High-throughput sequencing and degradome analysis reveal differential expression of miRNAs and their targets between cytoplasmic male-sterile cybrid pummelo and its fertile type (Citrus grandis)

High-throughput sRNA and degradome sequencing was applied in G1+HBP and its fertile type HBP to identify miRNAs and their targets during reproductive development. A total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2), transport inhibitor response 1 (TIR1), etc. Eight target genes were confirmed to be sliced by corresponding miRNAs using 5’RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between CMS line G1+HBP and fertile line HBP were discovered. Differential expression of miRNAs and their target genes was validated by quantitative RT-PCR and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulation mechanism of miR167a was elucidated by yeast one-hybrid and dual-luciferase assay that a dehydrate responsive element binding (DREB) transcription factor bind miR167a promoter and transcriptionally repress miR167 expression. Our study revealed altered expression of miRNAs and their target genes in cytoplasmic male sterile pummelo (CMS) line and highlighted that miRNA regulatory network may be involved in nucleus-cytoplasmic cross talk of citrus CMS. small RNA and degradome sequencing of seedy ‘HBP’ and its cytoplasmic male sterile and seedless line ‘G1+HBP’

本研究以细胞质雄性不育（cytoplasmic male sterile, CMS）柚品系“G1+HBP”及其可育型品系“HBP”为实验材料，采用高通量小RNA（small RNA, sRNA）和降解组测序（degradome sequencing）技术，对二者生殖发育过程中的microRNA（miRNA）及其靶基因进行鉴定。最终共鉴定得到184个已知miRNA、22个新miRNA以及86个靶基因。部分靶基因为参与花发育的转录因子，包括生长素响应因子（auxin response factors, ARFs）、SQUAMOSA启动子结合蛋白盒（SQUAMOSA promoter binding protein box, SBP-box）、MYB、碱性亮氨酸拉链（basic region-leucine zipper, bZIP）、APETALA2（AP2）、运输抑制剂响应蛋白1（transport inhibitor response 1, TIR1）等。借助5'快速扩增cDNA末端（rapid amplification of cDNA ends, 5'RACE）技术，证实有8个靶基因可被对应miRNA介导剪切。基于测序丰度分析，本研究在CMS系“G1+HBP”与可育系“HBP”之间筛选得到42个差异表达miRNA。通过定量实时聚合酶链反应（quantitative RT-PCR, qRT-PCR）验证了miRNA及其靶基因的差异表达模式，并证实部分miRNA与对应靶基因呈现反向表达趋势。通过酵母单杂交（yeast one-hybrid）和双荧光素酶报告基因实验（dual-luciferase assay）阐明了miR167a的调控机制：脱水响应元件结合蛋白（dehydrate responsive element binding, DREB）转录因子可结合miR167a的启动子区域，并对miR167的表达产生转录水平的抑制作用。本研究揭示了细胞质雄性不育（CMS）柚品系中miRNA及其靶基因的表达异常，并强调miRNA调控网络可能参与柑橘CMS的核质互作过程。本研究针对有籽“HBP”及其细胞质雄性不育无籽品系“G1+HBP”开展了小RNA与降解组测序。