Genome-wide identification and expression analysis of GRF gene family in banana

Abstract:【Objective】Accumulated evidences have revealed that the plant specific growth-regulating factor (GRF) plays very important roles in regulating plant growth and development, and stress respons- es. Banana widely grown in tropical and subtropical areas is one of the most important fruit trees world- wide. However, up to now, the roles of the banana GRF gene family has not been systematically stud- ied. Our present study aimed to identify the banana GRF gene family and to investigate their sequence characteristics, and expression patterns in fruits at different ripening stages under normal condition and ethylene treatment and in leaves under 4 °C low temperature treatment, in order to facilitate the under- standing of the roles of the banana GRF genes.【Methods】According to the Hidden Markov Model of the two GRF domains, QLQ (PF08880) and WRC (PF08879), HMMER software were used to identify the GRF gene family members from the genome of Musa acuminata var. DH-Pahang. Softwares such as ExPASy, SignaIP 3.0 Server, TMHMM Server v.2.0, Wolf PSORT, MCScanX, Circos, GSDS, MEME, MEGA6.0, PlantCARE, PlantTFDB et al. were used for the bioinformatic analysis of M. acum- inata GRFs (MaGRFs). By using the above mentioned softwares, characteristics of their nucleotide se- quences (including chromosome location, gene structure, distributions of cis-acting elements and tran- scription factor binding sites in promoter, prediction of miRNAs targeting MaGRF and so on) and char-acteristics of their encoded proteins (including the protein physicochemical properties, the existence of signal peptides and the transmembrane structure, the protein subcellular location, the conserved domains and motifs and phylogenetic analysis) were studied. Besides, to investigate the expression pat- terns of the MaGRFs, their expression values were extracted from our previous transcriptome data and were used to show their expression pattern in the fruits at different ripening stages with and without eth- ylene treatment, and in the leaves under low temperature (4 °C) treatment.【Results】We totally identi- fied twenty members of the MaGRF from the M. acuminata genome data (named as MaGRF1-20). Chromosome location analysis showed that these MaGRFs were unevenly distributed in all the M. acu- minata chromosomes except for chromosome 9. Among them, 5 MaGRFs were found to have ≥ 2 tran- scripts, for example, MaGRF5, 6 and 18 had two variable transcripts, and MaGRF12 and 14 had 3 vari- able transcripts. The exon number of the MaGRFs ranged from 2 to 6. The protein length of theMaGRFs ranged from 141 aa to 607 aa. Their isoelectric points were between 5.94-9.92, and their pro- tein molecular weights were between 16 042.66-65 554.44 Da, their instability coefficient ranged from 52.04 to 73.30. The 20 MaGRF family members were all hydrophilic proteins without signal peptide. Subcellular localization prediction result showed that all the MaGRFs were nucleus-located. Collinear analysis identified 14 pairs of fragment duplication events in the MaGRFs, but no gene tandem phenom- enon was found. According to the phylogenetic analysis results, the GRFs from Arabidopsis, rice and banana could be divided into 5 subfamilies (Subfamily I-V), among them, the MaGRFs existed in the 4 subfamilies (there were 8, 7, 2 and 3 members in Subfamily I, II, IV and V, respectively). Consis- tently, we found that the gene structure and the distribution of conserved motifs in their encoded pro- teins of the MaGRF members belonging to the same subfamily were similar. Promoter sequence analy- sis revealed that the MaGRF promoters contained a large number of growth and development, plant hor- mone and stress-responsiveness related cis-acting elements, as well as binding sites for nine types of transcription factors (AP2, BBR-BPC, DOf, MIKC-MADS, TALE, C2H2, ERF, MYB and GATA; the num- ber of the MaGRFs family members contained these transcription factor binding sites (TFBSs) is 11, 12, 12, 13, 9, 11, 7, 4 and 3, respectively). The promoter of the MaGRF6 was of the largest number of the TFBSs (103), however, no TFBS was found in the promoters of the MaGRF3 and MaGRF20. TheMaGRF1, 2, 4, 10, 18, and 19 were predicted to be targets of the same 7 miRNAs (mac-miR396, mac- miR396b, mac-miR396a.4, mac-miR396a-5p, mac-miR396b-5p, mac-miR396f and mac-miR396g-5p.1). The MaGRF9, 20 were predicted to be target gene of the mac-miR396, the mac-miR396f and the mac- miR396g-5p.1. The MaGRF3, 6, 7, 11, 13, 14 and 15 were predicted to be target genes of the mac- miR396b, the mac-miR396a.4, the mac-miR396a-5p and the mac-miR396b-5p. Interestingly, two cleav- age targeting sites of the mac- miR396b- 5p were found in the MaGRF1. Gene expression analysis showed that the MaGRF6, 7, 13 and 17 were highly expressed in banana fruits, and the MaGRF6 were highly expressed in banana leaves. The MaGRF7 and 13 were found to be highly expressed in banana fruits in natural ripening periods and the MaGRF13 also showed high expression in the ethylene treated fruits. The expression levels of the MaGRF6, 7 and 17 fluctuated during the fruit ripening stages. Com- pared with the natural ripening fruits, the expression of the MaGRF10 and 12 were found to be induced by the ethylene, while the expression of the MaGRF6, 7 and 17 were significantly inhibited by the ethyl- ene. Moreover, the expression levels of the MaGRF9, 14, 15, 18, 19 were found to be significantly in- duced by the low temperature, while the expression of the MaGRF2, 6, and 13 was suppressed by the low temperature stress.【Conclusion】We totally identified 20 GRF members from the M. acuminata ge- nome data, which is more than other plant species. The expansion of the MaGRF family might be caused by the fragment duplication events occurred during the evolution of banana. The expression pat-tern of each member varied greatly with fruits at different ripening stages and the low temperature treated leaves. And several MaGRF members belonging to the subfamily I and II of the plant GRFsshowed significant differential expression at different fruit ripening stages and the leaves under low tem- perature treatment. Moreover, more than a half of the MaGRFs were predicted to be targets of themiR396 members, indicating that the expression of the MaGRFs might be regulated by the miR396.