Effects of transgenic Cry1A＋CpTI cotton and Cry1Ac toxin on the parasitoid, Campoketis chlorideae （Hymenoptera：Ichneumonidae）

Abstract The effect of transgenic double genes, Cry1A + CpTI cotton and Cry1Ac toxin on the parasitoid, Campoketis chlorideae Uchida of cotton bollworm, Helicoverpa armigera (Hübner), was investigated in the laboratory. Helicoverpa armigera larvae when in the first, second and third instar could not survive if fed on transgenic cotton leaves. Consequently, C. chlorideae larvae could not complete their development if parasitizing on such hosts. After H. armigera larvae were reared on transgenic or traditional cotton leaves for 12J48 hours, they were parasitized by C. chlorideae females. Parasitized larvae continued to feed on transgenic or traditional cotton for 12–48 h. The present results showed that the body weight of larvae of the parasitoids were significantly reduced when parasitized hosts fed on transgenic cotton leaves compared to those fed on traditional cotton. Duration of egg and larvae stage were significantly prolonged, pupal and adult weight of C. chloridae was decreased when the host larvae fed on transgenic cotton leaves longer than 48 h. The development duration of C. chlorideae pupae on the hosts fed on transgenic cotton leaves in each treatment was not significantly different from those of controls. The longevity of parasitoid females and males fed with a solution containing Cry1Ac toxin was not significantly different with that of the control.     

Cry1Fa对Cry1Ac抗性棉铃虫的毒力评价

为了防治多种鳞翅目害虫, 表达Cry1Fa的转基因玉米和棉花已在美国商业化种植。明确棉铃虫Helicoverpa armigera对Cry1Fa与Cry1Ac的交互抗性及这两种杀虫蛋白之间的协同作用, 可以为表达 Cry1Fa+Cry1Ac的转双价抗虫棉花的合理应用提供依据。本实验测定了Cry1Fa对棉铃虫敏感品系（96S）及用Cry1Ac筛选的抗性品系（BtR, 抗性倍数2 194.15倍）的毒力, 发现Cry1Fa对敏感棉铃虫的毒力远低于Cry1Ac, LC₅₀值是Cry1Ac的504.80倍; 而且抗性品系BtR对Cry1Fa存在19.98倍的交互抗性。Cry1Fa与Cry1Ac混用可以提高Cry1Fa毒杀敏感棉铃虫的效果, 尤其是Cry1Fa浓度较低时, 加入Cry1Ac, 可以显著增加Cry1Fa的毒力; 但只有加入较高浓度的Cry1Fa时才能增加Cry1Ac的毒力。由于BtR品系已经对Cry1Ac产生抗性, Cry1Ac对抗性棉铃虫的毒力明显降低; 在较高浓度的Cry1Ac中加入Cry1Fa可以显著增加棉铃虫的死亡率（P=0.0015, F=6.88, df=6）, 但最高死亡率仅为58.33%。D-饱和最优试验的结果证实, Cry1Ac对于敏感棉铃虫的死亡率的影响达到显著水平（t₁=13.76﹥t_0.05）, Cry1Ac与Cry1Fa的交互作用对毒力的影响也达到显著水平（t₂₂=2.42﹥t_0.05; t₁₁=6.95﹥t_0.05; t₁₂=3.43﹥t_0.05）。Cry1Ac和Cry1Fa对抗性棉铃虫死亡率的影响都达到显著水平（t₁=3.03﹥t_0.05;t₂=2.59﹥t_0.05）, 但Cry1Ac是决定抗、 感棉铃虫死亡率的关键因素; Cry1Ac与Cry1Fa最佳浓度配比范围都是1.41~2.10 μg/cm2; 在抗性品系中, Cry1Ac和Cry1Fa的交互作用不显著。所以, 尽管Cry1F+Cry1A作物扩大了杀虫谱, 但棉铃虫对这两种蛋白存在交互抗性, 而且这两种蛋白混用对治理抗Cry1Ac棉铃虫的效果不理想, 因此不建议在中国种植表达Cry1F+Cry1A的棉花。关     

Expression and crystallization of a Cry3Aa–Cry1Ac chimerical proteinof Bacillus thuringiensis

