【作者】 朱勋；

【导师】 李建洪； 张友军；

【作者基本信息】 华中农业大学 ， 农药学， 2014， 博士

【摘要】 苏云金芽孢杆菌(Bacillus thuringiensis, Bt)是目前世界上用途最广、产量最大的微生物杀虫剂,其编码Bt杀虫毒蛋白基因作为主要的杀虫基因转入到多种作物中,在害虫防治中成效显著。但与此同时,Bt制剂的大量使用和转Bt作物的广泛种植,使得Bt的抗性问题越来越严重。十字花科重要害虫小菜蛾Plutella xylostella (L.)是最早被发现且目前仅有的6种在田间对Bt产生抗性的害虫之一。但到目前为止,Bt对小菜蛾的杀虫作用过程和小菜蛾对Bt的抗性机理,研究的仍不是完全清楚。在本研究中,我们建立了对Cry1Ac有高于5000倍抗性的近等基因系小菜蛾种群,同时研究了其相关生物学特性。同时,我们首次利用shotgun HPLC-ESI-MS/MS技术对小菜蛾幼虫中肠蛋白质进行了蛋白质基因组分析。同时为了更好的揭示小菜蛾抗Cry1Ac的分子机理,我们利用iTRAQ和MRM两种蛋白质组分析技术,对两个种群的中肠蛋白质组进行了定量分析,发现了大量差异表达蛋白质和可能的Bt受体。结果如下：1.小菜蛾抗CrylAc近等基因系种群的构建我们利用弗罗里达Florida抗Cry1Ac小菜蛾DBMlAc-R与纽约日内瓦Geneva敏感种群DBM1Ac-S,建立了两套近等基因系种群。经ISSR分子标记技术对其近等性进行评估发现,利用高世代回交法构建的近等基因系抗性种群BC7F3(更名为NIL-R),与轮回亲本敏感种群DBM1Ac-S之间的遗传相似度达到98.24%,具有较高的相似度,且其对Cry1Ac毒蛋白的抗性倍数保持在5000倍以上,是理想的实验材料。2.抗CrylAc近等基因系小菜蛾的抗性相关生物学研究我们利用以上建立的抗CrylAc小菜蛾近等基因系种群NIL-R开展相关生物学研究。我们通过3次生命表对NIL-R种群的抗性适合度研究发现：在卵的大小、寿命、幼虫发育、成虫寿命和产卵量上,抗性种群NIL-R与敏感种群DBM1Ac-S相比没有显著差别,说明NIL-R没有出现发育的延迟；利用两种群的内禀增长率(Ro)计算相对适合度,三次生命表结果分别为1.016、0.940和0.932,说明抗Cry1Ac小菜蛾近等基因系种群NIL-R没有抗性适合度代价。根据交互抗性谱分析发现：NIL-R对CrylAb毒蛋白和Cry1Ah蛋白具有一定的交互抗性,对Cry1Ca毒蛋白和Cry1Ie毒蛋白没有交互抗性,由此我们可以推断小菜蛾对CrylA类毒蛋白可能具有共同的受体位点,而与Cry1Ca毒蛋白和Cry1Ie毒蛋白的受体位点不同。利用单对杂交实验和剂量对数-期望死亡机率曲线两种方法研究其遗传方式发现：抗性近等基因系种群NIL-R对Cry1Ac的抗性由一个常染色体、不完全隐形的基因位点决定。3.利用shotgun ESI-MS对小菜蛾幼虫中肠进行蛋白质基因组研究我们利用shotgun HPLC-ESI-MS/MS对小菜蛾幼虫中肠蛋白质进行测定,结合其基因组数据,完成了首个小菜蛾中肠蛋白质基因组分析。通过研究,一共得到了876,341张质谱图,将这些质谱图与小菜蛾基因组的蛋白质数据库及全基因组6框翻译的蛋白数据相比对,共鉴定出15,887条肽段。其中,12,004条肽段与已经发表的小菜蛾基因组中的预测蛋白质信息相匹配,同时发现了2113新肽段。利用这些发现的新肽段鉴定得到491个新基因,修正了202个小菜蛾基因组注释基因。通过对小菜蛾中肠蛋白质组的功能注释分析我们发现脂质消化作用相关酶和基因在小菜蛾中肠蛋白质中占据了重要地位,这可能是小菜蛾与其主要寄主十字花科作物的共进化的又一证据。同时我们从这些蛋白质数据鉴定到了大量与小菜蛾杀虫剂抗性相关的蛋白质,为小菜蛾适应性及广泛抗药性等的分子机理、功能基因组研究供了必要的数据支持。4.抗CrylAc近等基因系小菜蛾的比较蛋白质组研究我们利用iTRAQ技术,对NIL-R和DBM1Ac-S两个种群的中肠BBMV进行蛋白质定量比较研究,共筛选出了128个差异表达蛋白质。其中发现了氨肽酶和ABC转运蛋白质两个已报道的Bt受体蛋白质,在抗性种群中的表达量显著低于敏感种群。这可能说明这两个蛋白质也是Cry1Ac在小菜蛾中的受体。我们还发现了一个葡糖基转移酶的显著升高。另外,我们还发现了细胞色素P450解毒酶和谷胱甘肽转移酶两个解毒酶在抗性种群中显著升高的现象。随后,我们利用MRM技术对找出的差异蛋白质及已报道的Bt受体进行质谱定量检测,比较以上蛋白质在两个种群的中肠样品的表达量差异情况。MRM技术检测的结果与iTRAQ结果基本一致。另外,我们还发现氨肽酶、ABC转运蛋白质、碱性磷酸酶、钙黏蛋白以及鞘糖脂等潜在受体蛋白质在抗性小菜蛾中都有显著的降低。这个结果说明在本研究的小菜蛾种群抗Cry1Ac过程中,这些潜在受体蛋白质可能都不同程度的发挥着作用。更多还原

