四种Bt毒素蛋白对二点委夜蛾幼虫毒力测定及中肠组织病理学变化

为更好地了解苏云金芽胞杆菌Bacillus thuringiensis毒素蛋白对二点委夜蛾Athetis lepigone的毒力以及作用机理,通过饲喂含有Cry1Ac、Cry1Ab、Cry2Ab和Vip3Aa四种不同Bt毒素蛋白饲料,测定Bt毒素蛋白对二点委夜蛾幼虫的毒力,并观察取食4种毒素蛋白后幼虫中肠组织的病理学变化。结果显示,二点委夜蛾幼虫取食毒素蛋白后72 h,Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫的杀虫活性较高,校正死亡率为84.7%和76.4%;Vip3Aa和Cry2Ab毒素蛋白的毒力较弱。二点委夜蛾幼虫取食4种Bt毒素蛋白后,中肠柱状细胞微绒毛脱落,杂乱地分散在肠腔内,杯状细胞变形和腔内微绒毛脱落,线粒体和内质网等变形破裂,细胞核的核膜消失、核质凝聚和形状发生变化,经Cry1Ab和Cry1Ac毒素蛋白处理后中肠细胞的病变症状和速度明显高于Cry2Ab和Vip3Aa毒素蛋白处理。表明Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫杀虫活性较高,显著高于Cry2Ab和Vip3Aa毒素蛋白,且对其中肠细胞的破坏作用也较强。     

转Bt基因玉米对粘虫的室内杀虫效果评价

在室内用生测法研究了表达Cry1Ab杀虫蛋白的Bt玉米MON810和Bt11小苗和心叶对粘虫Mythimna separata (Walker)初孵幼虫以及玉米叶片对暴食期4龄和5龄幼虫的杀虫效果.结果表明,2种Bt玉米小苗及心叶对粘虫初孵幼虫都具有很好的控制效果,取食MON810和Bt11小苗的粘虫初孵幼虫第1 天的存活率分别为69.44%和86.11%,到第3 天时,取食Bt11小苗的幼虫全部死亡,此时取食MON810小苗的幼虫存活率仅为4.17%,到第7 天全部死亡,而此时在各自相对应的对照玉米DK647和NX4906小苗上幼虫存活率分别为91.66%和88.89%.取食MON810和Bt11心叶的粘虫初孵幼虫第2 天时幼虫存活率急剧下降,分别为13.89%和19.45%,到第7 天时,取食两种Bt玉米心叶的初孵幼虫全部死亡,而取食对照玉米心叶幼虫存活率分别为94.45%和97.22%,差异极显著.两种Bt玉米对粘虫4龄幼虫有很好的杀虫效果,取食的第11天 4龄幼虫全部死亡,对照幼虫存活率均为100%;在Bt11和MON810叶片上取食的粘虫5龄幼虫也不能存活,分别在取食的第11天和12天全部死亡,对照存活率分别为96.00%和98.00%,差异极显著.表明Bt玉米对粘虫初孵幼虫和暴食期的4龄和5龄粘虫都具有显著的致死作用.     

斜纹夜蛾取食Cry1Ca蛋白后中肠组织病理变化

Cry1Ca对斜纹夜蛾、甜菜夜蛾等多种鳞翅目害虫具有很高的杀虫活性,它在转多种Bt蛋白基因策略中,可用于扩大杀虫谱,并可用于当前Bt蛋白的抗性治理,是极具潜力蛋白之一。为明确Cry1Ca对斜纹夜蛾的作用机制,我们利用透射电镜观察了幼虫取食Cry1Ca后中肠组织的病理变化。结果表明斜纹夜蛾取食含Cry1Ca蛋白的饲料2 h后中肠组织开始表现明显的病理变化,且随取食时间的增加病变越来越严重。具体表现为：中肠细胞微绒毛肿胀、扭曲、断裂和脱落;细胞核拉长或畸形、核膜模糊、染色质不均匀或聚集;线粒体变形、空泡化和内脊模糊不清;内质网杂乱无章、肿胀变形和数量变少。研究结果可为更好地利用Cry1Ca防治斜纹夜蛾提供理论依据。     

Bt对小地考虑呼吸作用的影响

利用红外CO2分析仪测定小地老虎幼虫呼吸节律的变化结果显示：小地老虎幼虫呼吸节律属于不规则波动型，取食含Bt烟叶的幼虫呼出的CO2量显著低于对照组，且呼吸节律的波动性也明显增大，说明小地考虑幼虫取食Bt后，呼吸代谢受到一定干扰，出现呼吸水平降低及代谢紊乱现象。     

