基于高通量测序发掘番木瓜果实成熟相关miRNA

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

【目的】番木瓜是典型的呼吸跃变型果实,外源乙烯处理使番木瓜呼吸跃变提前,促进果实成熟。分离番木瓜果实成熟相关miRNA,为深入了解呼吸跃变型果实的成熟分子机制奠定基础。【方法】利用高通量测序技术对乙烯(ETH)、1-MCP和清水对照(CG)处理的番木瓜果实进行miRNA和转录组高通量测序,然后对测序获得数据进行生物信息学分析,进行miRNA鉴定和靶基因预测,并与转录组测序结果进行关联分析。【结果】乙烯、1-MCP和对照处理分别获得10734196、16486803和16067290条纯净序列,共鉴定出523个miRNA。其中,已知miRNA个数为1-MCP(303)、CG(214)和ETH(239),新miRNA个数为1-MCP(184)、CG(188)和ETH(114)。与对照相比,在乙烯处理中上调和下调表达的miRNA分别是123和72条。靶基因预测共获得5053个靶基因,KEGG功能富集分析显示它们参与了戊糖、葡萄糖醛酸转换、淀粉和蔗糖代谢、卟啉和叶绿素代谢、类胡萝卜素合成等代谢途径。筛选出的番木瓜果实成熟衰老相关候选miRNA,包含4个果实软化调控相关miRNA(miR167-y、miR4993-x、miR3946-x和miR5059-x)、3个果实颜色调控相关miRNA(miR4993-x、miR815-y和miR7810-x)、3个激素调控相关miRNA(miR4993-x、miR8004-x和miR9722-x)和4个转录因子调控相关miRNA(miR5641-y、miR9722-x、miR838-y和miR319-y)。【结论】筛选的番木瓜果实成熟衰老相关miRNA为今后果实成熟衰老调控网络研究提供了可能的线索。

【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 1μL·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 18421155(1-MCP),17669301(CG)and 13178734(ETH)raw reads,and 16486803(1-MCP),16067290(CG)and 10734196(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 5053 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 lycopeneε-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.