvest) ; MdARF1 and MdARF10 had higher relative expression levels in the seeds; while MdARF5 and MdARF13 had higher relative expression levels in the roots. The expression levels of MdARFs (Malus domestica ‘Zihong Fuji') were detected under IAA, NaCl and mannitol treatments using qRT-PCR method. The results showed that, under 40 mg· L^-1 IAA treatment, the transcription levels of MdARF1, MdARF7, MdARF9 and MdARF10 increased;MdARF2, MdARF3, MdARF4, MdARF6, MdARF8, MdARF11 and MdARF12 transcriptional levels decreased.Under 150 mmol·L-1NaCl treatment, MdARF9 transcription level was induced, whereas MdARF1, MdARF5, MdARF7, MdARF9, MdARF10, MdARF12 and MdARF13 were down-regulated. Under 300 mmol·L^-1 mannitol treatment, MdARF6 and MdARF9 transcriptional levels were induced.【Conclusion】Ten MdARF genes were isolated and constitutively expressed in all examined tissues, and the relative expression levels were up-regulated or down-regulated under IAA, salt or mannitol treatments, which might be involved in the regulation of auxin signal transduction and stress responses, and might play important roles in the growth and development of apple.