Engineering modified Bt toxins to counter insect resistance

The evolution of insect resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins that are widely used in sprays and transgenic crops. Resistance to Bt toxins in some insects is linked with mutations that disrupt a toxin-binding cadherin protein. We show that susceptibility to the Bt toxin Cry1Ab was reduced by cadherin gene silencing with RNA interference in Manduca sexta, confirming cadherin's role in Bt toxicity. Native Cry1A toxins required cadherin to form oligomers, but modified Cry1A toxins lacking one alpha-helix did not. The modified toxins killed cadherin-silenced M. sexta and Bt-resistant Pectinophora gossypiella that had cadherin deletion mutations. Our findings suggest that cadherin promotes Bt toxicity by facilitating toxin oligomerization and demonstrate that the modified Bt toxins may be useful against pests resistant to standard Bt toxins.     

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.     

氨肽酶N(APN)与鳞翅目昆虫对Bt抗性的关系

使用Bt Cry毒素防治农业害虫是作物生产上的一个革命性的进步,受体与Bt杀虫蛋白结合能力的改变可能是昆虫对Bt产生抗性的主要原因。氨肽酶N(aminopeptidase N,APN)是一类存在于昆虫中肠内的Bt毒素受体蛋白,通过讨论APN与Bt毒素的结合作用,综述了APN基因变异与鳞翅目昆虫Bt抗性相关的分子机理,并介绍了(Bt)Cry毒素与APN相关的作用方式新模型。     

昆虫中肠Bt毒素受体氨肽酶N的研究进展

氨肽酶N(APN)是昆虫中肠中主要的Bt毒素受体,它与Cry毒素特异结合后,毒素插入细胞膜,在膜上形成孔洞,细胞裂解,最终导致昆虫死亡.APN的变异导致昆虫对Bt敏感性下降甚至产生抗性.就APN的结构特征、分类、APN与Cry毒素的相互作用机制以及APN与昆虫Bt抗性的关系作一综述.     

Identification of a gene associated with Bt resistance in Heliothis virescens

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used for pest control. Bt-resistant insect strains have been studied, but the molecular basis of resistance has remained elusive. Here, we show that disruption of a cadherin-superfamily gene by retrotransposon-mediated insertion was linked to high levels of resistance to the Bt toxin Cry1Ac in the cotton pest Heliothis virescens. Monitoring the early phases of Bt resistance evolution in the field has been viewed as crucial but extremely difficult, especially when resistance is recessive. Our findings enable efficient DNA-based screening for resistant heterozygotes by directly detecting the recessive allele.     

昆虫Bt作物抗性与中肠类钙粘蛋白的关系

Bt作物已在世界范围内广泛种植,为有效控制害虫的危害发挥了重要作用。靶标害虫的抗性问题是影响Bt作物长期利用的关键因素。通过分析Bt Cry1A毒素的主要受体类钙粘蛋白（cadherin-like）的生物学特性及其与Bt毒素的结合作用,讨论类钙粘蛋白基因突变与Bt抗性的关系,综述了基于基因突变的抗性分子检测技术的研究进展。     

苏云金芽孢杆菌cry1Ea基因的克隆及 生物信息学分析

不同来源的苏云金芽孢杆菌能产生多种多样的晶体(Cry)蛋白.基于这个特性,人们可以通过基因工程的手段向工程菌中转入编码多种Cry毒素的基因来控制虫害.通过DNA重组技术,从BtHZM2菌株中克隆出了cry1Ea基因,对其进行了生物信息学分析,同源比对结果表明.cry1Ea8基因的核苷酸序列与已知cry1Ea的同源性为99.77%～99.91%.对应的氨基酸序列同源性为99.49%～99.74%.对cry1Ea8基因的分析还揭示出了cry1Ea8及其编码蛋白的一些生物和理化性质.结构域预测表明,Cry1Ea8由3个结构域组成,其中N-末端螺旋状结构域与膜插入与孔隙形成有关,而第二和第三个结构域与受体的结合有关.该研究为转基因抗虫植物和微生物杀虫工程菌的构建提供了新的基因来源.     

Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and-susceptible strains of Helicoverpa armigera(Hübner)

The Bacillus thuringiensis vegetative insecticidal protein, Vip3 A, represents a new family of Bt toxin and is currently applied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa armigera is cross-resistant to Vip3 Aa protein, insecticidal activities, proteolytic activations and binding properties of Vip3 Aa toxin were investigated using Cry1Ac-susceptible(96S) and Cry1Ac-resistant H. armigera strain(Cry1Ac-R). The toxicity of Vip3 Aa in Cry1Ac-R slightly reduced compared with 96 S, the resistance ratio was only 1.7-fold. The digestion rate of full-length Vip3 Aa by gut juice extracts from 96 S was little faster than that from Cry1Ac-R. Surface plasmon resonance(SPR) showed there was no significant difference between the binding affinity of Vip3 Aa and BBMVs between 96 S and Cry1Ac-R strains, and there was no significant competitive binding between Vip3 Aa and Cry1 Ac in susceptible or resistant strains. So there had little cross-resistance between Vip3 Aa and Cry1 Ac,Vip3A+Cry proteins maybe the suitable pyramid strategy to control H. armigera in China in the future.     

棉铃虫氨肽酶N基因片段克隆、表达和内源蛋白检测

氨肽酶N（APN）是苏云金芽孢杆菌杀虫毒素Cry在昆虫中肠中的一个重要受体。研究氨肽酶N在昆虫中肠中的分布特征对于阐明Cry毒素的杀虫机理和昆虫对Cry毒素的抗性机理具有重要的意义。通过RT-PCR的方法从棉铃虫中肠上皮细胞中克隆得到氨肽酶N的基因片段APN1551,并诱导表达纯化得到其重组蛋白APN517。以此蛋白为抗原,制备其抗血清。用该抗血清能检测到棉铃虫中肠上皮细胞中的APN蛋白。为研究Cry毒素的作用机理奠定基础。     

Glycolipids as receptors for Bacillus thuringiensis crystal toxin

The development of pest resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins used in transgenic and organic farming. Here, we demonstrate that (i) the major mechanism for Bt toxin resistance in Caenorhabditis elegans entails a loss of glycolipid carbohydrates; (ii) Bt toxin directly and specifically binds glycolipids; and (iii) this binding is carbohydrate-dependent and relevant for toxin action in vivo. These carbohydrates contain the arthroseries core conserved in insects and nematodes but lacking in vertebrates. We present evidence that insect glycolipids are also receptors for Bt toxin.     

用Envirologix Cry1Ab/Cry1Ac试剂盒快速测定转基因水稻Bt杀虫蛋白含量的研究

 用 Envirologix Cry1Ab/Cry1Ac平板试剂盒检测了两个转 cry1Ab的水稻品系克螟稻 1号 ( KMD1)、克螟稻 2号 ( KMD2 ) ,及未转化的对照品种秀水 11米粒中的 Bt杀虫蛋白含量。结果表明 ,该试剂盒标样制作的标准曲线相关系数为 0 .985～ 0 .998,在 0 .0 5或 0 .0 1水平上显著。同批次试剂盒的不同试验及不同批次试剂盒的测定值均差异不显著。最低检测剂量达 0 .5 ng/g,每次测试时间比 Western dot blotting方法缩短约 2 h。试验表明 ,该试剂盒是定量检测转基因水稻 Bt毒蛋白表达的一种理想产品     

苏云金芽孢杆菌伴孢晶体的研究进展

伴胞晶体是苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)在芽孢形成过程中产生的一种蛋白晶体。作为生物杀虫农药之一,伴胞晶体/Bt由于具有高效、广谱、环保及生物安全等优势,在农林业中得到广泛应用。介绍了伴胞晶体的最新分类、命名情况,综述了其结构、杀虫机理及应用方面的研究进展,并对Bt生物农药的发展前景进行了展望。     

