Cry1Ba3蛋白对亚洲玉米螟杀虫活性测定及其与BBMVs的结合分析

提取并纯化Cry1Ba3蛋白截短片段,用饲喂人工饲料的方法对亚洲玉米螟（Ostrinia furnacalis）初孵幼虫进行生物活性测定。结果显示,Cry1Ba3截短片段对亚洲玉米螟有良好的杀虫活性（LC50=5.29 μg/g,95%置信限为3.96～7.10 μg/g）。而纯化的Cry1Ac蛋白60 ku的活性片段对亚洲玉米螟初孵幼虫的杀虫活性LC50为0.83 μg/g。提取亚洲玉米螟5龄幼虫的BBMVs,用纯化的Cry1Ba3和Cry1Ac蛋白进行竞争结合试验,结果显示Cry1Ba3与Cry1Ac在亚洲玉米螟中肠的结合没有竞争作用,说明两种毒素在亚洲玉米螟BBMVs上可能存在不同的受体。     

二化螟Cry1Ac汰选品系对不同Bt蛋白的敏感性

为明确靶标害虫对Bt蛋白的抗性及对不同类型Bt蛋白的交互抗性,采用生物测定法进行了二化螟Chilo suppressalis(Walker)敏感品系和Cry1Ac汰选品系在Cry1Ac毒饲料上的时间-死亡率反应、对不同Bt杀虫蛋白的敏感性及不同Bt蛋白复配组合对二化螟毒力效果的研究。结果表明,二化螟Cry1Ac汰选品系已对36.67μg/mL(LC_(50))的Cry1Ac杀虫蛋白产生了一定的适应性,但对308.69μg/mL(LC90)的Cry1Ac蛋白反应仍较敏感。Cry1Ac敏感品系和汰选品系对Cry1Ab蛋白的LC_(50)分别为1.40μg/mL和3.86μg/mL,二者差异显著,但对Cry1Ca(1.63μg/mL和1.73μg/mL)或Cry2Aa(127.48μg/mL和144.50μg/mL)的LC_(50)差异不显著,即Cry1Ac汰选品系与Cry1Ab存在明显的交互抗性,但与Cry1Ca和Cry2Aa不存在交互抗性。在不同Bt蛋白复配组合中(1∶1复配),Cry1Ab+Cry1Ca、Cry1Ab+Cry2Aa和Cry1Ca+Cry2Aa增效作用最为显著。表明抗虫基因cry1Ca和cry2Aa可作为双价转基因抗虫水稻研发的候选基因。     

亚洲玉米螟对Cry1Ab蛋白抗性的遗传规律与分子机理

意义与目的亚洲玉米螟[Ostrinia furnaca-lis(Guene)]是长期为害玉米、高粱、谷子、棉花等作物的重要害虫。转BtCry1Ab基因抗虫玉米、棉花等在整个生育期其植株能表达Cry1Ab杀虫蛋白,为玉米螟的防治开辟了新的途径。室内汰选条件下亚洲玉米螟能对Cry1Ab杀虫蛋白产生抗性,这将严重威胁到转基因玉米、棉花等的可持续利用。研究亚洲玉米螟对Cry1Ab蛋白抗性的遗传规律与分子机理,对田间抗性监测、延缓抗性产生及抗性治理具有重要的理论和实际意义。材料与方法采自陕西省田间的亚洲玉米螟自然种群,在室内用无琼脂半人工饲料中加入一定量的…     

