关键词: 意大利蜜蜂, piRNA, piR-ame-1128833, 中肠, 卵巢, 精巢, 靶基因

Abstract: 【Aim】 This study aims to uncover the regulatory network and function of piR-ame-1128833 in the Apis mellifera ligustica adult worker’s midgut, so as to provide a basis for clarifying its action mechanism. 【Methods】 The authenticity of expression and sequence of piR-ame-1128833 in the 6-day-old adult worker’s midgut, 7-day-old adult queen’s ovary and 12-day-old adult drone’s testis of A. m. ligustica were validated by stem-loop RT-PCR and Sanger sequencing. Relevant bioinformatic software was used to predict the target mRNAs of piR-ame-1128833 and perform GO and KEGG database annotation. The expression of the key target genes Wnt-1 and LAMP-1 of piR-ame-1128833 in the 6-day-old adult worker’s midgut, 7-day-old adult queen’s ovary and 12-day-old adult drone’s testis was verified using RT-PCR and Sanger sequencing. Dual-luciferase reporter assay was conducted to validate the binding relationships between piR-ame-1128833 and the key target genes Wnt-1 and LAMP-1. After feeding the newly emerged adult workers with piR-ame-1128833 mimic (mimic-piR) and negative control (mimic-NC), RT-qPCR was employed to detect the relative expression levels of piR-ame-1128833 in the midgut and those of the key target genes Wnt-1 and LAMP-1 in the midgut after overexpression of piR-ame-1128833 of adult workers. 【Results】 piR-ame-1128833 was existed and expressed in the 6-day-old adult worker’s midgut, 7-day-old adult queen’s ovary and 12-day-old adult drone’s testis. piR-ame-1128833 targeted 3 021 mRNAs annotated to 45 GO terms such as metabolic process and catalytic activity as well as 318 KEGG pathways such as lysosome and MAPK signaling pathways. Additionally, 51 target mRNAs were involved in seven growth and development-related signaling pathways including Wnt, mTOR, Hippo, AMPK, Hedgehog, MAPK and JAK-STAT, and 53 target mRNAs were engaged in two humoral immune pathways (MAPK and JAK-STAT signaling pathways) and three cellular immune pathways (endocytosis, lysosome and phagosome). Target genes Wnt-1 and LAMP-1 were expressed in the 6-day-old adult worker’s midgut, 7-day-old adult queen’s ovary and 12-day-old adult drone’s testis. There was the binding relationship between piR-ame-1128833 and target genes Wnt-1 and LAMP-1. The expression level of piR-ame-1128833 was extremely significantly up-regulated in the 2- and 5-day-old adult workers’ midguts and significantly up-regulated in the 3- and 4-day-old adult workers’ midguts in the mimic-piR group as compared with that in the mimic-NC group. After overexpression of piR-ame-1128833, the expression level of Wnt-1 was extremely significantly down-regulated in the 3-day-old adult worker’s midgut and significantly down-regulated in the 2-, 4- and 5-day-old adult workers’ midguts, while that of LAMP-1 was extremely significantly down-regulated in the midguts of 3-, 4- and 5-day-old adult workers’ midguts, and significantly down-regulated in the 2-day-old adult worker’s midgut. 【Conclusion】 piR-ame-1128833 is expressed in the adult worker’s midgut, adult queen’s ovary and adult drone’s testes of A. m. ligustica. The efficient overexpression of piR-ame-1128833 in the adult workers’ midguts could be achieved by feeding mimic-piR, and piR-ame-1128833 plays a potential role in the adult workers’ midguts through negative regulation of the expression of Wnt-1 and LAMP-1.

Key words: Apis mellifera ligustica; piRNA, piR-ame-1128833, midgut, ovary, testis, target gene