Identification and investigation of piRNAs in the midguts of Apis cerana cerana workers
Author of the article:ZANG He1, 2, 3** ZHANG Yi-Qiong1** YANG Xue1 KANG Jing1 DONG Shu-Nan1 ZHENG Yi-Di1 XU Wen-Hua1 CHEN Da-Fu1, 2, 3 QIU Jian-Feng1, 2, 3*** GUO Rui1, 2, 3***
Author's Workplace：1. College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. National & Local United Engineering Laboratory of Natural Biotoxin, Fuzhou 350002, China; 3. Apitherapy Research Institute of Fujian Agriculture and Forestry University, Fuzhou 350002, China
Key Words： Apis cerana cerana; midgut; piRNA; identify; target gene; regulation
Abstract：[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.