Cry类和Vip3Aa类Bt杀虫蛋白对亚洲玉米螟的毒力和增效作用

筛选对靶标害虫高效的Bt杀虫蛋白、评价蛋白组合对靶标害虫的杀虫作用,对于转基因抗虫新品种的研发具有重要的指导意义。本文采用人工饲料混合法评价了Cry1Ab、Cry1Fa、Cry1Ca、Cry1Ba、Cry2Aa、Vip3Aa11、Vip3Aa20、Vip3Aa19共8种Bt杀虫蛋白对亚洲玉米螟的毒力;同时评估了Cry1Ab/Vip3Aa19、Cry1Fa/Vip3Aa19混合蛋白对亚洲玉米螟的杀虫作用,筛选出毒力最佳的组合比例。结果表明：Cry1Ab、Cry1Fa、Cry2Aa对亚洲玉米螟的LC₅₀在0.24～0.72μg/g之间,其中Cry1Ab的毒力最强,显著高于Cry1Fa和Cry2Aa蛋白,Cry1Fa与Cry2Aa毒力无显著性差异。Cry1Ca、Cry1Ba、Vip3Aa11、Vip3Aa20、Vip3Aa19对亚洲玉米螟的LC₅₀均大于50μg/g。当Cry1Ab/Vip3Aa19、Cry1Fa/Vip3Aa19混合比例为1∶1时,增效作用最强,增效因子分别为3.18和1.58。本研究结果为多价转基因玉米的研发提供了重要依据。     

对黏虫具有杀虫活性的Bt蛋白筛选及分析

为筛选对黏虫[Mythimna separata(Walker)]具有毒杀作用的苏云金芽胞杆菌杀虫蛋白,本研究提取Cry1类、Cry2类、Cry9类及Vip3A类等11种蛋白,通过人工饲料喂毒方法对黏虫进行生物活性测定。结果表明,Cry1Ac、Cry1Ab、Cry2Ab、Cry1Be及Cry1Bb蛋白对黏虫具有杀虫活性,其致死中浓度依次为5.09、17.71、26.75、27.42及43.93μg/g;Cry9Aa、Cry9Eb、Cry9Ee蛋白对黏虫生长具有抑制作用,其浓度为10μg/g时的体重抑制率分别为78.4%、79.0%、86.9%,浓度为100μg/g时的体重抑制率分别为92.8%、95.7%、96.7%;Cry1Ba、Cry1Ca和Vip3Aa蛋白对黏虫无显著活性。本研究为黏虫的生物防治奠定了基础,并为抗虫转基因作物的研究提供优良的候选基因。     

Cry9Aa3蛋白对小菜蛾和亚洲玉米螟的最小活性区研究

为了确定Cry9Aa3蛋白对亚洲玉米螟(Ostrinia furnacalis)和小菜蛾(Plutella xylostella)的最小活性区,根据NCBI BLAST比对结果与SWISS-MODEL同源建模结果,设计了12对截短引物,以cry9Aa3全长序列为模板,扩增出了12种不同截短片段,与pEB载体重组后的阳性克隆,转入大肠杆菌(Escherichia coli)菌株Rosetta(DE3)诱导表达,提取蛋白。对小菜蛾和亚洲玉米螟的生物学活性测定,发现截短蛋白Cry9Aa3-45~700、Cry9Aa3-1~654,对小菜蛾和亚洲玉米螟有较高的活性,其中截短蛋白Cry9Aa3-45~700对小菜蛾和亚洲玉米螟的LC50分别为5.27、7.56μg/g,截短蛋白Cry9Aa3-1~654对小菜蛾和亚洲玉米螟的LC50分别为7.76、7.79μg/g,与阳性对照无显著差异,而截短蛋白Cry9Aa3-1~653和Cry9Aa3-46~700(LC50200μg/g)对幼虫几乎丧失生物学活性。这说明Cry9Aa3蛋白对小菜蛾和亚洲玉米螟的杀虫活性区相同,最小活性区位于45I和654P之间。     

