Abstract:

 【Aim】 Long non-coding RNAs (lncRNAs) play important roles in the regulation of development of the silkworm, Bombyx mori. In our previous study, a lncRNA BmlncR2036 was found near the silk fibroin gene P25 of B. mori. This study aims to further explore the molecular mechanism of BmlncR2036 regulating the expression of P25. 【Methods】 qPCR was used to detect the expression profiles of BmlncR2036 in different tissues (cuticle, brain, nerve, testis, ovary, silk gland, Malpighian tubules, hemolymph, fat body and midgut) of the day-3 5th instar larvae of B. mori and in the anterior silk gland, middle silk gland and posterior silk gland during different developmental stages of B. mori. miRNAs that can target both P25 and BmlncR2036 were predicted. The interaction between miRNA and P25 was verified by luciferase assay. Luciferase assay was used to assess the effect of BmlncR2036 on the transcription activity of P25 promoter. Finally, the expression of BmlncR2036 was knocked down in the day-3 5th instar larvae of B. mori by dsRNA injection, and phenotype changes of their silk glands and midgut were observed. 【Results】 The qPCR results showed that BmlncR2036 was highly expressed in the posterior silk glands of the day-3 5th instar larvae and mature 5th instar larvae, showing a expression pattern consistent with that of P25 gene. Meanwhile, BmlncR2036 was also highly expressed in the testis and midgut of the day-3 5th instar larvae of B. mori, while lowly expressed in other tissues, suggesting the diverse roles of BmlncR2036. miR-2739 and miR-279a can not only match with BmlncR2036, but also target the 3′UTR of P25 gene. Luciferase assay results showed that P25, however, was not the real target of miR-2739 and miR-279a, and BmlncR2036 had a negative effect on the promoter activity of P25, extremely significantly decreasing the luciferase activity by 52% after the co-transfection with pcDNA3.1(+)［BmlncR2036］ and pGL3-Enhancer［P25-promoter］ vector. After knocking down the expression of BmlncR2036 in the day-3 5th instar larvae of B. mori, the body color darkened, and a large amount of undigested food was retained in the midgut until its death, indicating that BmlncR2036 might be involved in the regulation of midgut development in B. mori. 【Conclusion】 We found that lncRNA BmlncR2036 can regulate the promoter activity of P25 gene and might also play roles in the regulation of midgut function in B. mori. This study lays the experimental foundation for further exploring the functional roles of lncRNAs in B. mori.

Key words: Bombyx mori; long non-coding RNA, silk fibroin, silk gland, metamorphosis, midgut