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.     

转基因玉米表达的Cry1Ab、PAT和EPSPS蛋白对日本通草蛉幼虫的安全风险评估

为评价我国自主研发的转基因玉米表达的Cry1Ab、PAT和EPSPS蛋白对日本通草蛉Chrysoperla nipponensis幼虫生长发育的安全风险,通过将外源蛋白混入日本通草蛉人工饲料中的方法,以加入砷酸二氢钾(KH_2AsO_4)的饲料为阳性对照,研究了日本通草蛉幼虫取食后的生长发育状况。结果表明:日本通草蛉取食含Cry1Ab和EPSPS蛋白饲料的幼虫发育历期、茧期、结茧率、羽化率及成虫体重等生物学参数与取食正常饲料处理相比均没有显著差异;而取食含PAT蛋白饲料的幼虫发育历期和结茧率分别为10.7 d和96.6%,与取食正常饲料的对照11.1 d和89.9%差异显著,即饲料中添加PAT蛋白显著提高了幼虫的存活率和发育速率;取食含KH_2AsO_4饲料的日本通草蛉幼虫不能存活到茧期,说明KH_2AsO_4具有显著的杀虫活性。ELISA检测结果表明,取食了分别添加有3种外源蛋白饲料的日本通草蛉幼虫体内可以检测到相应的Cry1Ab、EPSPS和PAT蛋白,含量分别为2 758.8~5 210.7、35 018.0~54 426.6、16.8~149.8 ng/g。表明转基因玉米所表达的Cry1Ab、EPSPS和PAT蛋白对日本通草蛉幼虫没有显著的不利影响。     

Cry1Ac杀虫蛋白对甜菜夜蛾飞行能力的影响

转Bt基因作物上害虫取食Bt杀虫蛋白后存活并羽化的成虫飞行能力对于其飞离Bt作物,寻找庇护所中敏感个体进行交配、产卵,从而稀释抗性基因频率,实现“高剂量/庇护所”抗性治理策略具有重要意义。本文对取食含不同浓度Cry1Ac杀虫蛋白人工饲料的初羽化（未取食补充营养）甜菜夜蛾飞行能力进行了测试,结果表明,幼虫取食含25、50、100 μg/g和200 μg/g Cry1Ac杀虫蛋白人工饲料存活并羽化的成虫飞行距离、飞行速度和飞行时间均差异显著（p<0.05）。取食含较低浓度Cry1Ac杀虫蛋白（25 μg/g和50 μg/g）的人工饲料有促进成虫飞行的倾向,尽管与取食不含Cry1Ac杀虫蛋白的对照相比,成虫飞行距离、飞行速度和飞行时间均未达到显著增加的水平,但强飞行个体比例明显增加。随着饲料中Cry1Ac杀虫蛋白浓度的升高,羽化的成虫飞行能力显著下降。雄蛾飞行能力下降的程度比雌蛾更加明显。这些数据为转Bt基因作物上迁飞性害虫的种群动态规律及其潜在的抗性治理策略提供了科学依据。     

Cry1Ab杀虫蛋白对淡足侧沟茧蜂生长发育的影响

以黏虫［〖WTBX〗Mythimna separata〖WTBZ〗 (Walker)］为寄主,研究了亚致死浓度的Bt杀虫蛋白Cry1Ab对淡足侧沟茧蜂［〖WTBX〗Microplitis pallidipes 〖WTBZ〗(Szepligeti)］生长发育的影响。结果表明,当寄主从淡足侧沟茧蜂寄生后开始取食含Cry1Ab杀虫蛋白浓度为1、2、4 μg/g和 8 μg/g的饲料时,淡足侧沟茧蜂卵 幼虫发育历期比以对照黏虫为寄主的显著延长,茧（蛹）重和成虫体重显著下降,但对茧历期及成虫寿命没有显著影响。淡足侧沟茧蜂的卵 幼虫发育历期随着寄主取食Cry1Ab杀虫蛋白浓度的增加而显著延长,茧重和成虫体重则随Bt蛋白浓度的上升而显著下降。因此,尽管Cry1Ab杀虫蛋白对淡足侧沟茧蜂的生长发育具有一定程度的负面影响,但其仍可以在取食Bt毒蛋白的黏虫上完成整个世代发育。     

