关键词: 中华蜜蜂, Mucin5AC, 原核表达, RNAi, 组织定位

Abstract: 【Aim】 The purpose of this study is to identify the structure, distribution and effect of intestinal mucin AcMucin5AC-1 on the midgut of Apis cerana cerana, so as to provide a theoretical basis for analyzing the physiological function of the honeybee midgut. 【Methods】 Bioinformatics was used to compare and analyze the sequence characteristics of Mucin5AC protein in A. cerana cerana. RT-qPCR was used to detect the expression levels of AcMucin5AC-1 in the midgut and cuticle of the 4-day-old larva of A. cerana cerana and in the midgut of the 2-day-old larva of A. cerana cerana at 0, 12， 24， 48 and 72 h after infection with Chinese sacbrood virus (CSBV). The prokaryotic expression system was used to express AcMucin5AC-1. The recombinant AcMucin5AC-1 was purified by Ni²⁺ affinity column and the polyclonal antibody to AcMucin5AC-1 was prepared. Western blot was used to detect the expression of AcMucin5AC-1 in the 3－6-day-old larvae, and the midgut, cuticle and peritrophic membrane of the 4-day-old larva of A. cerana cerana. Using indirect immunofluorescence assay, the localization of AcMucin5AC-1 in the 4-day-old larva of A. cerana cerana was analyzed. The interference efficiency of RNAi was analyzed at 12， 24， 48 and 72 h after silencing AcMucin5AC-1 in the 2-day-old A. cerana cerana larva by RNAi through feeding method. Hematoxylin-eosin (HE) staining method was used to explore the effects at 24, 48 and 72 h after silencing AcMucin5AC-1 in the 2-day-old larva by RNAi on the overall histomorphology of A. cerana cerana larva.【Results】 There are eight AcMucin5AC genes in the genome of A. cerana cerana, and their amino acid sequences all contain mucin domains. The results of RT-qPCR showed that the expression level of AcMucin5AC-1 was higher in the midgut of the 4-day-old larva of A. cerana cerana than in the cuticle, and was significantly down-regulated in the midgut of the 2-day-old larva at 12 and 72 h after infection with CSBV as compared to that in the control group. The recombinant protein AcMucin5AC-1 about 50 kD and polyclonal antibody were successfully obtained. Western blot result showed that AcMucin5AC-1 could be detected in the total protein of the 3－6-day-old larvae, and the midgut, cuticle and peritrophic membrane of the 4-day-old larva of A. cerana cerana. Indirect immunofluorescence experiment result showed that AcMucin5AC-1 was mainly localized in the midgut and peritrophic membrane of the 4-day-old larva. The detection result of RNAi efficiency showed that the expression level of AcMucin5AC-1 in A. cerana cerana was down-regulated by 92% at 24 h by 2.0 μg/individual dsAcMucin5AC-1 compared with the dsEGFP control group. HE staining detection result showed that the compactness between cells of the whole intestinal lumen of A. cerana cerana larva was weakened, and the morphological structure was disordered at 72 h after RNAi. 【Conclusion】 AcMucin5AC-1 is characterized as a mucin located in the midgut and peritrophic membrane of A.cerana cerana larvae. Down-regulation of AcMucin5AC-1 affects the morphology of the midgut of A. cerana cerana larva, suggesting that this gene may play an important role in the development of the A. cerana cerana larval midgut.

Key words: Apis cerana cerana; Mucin5AC, prokaryotic expression, RNAi, tissue localization