Cry2Ab杀虫蛋白对小地老虎幼虫致死效果及体内酶活性的影响

为治理小地老虎的危害,研究了Bt杀虫蛋白对小地老虎幼虫的控制作用.室内生物测定结果表明,Cry2Ab杀虫蛋白对小地老虎初孵幼虫的致死效果高于Cry1Ac杀虫蛋白.取食含有Cry2Ab杀虫蛋白人工饲料的小地老虎幼虫体内的中肠总蛋白酶和类胰凝乳蛋白酶活性高于对照,当人工饲料中Cry2Ab杀虫蛋白含量达到12μg/g时,小地老虎幼虫中肠氨肽酶活性显著升高;而取食含有Cry1Ac杀虫蛋白人工饲料的小地老虎幼虫中肠蛋白酶活性无显著变化.此外,小地老虎幼虫取食含有Cry2Ab杀虫蛋白的人工饲料后,体内谷胱甘肽-S-转移酶活性显著升高,而取食合有Cry1Ac杀虫蛋白的人工饲料后其变化不明显.     

取食转Bt基因棉等植物对甜菜夜蛾生长发育和药剂敏感性的影响

以转Bt基因棉(Bt棉)等6种寄主植物为材料,研究了对甜菜夜蛾Spodoptera exigua Hübner生长发育和药剂敏感性的影响,并对它们的后代体内乙酰胆碱酯酶和羧酸酯酶比活力进行了初步测定。结果表明,在25±1℃恒温、光照L:D=12:12条件下,甜菜夜蛾取食Bt棉与取食普通棉相比,幼虫历期延长了9.7d,龄数由5龄增加到7龄,单雌产卵力降低,幼虫存活率、蛹重、化蛹率、成虫羽化率和寿命略有降低。取食不同寄主植物间幼虫的存活率、蛹重和单雌产卵力较取食Bt棉和普通棉花间的差异更大。取食Bt棉的幼虫对顺式氯氰菊酯、灭多威、丙溴磷和氟啶脲的敏感性,较取食普通棉花的降低1～1.6倍,也较取食其它植物的敏感性明显降低。但该幼虫体内乙酰胆碱酯酶和羧酸酯酶的比活力却比取食普通棉花及其它植物的明显高。     

亚洲玉米螟和黏虫幼虫取食Cry1F蛋白后的中肠组织病理变化

为了明确Cry1F杀虫蛋白对亚洲玉米螟和黏虫两种重要玉米害虫的作用机制,本文通过透射电镜观察了亚洲玉米螟和黏虫4龄幼虫取食含Cry1F蛋白人工饲料后的中肠组织病理变化,并与其取食含Cry1Ab蛋白人工饲料后的组织病理变化进行了比较。亚洲玉米螟和黏虫4龄幼虫取食Cry1F蛋白48 h后中肠杯状细胞发生了明显的病变,主要表现为：细胞微绒毛肿胀、脱落;细胞核变形,质膜和核膜界限不清晰;染色质发生固缩、贴近核膜;线粒体拉伸变形数量减少,严重的发生空泡化;内质网肿胀与断裂、数量减少,与取食含Cry1Ab蛋白人工饲料相比,玉米螟和黏虫中肠组织发生的病变程度相似。本研究可为Cry1F作为转基因玉米重要的杀虫蛋白在未来亚洲玉米螟和黏虫防治中更好地发挥作用提供理论依据。     

取食转Bt基因棉对斜纹夜蛾幼虫存活的影响

以常规棉泗棉3号为对照,测定了取食转Bt基因棉GKl2的不同部位(花、蕾、铃和不同空间部位叶片)10d对斜纹夜蛾初孵幼虫存活率的影响。结果表明：棉花品种和植株部位对斜纹夜蛾幼虫存活率均产生显著影响,但两者之间没有交互作用。取食Bt棉顶部新叶(倒1叶)、中上部展开叶片(倒3叶和倒6叶)和蕾10d的斜纹夜蛾幼虫,其存活率与对照没有显著差异。取食Bt棉下部成熟叶片(倒14叶)4d后,幼虫存活率方显著低于对照。取食Bt棉花朵的幼虫初期(≤4d)存活率低于对照,但随着对照死亡率的增加,6～10d内两者差异不再显著。取食Bt棉棉铃的幼虫存活率始终低于对照,但两者差异基本不显著。根据GKl2对棉铃虫和斜纹夜蛾杀虫效果的比较,初步探讨了斜纹夜蛾为害趋势加重的原因。     