The insecticidal Cry1 proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene was constructed with the coding regions of the Cry3Aa protein's toxic domain, and of the Cry1Ac protoxin's C-terminal fragment. This chimerical construction expressed a truncated (70kDa) protein in the acrystalliferous strain 4Q7 of B. thuringiensis, assembled in spherical to amorphous parasporal crystals. This protein was recognized only by antibodies raised against the Cry3Aa protein. When the protease-deficient mutant BL21 of Escherichia coli was transformed with the same chimerical construction, a complete (140kDa) chimerical protein was expressed. However, the formation of a crystalline inclusion was unclear. This protein was recognized by antibodies raised against the proteins Cry1Ac and Cry3Aa. Both chimerical proteins showed toxicity against larvae of Leptinotarsa texana, being much more active when expressed truncated in B. thuringiensis. These results suggest that the formation of bipyramidal crystals requires more than just the presence of the C-terminal region of Cryl protoxins. They also suggest that proteolysis plays an important role during the post-translational processing of Cry proteins.     

Cry1Ac和Cry2Ab杀虫蛋白对粘虫幼虫生长发育的影响

【目的】为探究Bt杀虫蛋白对次要靶标害虫粘虫Mythimna separata (Walker)(鳞翅目:夜蛾科)的杀虫活性及对其生长发育的影响。【方法】本文通过浸叶法饲喂初孵及2龄末粘虫不同剂量的Cry1Ac及Cry2Ab杀虫蛋白后,观察其死亡率,称量幼虫重,并统计了幼虫历期、化蛹率、蛹重、蛹期、蛹的羽化率、畸形率等指标。【结果】初孵幼虫取食浸泡含16、64、128μg/mLCry1Ac及Cry2Ab的玉米叶片后,随着时间的延长及浓度的增加,死亡率逐渐增加,且Cry1Ac杀虫蛋白对粘虫的生物活性高于Cry2Ab蛋白,在128μg/mL浓度下,取食Cry1Ac和Cry2Ab蛋白13d时的死亡率分别达到了65%及60%。取食两种蛋白后,初孵幼虫和2龄末幼虫重量均受到显著抑制,短期取食两种蛋白对幼虫历期、化蛹率、蛹重、蛹期、蛹的羽化率、畸形率没有影响。【结论】取食Cry1Ac和Cry2Ab杀虫蛋白后,对初孵幼虫有很好的杀虫活性,且Cry1Ac杀虫活性高于Cry2Ab杀虫蛋白;短期饲喂两种杀虫蛋白时,对2龄粘虫后期生长影响不大。本文结果为转Bt基因作物更好的应用于粘虫的防治提供了理论基础。     

N546 in β18-β19 loop is important for binding and toxicity of the Bacillus thuringiensis Cry1Ac toxin

Our previous mutagenic analysis showed that the unique residue N546 in the apex of β18-β19 loop of Bacillus thuringiensis Cry1Ac toxin is important for its toxicity. In this study, trypsin digestion susceptibility, binding to BBMV and oligomer formation activity was therefore analyzed to determine the mechanism of toxicity change of these mutant toxins. The results showed that residue N546 was not involved in toxin oligomerisation and maintaining the stability of toxin, the enhanced toxicity of mutant N546A was just because of increased binding to BBMV, and reduction in toxicity of other mutants were caused by reduction in initial or irreversible binding to BBMV. This is the first report that revealed N546 in Cry1Ac domain III played an essential role in its insecticidal activity and binding to insect BBMV.     

转cry1Ab/cry1Ac基因玉米Cry1Ab/Cry1Ac融合蛋白表达及对亚洲玉米螟的室内杀虫效果

【目的】室内抗螟性评价是转Bt基因抗虫玉米研发和安全性评价的重要环节。【方法】采用酶联免疫吸附测定法(ELISA)测定了转cry1Ab/cry1Ac基因玉米ZZM030心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量;采用室内生测法测定了分别取食转基因玉米ZZM030和非转基因玉米X249心叶后亚洲玉米螟Ostrinia furnacalis敏感品系ACB-BtS、Cry1Ab抗性品系ACB-AbR和Cry1Ac抗性品系ACB-AcR初孵幼虫的存活率。【结果】转基因抗虫玉米ZZM030 4叶期和8叶期心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量分别是10.62和2.94 μg/g FW。敏感品系亚洲玉米螟初孵幼虫取食转基因玉米ZZM030心叶2 d的存活率仅为23.6%,4 d后存活率为0,而取食非转基因对照玉米X249心叶4 d的存活率高达93.1%。Cry1Ab抗性品系和Cry1Ac抗性品系初孵幼虫取食转基因玉米ZZM030心叶6 d后的存活率分别为11.1%和12.5%,而取食非转基因玉米X249心叶6 d后的存活率分别为81.9%和77.8%。【结论】转cry1Ab/cry1Ac基因玉米ZZM030心叶中高表达的Cry1Ab/Cry1Ac融合蛋白对亚洲玉米螟初孵幼虫具有极高的杀虫效果。     

