昆虫学报2021，Vol.64Issue(7)：809-816,8.
电穿孔法可用于家蚕基因功能分析
Electroporation can be used to explore the gene function in the silkworm,Bombyx mori
摘要
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-34th 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-34th 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.关键词
家蚕/电穿孔/转基因/Wnt1/RNAi/基因功能Key words
Bombyx mori/electroporation/transgenesis/Wnt1/RNAi/gene function分类
生物科学引用本文复制引用
周文林,藤原晴彦,上村望,叶爱红,武雪会,陈学东,张婷婷,曹锦如..电穿孔法可用于家蚕基因功能分析[J].昆虫学报,2021,64(7):809-816,8.基金项目
浙江省重点研发计划(2019C02044) （2019C02044）
浙江省农业(畜禽)新品种选育重大科技专项(2016C02054-17) （畜禽）
浙江省蚕蜂资源利用与创新研究重点实验室(2020E10025) （2020E10025）
国家蚕桑产业技术体系(CARS-18-SYZ05) （CARS-18-SYZ05）