转Bt基因玉米(瑞丰125、DBN9936、DBN9978)对亚洲玉米螟的抗虫效果研究

评价转Bt基因玉米对靶标生物亚洲玉米螟的杀虫作用是转基因玉米研发的重要一环。本文采用室内生测法对3种转Bt基因抗虫玉米‘瑞丰125’(表达Cry1Ab/Cry2Aj杀虫蛋白),‘DBN9936’‘DBN9978’(表达Cry1Ab杀虫蛋白)对亚洲玉米螟敏感品系ACB-S及抗Cry1Ab品系ACB-AbR、抗Cry1Ac品系ACB-AcR、抗Cry1F品系ACB-FR、抗Cry1Ah品系ACB-AhR、抗Cry1Ie品系ACB-IeR的杀虫活性进行测定,同时采用心叶期和抽丝期人工接虫法进行田间抗虫效果鉴定。结果表明,取食3种Bt玉米的ACB-S幼虫, 3 d死亡率100%,而取食对照常规玉米3 d存活率100%。取食3种Bt玉米的5个抗性品系幼虫除ACB-AbR和ACB-AcR有2%~6%的个体存活4~5 d, 6 d死亡率也达到了100%,其余品系均在3 d全部死亡,而取食对照玉米5~6 d的死亡率仅为4%~14%,差异显著。田间心叶期食叶级别及穗期活虫数、雌穗被害和茎秆被蛀等为害等级说明3种Bt玉米高抗亚洲玉米螟。明确了‘瑞丰125’‘DBN9936’和‘DBN9978’对亚洲玉米螟有很高的杀虫活性和田间防治效果。5个Bt蛋白抗性亚洲玉米螟品系幼虫在常规玉米上显示一定的适合度劣势。     

亚洲玉米螟中肠类钙黏蛋白Cry1A毒素结合区对Cry1Ac毒素杀虫活性的影响

通过原核表达及亲和层析获得亚洲玉米螟Ostrinia furnacalis中肠类钙黏蛋白Cry1A毒素结合区MPR及其在Cry1Ac抗性玉米螟中的突变体MPR-r2多肽片段。采用饲料混合法测定了MPR和MPR-r2及其分别与Cry1Ac毒素混合对亚洲玉米螟幼虫的杀虫效果。结果表明,MPR和MPR-r2多肽片段对亚洲玉米螟幼虫都有一定的杀虫作用;当MPR多肽片段与Cry1Ac毒素混合时,杀虫活性具有加性效应,而MPR-r2多肽片段与Cry1Ac毒素混合的杀虫效果则没有显著提高。     

黏虫对Cry1Ab蛋白的抗性遗传规律及与其他蛋白的交互抗性

为转Bt作物抗性治理策略的有效实施提供依据，采用遗传杂交和毒力生测方法评价黏虫Mythimna separata Cry1Ab蛋白抗性品系对Cry1Ab蛋白的抗性显性度，明确调控抗性基因的位置和数目以及对Cry1F和Vip3Aa19蛋白的敏感性。结果显示，Cry1Ab蛋白对黏虫正交F₁代和反交F₁代的LC₅₀分别为15.30μg/g和12.91μg/g，抗性为常染色体调控；在浓度2.5～50.0μg/g范围内黏虫对Cry1Ab蛋白的抗性显性水平随着浓度的变化而变化，即在2.5μg/g时黏虫对Cry1Ab蛋白的抗性为不完全显性遗传，在50.0μg/g时黏虫对Cry1Ab蛋白的抗性为不完全隐性遗传。黏虫对Cry1Ab蛋白的抗性受多基因调控。另外，黏虫Cry1Ab抗性品系对Cry1F和Vip3Aa19蛋白未产生交互抗性，抗性倍数分别为0.9和1.1。     

Cry1Ba3、Cry1Ia8蛋白对Cry1Ac抗性小菜蛾的杀虫活性研究

利用2种苏云金芽胞杆菌原毒素Cry1Ba3、Cry1Ia8及其组合,分别对Cry1Ac抗性种群小菜蛾幼虫进行生物活性测定。结果表明Cry1Ba3、Cry1Ia8对2种目标试虫均有高毒力,LC50分别为0.2175、0.6706μg/mL;Cry1Ba3毒力3倍于Cry1Ia8。2种蛋白混配的结果也表现出高毒力,LC50为0.4375μg/mL,没有显著的协同增效作用,也不存在拮抗。敏感与抗性小菜蛾种群生测结果统计分析比较,结果表明这2种蛋白及其组合与Cry1Ac并无交互抗性。     

