转Cry1Ac/1Ab基因棉花对棉铃虫的控制效果及对甜菜夜蛾和斜纹夜蛾生长发育的影响

为评估转Cry1Ac/1Ab基因棉花对棉铃虫Helicoverpa armigera的抗性及对非靶标害虫甜菜夜蛾Spodoptera exigua和斜纹夜蛾S.litura生长发育的影响,采用室内生测法测定其对棉铃虫的抗性及对甜菜夜蛾和斜纹夜蛾不同龄期幼虫存活率、营养代谢及中肠酶活性的影响。结果表明,转Cry1Ac/1Ab基因棉花对棉铃虫第2代幼虫的抗性程度最高,幼虫校正死亡率达91.33%,但对甜菜夜蛾和斜纹夜蛾的抗性程度较低,幼虫校正死亡率分别为15.33%和13.33%。甜菜夜蛾和斜纹夜蛾各龄期幼虫取食转Cry1Ac/1Ab基因棉花后,其存活率与取食常规棉花对照无显著差异;甜菜夜蛾对转Cry1Ac/1Ab基因棉花叶片的相对取食量、近似消化率分别为16.68和93.12%,均高于取食常规棉花对照的10.72和92.00%,但差异不显著,而斜纹夜蛾取食转Cry1Ac/1Ab基因棉花后的各项营养指标均低于取食常规棉花对照,差异也不显著。取食转Cry1Ac/1Ab基因棉花后,甜菜夜蛾的过氧化物酶活性为0.02 U/mg prot,显著低于取食常规棉花的0.05 U/mg prot;斜纹夜蛾的酸性磷酸酶活性为0.15 U/mg prot,高于取食常规棉花的0.10 U/mg prot,但差异不显著,其它中肠酶活性均低于对照,亦无显著差异。     

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。     

转基因棉和常规棉叶片对抗Cry1Ac近等基因系棉铃虫生长发育的影响

利用室内饲喂法,以抗Cry1Ac近等基因系棉铃虫为材料,比较转基因棉花33B和SGK321及其对照亲本DP5415和石远321对抗、感棉铃虫生长发育的影响。结果表明,抗性棉铃虫在取食常规棉叶后表现出一定的适合度代价。取食DP5415和石远321两种常规棉花后,抗性品系棉铃虫的幼虫存活率显著低于敏感品系,取食33B和SGK321两种转基因棉花的抗性棉铃虫,不仅其幼虫存活率显著高于敏感品系,而且致死中时间也比敏感品系延长。取食9天后,抗性品系在常规棉花石远321和DP5415上发育到3龄和4龄幼虫的比例显著低于敏感品系,取食33B和SGK321转基因棉花的抗性品系发育到3龄幼虫的比例均显著高于敏感品系。抗性品系在常规棉上的蛹重均显著低于敏感品系,部分取食转基因棉花的抗性品系棉铃虫可以化蛹,而敏感品系不能在转基因棉花上化蛹。     

转 Bt基因抗虫棉对棉大卷叶螟抗性的研究

:转Bt基因抗虫棉对棉铃虫有很好的抗性,对棉大卷叶螟(SyleptaderogataFabricius)的抗性未见报道。作者研究表明,棉大卷叶螟在棉田为聚集分布型,低龄幼虫有集中为害习性;抗虫棉对其有很好的抗性,平均虫株率为2.2%,较常规棉中棉所12降低96.6%;百株虫量为133.8头,较常规棉降低88.4%。     

