本文選題：家蠶　切入點(diǎn)：白僵菌　出處：《江蘇科技大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：白僵菌是家蠶重要的病原真菌,其感染家蠶引起的白僵病,往往對蠶桑業(yè)的生產(chǎn)造成巨大損失。目前,包括白僵菌在內(nèi)的真菌對家蠶的作用機(jī)理尚不明確。因此開展家蠶抗菌肽enbocin1在抵御白僵菌感染過程中的功能研究,可解析家蠶對白僵菌感染免疫應(yīng)答反應(yīng)的分子機(jī)理,為從分子水平上闡明家蠶對白僵菌感染的防御機(jī)制和白僵菌對家蠶的致病機(jī)理提供新的實(shí)驗(yàn)依據(jù)。用白僵菌分生孢子體表接觸感染家蠶P50五齡起蠶,隨機(jī)收集感染組和對照組幼蟲在感染后10個(gè)不同時(shí)間點(diǎn)的血淋巴、體壁、馬氏管、脂肪體。用real-time PCR檢測了enbocin1基因在血淋巴、體壁、馬氏管、脂肪體組織的時(shí)空表達(dá)模式,結(jié)果顯示enbocin1基因主要在脂肪體中上調(diào)表達(dá)明顯,而且上調(diào)表達(dá)持續(xù)至誘導(dǎo)后30h,而在馬氏管、體壁中差異表達(dá)不顯著,在血淋巴中幾乎檢測不到enbocin1的表達(dá),表明enbocin1對家蠶抵御白僵菌感染可能具有重要作用。構(gòu)建了重組表達(dá)載體PET-30a(+)-Enbocin1,轉(zhuǎn)化感受態(tài)大腸桿菌BL21(DE3),成功地誘導(dǎo)表達(dá)并用Ni-NTA親和層析純化獲得了高純度的重組蛋白enbocin1,經(jīng)SDS-PAGE檢測蛋白大小與預(yù)期的一致,約10.8kDa。測得重組蛋白濃度為10mg/mL。經(jīng)抑菌試驗(yàn)表明其對白僵菌的生長具有較強(qiáng)抑制作用。成功制備了抗enbocin1的多克隆抗體,可用于檢測enbocin1的表達(dá),為進(jìn)一步深入研究enbocin1的功能及其機(jī)制奠定了基礎(chǔ)。本研究可為闡明其他類型昆蟲真菌致病的分子機(jī)制提供有益參考,并為開發(fā)家蠶白僵病防治新方法提供了理論參考。同時(shí)也為進(jìn)一步研究家蠶enbocin基因的功能奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S884.31

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