不同Bt杀虫蛋白通过食物链对大草蛉的影响

为了明确目前转基因抗虫植物中表达的Bt蛋白对农田重要捕食性天敌大草蛉的生态安全性，采用三级营养食物链评价体系，分别以对供试Bt蛋白Cry1Ac、Cry1F和Cry2Ab具有抗性的靶标害虫或不敏感的非靶标害虫作为大草蛉的猎物，研究了供试3种蛋白对大草蛉的影响。结果表明，当大草蛉以抗性棉铃虫（抗Cry1Ac或Cry2Ab）或抗性玉米螟（抗Cry1F）为猎物时，供试3种Bt蛋白通过食物链对大草蛉幼虫（4龄幼虫重量和幼虫发育至蛹历期）、蛹（化蛹率、蛹重和蛹发育历期）及成虫（羽化率、雌虫重量、雄虫重量和单雌产卵量）的生长发育及产卵量均无显著影响；同样地，当大草蛉以对3种Bt蛋白不敏感的非靶标昆虫——绿盲蝽作为猎物时，通过食物链传递的Bt蛋白对大草蛉生长发育及繁殖也无显著影响。因此，目前转基因棉花、玉米或水稻中广泛表达的Cry1Ac、Cry1F和Cry2Ab蛋白，通过三级营养食物链，对大草蛉生长发育及繁殖均无潜在毒性。     

Cry1Ab杀虫蛋白在转Bt水稻中的表达及在害虫体内的积累动态

室内研究了Cry1Ab蛋白在转Bt水稻茎和叶片中的表达及在害虫稻纵卷叶螟(Cnaphalocrocis medinalis Gue-nee)幼虫体内的积累动态。结果表明,Cry1Ab蛋白在水稻各部位中均有表达。不同生育期及稻株不同部位中Cry1Ab蛋白的表达量有差异。总的趋势是生育前期的表达量较生育后期高。在时间上,主茎中的蛋白在分蘖期最高,成熟期最低,而叶片中蛋白的表达在整个生育期比较稳定。稻纵卷叶螟幼虫取食转Bt基因水稻后,在其体内及粪便中均能检测到Cry1Ab蛋白。其中取食6h前,虫体内的Cry1Ab蛋白含量高于粪便中的;到12 h时,粪便中Cry1Ab蛋白含量高于虫体内,至24 h时达显著水平(P0.05),直到48 h,粪便中的Cry1Ab蛋白含量一直维持较高水平。     

Cry1Ac毒蛋白对亚洲玉米螟生长发育和种群增长的影响

以含不同浓度的Cry1Ac毒蛋白的人工饲料(不加毒蛋白为对照)饲喂亚洲玉米螟幼虫,采用实验种群生命表的方法,测定了Cry1Ac毒蛋白对亚洲玉米螟的控害作用。结果显示,含2 000.0ng/g Cry1Ac毒蛋白的人工饲料处理该虫第5天的幼虫存活率为0;200.0、20.0 ng/g CrylAc毒蛋白处理第10、15天的幼虫存活率、化蛹率、成蛾率、净增长率和种群增长指数均显著低于对照,且高浓度处理显著低于低浓度处理;200.0 ng/g CrylAc毒蛋白处理的世代平均周期和种群加倍时间显著长于其它处理;而200.0 ng/g CrylAc毒蛋白处理的周限增长率显著小于2.0、0.2 ng/g处理。表明微量的Cry1Ac毒蛋白对亚洲玉米螟的种群增长有显著影响,且随着浓度的增加控制作用增强。     

