Re-identification of MaAP2/ERFs and their differential expression characteristics during postharvest banana fruit ripening

Abstract：【Objective】APETALA2/ethylene response factors (AP2/ERFs) are in a super transcription factor family involved in the terminal of ethylene signal transduction pathway, which plays important regulatory roles in plant growth and development, stress response, fruit ripening, quality formation and other biological processes. However, there is no systematically re- identification of Musa acuminate AP2/ERFs (MaAP2/ERFs). With the rapid development of sequencing technology, the quality of whole genome assembly is improving. The aims of the present study were to re-identify the MaAP2/ERFs family members in the whole genome-wide level and to determine the key genes involved in the regulation of banana fruit ripening.【Methods】MaAP2/ERFs family members were genome-widely analyzed. Thewhole AP2/ERFs protein sequences of banana and tomato were obtained from the Banana Genome Hub released January 2016 and reference’s report, respectively. To identify the MaAP2/ERFs family genes, BLAST searches were performed to check the predicted MaAP2/ERFs in banana database with all the tomato AP2/ERFs as queries. All candidate protein sequences were further examined by the CDD and PFAM databases. Then, multiple sequence alignments were applied to confirm the conserved domains of predicted MaAP2/ERFs proteins. Additionally, sequence alignments of the full-length MaAP2/ERFs proteins from banana and tomato were performed by Clustal X 2.0. The bootstrap neighbor-joining evolutionary tree was created by MEGA 5.0 software with 1000 bootstrap replicates based on the sequence alignments. The ExPASy proteomics server and TBtools were employed to detect the molecular weight and isoelectric points and gene structure, respectively. At 0 day postharvest (DPH), two main cultivars BaXi Jiao (BX) and Fen Jiao (FJ) fruits were obtained from the banana plantation of Institute of Tropical Bioscience and Biotechnology (Chengmai, Hainan, 20 N, 110 E). Postharvest banana hands at similar developmental stage were selected and allowed to ripen naturally. Samples at the 8 DPH and 14 DPH fruits for BX and at 3 DPH and 6 DPH fruits for FJ were obtained according to ethylene production, which occurred faster in FJ reaching full yellow degree earlier than in BX. Samples were collected to extract total RNA using plant RNeasy extraction kit for transcriptome analysis. The sequencing was performed with an Illumina GAII following manufacturer’s instructions. Gene expression levels were calculated as Fragments per Kilobase of exon model per Million mapped reads (FPKM).【Results】A total of 317 MaAP2/ERFs family members were identified. The 317 predicted MaAP2/ERFs proteins varied from 68 (MaERF68) to 716 (MaAP2-41) in amino acid residues and the relative molecular mass ranged from 7.5 (MaERF68) to 76.6 (MaAP2-34) kDa, with isoelectric points in the range of 4.6–10.3. The instability index varied from 44.0 (MaERF218) to 83.5 (MaERF240), with hydropathicity ranged from - 1.3 (MaERF52) to - 0.2 (MaERF134). MaAP2/ERFs could be divided into three subfamilies: AP2 (49), ERF (253) and RAV (15). ERFs were further divided into 10 subgroups, including a, b, c, d, e, f, h, i, j and k, according to the conserved domain and gene structure characteristics. The 317 AP2/ ERFs were unevenly distributed on 11 chromosomes. The maximum number of 41 genes (12.9%) localized on chromosome 4, followed by 37 (11.7%) on chromosome 3 and 33 (10.4%) on chromosomes 6 and 10, whereas chromosomes 1 had only 12 (3.8% ). The gene structure characteristics of MaAP2/ ERFs are similar among different members of the same subfamily. AP2 subfamily members contained 7-10 exons and 6-9 introns. 14 of a total of 15 MaRAVs contained only one exon. In ERF subfamily, most of the intronless genes were clustered in a, b, c, d, f, i, h and j subgroups and only 2 genes with a single intron. The pattern of two exons with one intron was found in all members in k subgroup. Most of the genes in e subgroup had 6 introns. This suggested that similar exon- intron organizations of MaAP2/ERFs exist in the same group and the gene structure might be meaningful for gene evolution and function. Conservative domain analysis showed that all AP2/ERFs family members had two conserved AP2 domains, which further supports the phylogenetic analyses. The expression patterns of MaAP2/ERFs were detected in fruits sampled from different ripening stages of BX and FJ. The results indicate 24.3% and 23.3% of MaAP2/ERFs were differentially expressed during postharvest ripening process of BX and FJ fruits, respectively. For BX, there were 77 MaAP2/ERFs differentially expressed. Specially, 14 genes (MaERF15, 36, 42, 44, 103, 115, 132, 156, 180, 181, 222, 242, AP2- 28 and MaRAV2) were highly expressed (FPKM value＞50). Among them, MaERF15, 36, 42, and AP2-28 displayed super expression levels (FPKM value＞100). For FJ, there were 74 MaAP2/ERFs differentially expressed. Among them, 4 genes (MaERF15, 42, AP2-28 and MaRAV2) were highly expressed with theexpression levels (FPKM value＞50). Among them, MaERF42 and AP2-28 displayed super expression levels (FPKM value＞100). 57 MaAP2/ERFs simultaneously expressed in BX and FJ. 20 and 17 MaAP2/ERFs specially expressed in BX and FJ, respectively. Among those differentially expressed genes, the expression patterns of 34 genes (MaERF5, 15, 22, 32, 42, 49, 63, 72, 77, 103, 109, 111, 131, 139, 140, 141, 142, 143, 165, 167, 174, 179, 180, 185, 193, 207, 213, 222, 228, 234, 242, 250, MaRAV2 and MaRAV4) were closely related to BX fruit ripening process, whose expression levels were quickly increased at 8 DPH and were 2-fold higher than at 0 DPH. The expression patterns of 29 genes (MaERF11, 32, 36, 45, 49, 50, 70, 72, 73, 97, 103, 111, 120, 122, 140, 142, 143, 164, 165, 174, 193, 204, 240, 242, 247, 250, 252, AP2-44 and MaRAV4) were closely related to FJ fruit ripening process, and their expression levels quickly increased at 3 DPH and were 2-fold higher than those at 0 DPH. These results suggested that these genes play important roles in BX and FJ fruits ripening.【Conclusion】317 MaAP2/ERFs family members were genome-widely re-identified. The key genes involved in BX and FJ fruit ripening were detected. These findings laid a foundation for the systematic and in-depth analysis of the function of MaAP2/ERFs, and provided target genes for the regulation of fruit ripening.