我国棉铃虫对Bt蛋白Cry1Ac抗性现状及分子机制研究进展

棉铃虫Helicoverpa armigera是一种全球性的重要农业害虫,主要为害棉花、玉米和大豆等作物。长期种植单价Bt棉花（表达Cry1Ac蛋白）会使棉铃虫田间种群承受单一、持续的选择压力,必然会导致棉铃虫对Cry1Ac的抗性发生演化。该文概述我国棉铃虫田间种群对Cry1Ac的抗性现状、自然庇护所对棉铃虫Cry1Ac抗性演化的延缓作用以及棉铃虫对Cry1Ac抗性的遗传多样性,并对今后我国关于棉铃虫Bt抗性的治理对策进行了展望。     

The effects of diet on the detection of resistance to Cry1Ac toxin in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

The effects of raw or heat-denatured soybean flour in an artificial diet on the detection of Cry1Ac resistance in Helicoverpa armigera were examined. Resistant neonate larvae reared on denatured soybean flour diet showed resistance factors of 7980 and 16,901 at the LC₅₀ and LC_99.9 levels, respectively. By comparison, resistance could not be detected in neonate larvae reared on raw flour diet. Third instar larvae reared on denatured flour diet showed resistance factors of 322 and 21,190 at the LC₅₀ and LC_99.9 levels. Resistance was not detected in third instar larvae reared on raw flour diet. There was 68% survival of resistant neonate larvae on Bollgard II cotton leaf feeding assays, compared to 100% mortality in a susceptible strain. We conclude that detection of CRY1Ac resistance in H. armigera from Australia can be masked, if an artificial diet gives chronic exposure to potent, protease inhibitors present in raw soy flour.     

V-ATPase H表达量下调增强棉铃虫对Cry1Ac的抗性

棉铃虫Helicoverpa armigera是世界性重要农业害虫。目前防治棉铃虫的主要手段是种植转苏云金芽胞杆菌Bacillus thuringiensis(Bt)杀虫蛋白的转基因作物。本文旨在研究棉铃虫V-ATPase H在Cry1Ac蛋白毒力和抗性中的作用。利用实时荧光定量qRT-PCR技术分析V-ATPase H在Cry1Ac抗、感品系棉铃虫幼虫中肠及敏感品系棉铃虫幼虫受Cry1Ac诱导后的表达情况;在昆虫Sf9细胞中过表达V-ATPase H对其进行细胞定位,通过细胞毒力试验验证其对Cry1Ac毒力的影响。结果发现棉铃虫V-ATPase H基因在抗性品系中低表达,并且V-ATPase H在受到Cry1Ac诱导时也低表达;在Sf9细胞内表达V-ATPase H蛋白发现其在整个细胞中都有分布,过表达该蛋白后增强了细胞对Cry1Ac蛋白的敏感性。结果表明V-ATPase H参与Cry1Ac蛋白的毒力。     

Insecticide Resistance in Field Strains of Pectinophora gossypiella (Saunders) in China and Effect of Synergists on Deltamethrin and Parathion-methyl Activity

Filter-paper residual toxicities of some insecticides used extensively in China were determined during 1994 using newly hatched (within 30 min) larvae of four Pectinophora gossypiella (Saunders) strains. The strains were field collections collected in the Yangtze River cotton-belt areas. Compared with the susceptible laboratory strain from Qunli (Lishui County, Jiangsu province), the four field strains from Anqing (Anhui province), Jiangling (Hubei province), Cixi(Zhejiang province) and Tongzhou(Jiangsu province) had developed 185-, 6·7-, 698- and 249-fold resistance, respectively, to deltamethrin. Cixi and Tongzhou field strains had also developed 103- and 94-fold resistance to fenvalerate, and 10- and 3·6- fold resistance to parathion-methyl. Percentage of survivors at diagnostic dosage for deltamethrin showed that the strains from Anqing, Jiangling, Cixi and Tongzhou had 87·2, 18·3, 90·1 and 74·6% resistant individuals respectively. Cixi and Tongzhou field strains had 88·9 and 65·3% resistant individuals after application of parathion-methyl, which was consistent with the corresponding resistance ratios. Studies of the effect of synergists piperonyl butoxide (PBO), triphenyl phosphate (TPP) with deltamethrin and parathion-methyl in Cixi, Anqing and Tongzhou field strains suggested that metabolic resistance mechanisms such as carboxylesterases (CarE) and mixed function oxygenases (MFO) were involved in parathion-methyl resistance, but not in deltamethrin resistance. © 1997 SCI.     

