The relationship between Bt toxin content and resistance to Sylepta derogata in various transgenic Bt cotton varieties

棉大卷叶螟Sylepta derogata Fabricius为近年来长江流域棉花中后期的一种重要害虫.在其危害盛期测定了8种转基因棉叶片Bt毒蛋白的含量与受害程度,在此基础上就转基因棉对棉大卷叶螟的抗虫性采用不同抗性指标相结合的方法进行了综合评估,同时对转基因棉对棉大卷叶螟的抗虫效果与毒蛋白含量的相关性进行分析.结果表明:各转基因棉品种的不同部位叶片的毒蛋白含量呈现顶叶最高,功能叶次之,老叶含量最低的趋势.抗蚜8017和SGK321 2个转基因棉品种的棉叶毒蛋白表达量相对较低,平均值在120μg/g以下,对棉大卷叶螟的抗性级别均为中抗.其它供试转基因棉棉叶毒蛋白表达量均值在150μg/g以上,抗性级别也均为高抗,而非转基因棉泗棉3号与石远321对棉大卷叶螟不具抗性.转基因棉对棉大卷叶螟的抗性水平与毒蛋白含量呈正相关.     

转Bt基因棉对棉大卷叶螟种群动态的影响

2年的研究发现,棉大卷叶螟SyleptaderogataFabricius的种群数量在不同棉花品种田均呈现上升趋势,但转基因抗虫棉“国抗2 2”的为害程度及百株虫量均小于亲本对照棉“泗棉3号”,其差异达显著或极显著水平;该虫的室内饲养结果表明,2个棉花品种均有利于棉大卷叶螟的增殖,但“国抗2 2”对棉大卷叶螟的抗性明显优于“泗棉3号”,且对棉大卷叶螟成虫存在一定的产卵排斥效应。     

低浓度Cry1Ac毒蛋白对棉大卷叶螟连续三代生长发育与繁殖的影响

棉大卷叶螟是棉花生长中后期的重要食叶性害虫.本研究以浸叶法测定低浓度(0.5 μg·g^-1)的Cry1Ac毒蛋白处理对棉大卷叶螟连续3个世代生长发育与种群增长的影响.结果表明: 处理后的棉大卷叶螟幼虫期比对照延长1.78～2.00 d,成虫寿命延长1.44～1.94 d,蛹体质量增加5.3%～11.8%;3个世代的单雌产卵量分别增加65.1%、47.3%和37.6%,种群趋势指数分别提高11.4%、17.5%和20.0%.这些指标与对照均存在显著差异.蛹历期、羽化率、性比和卵孵化率等与对照无显著差异.研究表明,转Bt基因棉长期种植后棉大卷叶螟的种群数量将会增加,需要进一步评估转基因棉种植的风险性,加强转基因棉田害虫的综合治理.     

取食转基因棉的棉大卷叶螟对中华通草蛉捕食功能与生长发育的影响研究

以GK-12、新棉33B和SGK321等转基因棉和常规棉泗棉3号为材料,研究了取食转基因棉的棉大卷叶螟对中华通草蛉生长发育和捕食功能的影响。结果表明,中华通草蛉捕食转基因棉花品种上的棉大卷叶螟后,幼虫死亡率增加2.8%-8.8%、茧重降低0.12 mg-0.50 mg/头、羽化率下降0.7%-10.1%,幼虫及茧的历期与对照相比差异达到显著或极显著水平。草蛉对棉大卷叶螟的捕食功能反应符合HollingⅡ型,其捕食量和捕食效率均高于对照,处理1头卷叶螟幼虫所用时间短于对照,瞬间攻击率大于对照。研究认为,草蛉更易捕获取食转基因棉田的棉大卷叶螟,但捕食了这些猎物后对捕食者本身的生长发育带来一定影响。     

转Bt基因抗虫棉(Gossyposium)伤流中Bt毒蛋白的运输

本文以常规棉品种‘石远321'和抗虫棉品种‘SGK321'与‘99B'为材料,通过考马斯亮蓝和ELISA方法分析了伤流中可溶性蛋白和Bt(苏云金芽孢杆菌)毒蛋白含量.结果发现,转基因抗虫棉伤流中可溶性蛋白含量显著高于常规棉,并在抗虫棉伤流中检测到了Bt毒蛋白,而常规棉伤流中没有Bt毒蛋白的存在.通过嫁接实验研究发现,将常规棉嫁接到转Bt基因抗虫棉砧木上,叶片中也有Bt毒蛋白的积累.这些结果说明,Bt毒蛋白可以通过木质部伤流液向地上部运输,并在叶片中积累,Bt毒蛋白由根系向地上部的运输对棉花的抗虫性是有益的.     

