Isolation of ripening-related miRNAs from Carica papaya fruit based on high-throughput sequencing

Abstract: 【Objective】Carica papaya is a typical climacteric fruit, and exogenous ethylene(ETH) applications can induce premature and quicker ripening. 1-methylcyclopropene(1-MCP) is one of ethylene perception inhibitors, which is used commercially to slow down the ripening of fruits. In previous studies, a lot of genes were found playing important roles in the process of papaya fruit ripening. However,the regulation mechanism of these genes is not clear. MicroRNAs(miRNAs) are a class of small noncoding RNAs that regulate the expression of target messenger RNAs(mRNAs), which are widely involved in the regulation of a variety of biological processes in plant, such as cell development and differentiation, biological and abiotic stress, maturation and senescence, etc. Isolation of fruit ripening-related miRNAs will lay a foundation for further understanding the fruit ripening regulation mechanism.【Methods】Papaya fruits(C. papaya L.‘Daqing No.7') at the green-mature stage were harvested from a local commercial plantation in Zhangzhou, China. Thirty-six fruits were incubated with 1 μL · L^-1 of 1-methylcyclopropene(1-MCP) gas for 18 h in a sealed box; thirty-six fruits were dipped into 0.5 g · L-1 of ethephon solution for 3 min, then dried and put in a sealed box for 2 h; thirty-six fruits(Control Group,CG) were dipped into water for 3 min, then dried and put in a sealed box for 2 h. After treatments, all fruits were stored at 25 ℃ and allowed to ripen. Fruits were taken randomly at 24 h after treatments.Three fruits were peeled, seeds were removed, and the flesh was cut into some pieces. The pieces of papaya flesh were mixed, frozen in liquid nitrogen, and stored at-80 ℃. The flesh samples of different treatments(ETH, 1-MCP and CG) were sent to Genedenovo Biotechnology Co., Ltd(Guangzhou, China) for miRNA sequencing and RNA-seq using Illumina Hiseq2500. The raw reads were filtered to remove the low quality reads, including the adaptor sequences, the sequences of shorter than 18 nt or longer than 30 nt. Then, the sequences of rRNA, scRNA, snoRNA, snRNA, tRNA, and degradation fragments were also removed. Through miRNA sequences searching, the structure prediction was undertaken to identify the known miRNA and new miRNA. Patmatch was used to conduct the target gene prediction. Then all of the target genes were annotated using the papaya reference genome database, NCBI non-redundant(Nr) database, Gene Ontology(GO) database, and the Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway database. Based on the differential miRNAs expression and the KEGG pathway enrichment of their target genes, fruit ripening-related miRNAs were selected.【Results】A total of 18 421 155(1-MCP), 17 669 301(CG) and 13178734(ETH) raw reads, and 16 486 803(1-MCP),16 067 290(CG) and 10 734 196(ETH) clean reads were obtained, respectively. Bioinformatics analysis identified 523 conserved miRNAs. Among them, the numbers of known miRNAs were 1-MCP(303),CG(214) and ETH(239), and the numbers of new miRNAs were 1-MCP(184), CG(188) and ETH(114). Compared with the untreated papaya, 123 miRNAs were up-regulated and 72 miRNAs were down-regulated with ETH-treatment; however, 29 miRNAs were up-regulated and 15 miRNAs were down-regulated with 1-MCP-treatment. Target gene prediction showed that a total of 5 053 target genes were predicted. The target genes were annotated using the GO database and genes were classed into three categories: molecular function, cellular component and biological process. The terms of binding and catalytic activity were observed to occur most frequently in the ontology of molecular function;while the terms of cell and cell part were observed to occur most frequently in the ontology of cellular component; and the terms of metabolic process and cellular process were observed to occur most frequently in the ontology of biological process. KEGG analysis revealed that these genes may be involved in pentose and glucuronate interconversions, starch and sucrose metabolism, porphyrin and chlorophyll metabolism, carotenoid biosynthesis pathway, etc. A total of eleven fruit ripening-related miRNAs were selected: four fruit softening regulation-related miRNAs including miR167-y, miR4993-x,miR3946-x and miR5059-x, with their corresponding target genes being UDP-glucuronate 4-epimerase(6 GAE6), cellulose synthase 6(CESA6), cellulose synthase-like protein G3 isoform X1(CSLG3) and expansin-A8-like(EXP8); three fruit coloring regulation-related miRNAs containing miR4993-x, miR815-y and miR7810-x, with their corresponding target genes being magnesium-protoporphyrin IX methyltransferase(CHLM), protoporphyrinogen oxidase 2(HEMG2) and lycopene ε-cyclase(LCYE); three hormone related miRNAs including miR4993-x, miR8004-x and miR9722-x, with their corresponding target genes being ethylene-responsive transcription factor RAP2-3-like(EBP), ERF domain protein 12(ERF12), gibberellin 2-β-dioxygenase 8(GA2 OX8) and growth-regulating factor 7(GRF7); and four transcription factors regulation-related miRNAs including miR5641-y, miR9722-x, miR838-y and miR319-y, with their corresponding target genes being zinc finger(CCCH-type) family protein(C3 H53), WRKY DNA-binding protein 23(WRKY23) and MYB domain protein 65(MYB). The expression of selected miRNAs was negatively correlated with their target genes' expression, indicating that these miRNAs may negatively regulate the expression of their target genes.【Conclusion】High throughput sequencing is a sound approach to isolating miRNAs. Based on the correlation analysis between the miRNA sequencing and RNA-seq of ETH/1-MCP-treated papaya, eleven fruit ripening-related miRNAs were selected. These miRNAs provide a useful resource for further elucidation of the regulatory roles of miRNAs that participate in papaya fruit ripening.