表达Cry1Ab和Cry2Ab蛋白的转基因玉米GAB-3对四种主要鳞翅目害虫的抗性评价

为评价表达Cry1Ab和Cry2Ab蛋白的转基因玉米GAB-3对黏虫Mythimna separata（Walker）、亚洲玉米螟Ostrinia furnacalis（Guenée）、棉铃虫Helicoverpa armigera（Hübner）和草地贪夜蛾Spodoptera frugiperda（J. E. Smith）的杀虫效果，采用室内生测的方法研究了GAB-3不同组织器官对以上四种害虫存活率的影响，并通过田间接虫试验进一步测试了GAB-3对黏虫和亚洲玉米螟的抗性。室内生测结果显示：转基因玉米GAB-3心叶期叶片对黏虫、亚洲玉米螟、棉铃虫和草地贪夜蛾具有很强的毒杀作用，苞叶、穗尖、穗轴、花丝、雄穗和籽粒对亚洲玉米螟和棉铃虫具有显著的抗性。田间接虫试验结果显示：心叶期GAB-3高抗黏虫和亚洲玉米螟，穗期GAB-3高抗亚洲玉米螟。上述结果表明，转基因玉米GAB-3对4种主要玉米鳞翅目害虫有很好的杀虫效果，具有较好的商业化应用前景。     

抗虫耐除草剂玉米杂交种瑞丰1号-双抗12-5对3种主要鳞翅目害虫的抗性

本试验用田间接虫和室内生测方法,研究国产转基因玉米杂交种瑞丰1号-双抗12-5(RF1-12-5)对3种主要的玉米鳞翅目害虫亚洲玉米螟、黏虫、棉铃虫的抗性。田间结果表明:RF1-12-5对亚洲玉米螟、棉铃虫的抗性在相应的鉴定时期均达到高抗水平,RF1-12-5心叶期对黏虫的抗性为抗性水平。室内鉴定结果表明:心叶期,RF1-12-5对亚洲玉米螟抗性起效较慢,饲喂48~72 h后校正死亡率分别为63.97%、77.14%、88.00%;吐丝期饲喂48 h后校正死亡率达100%,抗性显著;籽粒期饲喂24、48、72 h后校正死亡率分别为68.86%、83.96%、98.03%。心叶期饲喂24 h后对黏虫基本无抗性效果,但48、72 h后的校正死亡率分别达到94.64%、100%。吐丝期饲喂24 h后对棉铃虫抗性达到87.59%,48 h后对棉铃虫校正死亡率达到100%,抗性较强。总体来看,RF1-12-5田间对亚洲玉米螟、棉铃虫抗性较高,对黏虫抗性略低;室内心叶期对亚洲玉米螟抗性较弱,但在其他时期对亚洲玉米螟、黏虫、棉铃虫均表现了较好的抗性。因此RF-12-5具有推广应用的潜力。     

抗虫Cry2Ab基因的原核表达及玉米的遗传转化

玉米的产量深受虫害的影响,抗虫Cry2Ab基因对玉米黏虫抗性有重要意义。利用外源抗虫基因Cry2Ab构建到原核表达载体pET-22b中,对其进行SDS-PAGE检测和抗黏虫性鉴定。SDS-PAGE检测结果表明,Cry2Ab基因编码的杀虫蛋白在大肠杆菌BL21中得到正确的表达,条带大小为70.68 kD;抗黏虫性鉴定结果表明,当Cry2Ab蛋白浓度为100μg/g时,对东方黏虫致死率达到86.66%。同时构建pCAMBIA3301-Cry2Ab-Bar植物表达载体,通过农杆菌介导法将外源抗虫基因Cry2Ab转入玉米自交系GSH9901愈伤组织中,利用除草剂筛选及PCR技术检测得到T₁转基因阳性植株4株。获得的转基因抗虫玉米植株为抗虫转基因玉米的研发及抗虫玉米新种质的创制奠定了基础。     

