【作者】 张宏；

【导师】 程罗根；

【作者基本信息】 南京师范大学 ， 遗传学， 2013， 硕士

【摘要】 小菜蛾(Plutella xylostella L.)属于鳞翅目菜蛾科,是十字花科蔬菜的主要害虫之一。化学防治是控制害虫的一个重要手段。随着杀虫剂的大量使用,小菜蛾对多种杀虫剂产生了抗药性,杀虫剂抗性已成为病虫害防治工作中的一大障碍。发现和研究抗药性相关基因将有助于阐明抗药性的分子基础和寻找新的抗性靶标。本实验室采用cDNA代表性差异分析(cDNA representative difference analysis, cDNA RDA)发现泛素基因(ubiquitn)在小菜蛾溴氰菊酯抗性和敏感品系中出现差异表达。泛素(UBI)是真核细胞中普遍存在的小分子蛋白质,其基本功能是通过泛素-蛋白酶体途径(ubiquitin proteasome pathway, UPP)高效选择性降解蛋白质。泛素在调控细胞生长、细胞周期调控、细胞凋亡以及信号转导中发挥着重要作用。泛素基因是否在害虫抗药性中发挥作用以及发挥着何种作用,目前尚未见报道。本文在前期研究的基础上,以小菜蛾四龄幼虫为实验材料,克隆了小菜蛾泛素基因家族各成员(UBL40, UBS27a, polyubiquitin)的基因序列,通过活体水平RNA干扰(RNA interference, RNAi)后低表达和细胞水平的细胞转染等方法,研究泛素对溴氰菊酯抗性的影响。主要结果如下：(1)小菜蛾UBL40的开放阅读框为387bp,编码128个氨基酸残基；第1至228个碱基编码泛素蛋白(76个氨基酸),第229到387个碱基编码核糖体蛋白L40(52个氨基酸)。UBS27a的开放阅读框为468bp,编码155个氨基酸残基。前76个氨基酸为泛素蛋白,后79个氨基酸为核糖体蛋白S27a。UBL40和UBS27a同属于泛素核糖体融合蛋白。(2)实时荧光定量PCR结果显示,泛素核糖体融合基因在抗性品系四龄幼虫中的表达量显著上调。(3)RNAi沉默UBL40后,实时荧光定量PCR实验证明干涉效率约为60%。随后通过生测实验测定RNA干扰后的小菜蛾抗性品系抗药性变化情况,探讨二者之间可能存在的关系。结果显示RNA干扰UBL40后,与对照组相比,干扰组小菜蛾对溴氰菊酯的抗药性水平显著下降,从而揭示UBL40与小菜蛾溴氰菊酯抗性相关。(4)将UBL40转染进果蝇Kc细胞,使其在果蝇细胞中高表达,并使用CCK-8试剂盒进行细胞毒力实验。结果表明,在一系列溴氰菊酯浓度梯度的刺激下,UBL40转染组细胞的存活率相对于对照组细胞有明显升高。以上结果表明,UBL40与小菜蛾溴氰菊酯抗性有关,UBL40适度高表达会对细胞产生抗溴氰菊酯的保护作用。这是泛素基因与昆虫抗药性相关的首次报道,为进一步阐明杀虫剂抗性的分子机制和建立抗药性检测方法提供了新的科学依据,具有重要的理论意义和潜在的实际应用价值。更多还原

【Abstract】 Diamondback moth (Pluttela xylostella L.), which belonging to the family Plutellidae, order Lepidopteran, has become one of the most destructive pests in Cruciferous vegetables (CFV) all over the world. Chemical control is a major method to keep insects down. However, under natural selection, continuous and excessive application of insecticides has caused the development of insecticide resistance, which is now the major obstacle to pest control. Identification and characterization of genes related to insecticide resistance will undoubtly contribute to clarify the mechanism of insecticide resistance and find the new target of insecticide resistance.In our previous experiments, we had employed cDNA representative difference analysis (cDNA RDA) to identify differentially expressed genes between deltamenthrin-resistant and-susceptible strains of Pluttela xylostella (L.), and some differentially expressed genes were isolated. One of the highly expressed genes was UBI-homolog. UBI is a small protein that ubiquitously exist in the eukaryotic cells. The primary function of UBI is to selectively degenerate proteins. UBI plays important roles in cell growth, cell cycle regulation, cell apoptosis and signal transduction. Whether UBI plays a part in insecticide resistance or what function it plays, depends on our further research.Based on these, in this study, we acquired the open reading frame of all the numbers of ubiquitin gene family by RT-PCR using Pluttela xylostella fourth instar larvae as experimental materials, we detected which gene or genes has high expression in deltamethrin-resistant strain by qPCR, and explored the relationship between ubiquitin and deltamethrin resistance (DMR) by RNA interference (RNAi) and cell transfection. The main results are as followes:(1) The RNA from diamondback moth was reversed transcribed and ubiquitin genes were cloned by RT-PCR method. The open reading frame of UBL40was387bp, which encodes128amino acids.1to76amino acids consist ubiquitin monomer, and77to128consist ribosomal protein L40. The GenBank accession number was JX163118. The open reading frame of UBS27a was468bp, which encodes155amino acids, with GenBank number was JX437934.1to76amino acids consist ubiquitin monomer, and77to155consist ribosomal protein S27a. Both UBL40and UBS27a are ubiquitin ribosome fusion proteins.(2) The results of quantitative real-time PCR revealed that UBL40is highly expressed in the deltamethrin-resistant strain of diamondback moth.(3) RNAi to resistant Pluttela xylostella fourth instar larvae was applied to silence UBL40through microinjection in vivo, and After48h, the RNAi efficiency was60%by qPCR verification.48h post RNAi, we conducted bioassay on the fourth instar lavae of resistant Pluttela xylostella. The results showed that the resistance level of RNAi group was significantly decreased compared to the control group, which means UBL40is related to deltamethrin resistance.(4) To further testify the reliability of the above results, we transfected UBL40into Drosophila Kc cells, afterwards, cells were stimulated by deltamethrin in a series of concentration gradient. Following that, cell toxicity experiment was conducted using CCK-8kit. The results showed that the survive rate of UBL40-transfected group was declined compared with controls.In sum, the results showed that UBL40is related to deltamethrin resistance, and UBL40moderate overexpression would protect cells from deltamethrin damage. Our research firstly reported that ubiquitin a pesticide resistance related gene, which provides us with a new scientific basis for clarifying molecular mechanism of insecticide resistance and establish a detection method for pesticide resistance.更多还原