转Bt 基因抗虫棉(Gossyposium)伤流中Bt 毒蛋白的运输

本文以常规棉品种‘石远321’和抗虫棉品种‘SGK321’与‘99B’为材料, 通过考马斯亮蓝和ELISA方法分析了伤流中可溶性蛋白和Bt(苏云金芽孢杆菌)毒蛋白含量。结果发现, 转基因抗虫棉伤流中可溶性蛋白含量显著高于常规棉, 并在抗虫棉伤流中检测到了Bt毒蛋白, 而常规棉伤流中没有Bt毒蛋白的存在。通过嫁接实验研究发现, 将常规棉嫁接到转Bt基因抗虫棉砧木上, 叶片中也有Bt毒蛋白的积累。这些结果说明, Bt毒蛋白可以通过木质部伤流液向地上部运输, 并在叶片中积累, Bt毒蛋白由根系向地上部的运输对棉花的抗虫性是有益的。     

Bt棉与常规棉根际土壤Bt毒蛋白和植物激素变化动态

通过ELISA法对常规棉（石远321）和转基因抗虫棉（99B、SGK321）根际土壤苏云金芽孢杆菌（Bt）毒蛋白和植物激素含量进行了分析,结果表明抗虫棉根际土壤Bt毒蛋白含量显著高于常规棉,特别是从苗期到盛花期抗虫棉根际土壤Bt毒蛋白含量高出常规棉200-300ng／g。抗虫棉根际土壤中脱落酸（ABA）和生长素吲哚乙酸（IAA）含量变化趋势与常规棉相比表现出较大差异。常规棉与抗虫棉根际土壤中ABA变化趋势表现为相似的单峰曲线,而抗虫棉根际土壤中ABA含量变化幅度明显小于常规棉。在棉花生长发育后期抗虫棉根际土壤中赤霉酸（GA3）含量较高,而常规棉根际土壤中GA,含量升高较抗虫棉早且变化幅度大,在高峰处常规棉高于抗虫棉,低谷时抗虫棉高于常规棉。7月17日常规棉根际土壤IAA含量显著高于抗虫棉,8月27日抗虫棉又大大高于常规棉,其他时期变化不大。上述结果表明,外源Bt基因的插入或表达能引起棉花根际土壤中Bt毒蛋白和植物激素含量的较大改变,这种改变可能会影响到抗虫棉的生长发育,并对环境产生影响。     

高温对Bt棉盛蕾期蕾中Bt蛋白表达及氮代谢生理的影响

以来源于中国的Bt棉品种泗抗1号(常规种)、泗抗3号(杂交种)和来源于美国的Bt棉品种99B(常规种)、岱杂1号(杂交种)为材料,研究了不同高温对Bt棉盛蕾期蕾中Bt蛋白表达量变化及氮代谢生理特征的影响.结果表明: 与对照(32 ℃)相比,在38 ℃以上高温时,4个品种棉蕾中Bt蛋白表达量均显著下降,且随温度升高,Bt蛋白含量下降幅度增大.杂交种棉蕾中Bt蛋白表达量要高于常规种,且受高温影响较小.Bt杀虫蛋白表达量下降幅度大的品种,其可溶性蛋白含量、谷氨酸丙酮酸转氨酶(GPT)活性、谷氨酸草酰乙酸转氨酶(GOT)活性下降幅度也大,游离氨基酸含量、蛋白酶活性、肽酶活性增加幅度较大.     

转CpTI-Bt基因棉和转Bt基因棉对棉铃虫幼虫存活、生长及营养利用的影响

2000年7月中旬和8月中旬, 分别测定了采自田间的转CpTI-Bt基因双价抗虫棉（SGK321, 以下简称CpTI-Bt棉）和转Bt基因抗虫棉（中30,以下简称Bt棉）对棉铃虫Helicoverpa armigera幼虫存活、生长的影响。结果表明：7月中旬两种转基因抗虫棉抗虫效果均较好,尤其是CpTI-Bt棉棉叶和花瓣对4龄幼虫3天内致死率为92%以上;8月中旬两种转基因棉的抗虫活性均明显降低,且Bt棉的杀虫活性显著低于CpTI-Bt棉,其幼虫死亡率与对照受体棉中16的死亡率之间无显著差异,仅显著抑制了幼虫的生长;石远321(SGK321受体品系)的花瓣具有一定的抗虫活性,可显著降低取食幼虫的体重,甚至造成部分幼虫死亡; CpTI-Bt棉中,花瓣和棉叶的抗虫性明显高于蕾和铃心。对5龄幼虫取食棉铃1日后的营养指标测定结果显示： 两种转基因抗虫棉处理的幼虫相对生长率和相对取食量均显著低于石远321,但两者之间无显著差异; CpTI-Bt棉处理的幼虫近似消化率显著低于石远321和Bt棉,但其食物利用率显著高于石远321和Bt棉。     