抗Cry1Ac小菜蛾种群生命表及其适合度研究

本实验研究了小菜蛾Plutella xylostella L.高抗Cry1Ac种群DBM1Ac-R(抗性倍数大于1000)及敏感种群DBM1Ac-S的生长发育、繁殖等生物特征情况,组建了两种群的生命表,并对其适合度进行了研究。结果表明,DBM1Ac-R的产卵量、卵历期、孵化率和化蛹率、蛹重及雌雄比(♀:♂),均显著低于DBM1Ac-S。DBM1Ac-R相对于DBM1Ac-S的相对适合度为0.5762,表明Cry1Ac抗性种群在繁殖能力上存在明显的生存劣势。     

转cry1Ab/cry1Ac基因玉米Cry1Ab/Cry1Ac融合蛋白表达及对亚洲玉米螟的室内杀虫效果

【目的】室内抗螟性评价是转Bt基因抗虫玉米研发和安全性评价的重要环节。【方法】采用酶联免疫吸附测定法(ELISA)测定了转cry1Ab/cry1Ac基因玉米ZZM030心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量;采用室内生测法测定了分别取食转基因玉米ZZM030和非转基因玉米X249心叶后亚洲玉米螟Ostrinia furnacalis敏感品系ACB-BtS、Cry1Ab抗性品系ACB-AbR和Cry1Ac抗性品系ACB-AcR初孵幼虫的存活率。【结果】转基因抗虫玉米ZZM030 4叶期和8叶期心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量分别是10.62和2.94μg/g FW。敏感品系亚洲玉米螟初孵幼虫取食转基因玉米ZZM030心叶2 d的存活率仅为23.6%,4 d后存活率为0,而取食非转基因对照玉米X249心叶4 d的存活率高达93.1%。Cry1Ab抗性品系和Cry1Ac抗性品系初孵幼虫取食转基因玉米ZZM030心叶6 d后的存活率分别为11.1%和12.5%,而取食非转基因玉米X249心叶6 d后的存活率分别为81.9%和77.8%。【结论】转cry1Ab/...     

苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白及其分子设计

cry1Ac编码的杀虫晶体蛋白是苏云金芽孢杆菌(Bt)产生的多种杀虫晶体蛋白中对鳞翅目昆虫有很高毒性的蛋白.第一个Cry1Ac杀虫晶体蛋白最早在库斯塔克亚种HD73中以伴胞晶体形式分离获得,其编码区为3 534 bp,编码蛋白分子量为133 kD,含1 178个氨基酸,等电点为4.84.自此以来,Cry1Ac杀虫晶体蛋白结构、功能以及应用研究一直是Bt杀虫晶体蛋白研究的重要方向.本文介绍了苏云金芽孢杆菌中应用最广泛的Cry1Ac杀虫晶体蛋白家族的结构、功能及其基因分类,并进一步就基于苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白的基因工程研究做了分析,提出了持续利用BtCry1Ac杀虫晶体蛋白的一些见解.     

Cry2Ab及Cry1Ac杀虫蛋白对棉铃虫中肠蛋白酶活性的影响

为明确Cry2Ab和Cry1Ac2种Bt杀虫蛋白单用与混用对棉铃虫Helicoverpa armigera（Htibner）中肠主要蛋白酶活性的影响,本文测定了取食含不同Bt蛋白人工饲料后棉铃虫中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的差异。结果发现：Cry2Ab处理12h后对棉铃虫中肠总蛋白酶影响不大;对类胰蛋白酶的影响最大,除最高浓度处理外,其他浓度处理后棉铃虫类胰蛋白酶的活性明显高于对照;但对类胰凝乳蛋白酶活性的影响呈倒“V”字型,只有6．67ug／gCry2Ab处理后的棉铃虫酶活力显著高于对照,其他浓度处理与对照差异不显著或略低于对照;随着取食含Cry2Ab饲料时间的增加,棉铃虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性比对照显著增加;与对照相比,处理36h后类胰蛋白酶活性最高可增加到6．43倍。Cry1Ac处理棉铃虫12h后总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性都明显增加,而且与处理浓度呈正相关;但是24h后,处理后棉铃虫的总蛋白酶和类胰凝乳蛋白酶活性明显降低,只有类胰蛋白酶活性仍高于对照,但活性增长倍数低于12h时的处理。Cru2Ab和Cry1Ac2种蛋白混用处理棉铃虫后,2种酶的酶活力基本低于Cry1Ac和Cry2Ab单用的酶活力之和;只有2种蛋白浓度均为2．22ug／g混用时,处理12h后类胰蛋白酶和类胰凝乳蛋白酶的活性高于2种蛋白单用时酶活力之和,且都显著的高于对照。     