转Bt基因杨树对杨扇舟蛾的抗性

通过室内饲养和组织切片,研究了表达Cry1A杀虫蛋白的中嘉8号杨树转Bt基因植株对杨扇舟蛾的抗虫效果。结果表明：随着饲养时间的延长,幼虫死亡率和体重抑制率呈上升趋势,处理2d仅分别为10%和6.85%,处理4d分别增加到48.33%和27.93%,处理10d分别达81.67%和63.03%。此外,处理12d取食非转基因杨树幼虫化蛹率100%,而取食转Bt基因杨树幼虫的化蛹率仅为18.18%。组织切片观察表明,取食转Bt基因杨树叶片幼虫的中肠上皮细胞排列松驰,与肠壁基底膜分离,细胞界限较模糊,部分上皮细胞形成空泡。因此,初步认为转Bt基因杨树对杨扇舟蛾幼虫的抗性表现为毒杀作用和抑制其生长发育。     

转基因抗虫棉花对棉铃虫生长发育影响的系统评价

以我国研制的转基因抗虫棉(Bt棉)品系CK595-1为材料,系统研究了对棉铃虫1～5龄幼虫存活及生长发育的影响。结果表明,Bt棉对棉铃虫的杀虫活性和体重抑制率有明显的器官和幼虫龄期间差异,其活性顺序为叶>蕾>铃>花,其作用随着幼虫龄期的增大而降低。棉铃虫1～4龄幼虫连续取食Bt棉叶片和棉蕾不能正常化蛹,5龄幼虫可部分化蛹及羽化;1～4龄幼虫取食Bt棉的花及2～5龄幼虫连续取食Bt棉铃后均可部分化蛹及羽化,化蛹率随龄期增大而增加。     

荔枝害虫群落结构及其动态研究

以栽培品种ＮＣ８９为对照,通过室内饲养和组织切片,系统研究了转双基因抗虫烟草对烟夜蛾和小地老虎幼虫的抗性表现。结果表明,转双基因抗虫烟草对烟夜蛾幼虫具有稳定的杀虫作用,取食后２－３ｄ死亡,死亡率达１００％,不同龄期间表现一致;对小地老虎幼虫个有一定的杀虫作用,其抗性随龄期的增加而减弱,１龄死亡率为４８％,２龄为２８％,３龄以后死亡率为０,但对各龄幼虫的生长发育均具有明显的抑制作用。     

棉铃虫APN基因家族系统进化与功能分析

氨肽酶N(APN)是锌金属蛋白酶M1家族的成员,鳞翅目昆虫中肠刷状缘膜囊泡(brush border membrane vesicles,BBMV)细胞膜上的APN不仅在蛋白消化吸收过程中起着重要的作用,而且是Bt的重要受体蛋白.本研究通过PCR技术克隆得到7条棉铃虫APN基因全长序列,HaAPN1～7(Genbank...     

Interaction of Cry1Ac toxin (Bacillus thuringiensis) and proteinase inhibitors on the growth, development, and midgut proteinase activities of the bollworm, Helicoverpa zea

Potential resistance development to Bt cotton in certain lepidopterans has prompted research to develop strategies that will preserve this environmental-friendly biotechnology. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against lepidopteran pests and for expanding the spectrum of control for other insects. Interactions of Bt toxin from Bacillus thuringiensis and proteinase inhibitors were investigated by monitoring growth, development, and gut proteinase activities of the bollworm, Helicoverpa zea. Several proteinase inhibitors were combined with Bt protoxin Cry1Ac in artificial diet and fed to newly molted 3rd-instar bollworm larvae to determine effects on larval body weight and length, pupation progress, and mortality rate. Major midgut proteinase activities, including caseinase, tryptic, and chymotrypsin activities, were examined after treatment. A concentration of Bt at a level causing minimal mortality (<10%), was mixed with the following proteinase inhibitors: benzamidine, phenylmethylsulfonyl fluoride (PMSF), and N-α-tosyl-l-lysine chloromethyl ketone (TLCK). When compared with controls, the synergistic effect of Bt toxin and proteinase inhibitors caused significant decreases in mean larval weight and length over time. Midgut samples tested against the substrates azocasein, α-benzoyl-dl-arginine-p-nitroanilide (BApNA), and N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPFpNA) showed significant decreases in the protease activity of larvae fed Bt plus inhibitor versus control. Interaction of Bt and proteinase inhibitors significantly retarded larval growth and resulted in developmental delay and up to 20% mortality.     

