转Bt基因水稻表达的毒蛋白Cry1Ab在害虫及其捕食者体内的积累动态

在室内对转基因水稻KMD1中的Cry1Ab毒蛋白经食物链在几种主要害虫及其捕食性天敌体内的积累进行了研究。结果表明： 无论是水稻孕穗期还是成熟期,二化螟Chilo suppressalis连续取食KMD1或取食KMD1.36 h后移至对照品种秀水11上取食不同时间后,幼虫体内的Cry1Ab含量均随取食时间延长逐渐下降。稻眼蝶 Mycalesis gotama幼虫连续取食KMD1或在KMD1上取食两天后移至秀水11上继续取食不同时间,体内的Cry1Ab含量也都随取食时间延长而下降,但下降速度较二化螟更快。取食KMD1的二化螟和稻眼蝶幼虫的粪便中均检测到较高浓度的Cry1Ab,对照组中均无Cry1Ab。取食KMD1的二化螟幼虫血淋巴中检测到Cry1Ab,含量为3.5 ng/g。取食KMD1的褐飞虱 Nilaparvata lugens、稻蚜Sitobion avenae以及饲喂取食过KMD1的二化螟或稻眼蝶幼虫的拟水狼蛛Pirata subpiraticus体内都含有一定浓度的Cry1Ab,其中,拟水狼蛛体内的CrylAb含量以饲喂取食KMD1稻眼蝶幼虫的含量最高,约为饲喂取食KMD1二化螟幼虫的60倍。这些结果表明Cry1Ab可以沿水稻害虫天敌食物链传递。     

水稻-褐飞虱-拟水狼蛛食物链的定量研究

应用荧光物质稀土元素铕示踪法对水稻褐飞虱拟水狼蛛食物链进行了定量分析研究。所施荧光物质的三种浓度20 mg/L、50 mg/L和100 mg/L 均为有效浓度,对水稻生长无可见的影响,孕穗期、抽穗期、乳熟期和黄熟期四个生育期水稻、褐飞虱和拟水狼蛛的荧光物含量均随所施荧光物质浓度的增高而增高。褐飞虱对不同生育期水稻的取食量依次为抽穗期>孕穗期>乳熟期>黄熟期,分别为每克褐飞虱生物量在24 h内摄食水稻17.5910 g, 17.4510 g, 13.8290 g和8.7070 g。拟水狼蛛对褐飞虱的捕食量为乳熟期>孕穗期>抽穗期>黄熟期,分别为每克拟水狼蛛生物量24 h捕食褐飞虱3.6380 g, 3.0830 g, 3.0770和2.8000 g。根据田间调查数据换算为每头拟水狼蛛捕食褐飞虱在孕穗期、抽穗期、乳熟期和黄熟期分别为11头、11头、13头和10头。     

温度对Bt水稻及其非靶标害虫褐飞虱生态适应性的影响

【背景】温度是影响植物生长发育最为重要的环境因子之一。明确温度对Bt水稻及其非靶标害虫的影响有助于今后Bt水稻的推广应用。【方法】设置不同的温度梯度(26、30和34℃)分别处理Bt水稻T2A-1(表达Cry2A蛋白),在处理后不同天数内分别测定Cry蛋白、叶绿素、草酸、可溶性糖和总氮含量,同时评价不同温度处理下Bt水稻上褐飞虱的生态适应性。【结果】Cry蛋白含量显著受温度影响,随着温度升高Cry蛋白含量下降。温度、处理时间及两者相互作用均显著影响Bt水稻T2A-1的叶片叶绿素、叶鞘草酸、可溶性糖和总氮含量。叶绿素含量在26℃处理20 d时高于30、34℃的处理。随着温度的升高,Bt水稻叶鞘草酸含量升高。可溶性糖含量高温(30和34℃)处理后20 d低于26℃处理。Bt水稻叶鞘内可溶性氮含量随着温度的升高而升高。温度对非Bt水稻MH63上叶绿素、草酸、可溶性糖和总氮含量的影响与T2A-1趋势一致。高温(34℃)处理的Bt水稻上的褐飞虱雌雄虫寿命、每雌产卵量及卵孵化率显著低于26℃处理。温度对非Bt水稻MH63上褐飞虱生态适应性参数的影响与T2A-1趋势一致。【结论与意义】温度对Bt水稻具有显著影响,且对Bt水稻上非靶标害虫褐飞虱的生态适应性产生不利影响。本研究有利于今后Bt稻田的害虫综合治理措施的制定。     