【Abstract】 Bt crystalline toxins, produced by the bacteria Bacillus thuringiensis, are the most widely used biological insecticide in pest management. Genes encoding Bt insecticidal proteins are commonly incorporated into transgenic crops to protect plants from insect feeding damage. The widespread use of Bt foliar sprays and transgenic Bt crops provide the opportunity for the evolution of Bt resistance to arise in populations of targeted insects. Understanding resistance mechanisms will provide new insights into the mode of action of Bt toxins to develop the effective management of resistance, which is still controversial. Plutella xylostella (L.) was the first of six lepidopteran species to develop field resistance to insecticidal Bt toxins. Different populations selected for Bt resistance often show diverse responses in terms related to resistance mechanism.In order to elucidate the mechanism of resistance to Cry1Ac, the near-isogenic P.xylostella with>5,000-fold resistance were constructed. And the biology characterization of the near-isogenic resistant strain was also studied. In addition, we carried out an in-depth proteogenomic analysis using shotgun HPLC-ESI-MS/MS approach to identify genes and gene networks putatively involved in various physiological and toxicological processes in the P. xylostella larval midgut. And then iTRAQ and Multiple Reaction Monitoring (MRM) were used to discover different expression proteins between the two strains. Many different expression proteins and candidate receptors were discover. The results were as follows:1. Construction of near isogenic P.xylostella strains resistant to CrylAc toxinTo elucidate the mechanism of resistance to Bt CrylAc toxin, we developed near-isogenic line (NIL) strains with>5,000-fold resistance. Two pairs of NILs were generated using either backcross or recombinant inbred line methodologies and then evaluated for near isogenicity using ISSR markers. One backcross line, BC7F3(renamed NIL-R), showed98.24%similarity to the susceptible parental strain, DBM1Ac-S, suggesting its optimal use for resistance characterization studies.2. Characterization of near isogenic P.xylostella strain resistant to CrylAc toxinThe cross-resistance spectrum in the NIL-R strain suggested high levels of cross-resistance to Cry1Ab and Cry1Ah but no cross-resistance to CrylCa or Cry1Ie. The interpretation of the overall data seems to suggest the involvement of an alteration in the binding of CrylA toxins to a common receptor, not recognized by CrylCa or Cry1Ie toxins. Three times of life tables and comparisons of the biological parameters were investigated to minimize the effects of environmental factors and personal error to obtain more real and accuracy date on the fitness of resistance. There were no significantly difference in the development of egg, larvae, the longevity of adult and fecundity of every female adult between the near isogenic resistant strain NIL-R and susceptible strain with DBM1Ac-S, which indicated the Cry1Ac-resistant P.xylostella strain did not lead to developmental asynchrony in our stains. And then the net reproductive rate (R0), the gross reproduction rate (GRR), the finite rate of increase (λ) and the mean generation time (T) of the two strains were similar. The relative fitness of P.xylostella, measured as a ratio of Ro (the net reproductive rate) of resistant strain divided by Ro of the susceptible strain, were ratios of1.02,0.94and0.93respectively in repeats of life table study, which reflected the lack-of-fitness costs of Cry1Ac resistance in NIL-R strain. Single pair genetic cross analyses between the two strains revealed that Cry1Ac-resistance in DBM was controlled by a single, autosomal, recessive locus. The experiment of inheritance of resistance based on the log dose-probit line indicated that resistance might be controlled by a single locus and autosome and be incompletely recessive with degree of dominance of F1and F1’ progeny were-0.74and-0.71, respectively. These informations provide reference in the study of resistance to Cry1Ac proteins in P. xylostella.3. Proteogenomic analysis of the larval midgut by shotgun ESI-MS approachWe carried out an in-depth proteogenomic analysis using shotgun HPLC-ESI-MS/MS approach to identify genes and gene networks in the P. xylostella larval midgut. A total of876,341tandem mass spectra were searched against a database of predicted P.xylostella protein sequences generated from public databases, and a whole-genome six-frame translation. This search identified15,887distinct peptide sequences, total5270proteins (including58containment protein), and a comparison with the publicly available proteome from the published P.xylostella genome identified1,568new peptides suggesting corrections or additions to the current annotations. In-depth analysis identified proteins putatively involved in nutrient digestion and insecticide resistance. Presence and expression at high levels of numerous enzymes of absorption and transport of fatty acids and lipids and glycolysis pathway indicate that active metabolism processes of carbohydrates, and lipids occurred in the larval midgut of P. xylostella. 4. Comparative Proteomics Analysis on the near isogenic P.xylostella resistant to Cry1Ac toxinThe iTRAQ and multiple reaction monitoring (MRM) were used to discover different expression proteins between the near-isogenic resistant strain and susceptible strain. Based on iTRAQ,128different expression proteins were obtained. Notably, one aminopeptidase N and one ATP-binding cassette were found to express lower in resistant strain than susceptible strain, which were reported as Bt receptor before. This may make some evidences that aminopeptidase N and ATP-binding cassette are the receptors to CrylAc in P.xylostella. And then one glucosyltransferase and two enzymes related to medication (methyltransferases and phosphatase) were also found to express higher. There were also two detoxifying enzymes expressing in higher level. The results evaluating by MRM agreed with these from iTRAQ. In addition, some candidate Bt receptors reported in other species were analyzed in these two P.xylostella strains. Finally, all the candidate receptors including aminopeptidase N, ATP-binding cassette, Alkaline Phosphatase, cadherin and beta-1,4-Ga1NAc transferase bre were found had low expression in resistant strains, which may indicated that these receptors may all play some role in the process Cry1Ac resistance in P. xylostella.更多还原