转crylAh基因玉米花粉对龟纹瓢虫生长发育和成虫移动能力的影响

本文利用花粉饲喂法研究了转crylAh基因玉米花粉对龟纹瓢虫Propyleajaponica生长发育及成虫移动能力的影响。结果显示,与混有适量蚜虫的非转基因亲本对照玉米花粉（花粉与蚜虫重量比为2：1）相比,龟纹瓢虫幼虫取食混有适量蚜虫的转crylAh基因玉米花粉（花粉与蚜虫重量比为2：1）后,幼虫总发育历期、蛹期和成虫寿命均无显著差异,但l、3和4龄幼虫的发育历期显著高于对照,2龄幼虫的发育历期显著低于对照;2龄和4龄幼虫的存活率与对照无显著差异,但3龄幼虫的存活率显著增加;幼虫取食Bt花粉对该虫的蛹重、羽化率和雌雄比与对照无显著差异。幼虫期取食Bt玉米花粉而成虫期取食蚜虫对成虫的日产卵量无不利影响,与对照无显著差异。幼虫取食Bt玉米花粉羽化后雄虫和雌虫的步速和翻跃时间与对照均无显著差异。     

Ion regulation in the larval lepidopteran midgut and the response to Bacillus thuringiensis δ-endotoxin

Fourth-instar Bombyx mori (silkworm) was used as a model insect to study the effects of Bacillus thuringiensis (Bt) on ion homeostasis in the larval lepidopteran midgut. The K⁺ chemical gradient across the midgut of B. mori larvae is quite small, sustained by a lumen/haemolymph activity ratio of only 1.6. More than 95% of the driving force causing K⁺ flux from lumen to haemolymph is electrical. In contrast to K⁺, the H⁺ chemical gradient is exceedingly large, with luminal pH values of 11–12 and haemolymph/lumen H⁺ ratios as high as 10⁵. At equilibrium, the steep proton gradient is consistent with a passive distribution of H⁺ across the midgut epithelium as predicted from the Nernst equation. In B mori larvae, ingestion of a lethal dose of Bt δ-endotoxin produces an increase in K⁺ conductance in the midgut apical epithelium, causing a decrease in the electrical gradient and dissipation of the pH gradient. Larval morbidity can be correlated with a rise in haemolymph K⁺ and pH and a decline in luminal pH. Midgut K⁺ activity, however, remains unchanged. An important factor in the pathogenesis of Bt is irreversible alkalization of the epithelial cells as H⁺ is redistributed across the midgut to reach a new Nernst equilibrium.     

Cry1Ac的绿色荧光蛋白GFP标记及其在棉铃虫幼虫中肠的定位分布

为了明确Cry1Ac蛋白在棉铃虫体内与中肠组织的相互作用,采用重叠PCR方法将Bt-cry1Ac基因和绿色荧光蛋白GFP基因融合,构建含Cry1Ac毒蛋白和绿色荧光蛋白GFP原核表达载体,并在大肠杆菌大量表达。利用荧光显微镜观察发现,表达Cry1Ac-GFP融合蛋白的大肠杆菌在蓝光激发下发出绿色荧光。将含有融合蛋白的菌液拌入人工饲料饲喂3龄棉铃虫幼虫96h,取棉铃虫幼虫中肠做冰冻切片并在荧光显微镜下观察。结果显示,取食含有Cry1Ac-GFP融合蛋白饲料的棉铃虫幼虫中肠能够发出强烈荧光。比较Cry1Ac杀虫蛋白敏感和抗性棉铃虫幼虫中肠的发光部位,敏感棉铃虫幼虫的中肠围食膜已经消失,肠壁细胞发出强烈的荧光,而抗性棉铃虫的围食膜较健全并发出荧光。     

Note: Histology of the midgut and peritrophic membrane inLymantria dispar caterpillars fed on leaves ofQuercus cerris orRobinia pseudoacacia

The structure of the columnar cells, goblet cells and peritrophic membrane was studied in the midgut of gypsy moth (Lymantria dispar L.) caterpillars fed with a suitable (Quercus cerris L.) or an unsuitable (Robinia pseudoacacia L.) host. Columnar cells in the midgut of caterpillars fed onR. pseudoacacia leaves were elongated with small nuclei, partial loss of microvilli and vacuolated cytoplasm. The number and height of goblet cells and their nuclei were greater in caterpillars fed onQ. cerris leaves in comparison with larvae fed onR. pseudoacacia leaves. Peritrophic membranes were thin and delicate in the midgut of caterpillars fed onR. pseudoacacia leaves. http://www.phytoparasitica.org posting Nov. 8, 2005.