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

作　　者：周文林[1] 藤原晴彦 上村望 叶爱红[1] 武雪会 陈学东 张婷婷 曹锦如[1] ZHOU Wen-Lin;Haruhiko FUJIWARA;Nozomi UEMURA;YE Ai-Hong;WU Xue-Hui;CHEN Xue-Dong;ZHANG Ting-Ting;CAO Jin-Ru(Institute of Sericulture and Tea,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Department of Integrated Biosciences,Graduate School of Frontier Sciences,The University of Tokyo,Kashiwa,Chiba 277-8562,Japan)

机构地区：[1]浙江省农业科学院蚕桑与茶叶研究所,杭州310021 [2]东京大学大学院新领域创成科学研究科先端生命科学专攻,日本千叶县柏市277-8562

出　　处：《昆虫学报》2021年第7期809-816,共8页Acta Entomologica Sinica

基　　金：浙江省重点研发计划(2019C02044);浙江省农业(畜禽)新品种选育重大科技专项(2016C02054-17);浙江省蚕蜂资源利用与创新研究重点实验室(2020E10025);国家蚕桑产业技术体系(CARS-18-SYZ05)。

摘　　要：【目的】明确基于电穿孔的基因功能分析方法在家蚕Bombyx mori活体内的应用实效。【方法】针对调控家蚕幼虫体表斑纹黑色素合成的靶基因Wnt1(Wingless),人工合成特异性siRNA,向4龄第3天家蚕幼虫注射Wnt1 siRNA并进行电穿孔作为处理组(ERFA-RNAi),以注射Wnt1 siRNA但未进行电穿孔的幼虫作为阴性对照组(negative control,NC),解剖5龄幼虫斑纹区的表皮,用实时定量RT-PCR测定表皮中Wnt1的相对表达量,验证电穿孔介导的RNAi效果。带有增强型绿色荧光蛋白报告基因EGFP和红色荧光蛋白报告基因DsRed2表达元件的转座子载体pPIG-A3GR,通过电穿孔导入家蚕2龄幼虫;正常饲养72 h后,用荧光体视显微镜观察幼虫中EGFP和DsRed2的表达,验证当世代家蚕的嵌合体转基因。【结果】家蚕4龄第3天幼虫中导入Wnt1的特异性siRNA后,5龄幼虫体表特定部位斑纹的形成明显受到抑制,实时定量RT-PCR分析表明5龄幼虫表皮中Wnt1的表达水平显著降低。嵌合体转基因家蚕的阳性率达56.60%,并且两个荧光报告基因EGFP和DsRed2在幼虫、蛹及成虫期持续表达。【结论】电穿孔技术可快捷、高效地向家蚕活体内导入外源RNA或DNA,是家蚕乃至其他昆虫基因功能解析的有效方法。【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 

分 类 号：Q966[生物学—昆虫学]