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Home-Journal Online-2017 No.8

Identification and characterization of a trihelix transcription factor MaGTL1a from banana

Online:2017/10/24 14:52:13 Browsing times:
Author: LIANG Shumin, LUO Donglan, CHENG Yujin, YU Lean, YANG Yajie, CHEN Jianye, LU Wangjin, KUANG Jianfei
Keywords: Banana fruit; Ripening; Transcription factor; Trihelix; Expression;
DOI: 10.13925/j.cnki.gsxb.20160309
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Abstract:【Objective】The trihelix transcription factors, also known as GT factors, are plant-specific transcription factors with conserved trihelix DNA-binding domains that bind specifically to the GT elements in promoters of light regulated genes. The trihelix transcription factors can be further divided into five clades, namely GT-1, GT-2, SH4, GTγ and SIP1, on the basis of their sequence structure. To date, the trihelix transcription factors have been identified and characterized in various plant species, such as pea, Arabidopsis, rice, maize, tomato, and chrysanthemum. A large number of studies indicated that trihelix transcription factors played an essential role in the regulation light-responsive genes, and different developmental processes of growth and development of flowers, embryos, seeds, stomata and trichomes, as well as the adaptation to environmental stimuli like salt and pathogen stresses. However, little information was available on the involvement of the trihelix transcription factors in the ripening of fleshy fruits, especially in economical fruit crops such as banana. Therefore, the aims of this study were to isolate a trihelix tran-scription factor from banana fruit (MaGTL1a) , and investigate its subcellular localization and transcription-al activation activity. Moreover, the gene expression during banana fruit ripening and the promoter activity of MaGTL1 a were also analyzed, in an effort to elucidate the possible roles of MaGTL1 a in the ripening of banana fruit.【Methods】Total RNA was extracted from banana pulp using the hot borate method and the first strand c DNA was synthesized. RT-PCR was performed to isolate the full-length c DNA of MaGTL1 a.Bioinformatics analysis was conducted to analyze the sequence characteristics of MaGTL1 a. Phylogenetic tree was constructed using the MEGA 6.0 software to investigate the evolutional relationship between MaGTL1 a and other trihelix transcription factors from other plant species. Agrobacterium-mediated transient expression in Ben's tobacco leaves was used to assess the subcellular localization and transactivation activity of MaGTL1 a. Quantitative real-time PCR (RT-q PCR) was carried out to monitor the expression pattern of MaGTL1 a during fruit ripening of banana. Promoter of MaGTL1 a was isolated using banana genome DNA as template, and its promoter activity was tested using transient expression in tobacco BY2 protoplasts via the PEG method.【Results】A trihelix gene (GSMUAAchr4G24780001) was isolated from banana fruit using the RT-PCR technology. A BLAST search of Gen Bank revealed that the protein encoded by this gene shared 59%, 58%, 53%, 47% and 44% identity with Elaeis guineensis Eg GTL1-like isoform (XP010933772.1) , Phoenix dactylifera Pd GTL1-like isoform (XP008791630.1) , Nelumbo nucifera Nn GTL1-like isoform (XP010250883.1) , Populus tremula Pt GTL1 (AER42647.1) and Ricinus communis Rc GTL1-like isoform (XP002518968.1) , respectively, at amino acid level and thus the gene was designated as MaGTL1 a. The c DNA of MaGTL1 a had an open reading frame (ORF) of 2 283 bp in length which encoded a peptide of 761 amino acid residues with calculated molecular weight (MW) of82.70 ku and predicted isoeletric point (p I) of 6.07. Moreover, comparison of the protein sequence with other trihelix transcription factors from other plant species showed that MaGTL1 a consisted of two trihelix DNA-binding domains at its N-terminal and C-terminal regions respectively, and a typical feature of GT-2 clade of trihelix members, which implied that MaGTL1 a might belong to GT-2 clade. Phylogenetic analyses exhibited that the plant trihelix transcription factors could be grouped into five clades, namely GT-1, GT-2, SH4, SIP1 and GTγ, where MaGTL1 a of banana together with those of Oryza sativa Os GT-2, Populus tomentosa Pta GTL1 and Arabidopsi thalianas At GTL1 fell into GT-2 clade. Subcellular localization indicated that the green fluorescence signal of MaGTL1a-GFP fusion protein was exclusively observed in the nucleus of the tobacco epidermal cell, as other reported trihelix transcription factors, indicating that MaGTL1 a might function in nuclear compartments. Furthermore, transcriptional activation activity assays demonstrated that MaGTL1 a possessed transcriptional activation ability in yeast and in plant, implicating its role as a transcriptional activator. Importantly, RT-q PCR analyses revealed that the accumulation of MaGTL1 a transcript was obviously induced by ethylene, with increasing trends along with the progression of banana fruit ripening, which was in accordance with its expression pattern in our DGE data. In addition, the activity of MaGTL1 a promoter was activated by exogenous ethylene application when transiently expressed in tobacco BY2 protoplasts, further supporting the roles of MaGTL1 a involved in fruit ripening.【Conclusion】MaGTL1 a was an ethylene-inducible and nucleus-localized transcriptional activator likely involved in the modulation of banana fruit ripening, which expanded our understanding on trihelix transcription factors in regulating fruit ripening and the transcriptional controlling networks of ripening of banana fruits. Further experiments such as identification of MaGTL1a's target genes are needed to fully explore the biological functions of MaGTL1 a in relation to fruit ripening of banana.