球形芽胞杆菌Cry48Aa/Cry49Aa杀蚊毒素与致倦库蚊中肠上皮细胞复合物的结合特性

Cry48Aa/Cry49Aa毒素是近年来在球形芽胞杆菌IAB59中发现的新型双组分毒素,仅对库蚊具有较高的毒杀作用并能杀死二元毒素(Bin)抗性库蚊,是一种比较有潜力的新型杀蚊毒素蛋白,但目前对Cry48Aa/Cry49Aa毒素的作用模式还不清楚。本研究将cry48Aa和cry49Aa基因在苏云金芽胞杆菌无晶体突变株中表达,纯化毒素的生测结果表明该复合毒素对Bin毒素敏感和抗性致倦库蚊均表现较高的毒杀作用,毒力无显著差异。生物素标记的毒素与致倦库蚊BBMF特异性结合试验表明Cry48Aa和Cry49Aa毒素与两蚊虫品系BBMF都具有较高的结合特异性,Cry48Aa结合能力较高,其解离常数(Kd)分别为(9.5±1.8)和(13.9±2.3)nmol/L;Cry49Aa的解离常数分别为(25.4±3.8)和(28.1±4.2)nmol/L。异源竞争结合试验结果表明Cry48Aa毒素则可以有效地和Cry49Aa毒素竞争结合BBMF蛋白上的结合位点,其IC₅₀分别为(22.1±3.7)和(15.4±2.6)nmol/L,而Cry49Aa不能竞争封闭Cry48Aa毒素与两蚊虫品系BBMF蛋白的结合位点,其IC₅₀均大于17μmol/L。该研究结果可为揭示Cry48Aa/Cry49Aa毒素的作用机制提供一定的研究基础。     

Cry1Ac和Cry2Ab蛋白对大草蛉生长发育及酶活力的影响

为明确转Cry1Ac和Cry2Ab基因棉花对大草蛉的影响,运用Bt蛋白与人工饲料混合的方法,以大于转基因棉花叶片中蛋白含量20倍的剂量饲喂大草蛉初孵幼虫,初步研究了Cry1Ac和Cry2Ab对大草蛉生物学参数和消化酶、解毒酶活性的影响。结果表明：取食含Bt蛋白饲料的大草蛉幼虫的发育历期、体重、蛹重、成虫体重、羽化率等生物学参数与对照相比均没有显著差异;在大草蛉幼虫体内可以检测到含量较高的Cry1Ac和Cry2Ab蛋白,分别为974.92~1 282.39 ng/g鲜重和5 592.62~6 082.92 ng/g鲜重,而在大草蛉成虫体内检测到的Cry1Ac和Cry2Ab蛋白含量非常低,分别为0.29~0.39 ng/g鲜重和50.34~56.71 ng/g鲜重;取食含Bt蛋白的饲料对大草蛉幼虫的类胰蛋白酶、类胰凝乳蛋白酶、氨肽酶和谷胱甘肽-S-转移酶活性有一定的影响,但对大草蛉成虫影响与对照差异不显著。表明大草蛉取食含Bt蛋白的人工饲料后,虽然体内可以检测到一定含量的Bt蛋白,但对大草蛉的生长发育并没有显著的直接不利影响。     

球形芽胞杆菌Cry48Aa/Cry49Aa毒素在苏云金芽胞杆菌中的共表达及杀蚊活性

球形芽胞杆菌Cry48Aa/Cry49Aa复合杀蚊毒素对库蚊有高毒力,并能杀死二元毒素BinA/BinB抗性蚊虫,是一种比较有潜力的新型杀蚊毒素蛋白,但Cry48Aa在野生菌株中表达量较低限制了该毒素的开发与应用。本文将cry48Aa基因启动子置换,并将cry48Aa和cry49Aa在苏云金芽胞杆菌中进行共表达。SDS-PAGE检测重组蛋白表达,可见120 kD左右的Cry48Aa蛋白带和53 kD的Cry49Aa蛋白带,cry48Aa在自身启动子下表达量偏低,在置换强启动子Pcry1Aa后表达量明显提高,但Cry48Aa仍低于Cry49Aa的表达量。重组菌株BMB171-pBU-cry48Aa-cry49Aa和BMB171-pBU-Pcry1Aa-cry48Aa-cry49Aa产生典型的双锥型和方形2种晶体。两菌株发酵液对实验室饲养的二元毒素敏感(CqSL)和抗性致倦库蚊(CqRL/C3-41)品系4龄幼虫都表现出高毒力,LC₅₀分别为3.05和3.45以及0.65和0.82 μL/mL。     