转Bt基因棉花中Cry1Ac蛋白经烟粉虱途径向龟纹瓢虫的传递

【目的】烟粉虱Bemisia tabaci是转基因棉的非靶标害虫,对棉花生产造成严重影响。本文探讨转Bt基因棉花中Cry1Ac蛋白在棉花-烟粉虱-龟纹瓢食物链中的传递规律,以期为转基因棉的环境安全评价提供科学依据。【方法】在实验室条件下,以常规棉SM3号、33、SY321为对照,分析转Bt基因棉花GK12、XM33B、SGK321叶片、在这些棉花上取食的烟粉虱、以及捕食烟粉虱的瓢虫体内Cry1Ac蛋白含量。同时,将取食转Bt基因棉花上的烟粉虱的瓢虫转接到对应的受体亲本棉花上,分析瓢虫体内Cry1Ac蛋白含量变化规律。【结果】在转Bt基因棉花上取食的烟粉虱成虫和若虫以及它们的蜜露中均能检测到Cry1Ac蛋白,以转Bt基因棉花上的烟粉虱若虫为食料的龟纹瓢虫体Propylaea japonica内也能检测到Cry1Ac蛋白。龟纹瓢虫取食转Bt基因棉花上的烟粉虱若虫1 d后体内即能检测到Cry1Ac蛋白,并且随着取食时间的延长,体内Cry1Ac蛋白的含量逐渐增加,但到第6~8天后Cry1Ac蛋白的含量相对稳定。取食3个不同品种棉花上烟粉虱若虫的龟纹瓢虫体内Cry1Ac蛋白的含量存在明显差异,这种差异与棉花叶片上表达的Cry1Ac蛋白量呈正相关。但取食后6 d,在3个品种棉花上取食的龟纹瓢虫体内的Cry1Ac蛋白含量之间没有明显的差异。以转Bt基因棉花上的烟粉虱若虫为食料的龟纹瓢虫转移到对应的常规棉亲本上以后,体内的Cry1Ac蛋白的含量迅速下降,但10 d后仍能检测到微量的Cry1Ac蛋白。【结论】转Bt基因棉花中的Cry1Ac蛋白可以通过烟粉虱途径传递到龟纹瓢虫体内,龟纹瓢虫对食料中的Cry1Ac蛋白具有富集作用,并且Cry1Ac蛋白的富集存在饱和现象,富集饱和量与食料中的Cry1Ac含量无关;龟纹瓢虫脱离含有Cry1Ac蛋白的食料环境后,体内的Cry1Ac蛋白可以消减,但在10 d时间内龟纹瓢虫体内仍会有Cry1Ac残留。     

常见水稻病虫害胁迫对转cry1C基因抗虫水稻Bt蛋白表达量的影响

【目的】明确转Bt基因抗虫水稻在病虫害胁迫下对Bt蛋白表达量的影响。【方法】以Bt水稻T1C-19(表达Cry1C蛋白)为研究对象,探究褐飞虱、白叶枯病等6种水稻常见病虫害胁迫下其体内Bt蛋白的表达量变化。【结果】褐飞虱取食引起T1C-19水稻叶片中Bt蛋白含量降低,而黑尾叶蝉取食显著降低了T1C-19水稻叶鞘中的Bt蛋白含量,白叶枯病侵染导致Bt水稻叶鞘中的Bt蛋白含量显著上升。二化螟、水稻普通矮缩病、稻瘟病的胁迫对转基因水稻T1C-19叶片和叶鞘中的Bt蛋白含量均无显著影响。【结论】病虫害胁迫因种类不同对Bt水稻中Bt蛋白表达量的影响有所不同。这将为Bt水稻的抗虫效果评价提供数据基础,同时为Bt水稻病虫害综合治理提供科学依据。     

转基因水稻表达的Bt蛋白对拟环纹豹蛛(Pardosa pseudoannulata)生长发育的影响

为明确转基因水稻表达的Bt蛋白沿捕食性食物链的传递及其对生态系统中较高营养级生物的影响,在实验条件下(28±0.5 ℃,R.H=80%,L∶D=12h∶12h)以田间优势自然天敌拟环纹豹蛛为对象,以取食转Cry1Ab/Cry1Ac基因水稻汕优63(简称Bt水稻)叶片24 h的稻纵卷叶螟幼虫为猎物饲养拟环纹豹蛛,并测定了...     

