【作者】 符伟；

【导师】 张友军；

【作者基本信息】 湖南农业大学 ， 农药学， 2013， 博士

【摘要】 小菜蛾Plutella xylostella (L.),属于鳞翅目菜蛾科,是世界性的重要农业害虫。由于杀虫剂的广泛使用,小菜蛾几乎对所有防治药剂均产生了抗药性,极大增加了防治难度。苏云金芽孢杆菌(Bacillus thuringiensis, Bt)是目前使用范围最广的生物杀虫剂,小菜蛾在田间亦对其产生了抗药性,严重影响了其在防治小菜蛾方面的应用价值。本文一方面通过构建两性生命表研究了小菜蛾Bt抗性种群的生物学特性,利用实时荧光定量PCR技术研究了小菜蛾可能受体基因的基因表达水平,经分子克隆得到小菜蛾Bt毒素可能受体氨肽酶N (Aminopeptidase N4, APN4)基因全长,分析和预测了其分子结构和功能位点。另一方面,以小菜蛾田间抗性监测为基础,结合其它防治措施对小菜蛾抗性进行治理,以达到湖南地区小菜蛾综合治理(Integreted Pest Management, IPM)的目的。本文主要研究内容及结论如下：1小菜蛾荧光定量PCR内参基因的筛选本文首次系统评价了小菜蛾荧光定量PCR内参基因的筛选。选用了8个候选内参基因,试验条件分为生物因子(发育阶段、组织和品系)和非生物因子(温度、光周期、药剂和机械伤害),应用内参分析软件geNorm、Normfinder、Bestkeeper和RefFinder进行各候选内参基因稳定性分析。结果表明,不同试验条件下应选择的内参基因有所不同,相比而言,EFl和RPL32在大多数试验条件下均表现出良好的稳定性。该研究为小菜蛾基因表达相关研究奠定了良好的基础。2小菜蛾抗敏品系APN4基因全长克隆及结构和功能预测分析经分子克隆得到小菜蛾APN4基因全长序列,结果表明其开发阅读框包括2835bp,编码944个氨基酸残基。SignalP软件预测APN4在N端有一个长为16个氨基酸左右的信号肽结构。Big-PI Predictor软件预测APN4第496位氨基酸谷氨酰胺(Gln)为潜在的C端GPI修饰位点。ScanProsit软件预测APN4有13个N端酰基化位点、9个酪氨酸蛋白激酶2磷酸化位点、4个N端糖基化位点、9个蛋白激酶C磷酸化位点和2个酪氨酸激酶磷酸化位点。比对分析小菜蛾Cry1Ac抗敏品系氨基酸序列,抗性品系相对于敏感品系有5个氨基酸的改变。该研究为明确小菜蛾APN4基因的结构和功能及其在小菜蛾Bt抗性中的作用奠定了一定的基础。3小菜蛾Bt毒素可能受体基因的基因表达水平分析系统研究了Bt毒素小菜蛾敏感和抗性品系中3种Bt可能受体基因(钙粘蛋白、氨肽酶和碱性磷酸酶)的基因表达水平差异分析。结果显示,钙粘蛋白基因除1龄幼虫外,其余各龄期幼虫和中肠组织中敏感品系的基因表达量均显著高于抗性品系。APN1和APN2基因在敏感品系中的表达量一般要高于抗性品系,但APN3基因除3和4龄幼虫外,其余各龄期敏幼虫感品系表达量均显著低于抗性品系,但中肠组织中敏感品系高于抗性品系,而抗敏品系APN4基因各龄期和中肠组织表达量均无显著性差异。碱性磷酸酶基因各龄期幼虫敏感品系和抗性品系均无显著差异,但中肠组织敏感品系基因表达量显著高于抗性品系。该研究为小菜蛾Bt受体的确定及阐明其分子抗性机制提供了一定的依据。4小菜蛾Bt抗敏品系生物学特性研究通过分别构建小菜蛾Bt抗性和敏感品系的两性生命表,结果表明,小菜蛾敏感品系的内禀增长率、种群增长率和净殖长率均要高于抗性品系,而世代周期短于抗性品系。抗性品系相对于敏感品系种群适合度代价为0.78。通过抗性品系对不同Bt毒素蛋白的室内活性测定,发现其对Cry1Ab和Cry1Ac有较高的抗性水平,抗性倍数分别为59.68和67.68,而对Cry1Ca和Cry1Ah保持较高的敏感性,抗性倍数分别为3.32和1.64倍。该研究有助于明确小菜蛾Bt抗性品系的生物学特性及小菜蛾对Bt毒素不同蛋白的药剂敏感性。5湖南地区小菜蛾发生规律和抗药性监测通过湖南长沙和怀化地区小菜蛾种群动态调查发现,湖南平原代表地长沙地区小菜蛾发生一年中有两个高峰期,一个是4月中下旬和11月上旬,而湖南丘陵山区代表地怀化小菜蛾高峰期分别为5月上旬和10月中旬。在建立了小菜蛾对9药剂敏感基线的基础上,分别监测了长沙和怀化2011和2012年春秋两季的9种药剂田间抗性水平。结果发现,Bt、氯虫苯甲酰胺和丁醚脲处于中等水平抗性,多杀菌素、定虫隆和溴虫腈达到高抗水平,而阿维菌素、高效氯氰菊酯和茚虫威达到极高抗水平。该研究明确了湖南不同地区的小菜蛾种群动态规律和田间抗药性水平现状,为小菜蛾的抗性治理和综合防治奠定了基础。6湖南地区小菜蛾综合防控体系研究小菜蛾在甘蓝上的防治指标的研究结果表明：苗期、莲座期、结球时期和结球中后期的防治指标分别为53.65、90.84、67.21和607.51头每百株。通过联合毒力测定表明,甲氨基阿维菌素苯甲酸盐和氟啶脲不同配比的联合毒力的共毒系数均大于100,表现出相加作用,其中甲维盐：氟啶脲=1：10时,其共毒系数最大(154.58),为最佳配比。整合各单项小菜蛾防控技术,建立了湖南地区小菜蛾综合防治体系,通过经济效益评价,结果表明,综防区投资效益比为1：17.39,显著高于化防区(1：9.06)。该研究为湖南地区小菜蛾综合治理提供了参考模式。更多还原

【Abstract】 The diamondback moth, Plutella xylostella (L.), is one of the world wide agricultural pest of cruciferous vegetables, and has developed resistance to a wide array of insecticides. The environmentally friendly insecticide crystal proteins produced by Bacillus thuringiensis (Bt) are of great scientific interest because of their potency and specificity to a wide range of insect pests. The resistance mechanism of Bt-resistance strain of P. xylostella was studied by constructing the life table to compare the biological characteristics between the resistance and susceptible Bt strains, cloning the aminopeptidase N (APN4) gene of P. xylostella, and using quantitative real-time PCR to studied gene expression of potential Bt receptor of P. xylostella. On the other hand, the Integrated Pest Management (IPM) of P. xylostella in Hunan province was proposed on the basis of