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.     

Cry1Ba3、Cry1Ia8蛋白对Cry1Ac抗性小菜蛾的杀虫活性研究

利用2种苏云金芽胞杆菌原毒素Cry1Ba3、Cry1Ia8及其组合,分别对Cry1Ac抗性种群小菜蛾幼虫进行生物活性测定。结果表明Cry1Ba3、Cry1Ia8对2种目标试虫均有高毒力,LC50分别为0.2175、0.6706μg/mL;Cry1Ba3毒力3倍于Cry1Ia8。2种蛋白混配的结果也表现出高毒力,LC50为0.4375μg/mL,没有显著的协同增效作用,也不存在拮抗。敏感与抗性小菜蛾种群生测结果统计分析比较,结果表明这2种蛋白及其组合与Cry1Ac并无交互抗性。     

Cry1Ac和Cry2Ab蛋白对大草蛉生长发育及酶活力的影响

为明确转Cry1Ac和Cry2Ab基因棉花对大草蛉的影响,运用Bt蛋白与人工饲料混合的方法,以大于转基因棉花叶片中蛋白含量20倍的剂量饲喂大草蛉初孵幼虫,初步研究了Cry1Ac和Cry2Ab对大草蛉生物学参数和消化酶、解毒酶活性的影响。结果表明：取食含Bt蛋白饲料的大草蛉幼虫的发育历期、体重、蛹重、成虫体重、羽化率等生物学参数与对照相比均没有显著差异;在大草蛉幼虫体内可以检测到含量较高的Cry1Ac和Cry2Ab蛋白,分别为974.92~1 282.39 ng/g鲜重和5 592.62~6 082.92 ng/g鲜重,而在大草蛉成虫体内检测到的Cry1Ac和Cry2Ab蛋白含量非常低,分别为0.29~0.39 ng/g鲜重和50.34~56.71 ng/g鲜重;取食含Bt蛋白的饲料对大草蛉幼虫的类胰蛋白酶、类胰凝乳蛋白酶、氨肽酶和谷胱甘肽-S-转移酶活性有一定的影响,但对大草蛉成虫影响与对照差异不显著。表明大草蛉取食含Bt蛋白的人工饲料后,虽然体内可以检测到一定含量的Bt蛋白,但对大草蛉的生长发育并没有显著的直接不利影响。     

亚洲玉米螟对Cry9Ee与Cry1Ab蛋白的交互抗性分析

Cry9Ee是近年来发现的对亚洲玉米螟Ostrinia furnacalis(Guenée)等害虫具有高毒力的蛋白,具有良好的应用前景。为了明确亚洲玉米螟对Cry9Ee和Cry1Ab蛋白是否存在交互抗性,本文首先分别表达、活化、纯化了Cry9Ee和Cry1Ab蛋白,进而对敏感和Cry1Ab抗性亚洲玉米螟进行生物活性测定;随后对2种蛋白在敏感亚洲玉米螟中肠刷状缘膜囊泡(brush border membrane vesicles,简称BBMVs)上的结合进行分析比较。生物活性测定结果表明,Cry9Ee和Cry1Ab蛋白的活性片段对敏感亚洲玉米螟的LC₅₀分别为3.04和0.89 μg/g;而Cry9Ee活性片段在5.00 μg/g的浓度下,对Cry1Ab蛋白抗性亚洲玉米螟致死率高达96%,与对敏感种群活性相当。竞争结合试验表明,Cry9Ee和Cry1Ab两种蛋白间不存在竞争结合,推测它们在亚洲玉米螟中肠上存在不同的受体。综合生物活性测定及体外结合试验两方面结果得出结论：亚洲玉米螟对Cry9Ee与Cry1Ab蛋白不存在交互抗性。cry9Ee基因可以作为理想的候选基因,用于我国新一代转基因抗虫玉米的研制,为延缓和克服亚洲玉米螟对转基因抗虫玉米抗性的产生提供重要保证。     

对小菜蛾协同增效的Cry1和Cry9类蛋白组合的筛选

为筛选对小菜蛾具有协同增效作用的苏云金芽胞杆菌Cry蛋白组合,本研究在单独测定对重要蔬菜害虫小菜蛾具有高毒力的Cry1Ai、Cry1Ea和Cry9Aa蛋白杀虫活性的基础上,把Cry1Ai+Cry1Ea、Cry1Ea+Cry9Aa、Cry1Ai+Cry9Aa蛋白进行不同比例的混配,测定其对小菜蛾的毒杀作用.结果发现Cry1Ai+Cry1Ea有相加作用.Cry1Ea+Cry9Aa蛋白和Cry1Ai+Cry9Aa蛋白均在1∶1混配时增效因子最高,分别为6.67和9.06;后两种组合表现出了显著的协同增效作用.本研究获得的两种高效组合将为转基因抗虫植物的研发提供新的基因来源,有望在治理对Cry1A类蛋白产生抗性的害虫中发挥重要的作用.     

