基因Ran与溴氰菊酯耐药性关系的初步研究

发布时间：2018-12-26 10:52

【摘要】：拟除虫菊酯类农药是一类合成杀虫剂,具有高效和广谱性杀虫作用,在自然环境下容易分解,对哺乳动物毒性较低,因此被广泛使用,从而导致害虫对其产生了越来越严重的抗药性。昆虫抗药性机制涉及大量基因,因此发现和鉴定抗药性基因对害虫的防治有着关键作用。我们以前的研究表明,融合基因UBL40是一种溴氰菊酯抗药性相关基因,在另一项研究中,我们发现核糖体蛋白L40与溴氰菊酯抗性关系密切,因此我们推测核仁蛋白在溴氰菊酯抗性中发挥一定的作用。为进一步探索细胞核基因和溴氰菊酯抗性之间的关系,我们使用蛋白质双向电泳和质谱技术对核仁蛋白进行了分析。结果显示,细胞核基因Ran在溴氰菊酯处理后表达量显著提高。这意味Ran可能是参与溴氰菊酯应激反应的基因。已有很多的研究报道,三磷酸鸟苷(GTP)结合蛋白Ran在核运输、染色体定位、微管动力学、中心体复制、动粒微管的附件、核膜动力学和吞噬作用等具有重要作用。然而,直到现在,还没有报告显示Ran参与了溴氰菊酯抗药性。本试验的蛋白质双向电泳结果表明,在果蝇Kc细胞中,溴氰菊酯处理组Ran基因的表达水平显著高于对照组。此外,使用实时荧光定量PCR定量分析显示,在不同浓度的溴氰菊酯处理下Ran的表达量均表现出明显的上调,免疫印迹结果分析表明,30 ppm溴氰菊酯处理48 h, Ran的表达量是对照组的2.27倍。为进一步验证Ran在应激反应的作用,我们利用dsRNA干扰技术和细胞转染技术对Ran基因在溴氰菊酯应激中的作用进行验证。结果显示,细胞过表达Ran后,对细胞有一定程度的保护作用,而干扰Ran的表达后,细胞在一定程度上显著增加了对溴氰菊酯的敏感性。综合上述的研究结果,我们认为Ran参与溴氰菊酯的应激反应,并进而与溴氰菊酯抗性的形成相关。但是Ran能否作为溴氰菊酯抗性的靶标基因尚需进一步研究与证实。
[Abstract]:Pyrethroid pesticides are a class of synthetic insecticides, which have high efficiency and broad spectrum insecticidal activities. They are easy to decompose in natural environment and have low toxicity to mammals, so they are widely used. As a result, pests become more and more resistant to them. The mechanism of insect resistance involves a large number of genes, so the discovery and identification of resistance genes play a key role in pest control. Our previous studies have shown that the fusion gene UBL40 is a deltamethrin resistance-related gene. In another study, we found that ribosomal protein L40 was closely associated with deltamethrin resistance. Therefore, we speculate that nucleolar protein plays a role in deltamethrin resistance. In order to further explore the relationship between nuclear gene and deltamethrin resistance, we used two-dimensional electrophoresis and mass spectrometry to analyze nucleolus protein. The results showed that the expression of nuclear gene Ran was significantly increased after deltamethrin treatment. This suggests that Ran may be a gene involved in deltamethrin stress. It has been reported that guanosine triphosphate (GTP) binding protein Ran plays an important role in nuclear transport, chromosome localization, microtubule dynamics, centrosomal replication, attachment of microtubules, nuclear membrane dynamics and phagocytosis. However, until now, there have been no reports of Ran involved in deltamethrin resistance. The results of two-dimensional electrophoresis showed that the expression of Ran gene in Drosophila Kc cells treated with deltamethrin was significantly higher than that in control group. In addition, real-time quantitative PCR analysis showed that the expression of Ran was up-regulated under different concentrations of deltamethrin. Western blot analysis showed that 30 ppm deltamethrin was treated for 48 h. The expression of Ran was 2.27 times higher than that of the control. To further verify the role of Ran in stress response, we used dsRNA interference and cell transfection techniques to verify the role of Ran gene in deltamethrin stress. The results showed that the overexpression of Ran could protect the cells to a certain extent, but after interfering with the expression of Ran, the sensitivity of the cells to deltamethrin was significantly increased to a certain extent. Based on the above results, we conclude that Ran is involved in the stress reaction of deltamethrin and is related to the formation of deltamethrin resistance. However, whether Ran can be used as a target gene for deltamethrin resistance needs further study and confirmation.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S481.4

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