草地贪夜蛾与玉米田其他4种常见鳞翅目幼虫第8腹节背面毛片比较

为准确鉴定玉米田5种常见鳞翅目幼虫,弄清幼虫A8背面毛片区别,通过镜下测定,详细比较玉米田5种常见鳞翅目害虫草地贪夜蛾、玉米螟、黏虫、劳氏黏虫和棉铃虫老龄幼虫第8腹节（以下简称“A8”）背面4个毛片的排列方式、大小和形状。结果表明,草地贪夜蛾4个毛片的排列近似正方形,其他4种均为矩形。毛片大小依次为：草地贪夜蛾>玉米螟>棉铃虫>黏虫>劳氏黏虫,且草地贪夜蛾、玉米螟和棉铃虫前排毛片大于后排毛片,黏虫则相反,劳氏黏虫前、后排毛片大小相当。草地贪夜蛾和黏虫幼虫4个毛片形状均为椭圆形,劳氏黏虫和棉铃虫均为圆形,而玉米螟前排毛片为圆形,后排毛片为椭圆形。     

不同遗传背景的转Cry1C基因玉米品系的室内抗虫性鉴定

以转Cry1C基因的转基因玉米C492为供体,通过回交转育的方式将目标基因分别导入不同遗传背景的玉米自交系,获得转基因玉米自交系C492/N-76、C492/N202、C492/S273、C492/DH1901和C492/DH1904,采用室内离体组织生测的方法研究各品系对玉米螟和草地贪夜蛾的抗虫性。结果表明：转基因玉米C492和5个不同遗传背景自交系均表现出高抗玉米螟,虽对草地贪夜蛾的抗性有一定差异,但接虫6 d后幼虫死亡率均达到80%以上。这说明转基因玉米C492和5个不同遗传背景自交系可用于抗虫转基因玉米新品种培育,具有较好的商业化应用前景。     

漯河市夏玉米花粒期鳞翅目害虫的监测与分析

为明确夏玉米花粒期鳞翅目害虫的种群动态,于2019年和2020年在河南省漯河市对夏玉米花粒期鳞翅目害虫进行田间幼虫调查和测报灯下成虫监测。结果表明,漯河市夏玉米花粒期鳞翅目害虫分属2科6种,其中夜蛾科主要包括东方黏虫[Mythimna separata(Walker)]、劳氏黏虫[Mythimna loreyi(Duponchel)]、棉铃虫[Helicoverpa armigera(Hübner)]和草地贪夜蛾[Spodoptera frugiperda(J. E. Smith)],螟蛾科主要包括亚洲玉米螟[Ostrinia furnacalis(Guenée)]和桃蛀螟[Congethes punctiferalis(Guenée)]。8月上中旬至9月中下旬6种鳞翅目害虫幼虫在夏玉米田混合发生,8月中旬混合种群虫口密度最大,最高虫口密度为118头/百株;不同年份田间优势种略有差异,2019年优势种为桃蛀螟、亚洲玉米螟和棉铃虫,2020年优势种为亚洲玉米螟和桃蛀螟。同时期测报灯共诱集6种鳞翅目害虫8 850头,在8月中旬和9月上旬为诱虫高峰期;桃蛀螟、棉铃虫和劳氏黏虫在两年中均为优势种,亚洲玉米螟在2019年为丰盛种,在2020年为优势种。     

西安市夏玉米重要病虫害发生及防控对策

近年来，随着全球性气候变暖，玉米病虫害发生呈持续加重趋势，如2019年新入侵害虫草地贪夜蛾，对玉米生产构成直接威胁。作者于2017-2019年连续三年对西安地区进行了玉米病虫害调查，结果显示，目前玉米生产上主要虫害有亚洲草地贪夜蛾、玉米螟、桃蛀螟、棉铃虫、黏虫等鳞翅目钻蛀性害虫发生普遍；主要病害有穗腐病、茎腐病、大斑病。值得注意的是穗腐病，其病原菌产生的真菌毒素关系到人畜生命安全，需高度重视。此外，玉米南方锈病、瘤黑粉病、蚜虫、双斑长跗萤叶甲等次要病虫害在个别区县零星发生。对西安地区夏玉米主要病虫害发生及防控要点进行梳理与总结，旨在为今后西安市乃至陕西省玉米病虫害防控及可持续治理提供理论依据和技术支持。     

