抗菌肽Enbocin 1基因在家蚕抵御白僵菌感染过程中表达特征及其抗真菌活性

发布时间：2018-03-07 20:28

本文选题：家蚕　切入点：白僵菌　出处：《江苏科技大学》2016年硕士论文　论文类型：学位论文

【摘要】：白僵菌是家蚕重要的病原真菌,其感染家蚕引起的白僵病,往往对蚕桑业的生产造成巨大损失。目前,包括白僵菌在内的真菌对家蚕的作用机理尚不明确。因此开展家蚕抗菌肽enbocin1在抵御白僵菌感染过程中的功能研究,可解析家蚕对白僵菌感染免疫应答反应的分子机理,为从分子水平上阐明家蚕对白僵菌感染的防御机制和白僵菌对家蚕的致病机理提供新的实验依据。用白僵菌分生孢子体表接触感染家蚕P50五龄起蚕,随机收集感染组和对照组幼虫在感染后10个不同时间点的血淋巴、体壁、马氏管、脂肪体。用real-time PCR检测了enbocin1基因在血淋巴、体壁、马氏管、脂肪体组织的时空表达模式,结果显示enbocin1基因主要在脂肪体中上调表达明显,而且上调表达持续至诱导后30h,而在马氏管、体壁中差异表达不显著,在血淋巴中几乎检测不到enbocin1的表达,表明enbocin1对家蚕抵御白僵菌感染可能具有重要作用。构建了重组表达载体PET-30a(+)-Enbocin1,转化感受态大肠杆菌BL21(DE3),成功地诱导表达并用Ni-NTA亲和层析纯化获得了高纯度的重组蛋白enbocin1,经SDS-PAGE检测蛋白大小与预期的一致,约10.8kDa。测得重组蛋白浓度为10mg/mL。经抑菌试验表明其对白僵菌的生长具有较强抑制作用。成功制备了抗enbocin1的多克隆抗体,可用于检测enbocin1的表达,为进一步深入研究enbocin1的功能及其机制奠定了基础。本研究可为阐明其他类型昆虫真菌致病的分子机制提供有益参考,并为开发家蚕白僵病防治新方法提供了理论参考。同时也为进一步研究家蚕enbocin基因的功能奠定基础。
[Abstract]:Beauveria bassiana is an important pathogenic fungus of Bombyx mori. The white stiffness caused by infection of Bombyx mori often causes a great loss to the production of sericulture. At present, The action mechanism of fungi, including Beauveria bassiana, on Bombyx mori is not clear. Therefore, the study on the function of silkworm antimicrobial peptide enbocin1 in the process of resisting the infection of Beauveria bassiana can explain the molecular mechanism of the immune response of Bombyx mori to the infection of Beauveria bassiana. In order to elucidate the defense mechanism of Bombyx mori against Beauveria bassiana infection and the pathogenic mechanism of Beauveria bassiana to Bombyx mori at molecular level, the silkworm P50 was infected by surface contact with conidia of Beauveria bassiana. Hemolymph, body wall, Markov tube and fat body were collected randomly from infected group and control group at 10 different time points after infection. The expression patterns of enbocin1 gene in hemolymph, body wall, Markov duct and adipose tissue were detected by real-time PCR. The results showed that the expression of enbocin1 gene was mainly up-regulated in the fat body and continued until 30 hours after induction. However, the differential expression of enbocin1 gene was not significant in the Markov tube and the body wall, and the expression of enbocin1 was almost not detected in the hemolymph. It is suggested that enbocin1 may play an important role in silkworm resistance to Beauveria bassiana infection. A recombinant expression vector PET-30a (-Enbocin1) was constructed, which was transformed into the competent E. coli BL21DE-3. The recombinant protein was successfully induced and purified by Ni-NTA affinity chromatography. Enbocin1, the size of the protein detected by SDS-PAGE was the same as expected. About 10.8kDa. the concentration of recombinant protein was 10 mg / mL. The bacteriostatic test showed that the recombinant protein had a strong inhibitory effect on the growth of Beauveria bassiana. The polyclonal antibody against enbocin1 was successfully prepared, which can be used to detect the expression of enbocin1. This study may provide a useful reference for elucidating the molecular mechanisms of other insect fungi, which may contribute to the further study of the function and mechanism of enbocin1. It provides a theoretical reference for the development of a new method for the prevention and control of Bombyx mori white stiffness, and lays a foundation for the further study of the function of enbocin gene in silkworm, Bombyx mori.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S884.31

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