【作者】 张莹；

【导师】 李国清；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2013， 硕士

【摘要】 棉铃虫是棉花蕾铃期的害虫,寄主植物有20多科200余种,广泛分布在中国及世界各地,黄河流域棉区、长江流域棉区受害较重。甜菜夜蛾是一种世界性分布、间歇性大发生的以危害蔬菜为主的杂食性害虫。由于近年来中国的Bt棉商业化广泛种植,甜菜夜蛾凭借广泛的分布已经成为棉花上的主要害虫。苏云金芽孢杆菌(Bacillus thuringiensis)及其毒素蛋白转入植物或制成生物农药,在各地广泛应用于棉花害虫的防治。然而,在研究和应用过程中,一些尚未解决和新出现的问题逐渐积累,影响其进一步推广。本文拟比较研究两种毒素Cry1AC和Cry1Ca对甜菜夜蛾和棉铃虫幼虫和成虫的毒力,以及产生毒力差异的毒理学机理。1.棉铃虫和甜菜夜蛾成虫对Cry1Ac和Cry1Ca的毒理学研究将活化的Cry1Ac和Cry1Ca毒素溶解在10%蔗糖水溶液中,配制成浓度分别为500、100和20μm/ml的毒素溶液。饲喂500μm/ml的Cry1Ac和Cry1Ca溶液后,棉铃虫雌虫的寿命分别缩短了48.1%和48.9%,甜菜夜蛾雌虫的寿命分别缩短了43.5%和38.5%；棉铃虫雄虫的寿命分别缩短了37.8%和40.3%,甜菜夜蛾雄虫的寿命分别缩短了50.5%和47.4%。浓度为500μm/ml的Cry1Ac和Cry1Ca大幅降低了棉铃虫雌虫精包接受率50.0%和46.8%,以及甜菜夜蛾雌虫精包接受率的58.7%和57.3%；显著降低棉铃虫雄虫的精包形成率的40.0%和50.3%,以及甜菜夜蛾雄虫的精包形成率的61.3%和60.0%。在500μm/ml的Cry1Ac和Cry1Ca影响下,棉铃虫雌虫的产卵量显著降低57.5%和57.5%,甜菜夜蛾雌虫的卵块数量降低35.4%和45.8%。饲喂Cry1Ac和Cry1Ca都不影响卵孵化率。在浓度为100和20μm/ml时,Cry1Ac和Cry1Ca对昆虫寿命和交配基本不影响。2.3种Cry毒素与7种蛋白酶抑制剂对甜菜夜蛾的协同增效作用甜菜夜蛾幼虫中肠液可以有效降低Cry1Ac, Cry1Ab和Cry1Ca的活性。测定了3种Cry毒素和7种蛋白酶抑制剂(苯甲基磺酰氟PMSF、大豆胰蛋白酶抑制SBTL、乙二胺四乙酸EDTA、对甲苯磺酰-L-苯丙氨酸氯甲基酮TPCK、对甲苯磺酰-L-赖氨酸氯甲基酮TLCK、单宁酸Tanic acid,弹性蛋白酶抑制剂)混合使用对幼虫生长的影响。单独用抑制剂或毒素(Cry1Ac,31.30ng/cm2; Cry1Ab,3.2ng/cm2; Cry1Ca,0.6ng/cm2)连续六天饲喂二龄初幼虫只能轻微的影响幼虫生长。饲喂含有一种抑制剂和一种毒素的混合溶液,除EDTA外,均能够协同作用降低幼虫的体重。而且抑制剂浓度越高抑制效果越明显。表明甜菜夜蛾幼虫中肠内的胰蛋白酶,胰凝乳蛋白酶和类弹性蛋白酶与3种活化Cry毒素的水解相关。3.甜菜夜蛾丝氨酸蛋白酶基因的分子克隆及序列分析利用甜菜夜蛾转录组数据库和RT-PCR技术得到甜菜夜蛾中肠丝氨酸蛋白酶的6个基因全长序列,经过序列比对和系统进化树分析表明,这6个基因与家蚕丝氨酸蛋白酶家族的基因有很高的相似性,证明这6个基因序列是甜菜夜蛾中肠的丝氨酸蛋白酶基因序列。4.棉铃虫和甜菜夜蛾钙粘蛋白时间表达及丝氨酸蛋白酶活性测定测定了钙粘蛋白基因在棉铃虫与甜菜夜蛾各个虫态的表达,发现钙粘蛋白基因在甜菜夜蛾幼虫5龄期表达量最高,蛹期和成虫期基本不表达。钙粘蛋白基因在棉铃虫6龄幼虫期表达量最高,蛹期和成虫期基本不表达。推测Bt对棉铃虫和甜菜夜蛾的成虫和幼虫的毒力差异可能是由于Bt毒素初级受体钙粘蛋白的表达差异。检测了甜菜夜蛾幼虫与成虫中肠中丝氨酸蛋白酶的活性,幼虫中肠丝氨酸蛋白酶活性明显高于成虫,推测Bt毒素对昆虫成虫的作用方式存在目前还未发现的新的途径。更多还原