CrylAb蛋白在拟环纹豹蛛体内的富集及对其体内酶活性的影响

用Cry1Ab蛋白处理的果蝇喂养拟环纹豹蛛Pardosa pseudoannulata,在第1d、3d、5d、7d、9d、11d用酶联免疫(ELISA)检测技术测定实验组和对照组拟环纹豹蛛体内Cry1Ab蛋白含量,运用紫外分光光度法检测拟环纹豹蛛体内超氧化物歧化酶( SOD)、谷胱甘肽过氧化物酶(GSH-Px)、乙酰胆碱酯酶(AChE)的活性,探讨拟环纹豹蛛体内Cry1Ab蛋白的富集作用与其体内3种酶活性的关系.结果表明,从第1d至第7 d Cry1Ab蛋白在拟环纹豹蛛体内均具富集作用(p＜0 05),到第7d的时候达到最高值,达极显著差异(p＜0.01),然后逐渐降低,但仍显著高于对照(p＜0.05).SOD酶活性总体趋势是降低的,在第7d达到最低;用Cry1Ab蛋白处理拟环纹豹蛛后其体内的AChE和GSH-Px酶活性随饲养时间的延长而增加,并均高于对照组(p＜0.05).该研究证明Cry1Ab蛋白在拟环纹豹蛛体内具有富集效应,并且其体内Cry1Ab蛋白对其体内GSH-Px、AChE具有一定诱导作用,对SOD酶具有一定程度的抑制作用.     

粽管巢蛛和拟水狼蛛对稻纵卷叶螟和稻褐飞虱捕食作用研究

应用二次正交旋转组合设计方法建立粽管巢蛛和拟水狼蛛对稻纵卷叶螟和稻褐飞虱捕食作用的数学模型。结果表明,粽管巢蛛是稻纵卷叶螟的重要天敌,粽管巢蛛和拟水狼蛛均是稻褐飞虱的重要天敌。当稻褐飞虱密度较高时,随着蜘蛛密度的增加,由于蜘蛛之间的干扰作用,导致稻褐飞虱被捕食量下降。     

用ELISA方法评价捕食性天敌对白背飞虱的捕食效能

用酶联免疫吸附试验（ELISA）方法测定了9类捕食性节肢动物对白背飞虱的捕食阳性反应率,定量评价了拟水狼蛛对白背飞虱的捕食效能。结果表明：活动能力强,与稻飞虱生境相似的捕食性天敌种类表现出较高的阳性反应率;拟水狼蛛对白背飞虱的捕食效率随水稻的生长而加强,在水稻生长后期,分别为21.53％和43．08％。     

应用单克隆抗体评价拟环纹豹蛛对褐飞虱的控制作用

应用褐飞虱的单克隆抗体 4 B8研究了 1999年浙江大学华家池校区农场汕优 6 3单季晚稻田中拟环纹豹蛛对褐飞虱的捕食作用。采用盘拍法的调查结果表明 ,褐飞虱、拟环纹豹蛛种群数量高峰期均在水稻生长后期 (9月中旬 ) ,最大种群密度分别为12 6头 /丛和 1.83头 /丛。对每次捕获的每头拟环纹豹蛛样品的抗体夹心 EL ISA检测结果表明 ,拟环纹豹蛛对褐飞虱单克隆抗体的阳性反应率与田间褐飞虱的生物量及拟环纹豹蛛对褐飞虱的占有量显著相关。定量评估结果表明 ,在此密度条件下 ,拟环纹豹蛛对褐飞虱的最大捕食率仅为 2 .2 8%。拟环纹豹蛛对褐飞虱的平均捕食量、总捕食量和捕食率与田间褐飞虱的生物量显著相关。总捕食量、捕食率与拟环纹豹蛛种群密度极显著相关     