转Cry抗虫基因玉米对家蚕的安全性评价

家蚕可能通过取食漂移到桑叶上的玉米花粉从而受到转基因玉米中表达的Cry蛋白的影响。实验采用室内生物测试的方法评价了转cry1Ab和cry2Ab基因玉米GAB-3花粉对家蚕可能产生的影响,并采用在饲料中掺入蛋白的方式检测了转基因玉米中广泛使用的6种Cry杀虫蛋白对家蚕的毒性。当桑叶上GAB-3花粉密度为50粒·cm^-2时,对家蚕幼虫存活率无显著影响,达500粒·cm^-2时,家蚕14 d存活率为0;以10粒·cm^-2的GAB-3花粉饲喂家蚕幼虫7 d对其体重无显著不利影响,但延长饲喂时间或者当GAB-3花粉密度为50粒·cm^-2时,家蚕的体重显著低于对照组;转基因和非转基因玉米花粉处理14 d或更长时间的家蚕体重均低于无花粉处理组。与非转基因对照处理组相比,低于100粒·cm^-2的转基因玉米GAB-3花粉处理组家蚕的化蛹率、茧重、茧壳重和蛹重无显著差异,转基因玉米和非转基因玉米花粉处理均会延长家蚕幼虫的发育历期。GAB-3花粉对家蚕7 d的LC₅₀为261.18粒·cm^-2,随着处理时间延长,LC₅₀下降。测试的6种Bt杀虫蛋白对家蚕的毒性依次为Cry1C>Cry1Fa>Cry1B>Cry2A>Cry1Ab>Cry1Ac。目前,在中国接近商业化的转基因玉米中多采用的Bt杀虫蛋白为Cry1Ab和Cry2A,二者对家蚕的毒性较低,并且在实际生产中,家蚕接触到的转基因玉米花粉密度较低,因此转Cry抗虫基因玉米对家蚕的风险较低。     

国产两种转基因Bt玉米对草地贪夜蛾抗性较好

草地贪夜蛾是世界性重大农业害虫,种植转基因Bt 作物是主要的防治手段之一。近期,中国农科院植保所植物病虫害生物学国家重点实验室吴孔明院士团队利用室内生物测定方法,评价了国产Bt-Cry1Ab 玉米(转化体C0030.3.5)和Bt-(Cry1Ab+Vip3Aa)玉米(转化体DBN3601 和DBN3608)对草地贪夜蛾1 ~ 4龄幼虫的毒力。     

两种测报灯对浙中地区玉米田害虫的种群监测与动态分析

为研究浙中地区不同玉米害虫的发生规律,在东阳市同时使用自动虫情测报灯和高空测报灯对玉米田亚洲玉米螟、草地贪夜蛾、桃蛀螟、小地老虎、劳氏黏虫、黏虫、东方蝼蛄和斜纹夜蛾进行种群监测。结果表明:自动虫情测报灯和高空测报灯监测的种群动态基本一致,但峰值数量存在差别,高空测报灯诱集的成虫数量高于自动虫情测报灯;亚洲玉米螟、草地贪夜蛾、桃蛀螟、小地老虎、劳氏黏虫、黏虫和斜纹夜蛾在浙中地区每年均可发生4—5代,东方蝼蛄每年可发生1代;高空测报灯监测数量具有“突增突减”现象,亚洲玉米螟、草地贪夜蛾和黏虫的春季高峰均比自动虫情测报灯提早2—6 d。利用高空测报灯和自动虫情测报灯监测峰值的时间差可有效指示迁飞性昆虫的迁飞行为。     

