家蚕追寄蝇虾红素样精液蛋白酶EsSemp的基因克隆与特性分析  

Gene Cloning and Characteristic Analysis of the Seminal Astacin-Like Proteinase EsSemp from Exorista sorbillans

作　　者：王耒耒 王闪闪 浦月霞 刘吉银 王梅仙 朱娟 沈兴家[1,3] 沈中元 唐顺明[1,3] Wang Leilei;Wang Shanshan;Pu Yuexia;Liu Jiyin;Wang Meixian;Zhu Juan;Shen Xingjia;Shen Zhongyuan;Tang Shunming(Jiangsu Key Laboratory of Sericultural and Animal Biotechnology,School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212100,China;Guangxi General Station for Sericulture Technology Popularization,Nanning,530007,China;Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agriculture and Rural Affairs,Sericultural Scientific Research Center,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212100,China)

机构地区：[1]江苏科技大学生物技术学院,江苏省蚕桑与畜禽生物技术重点实验室,江苏镇江212100 [2]广西壮族自治区蚕业技术推广站,南宁530007 [3]农业农村部蚕桑遗传改良重点实验室,中国农业科学院桑蚕科学研究中心,江苏镇江212100

出　　处：《蚕业科学》2024年第3期222-231,共10页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(32072791,31372376);农业农村部蚕桑遗传改良重点实验室开放课题(KL202209);国家蚕桑产业技术体系蚕用兽药研制团队项目(CARS-18-ZJ0303);江苏省研究生科研与实践创新计划项目(SJCX22_1994)。

摘　　要：虾红素样金属蛋白酶广泛存在于各种昆虫中,参与受精繁殖、孵化及消化等生理功能。为鉴定家蚕追寄蝇生殖繁育相关基因,利用RACE技术克隆获得家蚕追寄蝇虾红素样精液蛋白酶基因全长cDNA序列,命名为EsSemp(GenBank登录号:OP777491),其全长为985 bp,ORF为771 bp,编码256个氨基酸残基。以染色体步移技术克隆获得EsSemp完整基因结构,由1个内含子和2个外显子组成。系统发生树结果显示,EsSemp与黑腹果蝇(Drosophila melanogaster)精液蛋白酶基因副本的产物CG15254高度同源。qRT-PCR结果显示,EsSemp在雄蝇副性腺表达较弱,在蝇蛆和成蝇的中肠和马氏管表达较强,在卵期0 h和卵后期大量表达,推测EsSemp参与了受精、孵化与消化代谢等过程。体外表达EsSemp蛋白,结果显示EDTA可抑制EsSemp酶活。以上研究结果为进一步阐明家蚕追寄蝇繁殖发育相关分子机制提供了参考,为开发家蚕蝇蛆病防治新途径提供了基础信息。Astacin-like metalloproteinases widely exist in various insects and are involved in physiological functions such as fertilization,reproduction,hatching and diges-tion.In order to identify the genes related to the reproduction of Exorista sorbillans,the full-length cDNA sequences of seminal astacin-like proteinase gene was cloned by RACE technology,and named as EsSemp(GenBank:OP777491),with cDNA lengths of 985 bp and ORF of 771 bp,encoding 256 amino acid residues.Complete gene structure of EsSemp was obtained by using genome walking technology,consisting of one intron and two exons.Phylogenetic tree analysis showed that EsSemp is highly homologous to CG15254,the product the seminal protease gene copy of the Drosophila melanogaster.The qRT-PCR results showed that EsSemp was weakly expressed in the male accessory gonad,highly expressed in the midgut and malpighian tubule of maggots and adult flies,while expressed abundantly at 0 h of egg stage and late egg stage,suggesting that EsSemp may be involved in fertilization,hatching and digestion metabolism.The EsSemp protein was expressed in vitro,and the results showed that EDTA can inhibit its enzyme activity.The above findings provide a reference for further elucidating the molecular mechanism related to the reproduction and development of E.sorbillans,and provide basic information for developing ways to control silkworm myiasis.

关 键 词：家蚕追寄蝇 虾红素样蛋白酶基因 RACE 繁殖发育 蛋白质表达 

分 类 号：S884.62[农业科学—特种经济动物饲养]