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

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.