抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1AC毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1C产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

Bt Cry1Ac抗性粉纹夜蛾离体细胞与敏感细胞mRNA的差异显示

为了研究昆虫对Bt的抗性机制 ,以苏云金芽孢杆菌Cry1Ac活性毒素对粉纹夜蛾离体细胞连续筛选 80代 ,获得了高抗性细胞。采用DDRT PCR技术比较了该抗性细胞与非选择敏感细胞mRNA的差异 ,经反向RNA斑点印迹杂交确证了 5个片段为两种细胞的显著差异表达序列标签(ESTs)。序列分析结果表明 ,敏感细胞特有的三种ESTs都位于cDNA的非编码区 ,两种ESTs(GenBank注册号 :S1 ,CF32 2 4 1 5 ;S3,CF32 2 4 1 7)与数据库中EST没有显著同源性 ,另一种S2(GenBank注册号 :CF32 2 4 1 6)与已登录的某些昆虫的ESTs具有一定的同源性。从抗性细胞特有的R1 (GenBank注册号 :CF32 2 4 1 3)推导出的氨基酸序列与磷酸烯醇式丙酮酸羧激酶有 60 %～63%的同源性 ,从抗性细胞特有的R2 (GenBank注册号 :CF32 2 4 1 4)推导出的氨基酸序列与黏蛋白类有约 30 %的同源性。这些差异表达基因可能与抗性的形成相关。     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1Ac 毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1c 产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响

为探讨苏云金芽孢杆菌Bacillus thuringiensis（Bt）杀虫晶体蛋白与昆虫细胞的相互作用,以Bt Cry1Ac毒素和对该毒素敏感的粉纹夜蛾Trichoplusia ni离体细胞BTI-TN-5B1-4为材料,研究了一些化学物质对Cry1Ac毒素与昆虫离体细胞相互作用的影响.结果表明：N-糖基化抑制剂衣霉素、蛋白质合成抑制剂放线菌酮、胞吞作用抑制剂莫能菌素和胰蛋白酶预处理,都能不同程度地提高BTI-TN-5B1-4细胞对Cry1Ac毒素的敏感性,其中胰蛋白酶预处理的作用最明显;而N-乙酰半乳糖胺不能抑制Cry1Ac毒素对这种离体细胞的毒力.     

Bt Cry1Ac毒素筛选的粉纹夜蛾离体抗性细胞与敏感细胞蛋白质组的差异分析

采用双向电泳技术和肽质量指纹图谱技术分析了Bt Cry1Ac毒素筛选的粉纹夜蛾Trichoplusia ni抗性BTI-Tn-5B1-4细胞与同源敏感细胞的蛋白质组的差。用Melanie ViewerⅡ软件在抗性细胞的双向电泳图谱上检测到的平均蛋白质点数为707±25个（n=3）,在敏感细胞的双向电泳图谱上检测到的平均蛋白质点数为637±19个（n=3）,其中分辨率高、重复性良好的显著差异点有10余个。对其中的一个敏感细胞特有的显著差异斑点进行了肽质量指纹图谱分析,经数据库 查询表明该蛋白质与胞质外周蛋白具有同源性。     

小菜蛾对Bt毒素Cry1Ac和Bt制剂抗性的选育及其抗性种群的生物学适应性

用Bt制剂和Bt毒素Cry1Ac分别对源自深圳田间的小菜蛾Plutella xylostella在室内进行抗性种群选育,获得相对抗性倍数分别为24.36、38.16倍的抗性种群DBM.1Ac-R30和DBM.Bt-R46。对这2个抗性种群及其敏感种群（DBM.Bt-S）的生长发育、存活及繁殖特征进行了详细地观察与比较,并以甘蓝为饲喂材料构建了2个抗性实验种群的生命表。结果表明,DBM.1Ac-R30和DBM.Bt-R46种群较DBM.Bt-S种群的产卵量和孵化率下降,幼虫发育历期延长,雌雄比显著降低,雌成虫数量、寿命减少。DBM.1Ac-R30和DBM.Bt-R46种群相对于DBM.Bt-S种群的相对适合度分别为0.75和0.65,抗性种群在繁殖能力上存在明显的生存劣势。     