转Bt基因玉米幼苗残体中Cry1Ab杀虫蛋白田间降解动态

【目的】研究间苗后留在田间地表的转Bt基因玉米幼苗残体中Cry1Ab杀虫蛋白的降解规律,比较两种Bt玉米幼苗残体中Cry1Ab杀虫蛋白的降解速度。【方法】以两种表达Cry1Ab杀虫蛋白的转Bt基因抗虫玉米MON810和Bt11为材料,采用ELISA方法测定各取样时期中幼苗残体中Cry1Ab杀虫蛋白残留量。【结果】转Bt基因玉米幼苗残体中杀虫蛋白降解是逐渐的,且降解速度较快,到50 d时幼苗残体已经完全腐烂,Bt11幼苗残体中的杀虫蛋白已经完全降解,在MON810中还能检测到微量的杀虫蛋白。两种转基因玉米幼苗残体中的Bt杀虫蛋白的初始含量差异不显著,但在同一时间段的Bt杀虫蛋白降解速度存在差异均显著,在30 d前MON810幼苗残体中Bt杀虫蛋白降解速度比Bt11降解的快,30d后,则降解趋势相反,到50 d取样结束时MON810和Bt11分别降解了初始含量的99.81%和100%。【结论】两种转Bt基因玉米间苗后留在田间的幼苗残体中的Cry1Ab杀虫蛋白降解速度不同,在50 d完全腐烂时,其中的杀虫蛋白完全降解或仅有微量残留。     

Studies on Rapid Quantitative Analysis of Bt Toxin by Using Envirologix Kits in Transgenic Rice

Investigations were done on the usefulness of Envirologix Cry1Ab/Cry1Ac Plate kits for quantitative analysis of Bt toxin content in transgenic rice grains. Two transgenic rice lines: Kemingdao 1 (KMD1) and Kemingdao 2 (KMD2), transformed with a cry1Ab gene, and their parental variety, cv.Xiushui 11, were used as positive and negative samples. Results showed that the correlation coefficients as high as 0. 985 - 0. 998, significant at probability level of 0.05 or 0.01, were obtained for linear regression equations by using the appended calibrators of the kits. No significant differences were detected for values of same rice sample obtained from different trials or by using different lots of the product (kit). The detectable Bt toxin content by this method could be as low as 0.5 ng/g. The Envirologix Kit could be useful for rapid quantitative detection of Bt toxin in rice grains because of its preciseness, simplicity and time saving.     

转Bt基因抗虫玉米根茬和根际土壤中Cry1Ab杀虫蛋白的田间降解动态

【目的】研究转cry1Ab杀虫蛋白基因玉米收获后玉米根茬及其根际土壤中Cry1Ab杀虫蛋白的降解动态,比较两种Bt玉米根茬和根际土壤中Cry1Ab杀虫蛋白的降解速度。【方法】以两种表达Cry1Ab杀虫蛋白的Bt抗虫玉米MON810和Bt11为材料,采用ELISA方法测定玉米收获后根茬残体和根际土壤中Cry1Ab杀虫蛋白的田间降解动态。【结果】转Bt基因玉米根茬残体和根际土壤中杀虫蛋白是逐渐降解的,Bt玉米MON810根茬中Cry1Ab杀虫蛋白含量较高,降解的速度也较慢,收获后8个月时还不能完全降解;Bt玉米Bt11根茬中Cry1Ab杀虫蛋白含量较低,降解速度比MON810根茬中Cry1Ab杀虫蛋白降解速度快,到7个月时已检测不到Cry1Ab杀虫蛋白。Bt玉米MON810根际土壤中Cry1Ab杀虫蛋白的降解较Bt11的慢,MON810和Bt11根际土壤分别在8个月和7个月时检测不到Cry1Ab杀虫蛋白。【结论】种植过Bt11和MON810抗虫玉米的田块,在第二年春播农作物已经出土时,其根茬和根际土壤中残留的Cry1Ab杀虫蛋白尚不能完全降解,还有少量残留。     

利用Dot-ELISA检测Bt棉杀虫蛋白的研究

用苏云金芽孢杆菌HD-1和HD-68晶体杀虫蛋白的碱溶产物作为抗原,制备相应的抗血清,建立了检测Bt杀虫蛋白的Dot-ELISA,可检测Bt杀虫蛋白的最低水平为87.5ng/ml。用Dot-ELISA对2个转基因抗虫棉和它们的亲本进行了检测,并与生物学测定的抗虫性进行比较,结果基本一致。因此,对测定大批量转基因抗虫棉的抗虫性,Dot-ELISA是一个快速、敏感、特异、有效的方法。     