转cry1Ac玉米对亚洲玉米螟的抗性评价

目标性状的有效性评价是转Bt基因抗虫玉米研发和安全性评价研究的重要内容之一。采用酶联免疫吸附剂测定（ELISA）、室内生测和田间人工接虫鉴定研究了转cry1Ac玉米纯合群体BT-X和分离群体BT-38、BT-181、BT-43、BT-105等转化事件Cry1Ac蛋白的表达量以及对亚洲玉米螟Ostrinia furnacalis（Guenée）的室内杀虫和田间抗虫效果。转cry1Ac玉米鲜组织中Cry1Ac蛋白含量在44.07～438.00 ng/g之间,不同转化事件及不同组织之间差异显著,其中BT-X鲜花丝中Bt蛋白表达量显著低于未展开心叶。室内生测试验中除BT-43与对照没有显著差异外,取食其它转基因玉米心叶的亚洲玉米螟初孵幼虫6或7天的存活率为0～15.3%,显著低于非转基因对照;取食夏播BT-X鲜花丝6天的幼虫存活率为62.5%,显著低于Bt11及非转基因对照。田间心叶期接虫鉴定显示BT-X高抗亚洲玉米螟。说明转cry1Ac玉米BT-X、BT-181、BT-38和BT-105对亚洲玉米螟有很高的杀虫作用,BT-X田间抗虫效果良好。     

转基因棉花对草间小黑蛛生长发育及其相关酶活性的影响

为评估转基因抗虫棉花对天敌的安全性,用取食常规棉(中棉所49)、单价棉(中棉所45)和双价棉(2009002)的棉铃虫幼虫饲喂草间小黑蛛,采用酶联免疫和分光光度计法研究转基因抗虫棉对草间小黑蛛生长发育及其相关酶活性的影响。结果显示,与常规棉花相比,单价棉花使草间小黑蛛4～5龄幼虫发育历期显著延长,对其余龄期影响不显著;双价棉花对草间小黑蛛1～3龄幼虫发育历期影响不显著,4～5龄幼虫发育历期显著延长,6龄后大量死亡。2种转基因棉对草间小黑蛛体重及其体内外源蛋白含量的影响均不显著,除谷胱甘肽-转移酶和单价棉处理的过氧化物酶外,对其它相关酶活性亦无显著影响。     

新疆南部棉区转Bt基因棉花对棉铃虫抗性的季节性变化规律

采用ELISA检测和室内生物测定方法,测定6个转Bt基因棉花品种(系)不同生育期叶片中的Bt毒蛋白含量及其对各世代棉铃虫的抗性水平。转Bt基因棉花叶片中的Bt毒蛋白含量总体上随着棉花生育期的推进而逐渐下降,6个品种(系)叶片的Bt毒蛋白含量子叶期最高,达1 210.03～1 733.15 ng/g,与子叶期相比,三叶期、七叶期、盛蕾期、花铃期、吐絮期Bt毒蛋白含量减少幅度为2.5%～96.0%。6个品种(系)对第4代棉铃虫幼虫抗虫性较低,毒杀效果仅为19.0%～41.3%。幼虫的校正死亡率逐代下降,各棉花品种对第4代幼虫的校正死亡率仅为12.0%～36.2%。     

棉铃虫危害对转基因棉花挥发物成分和释放速率的影响

采用顶空吸附法和气质联用对棉铃虫轻度危害的苗期转基因棉花33B释放的挥发物进行研究。结果表明:棉花挥发物的主要成分是绿叶气体、萜类及其衍生物,包括己醛,α-蒎烯、莰烯、β-蒎烯、柠檬烯、β-水芹烯、香芹醇、壬醛、顺3己烯醇乙酸酯、十四碳烯、β-石竹烯和柏木酮等;棉花的茎、叶及地上部分的释放速率分别为27.2、391.4、223.5 ng/g.h,棉株挥发物的总释放量主要由叶片所释放;棉花受棉铃虫幼虫轻度危害后,地上部分挥发物的释放速率明显增加,危害后1 h和12 h时的释放速率分别为622.54、69.6 ng/g.h,与对照棉花有明显差异;24h后其释放速率基本恢复;在受害棉花的挥发物组分中,主要是绿叶气体和萜类成分变化明显。     

