家蚕微孢子虫Ubc9蛋白的基因克隆与功能研究  

Gene Cloning and Functional Study of Ubc9 Protein from Nosema bombycis

作　　者：吕丁丁 沈瑞[2] 孙笑笑 祝慧婷 唐旭东 Lyu Dingding;Shen Rui;Sun Xiaoxiao;Zhu Huiting;Tang Xudong(Nursing School,Zhenjiang College,Zhenjiang Jiangsu 212028,China;Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agriculture and Rural Affairs,Zhenjiang Jiangsu 212100,China)

机构地区：[1]镇江市高等专科学校卫生护理学院,江苏镇江212028 [2]中国农业科学院蚕业研究所农业农村部蚕桑遗传改良重点实验室,江苏镇江212100

出　　处：《蚕业科学》2023年第6期515-526,共12页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(31972621);农业农村部蚕桑遗传改良重点实验室开放课题(KL202210);江苏省高校“青蓝工程”项目;江苏省高职院校教师专业带头人高端研修项目(2023TDFX013);镇江市科技创新资金项目(NY2023009,FZ2023118)。

摘　　要：SUMO(small ubiquitin-like modifier)修饰是一种重要的蛋白质翻译后修饰,Ubc9(ubiquitin-conjugating enzyme)是SUMO修饰靶蛋白过程中唯一的E2结合酶,在SUMO修饰过程中发挥关键作用。本研究克隆了家蚕微孢子虫(Nosema bombycis,Nb)NbUbc9基因的完整开放阅读框。序列分析表明,NbUbc9与所有来源于微孢子虫属(Nosema)的Ubc9聚类在同一大分支上,与同为Nosema属的西方蜜蜂微孢子虫(Nosema apis)和东方蜜蜂微孢子虫(Nosema ceranae)Ubc9同源性最高,说明NbUbc9蛋白在进化上高度保守。qRT-PCR分析表明,家蚕内源性Ubc9的表达量在Nb感染后有所下降,而Nb自身NbUbc9的表达显著增加。对NbUbc9基因RNAi后,Nb基因组DNA的复制水平显著下降,说明NbUbc9在Nb的细胞增殖中起着关键作用。经Pull-down和质谱分析,初步鉴定出有46个家蚕蛋白和8个Nb蛋白可能与NbUbc9相互作用,涉及蛋白质折叠与转运、去泛素化、信号转导等。研究结果为进一步研究NbUbc9在家蚕微孢子增殖中的作用提供了参考,也为开发微粒子病防治药物提供了新的方向。SUMO(small ubiquitin-like modifier) is an important post-translational modification protein. Ubc9(ubiquitin-conjugating enzyme)is the only E2 binding enzyme that plays a key role in the process of SUMO modification. In this study, the full-length open reading frame of NbUbc9 of Nosema bombycis(Nb) was cloned. Sequence analysis showed that NbUbc9 and all Ubc9 from Microsporidia were clustered on the same large branch and had the highest homology with Ubc9 from Nosema apis and Nosema ceranae, indicating that the sequence of NbUbc9 was highly conserved in evolution. qRT-PCR analysis showed that the expression of endogenous BmUbc9 decreased after Nb infection, while the expression of NbUbc9 increased significantly. The DNA copy number of Nb genome decreased significantly after NbUbc9 was knocked down by RNAi, indicating that NbUbc9 was critical for Nb proliferation. Pull-down and mass spectrometry analysis found that 46 silkworm proteins and 8 Nb proteins may interacted with NbUbc9, involving protein folding and transport, de-ubiquitination, signal transduction, etc. The results lay the foundation for further study on how NbUbc9 regulates the proliferation of Nosema bombycis, and also provide a new direction for the drug development of pebrine disease of Bombyx mori.

关 键 词：家蚕微孢子虫 类泛素化修饰 UBC9 表达分析 RNAI 

分 类 号：S884.21[农业科学—特种经济动物饲养]