中华蜜蜂工蜂中肠中piRNA的鉴定与分析
Identification and investigation of piRNAs in the midguts of Apis cerana cerana workers
臧 贺1, 2, 3** 张艺琼1** 杨 雪1 康 婧1 董舒楠1 郑一荻1 徐文华1 陈大福1, 2, 3 邱剑丰1, 2, 3*** 郭 睿1, 2, 3***
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DOI：10.7679/j.issn.2095-1353.2026.079
作者单位：1. 福建农林大学蜂学学院，福州 350002；2. 天然生物毒素国家地方联合工程实验室，福州 350002；3. 福建农林大学蜂疗研究所，福州 350002
中文关键词：中华蜜蜂；中肠；piRNA；鉴定；靶基因；调控
英文关键词： Apis cerana cerana; midgut; piRNA; identify; target gene; regulation
中文摘要：【目的】 本研究旨在通过鉴定和分析中华蜜蜂Apis cerana cerana工蜂中肠的piRNA，为深入开展中华蜜蜂piRNA的调控功能与作用机制研究提供资源和基础。【方法】 基于已获得的small RNA-seq数据鉴定中华蜜蜂7（Ac7）和10（Ac10）日龄工蜂中肠中的piRNA，再进行数量统计和结构特征分析。采用每百万标签序列（Tags per million, TPM）法计算piRNA的表达量并进行归一化。根据|log2 fold change| ≥1且P≤0.05的标准筛选差异表达piRNA (Differentially expressed piRNA, DEpiRNA)。使用相关软件预测DEpiRNA的靶mRNA并进行GO和KEGG数据库注释。采用stem-loop RT-PCR验证piRNA的表达。通过RT-qPCR验证测序数据的可靠性。【结果】 在Ac7和Ac10组中分别鉴定到534和495个piRNA，包括602个非冗余piRNA。上述两组中piRNA的长度分布均介于24-34 nt，分布在27 nt的piRNA数量最多，且首位碱基均表现出碱基C倾向。在Ac7 vs Ac10比较组中筛选到2个DEpiRNA，共靶向472个mRNA，可注释到生物调节、催化活性和细胞器等26个GO功能条目及内吞作用、Wnt信号通路和JAK-STAT信号通路等167条KEGG通路。有27和11条靶mRNA分别涉及到7条发育相关信号通路（Wnt, Notch, mTOR, AMPK, Hippo, TGF-beta和Hedgehog）和8条免疫通路（内吞作用、溶酶体、吞噬体、MAPK、PI3K-Akt、JAK-STAT、TGF-beta和FoxO）。Stem-loop RT-PCR结果显示随机选择的7个piRNA在工蜂中肠中均真实表达。RT-qPCR结果显示2个DEpiRNA的表达模式与前期测序结果一致，说明测序数据可靠。【结论】 中华蜜蜂工蜂中肠中表达的602个piRNA具有类似于其他动物piRNA的结构特征，通过靶向调控相关基因，具有参与调控工蜂中肠发育和免疫的潜力。
英文摘要：[Aim] To identify and analyze piRNAs in the midgut of Apis cerana cerana workers, thereby providing a foundation for the in-depth study of the regulatory function and mechanism of piRNAs in this species. [Methods] piRNAs in the midgut of 7- and 10-day-old A. c. cerana workers (Ac7 and Ac10) were identified based on the available small RNA-seq data, and their quantitative statistics and structural characteristics analyzed. The expression levels of piRNAs were calculated and normalized using the tag per million (TPM) method. Differentially expressed piRNAs (DEpiRNAs) were screened following the criteria of |log2 fold change| ≥ 1 and P ≤ 0.05. Relevant software was employed to predict target mRNAs of DEpiRNAs followed by GO and KEGG database annotation. The expression of piRNAs was validated using stem-loop RT-PCR. The expression trends of DEpiRNA were confirmed by RT-qPCR. [Results] A total of 534 and 495 piRNAs were discovered in the Ac7 and Ac10 groups, respectively, including 602 non-redundant ones. The length distribution of piRNAs in the above-mentioned two groups ranged between 24-34 nt, with most being 27 nt in length. The first base is biased toward C. Two DEpiRNAs were screened in the Ac7 vs Ac10 comparison group, targeting a total of 472 mRNAs, which could be annotated to 26 GO terms, including biological regulation, catalytic activity and organelle, and 167 KEGG pathways such as endocytosis, Wnt signaling pathway and JAK-STAT signaling pathway. There were 27 and 11 target mRNAs, respectively, involved in seven developmental signaling pathways (Wnt, Notch, mTOR, AMPK, Hippo, TGF-beta and Hedgehog), and eight immune pathways (endocytosis, lysosome, phagosome, MAPK, PI3K-Akt, JAK-STAT, TGF-beta and FoxO). The stem-loop RT-PCR results demonstrate that seven randomly selected piRNAs were actually expressed in the midgut of A. c. cerana workers. The results of RT-qPCR indicate that expression of the two DEpiRNAs is consistent with that observed in the sequencing data, confirming the latter’s reliability. [Conclusion] The 602 piRNAs expressed in the midgut of the A. c. cerana workers have structural features analogous to those of other animals. These piRNAs are potentially engaged in the development and immunity of the midgut through regulating the expression of related genes.