转基因棉对棉铃虫幼虫生长及中肠蛋白酶活性的影响

室内测定了转慈姑蛋白酶抑制素(API)基因棉对棉铃虫幼虫的抗虫活性及对中肠蛋白酶活性的影响。结果表明，以转基因棉饲养棉铃虫，对幼虫拒食率24h达76．49％，72h达80．57％，试虫体重在取食48h后显著下降，72h后较对照低54．04％。试虫取食转API基因棉后，中肠的强碱性胰蛋白酶和类胰凝乳蛋白酶活性显著降低，取食24、48h后的强碱性胰蛋白酶酶活性分别只有对照组的68．1l％和55．89％；类胰蛋白酶活性分别为对照组的85．87％和64．61％；对弱碱性类胰蛋白酶活性不明显。显示转API基因棉对棉铃虫幼虫中肠蛋白酶的抑制作用与对棉铃虫生长发育的阻滞有良好的相关性。     

Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India

BACKGROUND: The pink bollworm is one of the most destructive pests of cotton. Transgenic cotton producing Bt toxin Cry1Ac or a combination of Cry1Ac and Cry2Ab2 has been used effectively against this pest. However, some other insects have evolved resistance to Bt toxins in the field. During the 2007–2008 and 2008–2009 seasons, pink bollworm populations in India were surveyed to evaluate their responses to Cry1Ac and seed powder containing Cry1Ac and Cry2Ab2. RESULTS: The results provide evidence that resistance to Cry1Ac had evolved by 2008 in a population sampled from non‐Bt cotton in the Amreli district of Gujarat in western India. The median lethal concentration of Cry1Ac for five‐day‐old larvae (LC₅₀) was significantly higher for insects derived in 2008 from Amreli than for any of the other field populations tested from four locations in India. For Cry1Ac, the mean LC₅₀ for the strain derived from Amreli in 2008 was 44 times higher than for the most susceptible population. However, for seed powder of Bollgard II containing primarily Cry2Ab2, the 2008 Amreli population was only slightly less susceptible than the most susceptible population. CONCLUSIONS: The data reported here constitute the first evidence of field‐evolved resistance of pink bollworm to Cry1Ac. This initial evidence spurred more extensive evaluations during the 2009–2010 growing season, which confirmed field‐evolved resistance to Cry1Ac in Amreli. The lack of cross‐resistance to Cry2Ab2 suggests that plants producing this toxin are likely to be more effective against resistant populations than plants producing only Cry1Ac. Copyright © 2011 Society of Chemical Industry     

Selection and heritability of resistance to Bacillus thuringiensis subsp kurstaki and transgenic cotton in Helicoverpa armigera (Lepidoptera: Noctuidae)

Compared with an unselected susceptible population, a cotton bollworm, Helicoverpa armigera (Hübner), population selected for 22 generations with transgenic cotton leaves (modified Cry1A) in the laboratory developed 11.0-fold resistance to Cry1Ac (one single-protein product MVPII). Resistance to Bacillus thuringiensis Berliner subsp kurstaki (Btk) was selected for 22 generations with a 5.2-fold increase in LC50. The estimated realized heritabilities (h2) of resistance for transgenic-cotton- and Btk-selected populations were 0.1008 and 0.2341, respectively. This reflects the higher phenotypic variation in response to Cry1Ac in the transgenic-cotton-selected population. This variation may have been caused by differences in protein toxin levels expressed in different growth stages of the transgenic cotton. Because of the different slopes of the probit regression lines between Cry1Ac and Btk, the estimated realized h2 cannot be used visually to compare resistance development to Cry1Ac and Btk in H armigera. Thus, the response quotient (Q) of resistance was also estimated. The Q values of resistance for transgenic-cotton- and Btk-selected populations were 0.0763 and 0.0836, respectively. This showed that the rate of resistance development would be similar in both selection populations. This result indicates that the selection of resistance using transgenic cotton is different from that selected using the single toxin. Resistance risk to transgenic cotton and Btk in field populations was assessed assuming different pressures of selection by using the estimated h2. Assuming the h2 of resistance in a field population was half of the estimated h2, and the population received prolonged and uniform exposure to transgenic cotton or Btk causing >70% mortality in each generation, we predicted that resistance would increase 10-fold after <23 generations for Cry1Ac in transgenic cotton-selected-populations and after <21 generations for Btk in Btk-selected populations. Cross-resistance would be expected after <48 generations for Btk in transgenic-cotton-selected populations and after <21 generations for Cry1Ac in Btk-selected population. The results show that the potential to evolve resistance is similar in both transgenic-cotton- and Btk-selected populations, but that cross-resistance development to Btk is slower in transgenic-cotton-selected populations than cross-resistance development to Cry1Ac in Btk-selected populations.     

