关键词: 家蚕, 变态发育, 蜕皮激素, miRNAs, 靶基因, qRT-PCR, 表达分析

Abstract: 【Aim】 MiRNAs play very important roles in insect metamorphosis. The study of miRNAs and their target genes in Bombyx mori will help to elucidate the molecular mechanism of miRNAs involved in the metamorphosis of this insect. 【Methods】 The expression of miR-2769 in the fat body of B. mori was detected by qRT-PCR after injecting molting hormone, 20-hydroxyecdysone (20E), into the hemolymph of the day-2 5th instar larvae. The target genes of miR-2769 of B. mori were predicted by bioinformatics methods. The interaction between miR-2769 and its predicted target gene BmE75B was analyzed by dual luciferase reporter assay system. The expression levels of miR-2769 and different splice isoforms of its target gene BmE75 at different developmental stages (larva, pupa and adult) and larval tissues (head, epidermis, silk gland, fat body, testis, ovary, Malpighian tubules, midgut and hemolymph) of B. mori were detected by qRT-PCR. 【Results】 The results showed that miR-2769 could significantly inhibit the expression of luciferase reporter gene by interacting with the 3′UTR binding site of BmE75B of B. mori. The qRT-PCR results showed that miR-2769 and BmE75A/BmE75B presented the opposite expression trends in the fat body of B. mori post induction by 20E. The spatio-temporal expression analysis showed that miR-2769 and different splice isoforms of the target gene BmE75 had specific expression characteristics in different developmental stages and larval tissues of B. mori. The expression levels of miR-2769 and BmE75A were low in B. mori at different developmental stages; however, the expression level of BmE75B was relatively high in the pupal stage and that of BmE75C was extremely high in the late 5th instar larvae and early pupae. Furthermore, the expression of miR-2769 was negatively correlated with that of different splice isoforms of BmE75. miR-2769 promoted the expression of BmE75A in the hemolymph and BmE75B in the fat body of the 5th instar larvae of B. mori, while it inhibited the expression of different splice isoforms of BmE75 in other tissues. 【Conclusion】 MiR-2769 can negatively regulate the expression of different splice isoforms of BmE75 by interacting with the 3′UTR region of E75B in B. mori.

Key words: Bombyx mori, metamorphosis, molting hormone, miRNAs, target gene, qRT-PCR, expression analysis