Pesticidal and Receptor Binding Properties of Bacillus thuringiensis Cry1Ab and Cry1Ac δ-Endotoxin Mutants to Pectinophora gossypiella and Helicoverpa zea

Bacillus thuringiensis produces several larvicidal crystalline inclusions during sporulation. An understanding of their mechanisms of action is commercially important. In this study, two toxins, Cry1Ab and Cry1Ac, were compared that showed 98% amino acid identity in domain I and II, but differed significantly in domain III. Using site-directed mutagenesis techniques, two conserved loop 2 Arg's (³⁶⁸RR³⁶⁹) of Cry1Ab and Cry1Ac toxins were replaced with Ala (³⁶⁸AR³⁶⁹, ³⁶⁸RA³⁶⁹, ³⁶⁸AA³⁶⁹), Glu (³⁶⁸EE³⁶⁹), Phe (³⁶⁸FF³⁶⁹), His (³⁶⁸HH³⁶⁹), and Lys (³⁶⁸KK³⁶⁹). The effect of these mutants on structural stability, larvicidal potency, receptor binding, and ionic permeability towards two important cotton pests, pink bollworm (Pectinophora gossypiella) and bollworm (Helicoverpa zea) were analyzed. All seven mutants of Cry1Ab, excluding ³⁶⁸AR³⁶⁹, produced a stable protoxin, whereas for Cry1Ac all seven mutants yielded stable protoxin. Results showed that all the stable mutants behaved similarly to the wild type on incubation with trypsin and gut extract of both insect larvae. The Cry1Ab mutants, ³⁶⁸AR³⁶⁹, ³⁶⁸AA³⁶⁹, ³⁶⁸FF³⁶⁹, and ³⁶⁸HH³⁶⁹, lost toxicity; ³⁶⁸EE³⁶⁹ had reduced toxicity; whereas the more conserved change ³⁶⁸KK³⁶⁹ retained the toxicity similar to the wild type towards P. gossypiella. Double mutants of Cry1Ac, ³⁶⁸AA³⁶⁹ and ³⁶⁸FF³⁶⁹, abolished the toxicity. Double mutant ³⁶⁸KK³⁶⁹ of Cry1Ac retained its toxicity against P. gossypiella, whereas single mutants ³⁶⁸AR³⁶⁹, ³⁶⁸RA³⁶⁹, and ³⁶⁸HH³⁶⁹ retained only reduced toxicity. All the mutants of Cry1Ab lost their toxicity against H. zea except ³⁶⁸KK³⁶⁹. In Cry1Ac single mutants, ³⁶⁸AR³⁶⁹ and ³⁶⁸RA³⁶⁹, reduction in the toxicity was observed. A double mutant of Cry1Ac, ³⁶⁸KK³⁶⁹, also retained reduced toxicity. All the other double mutants lost their toxicity. Voltage clamping experiments on H. zea midguts provided an additional evidence about the insecticidal property and inhibition of I_sc across the transepithelial membrane of the insect midgut. Received: 5 June 2000 / Accepted: 5 July 2000     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1AC毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1C产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1Ac 毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1c 产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

田间棉铃虫种群对Cry1Ac蛋白的抗性基因频率

【目的】系统了解河南省新乡市田间棉铃虫Helicoverpa armigera(Hübner)种群对Cry1Ac蛋白的敏感性变化,可为该虫的抗性治理策略提供重要的科学依据。【方法】采用单雌系F1/F2代并结合诊断剂量法于2013-2016年连续监测了河南省新乡市棉铃虫种群对Cry1Ac蛋白的抗性基因频率以及种群敏感度的变化。【结果】2013-2016年河南省新乡市棉铃虫种群对Cry1Ac的抗性基因频率小于0.002 12,抗性基因频率处于较低水平;种群的相对平均发育级别由2013年的0.506分别下降到2015和2016年的0.448和0.442,表明棉铃虫对Cry1Ac蛋白的敏感度增加。【结论】河南省新乡市棉铃虫种群对转Cry1Ac基因棉花仍处于较为敏感阶段,转Bt基因棉花的种植面积在河南新乡地区的大面积缩小可能是其抗性发展缓慢的重要原因。     