棉花抗蚜性与可溶性糖含量的关系

为探讨可溶性糖含量与棉花抗蚜性的关系,在室内人工接蚜为害和田间自然蚜群两种条件下,测定不同抗蚜性棉花品种受棉蚜为害后叶片中可溶性糖含量及其对棉蚜生长发育和繁殖的影响。结果表明:未受棉蚜为害时,抗性品种中可溶性糖含量高于感性品种,棉蚜为害胁迫后,抗蚜棉花品种可溶性糖含量升高,感蚜品种的可溶性糖含量降低。棉蚜的生长发育和繁殖与棉花叶片中可溶性糖含量呈显著的相关,可溶性糖与棉蚜若蚜历期呈正相关,与内禀增长率呈负相关。本研究说明可溶性糖含量对棉花的抗蚜性有一定的影响,转基因棉可溶性糖含量低于常规棉品种可能是导致非目标害虫棉蚜猖獗的原因之一。     

转Bt基因植物对不同抗性棉铃虫的生长抑制作用

采用转Bt基因的烟草和棉花 ,对经转Bt单基因、转Bt与CpTI双基因烟草汰选 1 8代和常规烟草处理的棉铃虫种群进行生长抑制作用的比较测定。结果表明 ,转Bt烟草对以汰选后的抗Bt单基因汰选棉铃虫种群的体重抑制作用显著低于转双基因烟草汰选种群和对照种群 ;转Bt棉花对单基因种群的杀虫活性、体重、龄期发育以及化蛹率的抑制作用亦显著低于双基因种群和对照种群     

棉铃虫ABCA2作为Cry2Ab受体的功能分析

为明确棉铃虫Helicoverpa armigera ATP结合盒转运蛋白家族A蛋白亚家族2（ATP binding cassette transporter subfamily A member 2,ABCA2）在Cry2Ab杀虫机制中的作用,利用酵母双杂交技术研究棉铃虫ABCA2与Cry2Ab的结合特性,并利用RNA干涉（dsRNA和siRNA）降低ABCA2在细胞和幼虫中的表达,结合细胞毒理学试验分析ABCA2的功能。结果表明,棉铃虫ABCA2能与Cry2Ab特异性结合;棉铃虫的ABCA2序列与美洲棉铃虫H.zea相关序列的相似度为92.86%,利用dsRNA干涉美洲棉铃虫中肠细胞ABCA2的表达能显著降低Cry2Ab对中肠细胞的毒力;利用siRNA干涉技术降低ABCA2在棉铃虫幼虫活体中的表达也显著降低了Cry2Ab对幼虫的毒力。表明棉铃虫ABCA2不仅是Cry2Ab的特异性结合蛋白,而且参与Cry2Ab的杀虫过程,是Cry2Ab的一个重要功能受体。     

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.     