Laboratory and field assessments of prey-mediated effects of transgenic Bt rice on Ummeliata insecticeps (Araneida: Linyphiidae)

One major concern regarding the release of Bt rice is its potential impact through tritrophic interactions on nontarget arthropods, especially natural enemies. We studied the effects of two Bt transgenic rice varieties, TT9- 3 and KMD1, expressing Cry1Ab/Cry1Ac and Cry1Ab, respectively, on a predatory ground spider [Ummeliata insecticeps (B?senberg et Strand)] supplied with Bt rice-fed brown planthopper [Nilaparvata lugens (St?l)] nymphs. Although immunoassays confirmed that U. insecticeps ingested Bt insecticidal protein when supplied with Bt rice-fed N. lugens, no negative effects were found on its survival and development. Furthermore, the fecundity of U. insecticeps fed prey reared on Bt rice was not significantly different from that of those fed prey reared on non-Bt rice. A 3-yr field trial indicated that Bt rice did not significantly affect the population density of U. insecticeps in comparison with non-Bt rice. In conclusion, the Bt rice lines tested in this study had no adverse effects on the survival, developmental time, or fecundity of U. insecticeps in the laboratory or on population dynamics in the field.     

Multi-generation effects of Bt rice on Anagrus nilaparvatae, a parasitoid of the nontarget pest Nilapavarta lugens

Little is known about the potential cumulative long-term effects of transgenic crops on nontarget organisms. In the present laboratory study, the potential cumulative effects of transgenic Bacillus thuringiensis (Bt) rice on parasitoids in successive generations were observed for an egg parasitoid, Anagrus nilaparvatae parasitizing eggs of Nilaparvata lugens (St?l) (Hemiptera: Delphacidae) feeding on Bt rice. Enzyme-linked immunosorbent assay test confirmed that Cry1Ab insecticidal protein could be detected in newly eclosed parasitoid adults. However, no significant effect on the fecundity of Anagrus nilaparvatae Pang et Wang (Hymenoptera: Mymaridae) was observed between Bt and non-Bt rice. Developmental times of both genders of A. nilaparvatae parasitizing host eggs laid in Bt (KMD1 and KMD2) rice lines were significantly prolonged from first generation to second generation, but not always prolonged from third generation to 11th generation as compared with the control rice line. Furthermore, the sex ratio of A. nilaparvatae progeny from the first generation to 11th generation in three rice lines was not significantly different. In general, our results suggested that the effect of Bt rice on this parasitoid could be negligible.     

转Bt基因水稻对两种弹尾虫及尖钩宽黾蝽捕食作用的影响

转Bt基因水稻KMD1、KMD2和对照水稻XS11稻田主要有两种弹尾虫：灰橄榄长角跳虫 Entomobryagriseoolivata (Packard) 和钩圆跳虫 Bourletiella christianseni Banks。两种Bt稻田中灰橄榄长角跳虫种群密度均显著高于对照XS11稻田;在以KMD1和KMD2腐烂 茎叶为食的灰橄榄长角跳虫成虫中检测到微量Cry1Ab杀虫蛋白。室内测定结果表明,不管是单 头捕食还是多头协同捕食,尖钩宽黾蝽Microvelia horvathi Lundblad 成虫对用3种供试 水稻残体饲养的灰橄榄长角跳虫的捕食量和功能反应均符合HollingⅡ型方程,其日捕食量、瞬 时攻击率（a）和处理时间（T_h）均无显著差异。     

Expression of Cry1Ab and Cry2Ab by a Polycistronic Transgene with a Self-Cleavage Peptide in Rice

Insect resistance to Bacillus thuringiensis (Bt) crystal protein is a major threat to the long-term use of transgenic Bt crops. Gene stacking is a readily deployable strategy to delay the development of insect resistance while it may also broaden insecticidal spectrum. Here, we report the creation of transgenic rice expressing discrete Cry1Ab and Cry2Ab simultaneously from a single expression cassette using 2A self-cleaving peptides, which are autonomous elements from virus guiding the polycistronic viral gene expression in eukaryotes. The synthetic coding sequences of Cry1Ab and Cry2Ab, linked by the coding sequence of a 2A peptide from either foot and mouth disease virus or porcine teschovirus-1, regardless of order, were all expressed as discrete Cry1Ab and Cry2Ab at high levels in the transgenic rice. Insect bioassays demonstrated that the transgenic plants were highly resistant to lepidopteran pests. This study suggested that 2A peptide can be utilized to express multiple Bt genes at high levels in transgenic crops.     

