溴氰菊酯抗性品系和敏感品系小菜蛾成虫蛋白质组的比较与分析

【作者】 张娜娜；

【导师】 程罗根；

【作者基本信息】 南京师范大学 ， 遗传学， 2011， 硕士

【摘要】 小菜蛾Plutella xylostella(L.)属鳞翅目菜蛾科,是十字花科蔬菜的主要害虫之一。由于在生产上用药频繁,加之小菜蛾生活史的特殊性,它对多种杀虫剂都产生了抗药性,已成为世界上最难防治的害虫之一。因此,为了找出抗药性相关的蛋白并阐明抗药性机理,本实验采用了比较蛋白质组学的方法研究了溴氰菊酯抗性品系和敏感品系小菜蛾成虫在蛋白水平上面的表达差异。在本实验中,通过双向凝胶电泳,每块凝胶上得到600个左右的蛋白点,其中溴氰菊酯抗性品系和敏感品系小菜蛾成虫之间有23个蛋白点存在表达差异,将这些差异蛋白进行串联质谱鉴定,有8个蛋白鉴定成功。相对于敏感品系来说,在抗性品系中有5个蛋白表达量上升,3个蛋白表达量下降,鉴定成功的蛋白包括成虫盘因子,丙酮酸脱氢酶E1β亚基,凝血酶抑制剂前体,化学感应蛋白CSP2,Cu/Zn超氧化物歧化酶(SOD1),类过氧化氢酶等,这些蛋白涉及到生长因子,代谢酶,蛋白酶抑制剂,感受蛋白,表皮蛋白和保护酶等；利用western blotting对双向电泳和质谱鉴定出来的差异蛋白点Cu/Zn超氧化物歧化酶(SOD1)进行了验证,结果跟双向电泳中的结果是一致的；利用双向电泳筛选出来的具有差异表达的某些候选蛋白,利用荧光定量的试验方法研究了基因在RNA表达水平上的变化：对5个基因进行了相关的研究,相对于敏感品系来说,在小菜蛾抗溴氰菊酯品系中,Cu/Zn超氧化物歧化酶(SOD1)表达水平下调,化学感应蛋白CSP2和成虫盘因子表达水平上调,糜蛋白酶和凝血酶抑制剂在表达水平上没有显著差异；在RNA和蛋白表达水平上来看,Cu/Zn超氧化物歧化酶(SOD1)和化学感应蛋白CSP2基因在RNA上的表达水平和蛋白表达水平是一致的,成虫盘因子是相反的,糜蛋白酶和凝血酶在RNA水平上面没什么差异,但是在蛋白水平上面有明显的表达差异。由于本研究使用的抗性品系是在敏感品系的基础上通过多代的药剂选择培养而成的,因此这些差异表达的蛋白可能在昆虫的抗药性过程中起到一定的作用。所以,本实验有助于加深对昆虫抗药性机理的认识,有助于制定新的策略去预防减少抗性的进一步发展。更多还原

【Abstract】 The diamondback moth, Pluttela xylostella (L.) has become one of the most destructive pests of cruciferous crops all over the world whose high insecticide resistance has received much attention to date. So it is important to clarify the mechanism of insecticide resistance. In order to gain an insight into the insecticide resistance at protein level, we employed comparative proteomics approach to investigate the proteomic differences between deltamethrin-resistant strain and deltamethrin-sensitive strain (DR and DS) of adult diamondback moth.In this study, a large number of proteins (about 600) were detected on each gel,23 spots were differently expressed and 8 proteins were indentified by MALDI-TOF-TOF MS successfully, including imaginal disk growth factor, pyruvate dehydrogenase E1 component beta subunit, thrombin inhibitor infestin precursor, chemosensory protein CSP2, Cu/Zn superoxide dismutase (SOD1) and peroxiredoxin-like protein etc. These proteins can be classified into the following function groups:growth factor, metabolic enzyme, protease inhibitor, olfactory protein, cuticle protein and protective enzyme; Western blotting was used to validate the proteomic result of SOD1 and we found that the expression of SOD1 has the same tendency in the proteomic and western level; Five proteins were selected for mRNA expression analysis using real-time RT-PCR. Compare with DS, the expression of Cu/Zn superoxide dismutase (SODl) was down-regulated, chemosensory protein CSP2 and imaginal disk growth factor were up-regulated, chymotrypsin and thrombin inhibitor infestin precursor has no significant difference; The expressions of SOD1 and chemosensory protein CSP2 at mRNA level were in accordance with their expressions at protein level, imaginal disk growth factor was opposite to that observed for protein abundance, chymotrypsin and thrombin inhibitor infestin precursor have no significant difference at mRNA level, but they have significant difference at protein level.As resistence strain was obtained from the sensitive strain through several generations of selection, so the differentially expressed proteins were proposed to be involved in insecticide resistance, therefore this study is helpful to understand the mechanism of insecticide resistance and design novel strategies to prevent, or minimize the spread of resistance.更多还原