Evaluation of food sprays and relay strip crops for enhancing biological control of bollworms and cotton aphids in cotton

Studies were conducted at the Texas Agricultural Experiment Station research farms located at Munday (1996 test) and Chillicothe (1997 test) to evaluate relay strip crops in combination with a food spray to enhance biological control of bollworms, Helicoverpa zea (Boddie), and cotton aphids, Aphis gossypii Glover, in cotton. The relay crops included fall plantings of hairy vetch, Vicia villosa Roth, and canola, Brassica napus L., and a spring planting of grain sorghum, Sorghum bicolor L. Cotton, Gossypium hirsutum L., was planted between the relay crops or was isolated from the relay crops. Treatments within the two cotton systems included an untreated check plot, a plot sprayed with sugar+yeast (food spray) during summer to attract and hold predator insects, a plot sprayed with biological ('soft') insecticides for bollworm and cotton aphid control (Bacillus thuringiensis and pymetrozine, respectively), and a plot sprayed with harsh insecticides for bollworm and cotton aphid control (zeta cypermethrin and dicrotophos or profonofos, respectively). A split-plot experimental design, with three replications, was used where whole plots included relay and isolated cotton systems and subplots were the four food/chemical treatments. Predator numbers were monitored with a vacuum sampler once a week in relay crops and cotton. Bollworms and cotton aphids were monitored visually once a week in cotton during July and August. Total predator numbers were higher in cotton adjacent to relay crops in 1996, but not in 1997. The food spray did not enhance attraction and retention of predators either year. Bollworm larval numbers were significantly higher in relay cotton, food spray plots in 1996. Bollworm larval numbers were similar in relay and isolated cotton, and larval numbers were significantly reduced only in the plot where zeta cypermethrin (harsh insecticide plot) was used. In 1996, cotton aphid numbers in the relay cotton system were significantly higher in the untreated check plots in relation to numbers in the food spray, soft insecticide, and harsh insecticide plots, which were statistically similar. In the isolated cotton system, aphid numbers were highest in untreated plots, intermediate in food spray and soft insecticide plots, and lowest in the harsh insecticide plots. Aphid numbers increased more rapidly in the harsh insecticide plots that had been treated previously for bollworm control. The food spray and pymetrozine treatments reduced cotton aphids more effectively in the relay cropping system than in the isolated cotton system. Bollworms and cotton aphids did not reach pest status in 1997. The combination of a relay cropping system with a food spray did not enhance predator numbers and did not aid in retention of predators in cotton during August. Sucrose in the food spray attracted high numbers of bollworms in 1996.     

甜菜夜蛾幼虫对常规棉和转基因棉的取食选择行为

本文研究了甜菜夜蛾Spodoptera exigua幼虫在转基因棉和常规棉上的取食行为反应,以及有/无学习经历对其取食选择的影响及选择行为对蛹重的影响。结果表明,用人工饲料饲养至3龄5、龄的甜菜夜蛾幼虫在常规棉品种33和转基因棉品种33B上、常规棉品种石远321和转基因棉品种SGK321上的取食选择行为除24h内更多的5龄幼虫选择了石远321,在其它时段,幼虫并没有表现出对转基因叶片的规避行为,甚至有些时段更多地选择在转基因叶片上取食;分别用33叶片、33B叶片饲喂至3龄的甜菜夜蛾幼虫进行33和33B的取食选择,以及分别用石远321S、GK321的叶片饲喂至3龄的甜菜夜蛾幼虫进行石远321和SGK321的取食选择均未表现出对转基因棉的规避行为。上述所有处理中,甜菜夜蛾在常规棉上化蛹的蛹重与转基因棉上化蛹的蛹重无显著差异。结果表明,甜菜夜蛾对单价转基因棉33B和双价转基因棉SGK321均未表现出行为抗性。因此,在转基因棉花的害虫治理中要特别关注甜菜夜蛾的防治。     

不施药条件下转基因抗虫棉的种植表现

通过对8个试种的转基因抗虫棉品种对棉铃虫抗虫性的对比试验和系统观察,表明中29F1、南抗3号和GK22等品种对棉铃虫的抗性较强,且丰产性能好,可作为治理棉铃虫的一项重要措施进行推广;杂交抗虫棉只有其F1代种有较高的使用价值,F2代性状分离严重,不宜在生产上推广使用。     

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。     

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。