二化螟Cry1Ac汰选品系对不同Bt蛋白的敏感性

为明确靶标害虫对Bt蛋白的抗性及对不同类型Bt蛋白的交互抗性,采用生物测定法进行了二化螟Chilo suppressalis(Walker)敏感品系和Cry1Ac汰选品系在Cry1Ac毒饲料上的时间-死亡率反应、对不同Bt杀虫蛋白的敏感性及不同Bt蛋白复配组合对二化螟毒力效果的研究。结果表明,二化螟Cry1Ac汰选品系已对36.67μg/mL(LC_(50))的Cry1Ac杀虫蛋白产生了一定的适应性,但对308.69μg/mL(LC90)的Cry1Ac蛋白反应仍较敏感。Cry1Ac敏感品系和汰选品系对Cry1Ab蛋白的LC_(50)分别为1.40μg/mL和3.86μg/mL,二者差异显著,但对Cry1Ca(1.63μg/mL和1.73μg/mL)或Cry2Aa(127.48μg/mL和144.50μg/mL)的LC_(50)差异不显著,即Cry1Ac汰选品系与Cry1Ab存在明显的交互抗性,但与Cry1Ca和Cry2Aa不存在交互抗性。在不同Bt蛋白复配组合中(1∶1复配),Cry1Ab+Cry1Ca、Cry1Ab+Cry2Aa和Cry1Ca+Cry2Aa增效作用最为显著。表明抗虫基因cry1Ca和cry2Aa可作为双价转基因抗虫水稻研发的候选基因。     

水稻螟虫对4种Bt杀虫蛋白的敏感性测定

采用人工饲料喂毒法和浸叶喂毒法测定了二化螟、大螟和稻纵卷叶螟初孵幼虫对4种Bt杀虫蛋白的敏感性。二化螟对Cry1Ab、Cry1Ac、Cry1C和Cry2A的LC50分别为1.739、2.6452、.794 ng/mg和15.965 ng/mg;大螟依次为2.455、10.046、11.803 ng/mg和35.359 ng/mg;稻纵卷叶螟依次为4.000、1.611、69.031μg/mL和12.708μg/mL。作者就不同水稻螟虫对不同Cry蛋白的敏感性差异及其在转基因抗虫作物中的应用作了讨论。     

Cry1Ah蛋白对多异瓢虫和日本通草蛉的安全性评价

非靶标生物在转基因抗虫作物的环境安全性评价中具有非常重要的地位。本研究系统评估了新型Bt杀虫蛋白Cry1Ah对多异瓢虫幼虫、成虫和日本通草蛉成虫各项生物学指标的影响。结果表明,取食体表布满Cry1Ah蛋白的桃蚜后,多异瓢虫幼虫存活率、幼虫历期、蛹历期、体质量、雌雄成虫寿命、雌虫产卵量与对照相比均差异不显著。日本通草蛉成虫取食含有Cry1Ah蛋白的蜂蜜水后,雌雄成虫寿命、雌虫产卵量与对照间均差异不显著。因此,Cry1Ah蛋白对多异瓢虫和日本通草蛉的各项生命指标无显著的不利影响,这为转Cry1Ah基因抗虫棉花商业化释放前的安全性评价提供重要的技术支撑。     

Artificial diet based investigation on the impact of purified Cry1Ac,Cry1Fa and Cry2Ab on the survival and reproductive performance of adult green lacewing, <Emphasis Type="Italic">Chrysopa pallens</Emphasis> (Rambur) (Neuroptera: Chrysopidae)