不同棉花品种对棉盲蝽的抗性鉴定

随着转基因棉花在中国的大面积推广,棉盲蝽逐渐成为我国棉花生产中重要的害虫之一。我国对棉盲蝽研究基础比较薄弱,棉盲蝽的综合治理工作迫在眉睫。本实验通过田间调查,系统研究了棉盲蝽在不同时期对美中棉、荆7516-1、皖棉小黄花、海7124、SGK321、N73DelpatineF0、棕絮×浅绿红叶鸡脚叶棕絮和池州红叶棉8个不同棉花品种的为害情况,得到如下结论:皖棉小黄花对棉盲蝽的抗性较高且比较稳定,而N73DeltapineF0、棕絮×浅绿红叶鸡脚棉棕絮这2个棉花品种对棉盲蝽的抗性较低。     

转基因抗虫棉Bt基因不同剂量的聚合与抗虫性表现

通过有性杂交手段培育出聚有不同数目Bt基因的植株,在不同生育期进行抗虫性测定和Bt毒蛋白表达的ELISA检测,旨在揭示聚合不同数目Bt基因的植株抗虫性的互作表达机理。聚合有1－4个Bt基因的植株在整个生育期的抗虫性、毒蛋白表达特性和单价抗虫棉时空表达一致,生育前期抗虫性好、毒蛋白表达量高;生育中、后期抗虫性有所下降,毒蛋白表达量降低。聚合有4个Bt基因的纯合材料并未因Bt基因的增加而起到抗性增强的效果,相反还因同源抑制而有所降低。不同来源的Bt基因处于杂合状态时其抗虫性和Bt毒蛋白量均得到充分表达。     

Bt毒素在转基因棉花与土壤系统中的分布

研究了转Bt基因棉花与土壤系统中Bt毒素的分布.结果表明,两种转Bt基因棉花地上部(叶片、茎秆)的毒素表达量(103.5～134.1 ng·g^-1)显著高于地下部分(根系)(44.7～21.2 ng·g^-1),土壤中Bt毒素总量可通过转基因棉花地上部分秸秆的处理得到控制;Bt毒素在转Bt基因棉花根系分泌物中的含量极低,如果控制Bt毒素的其它导入来源,将显著降低转Bt基因作物释放中因Bt毒素导入而引发的对土壤生态系统的扰动.     

Feeding of fall armyworm,Spodoptera frugiperda,on Bt transgenic cotton and its isoline

Studies on insect food intake and utilization are important for determining the degree of insect/plant association and host species’ resistance, and also for helping design pest management programs by providing estimates of potential economic losses, techniques for mass breeding of insects, and identifying physiological differences between species. We studied the feeding and development of fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), on transgenic (Bt) and non‐transgenic (non‐Bt) cotton. The larvae of S. frugiperda fed on Bt cotton had a longer development period (23.0 days) than those fed on non‐Bt cotton (20.2 days). Survivorship of S. frugiperda larvae fed on Bt cotton (74.1%) was lower than that of larvae fed on non‐Bt cotton (96.7%). Pupal weight of larvae fed on Bt cotton (0.042 g) was lower than that of larvae fed on non‐Bt cotton (0.061 g). The cotton cultivar significantly affected food intake, feces production, metabolization, and food assimilation by S. frugiperda larvae. However, it did not affect their weight gain. Intake of Bt‐cotton leaf (0.53 g dry weight) per S. frugiperda larva was lower than the intake of non‐Bt‐cotton leaf (0.61 g dry weight). Larvae fed on Bt‐cotton leaves produced less feces (0.25 g dry weight) than those fed on non‐Bt‐cotton leaves (0.37 g dry weight). Weight gain per S. frugiperda larva fed on Bt‐cotton leaves (0.058 g dry weight) was similar to the weight gain for larvae fed on non‐Bt‐cotton leaves (0.056 g dry weight). The cotton cultivar significantly affected the relative growth, consumption, and metabolic rates, as well as other nutritional indices: the figures were lower for larvae fed on Bt‐cotton leaves than for larvae fed on non‐transgenic cotton leaves.     