Uptake of Bt-toxin by herbivores feeding on transgenic maize and consequences for the predator Chrysoperla carnea

Abstract  1. Chrysoperla carnea is an important predatory insect in maize. To assess the ecological effects of Bt-maize, expressing the Cry1Ab protein, on larvae of this predator, the following factors were examined: (1) the performance of three prey herbivores ( Rhopalosiphum padi , Tetranychus urticae , and Spodoptera littoralis ) on transgenic Bt and non‐transgenic maize plants; (2) the intake of the Cry1Ab toxin by the three herbivores; and (3) the effects on C. carnea when fed each of the prey species.
2. The intrinsic rate of natural increase (r_m) was used as a measure of performance for R. padi and T. urticae . No difference in this parameter was observed between herbivores reared on Bt or non‐transgenic plants. In contrast, a higher mortality rate and a delay in development were observed in S. littoralis larvae when fed Bt-maize compared with those fed the control maize plants.
3. The ingestion of Cry1Ab toxin by the different herbivores was measured using an immunological assay (ELISA). Highest amounts of Cry1Ab toxin were detected in T. urticae , followed by S. littoralis , and only trace amounts detected in R. padi .
4. Feeding C. carnea with T. urticae , which were shown to contain the Cry1Ab toxin, or with R. padi , which do not ingest the toxin, did not affect survival, development, or weight of C. carnea . In contrast, a significant increase in mortality and a delay in development were observed when predators were fed S. littoralis larvae reared on Bt-maize.
5. A combined interaction of poor prey quality and Cry1Ab toxin may account for the negative effects observed on C. carnea when fed S. littoralis . The relevance of these findings to the ecological risks of Bt-maize on C. carnea is discussed.     

Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay

Laboratory bioassays were conducted to evaluate the response of first instar larvae of the monarch butterfly, Danaus plexippus L. (Lepidoptera: Danaidae), a non‐target species, to pollen from corn, Zea mays L. (Commelinales: Poaceae), from two new corn hybrids genetically modified to express different types of insecticidal proteins derived from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt). One hybrid expresses both Cry1Ab and Cry2Ab2 proteins (MON 810 × MON 84006), active against lepidopteran pests, and the other expresses Cry3Bb1 protein (MON 863), targeted against coleopteran pests. First instar larvae were placed on milkweed leaves (Asclepias syriaca L.) (Gentianales: Asclepiadaceae) dusted with doses of either Bt pollen or its nonexpressing (isoline) pollen counterpart ranging from 50 to 3200 grains cm^?2 of milkweed leaves, or no pollen at all. Larvae were exposed to pollen for 4 days, then moved to pollen‐free leaves and observed for another 6 days. Survival was observed after 2, 4, and 10 days. Weight gain was estimated after 4 and 10 days, leaf consumption after 2 and 4 days, and larval development after 10 days. Exposure to pollen of the Cry1Ab/Cry2Ab2‐Bt expressing hybrid reduced larval survival approximately 7.5–23.5% at the dose ranges tested relative to a no pollen control. Larval weight gain and consumption were reduced for larvae exposed to pollen of this hybrid and a small minority of larvae (3.1%) never developed past the third instar after 10 days of observation. Exposure to pollen of the Cry3Bb1‐Bt expressing hybrid had no negative effects on larval mortality, weight gain, consumption, or development relative to the consumption of Bt‐free corn pollen. The relevance of these findings to the risk that these Bt corn hybrids pose to monarch populations is discussed.     