The green lacewing Chrysopa pallens (Rambur) (Neuroptera: Chrysopidae) is a common and abundant predator in many cropping systems in palearctic realm and it’s conservation is helpful in sustainable pest management in agro-ecosystem. Prior to commercialization of Bt crops in any agro- ecosystem, it is necessary to evaluate the impact of Cry proteins upon non-target organisms especially biological control agents (BCA). In present study an artificial diet consisting of shrimp, beef, beef liver and egg yolk was developed to mass-rear C. pallens for its use as biological control agents in sustainable pest management. Moreover, an artificial diet based risk assessment protocol was developed to investigate the impact of Cry1Ac, Cry1Fa and Cry2Ab on the survival and reproductive performance of C. pallens adults. C. pallens was fed on diets incorporated with Cry proteins and without addition of Cry proteins (control). The same diet containing boric acid was served as a positive control. Temporal stability, bioactivity and intake of Cry proteins by C. pallens were confirmed using double-antibody sandwich, enzyme-linked immunosorbent assay and bioactivity verification bioassays. Survival and reproductive performance of C. pallens, e.g., pre-oviposition period, daily fecundity, total fecundity and 30-day old adults dry weights, exhibited non-significant differences (p?>?0.05) for the diets containing Cry1Ac, Cry1Fa and Cry2Ab (50 μg/g) against Control. However, significant reduction in survival and reproductive performance (p?<?0.05) was observed in positive control. Our findings reveal that artificial diet is a good source of nutritional requirement with enhanced survival and reproductive performance of C. pallens and can be used for mass rearing of predator in case of natural diet scarcity and Cry proteins are safe for adult C. pallens and Bt crops cultivation help in predators conservation in sustainable agriculture.     

荧光增白剂对Cry1Ac抗感棉铃虫围食膜的影响及增效作用

为了提高Bt的杀虫活性,运用扫描电镜、SDS-PAGE电泳和生物测定技术,研究了荧光增白剂FB28对棉铃虫Cry1Ac抗性和敏感品系围食膜的影响及对Cry1Ac杀虫蛋白的增效作用。结果表明,正常棉铃虫围食膜表面光滑致密,没有空洞和缝隙。用含1%FB28的人工饲料饲喂刚蜕皮的5龄棉铃虫幼虫2 h后,抗感棉铃虫围食膜表面均出现许多空洞,且敏感棉铃虫的空洞明显大于抗性棉铃虫;随着处理时间的延长,围食膜上的空洞逐渐变小,13 h后恢复正常。用1%FB28离体或活体处理抗、感品系棉铃虫,均可造成围食膜蛋白被降解。用1%FB28与Cry1Ac杀虫蛋白混合饲喂抗、感棉铃虫初孵幼虫,可明显提高棉铃虫的死亡率,且对抗性品系的增效作用明显大于敏感品系,对敏感和抗性品系棉铃虫的增效比分别为2.23和9.34;1%FB28还可以明显增加对抗、感品系3龄棉铃虫幼虫的生长抑制作用。     

Binding of three Cry1A toxins in resistant and susceptible strains of cotton bollworm (Helicoverpa armigera)

Evolution of resistance by pests is the greatest threat to the continuous success of theBacillus thuringiensis (Bt) toxins used in conventional sprays or in transgenic plants. The most common mechanism of insect resistance to Bt is reduced binding of toxins to target sites in the brush border membrane of the larval mid-gut. In this paper, binding experiments were performed with three ¹²⁵I-Cry1A toxins and the brush border membrane vesicles from Cry1Ac resistant or susceptible strains of Helicoverpa armigera. The homologous competition test showed that there was no significant difference in Cry1Ac-binding affinity, but the concentration of Cry1Ac-binding sites dramatically decreased in the resistant strain (R_t decreased from 5.87 ± 1.40 to 2.23 ± 0.80). The heterologous competition test showed that there were three Cry1Ac-binding sites in the susceptible strain. Among them, site 1 bound with all three Cry1A toxins, site 2 bound with both Cry1Ab and Cry1Ac, and site 3 only bound with Cry1Ac. In the Cry1Ac resistant strain, the binding capability of site 1 with Cry1Ab decreased and site 2 did not bind with Cry1Ac. It is suggested that the absence of one binding site is responsible for H. armigera resistance to Cry1Ac. This result also showed that the resistance fitted the “mode 1” pattern of Bt resistance described previously.     

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.     