粉纹夜蛾离体细胞对Bt Cry1Ac毒素的抗性发展及其交互抗性

以Cry1Ac活性毒素逐代筛选抗性粉纹夜蛾离体细胞BTI-TN-5B1,抗性发展速度呈S形曲线,经56代选择后,抗性比上升至1 280倍.在去除选择压力后,抗性比逐渐下降.低水平抗性细胞抗性衰退较快,而高水平抗性细胞抗性衰退较缓慢.抗性比衰退至1.5倍的细胞在复筛后抗性比恢复较快.抗性细胞对苏云金杆菌工程菌GC-91(Bt GC-91)、苏云金杆菌鲇泽亚种(B.t.awazai)和苏云金杆菌库斯塔克亚种(B.t.kurstaki)的复合毒素晶体的活性毒素都具有较高的交互抗性,但对农药无交互抗性,对杆状病毒的敏感性也没发生变化.     

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。     

钙粘蛋白氨基酸点突变介导红铃虫对Cry1Ac的抗性

为明确室内筛选的红铃虫Pectinophora gossypiella抗性品系AQ-R对Cry1Ac的抗性机制,采用室内生物测定法明确该品系对Cry1Ac和Cry2Ab的敏感性,通过遗传杂交和基因克隆分析抗性基因的显隐性及突变位点,并进行细胞学试验分析突变蛋白的亚细胞定位。结果显示：红铃虫AQ-R抗性品系对Cry1Ac的抗性倍数为181.67倍,对Cry2Ab没有交互抗性;该品系携带了一种新型的隐性钙粘蛋白抗性等位基因PgCad1,其编码蛋白的钙粘蛋白重复区、前蛋白区和近膜区共发生了17个氨基酸替换。表达野生型PgCad1-s基因的Hi5细胞对Cry1Ac敏感,且钙粘蛋白定位于细胞膜;而表达抗性PgCad1-r基因的Hi5细胞则对Cry1Ac不敏感,且钙粘蛋白错误定位到内质网。表明钙粘蛋白氨基酸点突变能导致其定位错误,从而促成红铃虫AQ-R品系对Cry1Ac产生抗性。     

棉铃虫V-ATPase亚基B是Cry1Ac的功能受体

目前对转苏云金芽胞杆菌Bacillus thuringiensis（Bt）的研究发现,液泡型ATP酶（Vacuolar-type proton ATPase,V-ATPase）可能是Bt的一类新型受体。我们前期通过构建棉铃虫的中肠酵母文库筛选Cry1Ac的结合蛋白发现棉铃虫V-ATPase亚基B（V-ATPase B）可以与Cry1Ac结合。为明确V-ATPase B在Cry1Ac毒力和昆虫对Cry1Ac抗性机制中的作用,本研究首先采用实时荧光定量PCR技术分析了该基因在抗感Cry1Ac棉铃虫幼虫中及其受到Cry1Ac诱导时的基因表达情况;通过Ligand blot进一步地证实了其与Cry1Ac的结合特性;并通过在Sf9细胞中表达V-ATPase B的试验验证了其功能。结果表明V-ATPase B在抗性品系及受到Cry1Ac诱导时均下调表达,Ligand blot证实了V-ATPase B与Cry1Ac特异性结合;并且在昆虫细胞内过表达该基因,会增强Cry1Ac的细胞毒力。研究结果表明棉铃虫V-ATPase B是Cry1Ac的功能受体,并有可能通过降低基因表达来参与棉铃虫对Cry1Ac的抗性形成。     

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.