Genome editing of the SfABCC2 gene confers resistance to Cry1F toxin from Bacillus thuringiensis in Spodoptera frugiperda

ATP-binding cassette transporter C2(ABCC2) is known to be a receptor for Bacillus thuringiensis(Bt) toxins in several lepidopteran insects. Mutations in the ABCC2 gene have been genetically linked to field-evolved resistance to the Cry1 F toxin from Bt in Spodoptera frugiperda. Here we generated a SfABCC2 knockout strain of S. frugiperda using the CRISPR/Cas9 system to provide further functional evidence of the role of this gene in susceptibility and resistance to Cry1 F. Results from bioassays showed that the SfABCC2 knockout S. frugiperda strain displayed 118-fold resistance to Cry1 F compared with the parental DH19 strain, but no resistance to Vip3 A toxin from Bt. These results provide the first reverse genetic evidence for SfABCC2 as a functional receptor for Cry1 F.     

Expression profiles of Cry1Ab protein and its insecticidal efficacy against the invasive fall armyworm for Chinese domestic GM maize DBN9936

The fall armyworm(FAW) Spodoptera frugiperda, which originated in the Americas, is advancing across China and threatening the nation's maize crops. Currently, one widely used tool for its control is genetically modified(GM) Bacillus thuringiensis(Bt) maize. Sufficient content of Bt protein in appropriate plant parts is crucial for enhancing resistance against insect pests. In this study, we conducted a systematic investigation of Cry1 Ab levels in Chinese domestic GM maize DBN9936, which has recently obtained a biosafety certificate, and evaluated its efficacy against FAW. Quantification of expression levels of Cry1 Ab, via ELISA, indicated a spatio-temporal dynamic, with significant variation of mean Cry1 Ab, ranging from 0.76 to 8.48 μg g–1 FW with the Cry1 Ab protein level ranked as: V6–V8 leafR1 leafR4 leafR1 silkVT tasselR4 kernel. Among the nine locations, the Cry1 Ab levels in DBN9936 of the Xinxiang, Langfang, and Harbin fields were significantly lower than those from Wuhan and Shenyang, and were slightly, but not significantly lower than those from the other four fields. Furthermore, the artificial diet–Cry1 Ab mixture and plant tissue feeding bioassays revealed that DBN9936 has high efficacy against FAW. The insecticidal efficacy of different tissues against FAW larvae reached 34–100% with a descending order of lethality as follows: VT leafR4 leafR1 huskR1 silkVT tasselR4 kernel. Taken together, our results showed that Bt-Cry1 Ab maize DBN9936 has potential as a promising strategy to manage FAW.     

苏云金杆菌新菌株WZ-7的PCR-RFLP分析及生物活性研究

利用PCR—RFLP技术分析了自河北省土壤中分离的苏云金杆菌新菌株WZ-7，结果表明该菌株含有cryl、cry2Ab、cryllal型基因，其中cryl型基因的酶切片段类型不同于已发表的基因类型，有可能含有新的基因。SDS—PAGE分析结果出现130、79、73、66、60、58kD左右的6种晶体蛋白，毒素蛋白类型属于Ⅱ型。室内生测结果显示，该菌株对重要的农业害虫棉铃虫、甜菜夜蛾、小菜蛾、菜青虫、玉米螟等均有较高的杀虫毒力。     

苏云金芽孢杆菌杀虫晶体蛋白对棉铃虫活性分析

利用已克隆的5种Bt Cry基因Cry2Ab4、Cry1Ia8、Cry1Ie1、Cry1Ca7、Cry1Cb2和1种野生菌株HD-73(Cry1Ac)表达的6种Bt Cry杀虫晶体蛋白,对棉铃虫进行生物活性分析,并将Cry1Ac杀虫晶体蛋白分别与其它5种Cry蛋白按1:1的比例组合,对棉铃虫进行生物活性测定。结果表明,单独使用Cry1Ac时对棉铃虫活性最高,LC50为3.16μg·mL-1,其次为Cry2Ab4。Cry1Ac与Cry2Ab4组合对棉铃虫也有较高的活性,LC50为48.70μg·mL-1,该组合对棉铃虫的共毒系数为1.21,有相加作用。Cry1Ac与这5种蛋白的组合对棉铃虫都有较高的毒力。     