Toxicity of chlorantraniliprole to Cry1Ac-susceptible and resistant strains of Helicoverpa armigera

Transgenic Bt cotton expressing Cry1Ac is important in controlling various agricultural pests, including Helicoverpa armigera. Especially for transgenic crops that are cultivated in large expanses, avoiding resistance development is a key for ensuring sustainability of Bt technologies. Integrated pest management, in which transgenic crops are strategically combined with rational pesticide use, may help to prevent H. armigera resistance acquisition in Bt cotton. In this study, we evaluated the toxicity of a novel insecticide (chlorantraniliprole) on Cry1Ac-susceptible and resistant individuals of H. armigera. More specifically, we assessed the effect of chlorantraniliprole on the activity of two enzymes and conducted laboratory bioassays to determine its toxicity on H. armigera larvae. Chlorantraniliprole increased esterase and glutathione-S-transferase activities in Cry1Ac susceptible and resistant populations of H. armigera. Cry1Ac resistant populations XJ-F (Cry1Ac resistance ratio 21.8-fold), XJ-10.0 (95.8-fold) and BTR (3536.5-fold) did not show cross-resistance to chlorantraniliprole, with LC₅₀ values of 0.0733 (μg/mL) in XJ-F, 0.0545 (μg/ml) in XJ-10.0 and 0.0731 (μg/mL) in BTR, which were close to that in the susceptible strain 96S (0.0954 μg/mL). Our work shows that chlorantraniliprole could be considered to be integrated in Bt cotton management schemes to delay the H. armigera resistance development.     

Bacillus thuringiensis Cry1Ab, but not Cry1Aa or Cry1Ac, disrupts liposomes

We evaluated the ability of Cry1Aa9, Cry1Ab4, and Cry1Ac1 insecticidal toxins from Bacillus thuringiensis to destroy liposomes. Cry1A toxins are thought to form pores in midgut apical cell membranes (BBMV), thereby disrupting midgut cells. Liposomes containing fluorescent calcein were prepared using phosphatidylcholine (PC) and phosphatidylserine (PS) (PC/PS-Liposomes) or PC alone (PC-Liposomes). Cry1Ab (1.4 μM), but not Cry1Aa or Cry1Ac, disrupted PC/PS-Liposomes and PC-Liposomes. PC/PS-Liposomes containing cholesterol and oligosaccharylceramide from Plutella xylostella midgut were damaged even more extensively by Cry1Ab, but the inclusion of either lipid alone had no effect. The initial velocity of Cry1Ab-mediated liposome disruption increased 17-fold when liposomes were prepared with Triton X-100-soluble proteins from Bombyx mori BBMV and PC (PC/Proteo-Liposomes), and Cry1Aa and Cry1Ac also caused slight disruption. These data suggest that Cry1Ab achieves higher penetration into PC/PS-Liposomes, PC-Liposomes, and PC/Proteo-Liposomes compared with Cry1Aa or Cry1Ac and that Cry1Ab may interact with membrane proteins.     

棉铃虫对Cry1Ac抗性的稳定性及其对适合度的影响

为明确棉铃虫Helicoverpa armigera（Hübner）对苏云金芽胞杆菌Bacillus thuringiensis（Bt）Cry1Ac毒蛋白抗性的稳定性及其适合度变化,利用生物测定的方法研究了Cry1Ac抗性品系棉铃虫转到正常饲料饲养后的抗性衰退及再次筛选后抗性的恢复情况,并比较了敏感、抗性和抗性衰退后各品系间的适合度差异。在失去选择压的情况下,高抗品系棉铃虫对Cry1Ac的抗性迅速衰退,经过4代后抗性水平由最初的3626.67倍下降到1436.67倍;到第12代时抗性水平已低于10倍,随后品系保持较稳定的低抗水平;当重新进行抗性再筛选时,其抗性水平可快速恢复,抗性倍数快速回升,5代后恢复到1123.33倍。与敏感品系相比,高抗棉铃虫品系的适合度明显降低,相对适合度仅为0.33,但转到正常饲料连续饲养14代后,棉铃虫适合度明显上升,相对适合度为0.87,主要表现为卵孵化率和幼虫存活率等显著提高。