the occurrence rules and resistance monitoring of P. xylostella. The main contents and conclusions are as follows:1. Exploring valid reference genes in P. xylostellaIn this study, a total of eight candidate reference genes were evaluated under various experimental conditions throughout the entire qRT-PCR workflow. Based on the comprehensive analysis, a suite of internal references are recommended to accurately normalize and quantify gene expression in P. xylostella. The results showed that under the test conditions should be selected different reference gene, compared, EF1and RPL32in most experimental conditions showed a good stability. This study not only provides a standardized procedure for quantification of gene expression in the diamondback moth, but also lays a solid foundation for the genomics and functional genomics research in this emerging insect model.2. Cloning and sequencing of Aminopeptidase N (APN4) gene from P.xylostella susceptible and resistant to CrylAc toxincDNA of aminopeptidase N (APN4) from CrylAc toxin susceptible and resistant strains of P.xylostella was cloned and sequenced. It contained a2835bp open reading frame that encoded944amino acids. The sequences had a N-terminal siganal peptide with16amino acid. GPI-Anchor sites showed that the first496amino acid glutamine (Gln) is the potential C-terminal GPI modification sites. ScanProsit showed that APN4has13N-terminal acylated sites,9tyrosine kinase2phosphorylation sites,4N-glycosylation sites,9protein kinase C phosphorylation sites and2tyrosine kinase phosphorylation sites. There was5amino acids mutation in APN4from susceptible strain of P. xylostella relative to that of resistant strain. This study provides a certain foundation for studing the system structure and function of APN4in P.xylostella.3. The gene expression analysis of Bt potential receptors of P. xylostellaThe gene expression of Bt potential receptors (cadherin (CAD), aminopeptidase N (APN) and alkaline phosphatase (ALP)) in P. xylostella was studied. The results showed that the cadherin gene expression of susceptible strain of P. xylostella were higher than resistant strain except for the1st larvae. The gene expression of APN1and APN2of susceptible strain were higher than resistant strain in the most developmental stage larvae. The gene expression of APN3of susceptible strain were lower than resistant strain except for the3rd and4th larvae, while expression of susceptible strain were higher than resistant strain in the midgut.There were no significant difference between the gene expression of APN4of susceptible and resistant strain. There were no significant difference between the gene expression of ALP of susceptible and resistant strain in different development stages, while the gene expression of susceptible strain were higher than resistan strain in the midgut. This study contributes to the determination of the Bt receptor of P. xylostella and its molecular mechanism of resistance.4. The life characteristic of resistant P. xylostella populationObjective to compare biological characteristics and population dynamics of Bt-resistant and