转基因(Cry1Ac)抗虫棉对土壤微生物的影响

对不同种植年限的转基因(Cry1Ac)抗虫棉田土壤中主要微生物数量变化进行了测定,结果表明：（1）种植转基因抗虫棉后对土壤微生物细菌、放线菌和真菌数量的影响趋势基本相似;（2）种植抗虫棉1年后,土壤中细菌、放线菌、真菌数量将有所增加,连续4年达到高峰,然后数量开始下降,连续种植7年后,棉田微生物数量接近于种植1年的棉田;（3）种植1年后又种植非转基因棉花的棉田,土壤微生物数量低于种植1年抗虫棉的棉田,与种植非转基因棉的数量无明显差异。由此可见,种植转基因抗虫棉对土壤微生物数量有一定的影响。     

二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合

【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。     

棉铃虫中肠氨肽酶APN4与Cry1Ac、Cry2Aa结合能力的比较

【目的】Bt杀虫蛋白(Bacillus thuringiensis)具有高度的靶标特异性,已经被广泛用于农业害虫防治。Bt杀虫蛋白要发挥杀虫活性,必须首先与其受体蛋白结合,氨肽酶N(Aminopeptidase N)是一类重要的Bt受体蛋白。因此,分析该受体与Bt杀虫蛋白的结合能力,可为进一步明确不同Bt的分子作用机制、Bt的抗性治理以及新Bt的开发应用等提供借鉴。【方法】本文利用Ligand blot和Elisa方法比较了棉铃虫Helicoverpa armigera中肠APN4(Aminopeptidase N4,APN4)与Cry1Ac、Cry2Aa的结合能力。【结果】原核表达的APN4片段与活化的Cry1Ac、Cry2Aa都可以结合,解离常数(Kd)分别是48.59 nmol/L和21.73 nmol/L。【结论】APN4片段与Cry1Ac、Cry2Aa的结合能力在数量级上不存在显著性差异。     

大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析

对利用苏云金芽胞杆菌(Bacillus thuringiensis,Bt)强启动子——cry1Ac基因启动子p1Ac指导cry基因在大肠杆菌中的表达进行了研究。结果显示,大肠杆菌中由启动子p1Ac指导表达的Cry1Ac蛋白与苏云金芽胞杆菌来源的Cry1Ac蛋白在碱溶性、胰蛋白酶活化、杀虫活性等方面有较好的一致性,从而解决了目前商业化载体大肠杆菌表达cry基因时形成不易溶解的包涵体问题。同时,还对p1Ac指导的cry1Ac基因在大肠杆菌中表达的发酵条件进行了初步探索。     

Changes of inheritance mode and fitness in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) along with its resistance evolution to Cry1Ac toxin

The changes of inheritance mode and fitness of resistance in Helicoverpa armigera (Hübner) along with its resistance evolution to Cry1Ac toxin were evaluated in the laboratory. The resistance levels reached 170.0-, 209.6- and 2893.3-fold, on selection of the field population in the 16th (BtR-F(16)), 34th (BtR-F(34)) and 87th (BtR-F(87)) generation with artificial diet containing Cry1Ac toxin, respectively. As the resistance levels increased, more larvae feeding on the Bt cotton expressing Cry1Ac toxin survived. Most larvae of BtR-F(87) could develop to the 5th instar and about 3% individuals reached the adult stage. The inheritance of Cry1Ac resistance trait at three resistant levels was autosomal and incompletely recessive, but the degree of dominance decreased as the resistance increased. The resistance was primarily monogenic in BtR-F(16) strain, but polygenic as resistance increased. The relative fitness of H. armigera, measured as a ratio of R(0) (the net replacement rate) of resistant strain divided by R(0) of the susceptible strain, decreased with an increase of the resistance levels, with ratios of 0.79, 0.64 and 0.59 in their respective BtR-F(16), BtR-F(34) and BtR-F(87) strains.