Effect of Bt cotton expressing Cry1Ac and Cry2Ab, non-Bt cotton and starvation on survival and development of Trichoplusia ni (Lepidoptera: Noctuidae)

Effects of Bollgard II cotton containing two Bacillus thuringensis var. kurstaki Berliner (Bt) toxin proteins (Cry1Ac and Cry2Ab), non-Bt cotton (DPL 491) and starvation on survival and development of cabbage looper, Trichoplusia ni (Hübner), were determined in the laboratory. Larvae of the first four larval instars died when they fed on the terminal leaves of Bt cotton plants at 50 days after planting (DAP). However, 51.3% of fifth instars that fed on 50 DAP Bt cotton leaves pupated, and 87.1% of the pupae successfully developed into adults. Of the unfed fifth instars (starved), 55.6% pupated and 88.1% of the pupae emerged. Pupae that developed from larvae fed on Bt cotton leaves and unfed were significantly smaller, being 89.7 and 73.2% of the weight of the pupae that developed from larvae fed on non-Bt cotton leaves. Leaves of 120 DAP Bt cotton were less toxic to T. ni larvae. When the first instars continuously fed on 120 DAP Bt cotton leaves, 75.9, 60.6, 56.4 and 38.4% of larvae survived to second, third, fourth and fifth instars respectively, and 20.9% pupated and 17.9% successfully became adults. However, it took the surviving first instars 37.1 days to become adults, which was 7.2 and 8.9 days longer than those fed on 50 and 120 DAP non-Bt cottons respectively. Pupae that developed from larvae that fed on 120 DAP Bt cotton leaves were only 50.9 and 52.6% of the weight of those developed from larvae that fed on 50 and 120 DAP non-Bt cotton respectively. Non-Bt cotton, both 50 and 120 DAP, did not exhibit significant effects on larval survival and development, except that the pupae in the 50 DAP non-Bt cotton treatments developed over a significantly longer time than those in the 120 DAP non-Bt cotton treatment.     

Microarray analysis of global gene regulation in the Cry1Ab-resistant and Cry1Ab-susceptible strains of Diatraea saccharalis

BACKGROUND: Extensive adoption of transgenic Bt corn in recent years for stalk borer control has increased risk of resistance evolution in the target pest populations. A Bt‐resistant strain of the sugarcane borer, Diatraea saccharalis, was approximately 100‐fold more tolerant to Cry1Ab toxin than the susceptible counterpart. To gain a better understanding of the molecular mechanisms of Bt resistance, the Cry1Ab‐susceptible (Cry1Ab‐SS) and Cry1Ab‐resistant (Cry1Ab‐RR) strains of D. saccharalis were subjected to a microarray analysis. RESULTS: Results showed that the expression levels of many genes were significantly different between the Cry1Ab‐RR and Cry1Ab‐SS strains. Microarray analysis of 7145 cDNAs revealed 384 differentially expressed genes. A total of 273 genes were significantly upregulated 2–51.6‐fold, and 111 genes were significantly downregulated 2–22.6‐fold in the Cry1Ab‐RR strain. The upregulation of three potential resistance‐related genes, coding for a glutathione S‐transferase (GST), a chymotrypsin‐like protease (CHY) and a lipase (LP), was confirmed using real‐time PCR, indicating a reproducibility of the microarray data. Ontology analysis revealed that more than twice the number of metabolic‐related genes were upregulated compared with downregulated genes with the same biological function. Up to 35.2% of the upregulated genes in the resistant strain were associated with catalytic activity, while only 9.5% of the downregulated genes were related to the same catalytic molecular function. CONCLUSION: The large portion of metabolic‐ or catalytic‐related genes with significant upregulations indicated a potential large increase in metabolic or catalytic activities in the Cry1Ab‐RR strain. This cDNA microarray gene expression data could be used to characterize and identify new genes that may be associated with Bt resistance in D. saccharalis. Copyright © 2012 Society of Chemical Industry     

Bt cotton producing Cry1Ac and Cry2Ab does not harm the parasitoid Aenasius arizonensis (Girault): a host-mediated tritrophic assay

Phytoparasitica - Transgenic crops with genes from Bacillus thuringiensis (Bt) Berliner, a soil bacterium producing δ-endotoxin that is lethal to several phytophagous insects. Concerns related...     

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.     

苏云金芽孢杆菌Cry1Ie1蛋白末端缺失的研究

通过PCR扩增法 ,以cry1Ie1全长基因为模板 ,分别得到不同末端缺失的基因片段 ,转化大肠杆菌BL21(DE3)中诱导表达 ,得到 6种末端缺失蛋白。对小菜蛾生物测定结果表明 ,缺失蛋白IE659、IE656分别缺失了C末端60个和63个氨基酸残基 ,对小菜蛾的杀虫活性与Cry1Ie1全长蛋白相当 ;IE648、IE045分别缺失了C末端 71个氨基酸残基和N末端缺失44个、C末端缺失63个氨基酸残基 ,对小菜蛾的活性提高了50% ;IE633、IE105分别缺失了C末端 86个氨基酸残基和N末端缺失 104个、C末端63个氨基酸残基 ,丧失了对小菜蛾的活性。试验明确Cry1Ie1蛋白的最小活性区N端在45～104氨基酸位点之间 ,C端在633～648氨基酸位点之间。     

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。     

黏虫MagR和Cry2基因的时空表达分析

为了明确黏虫MagR、Cry2基因在定向行为中的作用,本研究采用基因序列分析及实时荧光定量技术,研究了黏虫MagR、Cry2基因的基因序列特征及其表达模式.结果 表明,黏虫MagR基因编码131个氨基酸,蛋白分子量为14.17 kD,等电点为9.3,具有多个铁硫簇结合位点;黏虫Cry2基因编码757个氨基酸,蛋白分子量...