susceptible strain of P. xylostella in the laboratory, the two sex life table was established. The results showed that the intrinsic rate of increase (r), the net reproductive rate (R0) and the the gross reproduction rateis (GRR) of susceptible strain is higher than resistant strain, while the mean length (T) of susceptible strain is shorter then Bt-resistant strain. The fitness cost of restant strain is0.78compared to the susceptible strain.This study found that Bt-resistant strain has a higher resistance to CrylAb (59.68-fold) and CrylAc (67.68-fold), lower resistance to CrylCa (3.32-fold), and no complete resistance to CrylAh (1.64-fold), respectively.This study helps to define the biological characteristics of Bt-resistant P. xylostella and the resistance of resistant strain to different Bt toxin protein.5. The occurrence rules and resistance monitoring of P. xylostella in Hunan provinceThe occurrence rules of P. xylostella in Changsha and Huaihua was ascertained with systemic investigation and field census. The results showed that the maximum occurrence of the adult of was diamondback moth in the late mid-April and the early November in Changsha and in the early May and the mid October in Huaihua, respectively.The relative susceptible toxicity baselines of P. xylostella to major insecticides in the laboratory condition were established with leaf dipping method. The Changsh and Huaihua population showed a low-level resistance to Bt, Chlorantraniliprole and Diafenthiuron, a middle-level resistance to Spinosad, Chlorfluazuron and Chlorfenapyr, and a high-level resistance to Abamectin, Cypermethrin and Indoxacarb, respectively.Studies to detect incripient resistant in the filed are very important to design effective strategies to avoid resistance development.6. The IPM of P.xylostella in Hunan provinceBased on the experiment between the yield loss and the the population density of P. xylostella, it is suggested that the economic threshold is53.65in seedling,90.84in rosette stage,67.21in early heading stage and607.51in the late period of the heading stage imported cabbage larvae on one hundred cabbages.The toxicities of emamectin benzoate, chlorfluazuron and their mixture on3rd instar larvae of P.xylostella were tested in laboratory. It was found that the synergism was the most significant when the proportion of emamectin benzoate and chlorfluazuron was1:10and the co-toxicity coefficient was154.58.Six different control systems for P. xylostella on autumn wild cabbage were established, and the population dynamics of P. xylostella, quantity of natural enemy, quality of wild cabbage and economic benefit in each control system were studied. The results showed that the effects of pest control, natural enemy protection, crop yield and crop quality in integrated control area were greater than that in other control areas, and the rate of return on investment was the best (1:17.39).The study evaluated the single system control techniques and integrated control system for the effective control of P. xylostella in Hunan Province which provide a model for IPM of P. xylostella in Hunan.更多还原