摘要
自噬是生物体内维持细胞环境稳定的重要机制,在生物体面临外界刺激时会被诱导以进行适应性反应。对家蚕自噬相关蛋白BmATG6进行基因克隆,制备多克隆抗体,分析呋虫胺暴露后BmN细胞与家蚕5龄第3天幼虫BmATG6的表达变化。氨基酸序列比对表明BmATG6与鳞翅目昆虫的ATG6/Beclin 1相似度较高。原核表达并纯化了BmATG6抗原蛋白,随后接种新西兰白兔,获得效价1∶64 000的多克隆抗体,Western blot在家蚕中肠样品中检测到大小约为50 kD的条带。呋虫胺处理2 h后BmN细胞中的BmATG6蛋白表述显著上调;呋虫胺处理24 h后,家蚕5龄第3天幼虫中肠和脂肪体中的BmATG6蛋白表达上调。结果表明,呋虫胺显著诱导BmN细胞和家蚕组织中的BmATG6表达上调。研究结果为解析新烟碱杀虫剂对非靶标生物的毒性机制和研究家蚕自噬调控机制提供了理论基础。
Autophagy is an important mechanism for maintaining the stability of cellular environment in organisms,which can be induced to respond to external stimuli.In this study,we cloned the coding gene of autophagy-related protein BmATG6 of Bombyx mori,and prepared a polyclonal antibody.The expression changes of BmATG6 in BmN cell and the 3rd day of the 5th instar silkworm larvae after exposure to dinotefuran were analyzed.Amino acid sequence alignment showed that BmATG6 was highly similar to ATG6/Beclin 1 of lepidopteran insects.The BmATG6 antigen protein was expressed by prokaryotic expression and purified,and then used to immunize New Zealand white rabbits to prepare polyclonal antibody.The obtained polyclonal antibody had a titer of 1:64000.In western blot analysis,a band of about 50 kD was detected in the midgut sample of silkworm.The expression of BmATG6 protein in BmN cell was significantly up-regulated after treatment with dinotefuran for 2 h.The expression of BmATG6 protein in the midgut and fat body of the 3rd day of the 5th instar silkworm larvae was up-regulated after 24 h of dinotefuran treatment.The results demonstrated that dinotefuran significantly induced the expression of Bm-ATG6 in BmN cells and silkworm organs.This study provides a theoretical basis for understanding the toxicity mechanism of neonicotinoid insecticides to non-target or ganisms and the regulation mechanism of autophagy in sikworm.
作者
许诗亮
郝志华
马焕艳
李颖慧
荆文辉
徐豫松
王华兵
Xu Shiliang;Hao Zhihua;Ma Huanyan;Li Yinghui;Jing Wenhui;Xu Yusong;Wang Huabing(College of Animal Sciences,Zhejiang University,Hangzhou 310058,China;Extension Center for Agricultural Technology of Zhejiang Province,Hangzhou 310020,China)
出处
《蚕业科学》
CAS
CSCD
北大核心
2023年第1期45-50,共6页
ACTA SERICOLOGICA SINICA
基金
浙江省自然科学基金项目(LR22C040001)
浙江省科技厅项目(LGN19C170002,2021C02072-6)
浙江省“三农九方”科技协作计划项目(2022SNJF043)。