Thrips-mediated impacts from transgenic rice expressing Cry1Ab on ecological fitness of non-target predator Orius tantilus(Hemiptera:Anthocoridae)

Various rice lines have been genetically modified with genes from Bacillus thuringiensis(Bt) to continuously produce Bt insecticidal proteins against lepidopteran larvae. The Bt insecticidal protein constantly expresses in the plants to create an opportunity for non-target herbivores to acquire and convey the protein to their predators or parasitoids across trophic levels. This paper evaluates the effects of Bt rice(namely, Kemindao 1(KMD1) and Kemindao 2(KMD2)) expressing Cry1 Ab as compared to its non-Bt control line, Xiushui 11 on non-target predator Orius tantilus(a generalist predatory anthocorid of thrips) under laboratory and field conditions. To measure several biological parameters such as total nymphal duration and fecundity of this bug, it was reared on thrips and pollens of KMD1 and KMD2 as compared to their control under laboratory conditions. By comparison with the control, Bt rice did not significantly affect main life-history characteristics(total nymphal duration, female adult longevity, oviposition period and fecundity) of this anthocorid preying on Bt rice-fed thrips along with Bt rice pollens, except that the fecundity of this predator for KMD1 was distinctly lower as compared with KMD2 or the control. Enzyme-linked immunosorbent assay(ELISA) results showed that no Cry1 Ab protein was detected in this predator fed on thrips or rice pollen from Bt rice but was in Bt rice pollens. With the beat plate, plastic bag and color trap sampling methods, two-year field monitoring of O. tantilus abundance demonstrated that Bt rice had no significant detrimental effects on the population dynamics and seasonal average densities of this predatory anthocorid as compared with the control. Thus, it is suggested that growing our tested Bt rice(KMD1 and KMD2) producing Cry1 Ab will pose a negligible risk to the anthocorid, O. tantilus.     