对Cry1Ac毒素敏感性不同的三种BT1-Tn-5B1细胞品系基因组DNA的RAPD及SSR分析

用Operon公司W系列和F系的 40个随机引物列对BT1 Tn 5B1Cry1Ac敏感细胞、抗性细胞和抗性衰退细胞的基因组DNA进行PCR扩增。经琼脂糖凝胶电泳和EB显色总共扩增出了 2 5 9条清晰带 ,其中有 2 5 0条带为三种细胞所共有 ,有 9条为特异性扩增带。其中 3条特异性带为敏感细胞和抗性衰退细胞样品共有 ,3条特异性带为抗性细胞和抗性衰退细胞样品所特有 ,1条特异性带为抗性衰退细胞样品特有 ,2条特异性带为抗性细胞和敏感细胞样品共有 ,另外某些带在三种细胞之间还存在明显的强弱和宽窄差异。用三个微卫星引物序列分别对三种细胞的总DNA进行扩增 ,得到了 14条清晰的扩增带 ,发现它们在带形上并无太大差异 ,但有 1条带存在明显的强弱差异。这表明BT1 Tn 5B1细胞对Cry1Ac抗性的产生和衰退与基因组DNA的多态性有关。     

二化螟Cry1Ac敏感和耐受品系的适合度差异及中肠蛋白酶活性比较

抗性/耐受种群的适合度代价和中肠蛋白酶活性研究是了解昆虫抗性进化、抗性产生的生理生化基础及抗性治理的重要内容。本研究以室内继代汰选的具有相同遗传背景的Cry1Ac耐受和敏感品系为研究对象,系统研究了两品系的适合度和中肠蛋白酶活性差异。研究结果表明,与敏感品系相比,耐受品系幼虫的存活率显著下降,且耐受种群不同日龄幼虫体重均低于敏感种群,20日龄时达显著差异;但两品系不同虫态的发育速度、蛹重、化蛹率、羽化率和成虫繁殖力均没有显著差异。中肠蛋白酶活性研究表明,耐受种群的类胰蛋白酶活性显著低于敏感种群,但两种群的总蛋白酶和类胰凝乳蛋白酶活性没有显著差异。该研究对于识别蛋白酶介导的Bt抗性机制和昆虫Bt抗性治理均具有重要意义。     

印楝素A与印楝素B对粉纹夜蛾BTI-Tn-5B1-4的细胞毒性

为了明确印楝素A和B活性差异的机理,本研究比较了印楝素A和印楝素B对粉纹夜蛾Trichoplusia ni离体培养胚胎细胞系BTI-Tn-5B1-4的毒性。结果表明：印楝素A与印楝素B对BTI-Tn-5B1-4细胞具有良好的增殖抑制活性,处理后3 d,其IC₅₀值分别为2.9 μg/mL和9.85 μg/mL,印楝素A的细胞毒力显著高于印楝素B。倒置显微镜观察发现,印楝素A和印楝素B处理可导致细胞变形,贴壁能力下降,并出现明显空泡,印楝素A的影响明显高于印楝素B。流式细胞仪检测结果表明,印楝素可导致BTI-Tn-5B1-4细胞体积显著膨大,印楝素A处理细胞体积增大程度显著高于印楝素B;印楝素可以明显影响BTI-Tn-5B1-4细胞膜电位,1.25 μg/mL印楝素A和印楝素B处理后3 d,细胞DiBAC4（3）荧光强度分别增加88.12%和55.37%,印楝素A的影响显著高于印楝素B。荧光显微镜观察发现,印楝素对BTI-Tn-5B1-4细胞核具有明显影响,印楝素B的影响明显高于印楝素A,印楝素B处理后,细胞核受损细胞数更多,受损程度更严重。结果显示印楝素A和印楝素B的细胞作用机理存在差异,本研究从细胞学水平解释了印楝素的生长发育抑制作用机理。     

Production of recombinant endostatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED₅₀) for recombinant endostatin was approximately 0.35 g ml^–1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l^–1 at 6 days of cultivation.     

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins’ mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD₅₀ concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.     

Binding of Bacillus thuringiensis Cry1A toxins to brush border membrane vesicles of midgut from Cry1Ac susceptible and resistant Plutella xylostella

Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 μg Cry1Ac/g diet but killed by Cry1Ab at 0.5 μg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 μg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.     

Differential proteomic analysis of Trichoplusia ni cells after continuous selection with activated Cry1Ac toxin

Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the sustained successful application of Bt-based biological control tactics. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance. Here, the Cry1Ac-selected Trichoplusia ni TnH5 cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. The differential proteins included transporter, molecular chaperon, structural molecules and many other molecules involved in protein metabolism, signal transduction, nucleotide binding, lipid biosynthesis, carbohydrates metabolism and energy production, suggesting that a complex mechanisms involved in the development of insect resistance to Bt Cry1Ac toxins at cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might contribute to the development of resistance in the selected cells. Our results provide some new cues for understanding the mechanism of Bt resistance.