蜕皮激素氧化酶基因在家蚕丝腺中的表达特征及重组表达分析  

Expression Analysis and Recombinant Expression of Ecdysone Oxidase of Silk Gland in Silkworm

作　　者：刘雪冰 李珊[1] 张基开朗 杨宏国 程廷才[1,2] 刘春 LIU Xue-bing;LI Shan;ZHANG Ji-kailang;YANG Hong-guo;CHENG Ting-cai;LIU Chun(State Key Laboratory of Resource Insects,Chongqing 400716,China;Chongqing Engineering and Technology Research Center for Silk Biomaterials and Regenerative Medicine,Chongqing 400716,China)

机构地区：[1]资源昆虫高效养殖与利用全国重点实验室(西南大学),重庆400716 [2]重庆市蚕丝生物材料与再生医学工程技术中心,重庆400716

出　　处：《蚕学通讯》2023年第2期1-9,共9页Newsletter of Sericultural Science

基　　金：国家自然科学基金面上项目(31872296);重庆市英才计划(cstc2021ycjh-bgzxm0048)。

摘　　要：昆虫蜕皮激素氧化酶(EO)是分解蜕皮激素的关键酶之一,该酶通过蜕皮激素3位异构化途径将蜕皮激素分解成3异构化蜕皮激素,从而调控蜕皮激素的滴度。课题组前期通过家蚕转录组学分析发现一个在丝腺组织特异表达的蜕皮激素氧化酶基因(EOs),为了研究该基因的时空表达模式及蛋白质功能,对丝腺蜕皮激素氧化酶基因的表达谱进行了分析,同时利用原核表达系统对其进行重组表达及分离纯化。RT-PCR和qRT-PCR检测表明,EOs在家蚕幼虫5龄期的丝腺组织特异表达,且主要在中部丝腺的前部表达;免疫荧光实验结果表明,EO不仅存在于家蚕中部丝腺细胞,而且还会分泌到丝腺腺腔中。将EOs全长cDNA克隆到pMD-19T载体中,再进一步亚克隆到原核表达载体PET-28a,然后将构建好的pET28-BmEOs质粒转入大肠埃希菌BL21中进行表达,通过SDS-PAGE检测发现在1mmol/LIPTG诱导后的上清中有一条大小约60kD的蛋白质条带,经Westernblot检测该蛋白质可与抗体发生特异性结合,表明BmEOs被成功表达。将大量上清经过Ni亲和柱纯化后,获得了纯化的目的蛋白质。研究结果为进一步分析丝腺中蜕皮激素氧化酶基因及蛋白质的功能奠定了基础。Ecdysone oxidase(EO)is one of the key enzymes in the degradation of ecdysone,which can convert ecdysone into 3-isomerized ecdysone through the 3-isomerized pathway,and then regulate the titer of ecdysone.In order to study the temporal and spatial expression pattern and protein function of ecdysone oxidase gene,the expression profile of ecdysone oxidase gene was analyzed,and the recombinant expression and purification of ecdysone oxidase gene were carried out by prokaryotic expression system.RT-PCR and RT-qPCR analysis showed that EOs were specifically expressed in the silk glands of the 5th instar larvae and mainly in the anterior part of the middle silk gland.The results of immunofluorescence showed that EO was not only present in the middle silk gland cells,but also secreted into the lumen of the silk gland.In order to obtain the protein product,the full-length cDNA of EOs was cloned into pMD-19T vector,and then subcloned into prokaryotic expression vector PET-28a.The constructed pET28-BmEOs plasmid was transformed into BL21 E.coli for expression.A protein band of about 60 kD was found in the supernatant induced by 1 mmol/L IPTG.After a large amount of supernatant was purified by Ni affinity column,the target protein was obtained.These results lay a foundation for further study on the function of ecdysone oxidase gene and protein in silk gland.

关 键 词：家蚕 蜕皮激素氧化酶 丝腺 基因表达 免疫荧光定位 原核表达 蛋白质纯化 

分 类 号：S881.2[农业科学—特种经济动物饲养]