【Abstract】 Helicoverpa armigera Hubner is a pest of cotton. It damages more than200plant species belonging to more than20families. It widely distributed in China and around the world, has occurred at China’s cotton-growing and vegetable growing areas, such as the Yellow River and the Yangtze River areas. In recent years, the pest has also occurred in Xinjiang. The Spodoptera exigua Hubner is a highly polyphagous pest and causes extensive damage to many field and truck crops, and even weeds and grasses. S. exigua has become a major economic pest of cotton across a wide distribution since the commercialization of Bt cottons in1997in China.Being delivered as sprays or expressed in plant, Bacillus thuringiensis (Bt) crystalline proteins (Cry toxins) display insecticidal activities against numerous Lepidopteran, Dipteran and Coleopteran larvae. However, the widespread and sustained use of Bt cotton and Bt sprays inevitably results in some negative effects. Comparative study of toxicities of Bt Cry toxins between larvae and adults may afford important new insights into the interactions of the toxins with receptor proteins in host insect, and represent intriguing targets for the control of insect pests.1. The toxicities of CrylAc and Cry1Ca to Helicoverpa armigera and Spodoptera exigua adultsThe effectiveness of CrylAc and CrylCa on lifespans and reproductive performance of H. armigera and S. exigua adults were evaluated by in vivo experiments, at the concentraion of500,100and20μg/ml in a10%sucrose aquous solution. At the highest concentration, Cry1Ac and Cry1Ca shortened48.1%and48.9%of H. armigera female lifespan, and43.5%and38.5%of S. exigua female lifespan, and they reduced37.8%and40.3%, and50.5%and47.4%of H. armigera and S. exigua male lifespans respectively. Bt toxins negatively affected copulation. Exposure to500μg/ml of Cry1Ac and Cry1Ca greatly reduced50.0%and46.8%, and58.7%and57.3%spermatophore acceptance by H. armigera and S. exigua females respectively. Similarly, CrylAc and Cry1Ca exposure decreased40.0%and50.3%, and61.3%and60.0%of spermatophore transfer by H. armigera and S. exigua males respectivly. Moreover, exposure females rather than males to500μg/ml of Cry1Ac and CrylCa significantly droped57.5%and57.5%of the number of eggs laid by H. armigera, and35.4%and45.8%of the number of egg masses deposited by S1. exigua. In contrast, both Cry1Ac and CrylCa did not negatively influence the egg hatchabilities. At the middle and the lowest concentrations, however, the biological effects of CrylAc and Cry1Ca on lifespans and reproductive performance partially or completely disappeared.2. Combined effects of three crystalline toxins with seven proteinase inhibitors on S. exiguaInteractions between the3Cry toxins and7proteinase inhibitors were investigated by monitoring larval growth. The proteinase inhibitors were phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, tannic acid, N-a-tosyl-L-phenylalanine chloromethyl ketone, N-a-tosyl-L-lysine chloromethyl ketone, elastatinal and ethylenediaminetetraacetic acid (EDTA). A6-day dietary exposure of the newly molted2nd instars to either the inhibitors (3test concentrations) or the toxins (CrylAc,31.30ng/cm2; Cry1Ab,3.2ng/cm2; Cry1Ca,0.6ng/cm2) alone only slightly affected larval growth. In contrast, exposure to the mixtures containing an inhibitor and a toxin, with the exception of those containing EDTA, synergistically reduced larval weight, In general, the synergisms were more obvious at higher inhibitor concentrations. These results indicated that trypsin-, chymotrypsin-. and elastase-like proteinases in S. exigua larval midgut were involved in proteolytical hydrolyzation of the3activated Cry toxins.3. Cloning and characterization of serine proteinase genesBased on the Spodoptera exigua transcriptome database and RT-PCR technology,6full-length cDNA sequences encoding serine proteinases were obtained from the midgut template of Spodoptera exigua. Sequence alignment and phylogenetic analysis showed that these six proteins showed high identify with corresponding serine protease-like proteins from Bombyx mori.4. Cadherin temporal expression and serine protease activity assay of H. armigera and S. exigua Cadherin gene expression level in different development stages was estimated by qPCR. It was found that cadherin protein mRNA level was higher in fifth and sixth instar larvae of H. armigera and S. exigua respectively. In contrast, cadherin genes did not expressed in pupa and adult stages. The intestinal serine protease activity in S. exigua larvae and adult was examined. Larvae serine protease activity was significantly stronger than the adult.更多还原