Presence of Cry1Ab in the Bt maize – aphid (Rhopalosiphum maidis) – ladybeetle (Propylea japonica) system has no adverse effects on insect biological parameters

Transgenic Bacillus thuringiensis Berliner (Bt) crops receive particular attention because they carry genes encoding insecticidal proteins that might negatively affect non‐target arthropods. Here, laboratory experiments were conducted to evaluate the impact of Cry1Ab‐expressing transgenic maize [5422Bt1 (event Bt11) and 5422CBCL (MON810)] on the biological parameters of two non‐target arthropods, the aphid Rhopalosiphum maidis (Fitch) (Hemiptera: Aphididae) and its predator the ladybeetle Propylea japonica (Thunberg) (Coleoptera: Coccinellidae). In a long‐term assay (three generations), no significant differences were found between R. maidis fed Bt maize and those fed a near‐isogenic line (5422) when individual parameters were compared, including nymph development time, adult longevity, aphid spawning period, and fecundity. No negative effects were detected throughout the life cycle of P. japonica in aphids’ feeding amount, development (nymphs, pupae, adults, and progeny eggs), fecundity, or egg hatching when they preyed on Bt maize‐fed aphids compared with non‐Bt maize treatments. A tritrophic assay revealed that Cry1Ab was highly diluted through the food chain (Bt maize leaves, R. maidis, and P. japonica), as detected by an enzyme‐linked immunosorbent assay (ELISA). In conclusion, although Cry1Ab concentrations in maize leaves increased as the plants developed, Cry1Ab levels were significantly reduced in the aphid R. maidis, and no traces of Cry1Ab were detected in P. japonica preying on Bt maize‐fed aphids. The two hybrids of Bt maize expressing Cry1Ab had no negative effects on the measured biological parameters of the aphid R. maidis or its predator, the ladybeetle P. japonica.     

转Bt基因稻谷对印度谷螟生长发育的影响

为建立仓储阶段转Bt水稻安全性评价中靶标害虫抗性汰选研究体系,配制了含不同比例(70%,50%,30%,10%)转Bt基因(Cry1Ab/Cry1Abc)明辉63水稻谷粉(简称Bt谷粉)的人工饲料饲喂印度谷螟Plodia interpunctella(Hübner),测定其对1～3龄幼虫在72h内的急性毒力,及对印度谷螟种群生长发育的影响,并采用ELISA法检测转基因稻谷和末龄幼虫体内Bt蛋白含量。结果发现:4种比例人工饲料对幼虫的毒力作用均发生在取食48h后,72h后剂量效应明显。含Bt水稻较高比例的饲料对印度谷螟发育的负面效应明显:幼虫死亡率高,发育历期延长。Bt蛋白在幼虫体内含量与对应饲料中的含量基本成正比。综合考虑,将Bt杀虫蛋白含量2.35μg/g作为转Bt基因稻谷对印度谷螟的亚致死剂量最为合适。     

Bacillus thuringiensis plants expressing Cry1Ac,Cry2Ab and Cry1F are not toxic to the assassin bug,Zelus renardii

Cotton‐ and maize‐producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non‐target organisms, including natural enemies that suppress pest populations. Here, we used Cry1F‐resistant Spodoptera frugiperda (J.E. Smith) and Cry1Ac and Cry2Ab‐resistant Trichoplusia ni (Hübner) as prey for the assassin bug, Zelus renardii (Kolenati), a common predator in maize and cotton fields. In tritrophic studies, we assessed several fitness parameters of Z. renardii when it fed on resistant S. frugiperda that had fed on Bt maize expressing Cry1F or on resistant T. ni that had fed on Bt cotton expressing Cry1Ac and Cry2Ab. Survival, nymphal duration, adult weight, adult longevity and female fecundity of Z. renardii were not different when they were fed resistant‐prey larvae (S. frugiperda or T. ni) reared on either a Bt crop or respective non‐Bt crops. ELISA tests demonstrated that the Cry proteins were present in the plant at the highest levels, at lower levels in the prey and at the lowest levels in the predator. While Z. renardii was exposed to Cry1F and Cry1Ac and Cry2Ab when it fed on hosts that consumed Bt‐transgenic plants, the proteins did not affect important fitness parameters in this common and important predator.