光杆状菌属细菌杀虫毒素研究进展

Bt制剂的大量使用及表达Bt毒素的转基因植物的应用已引发了抗性昆虫种群的发展,光杆状菌属细菌产生的杀虫毒素蛋白是一类高效、杀虫谱广的新型杀虫蛋白,使得有限的用来开发转基因抗虫作物的基因资源得到了补充。对近年来发现的Tc蛋白毒素复合体、PirAB双元毒素及其致软毒素(Mcf)的最新研究进展进行了综述,并对今后的研究方向提出了见解。     

利用分子标记辅助选择技术培育抗虫水稻品种的研究

Bt基因是从苏云金芽孢杆菌中克隆出的针对鳞翅目害虫的抗虫基因,经过基因工程方法人工改造已经获得Cry1Ab/Ac、Cry1C等抗虫基因。以云南主栽水稻品种楚粳28为受体,以携带Bt抗虫基因的"华恢1号"、"RJ-5"和"T1C-19"分别作为供体,利用分子标记辅助选择(MAS)技术进行回交育种,选育成携带Bt抗虫基因水稻新品系。结合农艺性状表现,获得了云抗虫稻1号、云抗虫稻2号、云抗虫稻3号Bt蛋白高表达的水稻抗虫品系。这些抗虫品系为云南省抗虫水稻的遗传改良和生产应用提供了基因资源和应用基础。     

Effect of three insect-resistant maizes expressing Cry1Ie,Cry1Ab/Cry2Aj and Cry1Ab on the growth and development of armyworm Mythimna separata(Walker)

Three transgenic maize events(IE09 S034, Shuangkang 12–5 and C0030.3.5) produced Cry1 Ie, Cry1 Ab/Cry2 Aj and G10-EPSPS, Cry1 Ab and EPSPS, respectively, all of which target the Asian corn borer. The oriental armyworm Mythimna separata(Walker) is the secondary target. In this study, the effects of the three Bt maizes on the development and survival of armyworm were studied. The results showed that IE09 S034 had insecticidal activity against 1 st instar larvae, and the survival rate of armyworm fed with Bt maize for 10 days was 46.2%, significantly lower than that of the control. The larvae at 3 rd–6 th instar were more tolerant of the Bt toxin than the early instar larvae. However, Shuangkang 12-5 had good insecticidal activity against 1 st–5 th instar larvae. The mortality was nearly 100% when the larvae were fed with Shuangkang 12-5 before 3 rd instar, and the toxin had quick-acting efficacy. This event significantly inhibited the development of armyworm; that is, the larval duration of the 3 rd and 4 th instar larvae fed with Shuangkang 12-5 was prolonged by 4.5 and 3.0 days, respectively. The pupal weight and egg number were also significantly lower than those of the control. For C0030.3.5, it could control 1 st–5 th instar larvae effectively. The mortality rates were all over 50% if 1 st–3 rd larvae were fed with this event. The pupal weight of 4 th–6 th instar larvae fed with Bt maize were only 53.9, 56.8 and 54.6%, respectively, compared to that of the control. The number of eggs laid was significantly less than the control. The results indicate that all three transgenic maize events exhibit the potential to provide effective control of early instar larvae of armyworm, which can be commercialized in future to control lepidoptera pests such as Asian corn borer and armyworm.     

利用枯萎病菌粗毒素筛选香蕉抗性突变体

在香蕉(Musa AAA)组织培养过程,将枯萎病菌(Fusarium oxysporum f.sp.cubense)粗毒素添加到组织培养基中。结果表明,毒素对香蕉组培芽的分化和存活具有较强的抑制作用,粗毒素的添加剂量与组培芽存活率成反相关,致枯萎50%的粗毒素为36.3038μg.mL-1。添加粗毒素的多种筛选法均获得了香蕉抗枯萎病突变体。突变体再生苗用病菌分生孢子接种结果表明,其相对抗病性均显著高于亲本组织培养再生苗。本研究中,多步正筛选方案Ⅱ是利用粗毒素筛选抗香蕉枯萎病突变体的最佳方案。