吡虫啉悬浮种衣剂对玉米田节肢动物群落及主要非靶标害虫的影响

【目的】明确吡虫啉种衣剂对玉米田节肢动物群落及主要非靶标害虫的影响,为种衣剂的科学使用及玉米害虫防控提供理论依据。【方法】分别于2015年和2016年6—9月,在河南农业大学许昌校区试验田,设置600 g·L-1吡虫啉悬浮种衣剂推荐最高使用剂量及其2倍和4倍剂量进行种子包衣,以清水为对照,每个处理重复4次,随机区组排列。每小区采用5点取样方法,每点连续标记10株玉米,分别在玉米苗期、喇叭口期、孕穗期、灌浆初期和灌浆中期调查样点内玉米植株上全部节肢动物的种类及其数量,分析不同处理区节肢动物的群落特征。在玉米灌浆期,调查样点内玉米穗部害虫种类及数量;玉米成熟时每小区收获两行,每行连续收取50株,调查雌穗上和茎秆中的鳞翅目幼虫种类和数量,分析吡虫啉种衣剂对主要非靶标害虫的影响。【结果】种衣剂处理不影响玉米田节肢动物群落物种种类,处理区与对照区的物种丰富度随时间的变化趋势基本相似,同一时间内不同处理区物种丰富度的差异与有些物种的密度低而在调查样点内的分布不均匀有关。吡虫啉种衣剂处理不会造成玉米田节肢动物群落多样性和均匀度下降,在靶标害虫玉米蚜发生量大时还能通过抑制玉米蚜的数量而使多样性和均匀度增加。种衣剂处理对玉米田节肢动物优势种的影响不仅年份间有差异而且不同玉米生育期间也有差异,在靶标害虫玉米蚜种群数量较高的2015年,玉米生长前期种衣剂处理区的植食性昆虫和捕食性天敌优势种与空白对照区基本一致,灌浆期黏虫和草间小黑蛛分别成为种衣剂2倍和4倍剂量处理区的植食性昆虫和捕食性天敌的优势种;在玉米蚜种群数量较小的2016年,孕穗期以后亚洲玉米螟和棉铃虫为各处理区植食性昆虫优势种,龟纹瓢虫和异色瓢虫成为捕食性天敌优势种。玉米不同生育期各处理区优势种的优势度指标与其优势集中度指标变化趋势相似。种衣剂处理对穗部鳞翅目幼虫的影响程度较复杂,如玉米灌浆初期和灌浆中期种衣剂处理区亚洲玉米螟幼虫种群数量均高于或显著高于对照区;灌浆初期种衣剂处理区桃蛀螟幼虫种群数量均显著高于对照,灌浆中期则低于或显著低于对照;灌浆中期种衣剂处理区黏虫幼虫种群密度均高于对照;种衣剂处理区棉铃虫幼虫密度与对照区虽有不同,但差异不显著。综合两年的调查结果,玉米灌浆期种衣剂处理区亚洲玉米螟、桃蛀螟和黏虫幼虫种群密度有增加的现象,但其种群密度与600 g·L-1吡虫啉悬浮种衣剂处理剂量没有明显的相关性。【结论】建议吡虫啉种衣剂处理玉米田应注意防控鳞翅目穗部害虫。     

Selection,effective dominance,and completeness of Cry1A.105/Cry2Ab2 dual-protein resistance in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)

In the U.S., Helicoverpa zea (Boddie) is a major pest targeted by both transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins. Resistance of insect to Bt maize and cotton containing cry1A and cry2A genes has widely occurred in the U.S. In this study, two trials were performed to investigate larval survival and development of a Cry1A.105/Cry2Ab2 dual-protein resistant (VT2P-RR), a susceptible, and an F₁ heterozygous (VT2P-RS) populations of H. zea on ears of nine Bt and three non-Bt maize hybrids. The Bt maize hybrids evaluated represent five common pyramided traits expressing two or three of the Cry1A.105, Cry1Ab, Cry1F, Cry2Ab2, and Vip3Aa20 proteins. In the laboratory, neonates of the three H. zea populations were inoculated on silks of ears collected from maize at R1–R2 plant stages; and larval survivorship was checked 10 d after neonate release. All three insect populations survived normally on non-Bt maize ears. Varied numbers of VT2P-RR and VT2P-RS survived on ears of Cry1A.105/Cry2Ab2 maize, while all larvae of the three populations died or could not develop on ears of Vip3Aa20-expressing maize. The results demonstrated that the dual-protein resistant H. zea was not cross-resistant to Vip3Aa20-expressing maize, and thus traits with vip3Aa20 gene should be effective to manage Cry1A.105/Cry2Ab2-resistant H. zea. The resistance in VT2P-RR was determined to be incomplete on Cry1A.105/Cry2Ab2 maize. The effective dominance levels varied greatly, from recessive to incompletely dominant, depending on maize hybrids and trials, suggesting that proper selection of maize hybrids could be important for mitigating the Cry1A.105/Cry2Ab2 resistance. The data generated should aid in modeling multiple-protein Bt resistance in H. zea.     

海南鲜食玉米蛀果性害虫种类与发生为害调查

对琼海、乐东、三亚等主要鲜食玉米种植区为害玉米的蛀果性害虫进行调查。结果表明：海南鲜食玉米蛀果性害虫主要有4种,隶属鳞翅目夜蛾科和螟蛾科。其中为害最严重的是亚洲玉米螟[Ostrinia furnacalis（Guenée）],田间为害率最高达72．2％,其次顺序为粘虫、棉铃虫和条螟。