关键词: 家蚕, 电穿孔, 转基因, Wnt1, RNAi, 基因功能

Abstract:

【Aim】 To confirm the effectiveness of electroporation-mediated functional analysis system in the silkworm, Bombyx mori. 【Methods】 siRNAs were synthesized for the target gene Wnt1 (Wingless), which is known to be involved in larval melanin coloration in B. mori. The day-3 4th instar larvae of B. mori were injected with Wnt1 siRNAs and subjected to electroporation as the treatment group (ERFA-RNAi) and those injected with Wnt1 siRNAs but without subjected to electroporation were used as the negative control group, the epidermis of the corresponding speckled area of the 5th instar larvae was dissected, and the relative expression level of Wnt1 in the epidermis was detected with real-time quantitative RT-PCR (qRT-PCR) to verify the effect of electroporationmediated RNAi. The transposon vector pPIG-A3GR with the enhanced green fluorescent protein reporter gene (EGFP) and the red florescence protein (RFP) reporter gene (DsRed2) expression cassettes, was introduced into the 2nd instar larvae of B. mori by electroporation. After 72 h of normal rearing, the expression of EGFP and DsRed2 in the larvae was observed under a fluorescent stereo microscope, to verify the somatic transgenesis of the silkworm. 【Results】 After the introduction of Wnt1 siRNAs into the day-3 4th instar larvae of B. mori, the formation of a speckle pattern of the 5th instar larvae was prevented on the larval body surface, and the qRT-PCR analysis showed that the expression level of Wnt1 in the epidermis of the 5th instar larvae was significantly decreased. The positive rate of somatic transgenic silkworm was 56.60%, and two fluorescent reporter genes EGFP and DsRed2 were continuously expressed in larval, pupal and adult stages. 【Conclusion】 Electroporation is an efficient technology for exploration of gene function in vivo, by efficiently introducing exogenous RNA or DNA into silkworm.

Key words: Bombyx mori; electroporation, transgenesis, Wnt1, RNAi, gene function