Effect of pyramiding Bt and CpTI genes on resistance of cotton to Helicoverpa armigera (Lepidoptera: Noctuidae) under laboratory and field conditions

Transgenic cotton (Cossypium hirsutum L.) varieties, adapted to China, have been bred that express two genes for resistance to insects, the CrylAc gene from Bacillus thuringiensis (Berliner) (Bt), and a trypsin inhibitor gene from cowpea (CpTI). Effectiveness of the double gene modification in conferring resistance to cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), was studied in laboratory and field experiments. In each experiment, performance of Bt+CpTI cotton was compared with Bt cotton and to a conventional nontransgenic variety. Larval survival was lower on both types of transgenic variety, compared with the conventional cotton. Survival of first-, second-, and third-stage larvae was lower on Bt+CpTI cotton than on Bt cotton. Plant structures differed in level of resistance, and these differences were similar on Bt and Bt + CpTI cotton. Likewise, seasonal trends in level of resistance in different plant structures were similar in Bt and Bt+CpTI cotton. Both types of transgenic cotton interfered with development of sixth-stage larvae to adults, and no offspring was produced by H. armigera that fed on Bt or Bt+CpTI cotton from the sixth stage onward. First-, second-, and third-stage larvae spent significantly less time feeding on transgenic cotton than on conventional cotton, and the reduction in feeding time was significantly greater on Bt+CpTI cotton than on Bt cotton. Food conversion efficiency was lower on transgenic varieties than on conventional cotton, but there was no significant difference between Bt and Bt+CpTI cotton. In 3-yr field experimentation, bollworm densities were greatly suppressed on transgenic as compared with conventional cotton, but no significant differences between Bt and Bt+CpTI cotton were found. Overall, the results from laboratory work indicate that introduction of the CpTI gene in Bt cotton raises some components of resistance in cotton against H. armigera, but enhanced control of H. armigera under field conditions, due to expression of the CpTI gene, was not demonstrated.     

Amino acid composition and level affect Bt protein concentration in Bt cotton

Low insect resistance has been widely observed in Bacillus thuringiensis (Bt) transgenic cotton. The objective of this study was to investigate the effects of amino acid compositions and their concentration on the Bt toxin content in Bt cotton to gain a better physiological understanding of the observed phenomenon. In the 2010 study, the leaf amino acid composition, contents, and Bt protein concentrations were investigated under high temperature (36?°C) and low temperature(18?°C) at both square and boll stage respectively. In 2011, the leaves were sprayed with ethionone sulfoximine (MSO), aminooxyacetic acid (AOA) or combination of MSO and AOA at the two developing stages, and the leaf amino acid composition and Bt protein concentrations were investigated. In 2012, 17 amino acid compositions were used to spray cotton plants at the same two stages, and the Bt protein concentration were measured. In comparison to the control, more amino acids changed and greater concentration changes had been observed under the extreme temperatures at boll stage compared to that at square stage, which were conformity to the changes of the leaf Bt protein concentration. More amino acids reduced and greater reduction was noted with the application of MSO, AOA or combined MSO and AOA at boll period than that at square period, which resulted in the reduction of the leaf Bt toxin concentration. The external uses of Aspartic acid, Glutamic acid, Glycine, Proline, Tyrosine, Methionine, Phenylalanine, Histidine and Arginin enhanced the leaf Bt toxin level significantly at boll stage. However, only Aspartic acid, Glutamic acid, Proline, Methionine, Arginin could increase Bt protein concentration markedly at square period and the bolstered extent were low. Therefore, it was concluded that cotton amino acid composition and concentration was more sensitive to external factors at boll stage. In addition, there are close relationship between leaf Bt toxin content and Aspartic acid, Glutamic acid, Glycine, Proline, Tyrosine, Methionine, Phenylalanine, Histidine and Arginin and their concentrations.     

Fitness of Bt‐resistant cabbage loopers on Bt cotton plants

Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt‐resistant insects on Bt and non‐Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt‐resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt‐resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non‐Bt cotton and on transgenic Bt cotton leaves expressing a single‐toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R₀) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4‐day asynchrony of adult emergence between the susceptible T. ni grown on non‐Bt cotton leaves and the dual‐toxin‐resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.     

