Luo Jin, Ren Qiaoyun, Liu Wenge, Qiu Xiaofei, Zhang Gaofeng, Tan Yangchun, Cao Runlai, Yin Hong, Luo Jianxun, Li Xiangrui, Liu Guangyuan
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Ministry of Education (MOE) Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Front Physiol. 2021 Apr 8;12:596289. doi: 10.3389/fphys.2021.596289. eCollection 2021.
MicroRNAs act as mRNA post-transcriptional regulators, playing important roles in cell differentiation, transcriptional regulation, growth, and development. In this study, microRNA expression profiles of ticks at different developmental stages were detected by high-throughput sequencing and functionally assessed. In total, 2,585,169, 1,252,678, 1,558,217, and 1,155,283 unique reads were obtained from eggs, larvae, nymphs, and adults, respectively, with 42, 46, 45, and 41 conserved microRNAs in these stages, respectively. Using eggs as a control, 48, 43, and 39 microRNAs were upregulated, and 3, 10, and 9 were downregulated in larvae, nymphs, and adults, respectively. MicroRNA-1 (miR-1) was expressed in high abundance throughout development, with an average of nearly one million transcripts, and it is highly conserved among tick species. Quantitative real-time PCR (qPCR) showed that miR-1 expression gradually increased with tick development, reaching the highest level at engorgement. Differential tissue expression was detected, with significantly higher levels in the salivary glands and epidermis than in the midgut. Inhibition assays showed no significant change in body weight or spawning time or amount between experimental and control groups, but there was a significant difference ( < 0.01) in engorgement time. With miR-1 inhibition, ticks displayed obvious deformities during later development. To more fully explain the microRNA mechanism of action, the miR-1 cluster was analyzed according to the target gene; members that jointly act on Hsp60 include miR-5, miR-994, miR-969, and miR-1011. Therefore, microRNAs are critical for normal tick development, and the primary structure of the mature sequence of miR-1 is highly conserved. Nonetheless, different developmental stages and tissues show different expression patterns, with a certain role in prolonging feeding. miR-1, together with other cluster members, regulates mRNA function and may be used as a molecular marker for species origin, evolution analysis, and internal reference gene selection.
微小RNA作为mRNA转录后调节因子，在细胞分化、转录调控、生长和发育中发挥着重要作用。在本研究中，通过高通量测序检测了蜱虫不同发育阶段的微小RNA表达谱，并进行了功能评估。分别从卵、幼虫、若虫和成虫中获得了2,585,169、1,252,678、1,558,217和1,155,283条独特读段，这些阶段分别有42、46、45和41个保守微小RNA。以卵为对照，幼虫、若虫和成虫中分别有48、43和39个微小RNA上调，3、10和9个微小RNA下调。微小RNA-1（miR-1）在整个发育过程中高丰度表达，平均转录本近100万个，并且在蜱虫物种中高度保守。定量实时PCR（qPCR）显示，miR-1表达随蜱虫发育逐渐增加，饱血时达到最高水平。检测到不同组织表达存在差异，唾液腺和表皮中的水平显著高于中肠。抑制试验表明，实验组和对照组之间体重、产卵时间或产卵量无显著变化，但饱血时间存在显著差异（<0.01）。抑制miR-1后，蜱虫在后期发育过程中出现明显畸形。为了更全面地解释微小RNA的作用机制，根据靶基因对miR-1簇进行了分析；共同作用于Hsp60的成员包括miR-5、miR-994、miR-969和miR-1011。因此，微小RNA对蜱虫的正常发育至关重要，miR-1成熟序列的一级结构高度保守。尽管如此，不同发育阶段和组织表现出不同表达模式，在延长取食方面具有一定作用。miR-1与其他簇成员一起调节mRNA功能，可作为物种起源、进化分析和内参基因选择的分子标记。