转基因棉花Bt毒蛋白的表达及其生态学效应

苏云金杆菌毒蛋白 (Bacillusthuringiensisgtoxicprotein)基因导入棉花植株后获得的转Bt棉可以特异性地毒杀棉铃虫及鳞翅目的一些其它害虫 ,有效地保护棉花植株不受此类棉花害虫的危害。Bt毒蛋白在棉花植株中的表达受一些内外界因素的影响而呈明显的时空变化。转Bt棉除了严重影响靶标害虫自身外 ,还能对其它一些非靶标昆虫和环境产生影响。另外 ,该文还对害虫对Bt棉的抗性以及防止害虫产生抗性的治理对策进行了综述     

Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis

Various studies have been conducted to assess the damage caused by secondary lepidopteran pests to transgenic Bt maize expressing Cry1Ab. However, to date little is known on the effects of transgenic maize on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a polyphagous herbivore which is considered a pest in Mediterranean maize growing areas. Here we present results on the effects of Bt maize (Bt‐11) and Bt spray (Dipel) on the various life stage parameters of this herbivore. We further assess the expression of Cry1Ab in different leaves and leaf parts in maize at a given plant growth stage, and determine whether the feeding damage of 3rd instar S. littoralis is influenced by Bt toxin expression. Contrary to previous literature reporting that S. littoralis is not sensitive to Bt Cry1Ab toxin, our results show that insects fed on either transgenic or Bt sprayed plants were negatively affected. Young S. littoralis larvae (1st and 2nd instars) were found to be the most sensitive to the Bt toxin. This was represented by a higher mortality and a slower developmental time of larvae maintained on transgenic or sprayed plants when compared to insects maintained on control plants. Moreover, Bt maize had a stronger and prolonged detrimental effect on insects when compared to Bt spray in maize. This was revealed by the fact that insects maintained on transgenic plants from 3rd instar to pupation took longer to reach adult emergence compared to insects that were maintained on sprayed plants. This was likely due to the continuous exposure of insects to the toxin when kept on transgenic maize. ELISA results showed a variation in the amount of Bt toxin among different leaf sections in transgenic maize at a given plant growth stage. These differences in Bt toxin were primarily found in the youngest leaf of growing plants. Although the lowest amounts of Bt toxin were detected in the growing leaf section of young leaves, this difference did not appear to influence the feeding behavior of 3rd instar S. littoralis.     

Bt毒蛋白在转Bt基因棉中的表达及其在害虫-天敌间的转移

以常规棉泗棉3号作为对照,采用酶联免疫生测法（ELISA）和室内生物测定法,研究了转Bt基因棉新棉33B和GK-12不同组织器官中Cry1Ac或Cry1Ab毒蛋白的表达及其向靶标害虫（棉铃虫）、非靶标害虫（棉蚜）以及天敌（龟纹瓢虫）的传递和影响。研究结果表明,新棉33B各组织器官中Bt毒蛋白的表达量较高,为79.7～1 390.0 ng/g鲜重,GK-012较低为165～2640 ng/g鲜重。在花盛期,新棉33B各组织器官中Bt毒蛋白的表达量依次为：柱头、花 >子房、花瓣>群尖;而5～7叶期的初展嫩叶、现蕾初期的幼蕾及花铃期的幼铃表达量相当,而且与花盛期群尖的表达量没有明显区别。同样处于花盛期的GK-12,其各组织器官中Bt毒蛋白的表达量依次为：花药>柱头>花瓣>群尖>子房;而5～7叶期的初展嫩叶、现蕾初期的幼蕾及花铃期的幼铃表达量相当,而且与花盛期群尖的表达量没有明显差异。常规对照棉的幼铃、花药、柱头以及子房中痕量Bt毒蛋白的存在可能与传粉昆虫等的活动有关。在转Bt基因棉田采集的棉蚜和棉铃虫老龄幼虫,其体内均可检测到Bt毒蛋白;在新棉33B棉田采集的龟纹瓢虫幼虫和成虫体内也可检测出Bt毒素。当以Bt棉田的棉蚜饲喂龟纹瓢虫时,龟纹瓢虫的生长发育、存活以及繁殖等基本没有受到影响。     

Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to promote survival of susceptible pests. To delay pest resistance to transgenic cotton producing Bt toxin Cry1Ac, farmers in the United States and Australia planted refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. Here we report data from a 2010 survey showing field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera), in northern China. Laboratory bioassay results show that susceptibility to Cry1Ac was significantly lower in 13 field populations from northern China, where Bt cotton has been planted intensively, than in two populations from sites in northwestern China where exposure to Bt cotton has been limited. Susceptibility to Bt toxin Cry2Ab did not differ between northern and northwestern China, demonstrating that resistance to Cry1Ac did not cause cross-resistance to Cry2Ab, and implying that resistance to Cry1Ac in northern China is a specific adaptation caused by exposure to this toxin in Bt cotton. Despite the resistance detected in laboratory bioassays, control failures of Bt cotton have not been reported in China. This early warning may spur proactive countermeasures, including a switch to transgenic cotton producing two or more toxins distinct from Cry1A toxins.