Bacillus thuringiensis Cry1Ab, but not Cry1Aa or Cry1Ac, disrupts liposomes

We evaluated the ability of Cry1Aa9, Cry1Ab4, and Cry1Ac1 insecticidal toxins from Bacillus thuringiensis to destroy liposomes. Cry1A toxins are thought to form pores in midgut apical cell membranes (BBMV), thereby disrupting midgut cells. Liposomes containing fluorescent calcein were prepared using phosphatidylcholine (PC) and phosphatidylserine (PS) (PC/PS-Liposomes) or PC alone (PC-Liposomes). Cry1Ab (1.4 μM), but not Cry1Aa or Cry1Ac, disrupted PC/PS-Liposomes and PC-Liposomes. PC/PS-Liposomes containing cholesterol and oligosaccharylceramide from Plutella xylostella midgut were damaged even more extensively by Cry1Ab, but the inclusion of either lipid alone had no effect. The initial velocity of Cry1Ab-mediated liposome disruption increased 17-fold when liposomes were prepared with Triton X-100-soluble proteins from Bombyx mori BBMV and PC (PC/Proteo-Liposomes), and Cry1Aa and Cry1Ac also caused slight disruption. These data suggest that Cry1Ab achieves higher penetration into PC/PS-Liposomes, PC-Liposomes, and PC/Proteo-Liposomes compared with Cry1Aa or Cry1Ac and that Cry1Ab may interact with membrane proteins.     

亚洲玉米螟和黏虫幼虫取食Cry1F蛋白后的中肠组织病理变化

为了明确Cry1F杀虫蛋白对亚洲玉米螟和黏虫两种重要玉米害虫的作用机制，本文通过透射电镜观察了亚洲玉米螟和黏虫4龄幼虫取食含Cry1F蛋白人工饲料后的中肠组织病理变化，并与其取食含Cry1Ab蛋白人工饲料后的组织病理变化进行了比较。亚洲玉米螟和黏虫4龄幼虫取食Cry1F蛋白48 h后中肠杯状细胞发生了明显的病变，主要表现为：细胞微绒毛肿胀、脱落；细胞核变形，质膜和核膜界限不清晰；染色质发生固缩、贴近核膜；线粒体拉伸变形数量减少，严重的发生空泡化；内质网肿胀与断裂、数量减少，与取食含Cry1Ab蛋白人工饲料相比，玉米螟和黏虫中肠组织发生的病变程度相似。本研究可为Cry1F作为转基因玉米重要的杀虫蛋白在未来亚洲玉米螟和黏虫防治中更好地发挥作用提供理论依据。     

入侵云南草地贪夜蛾种群对5种常用Bt蛋白的敏感性评价

草地贪夜蛾Spodoptera frugiperda(J. E. Smith)是原分布于美洲大陆热带和亚热带地区的一种重要玉米害虫。在当地,种植抗虫转基因玉米是防控草地贪夜蛾危害的主要手段。该虫于2019年1月入侵我国云南省,为明确入侵我国云南的草地贪夜蛾种群对常用Bt蛋白的敏感性水平,本文通过饲料表面涂抹法测定了瑞丽草地贪夜蛾幼虫对Cry1Ab、Cry1Ac、Cry1F、Cry2Ab以及Vip3A等5种Bt蛋白的敏感性。结果表明:几种常用Bt蛋白对瑞丽草地贪夜蛾致死作用顺序为Vip3ACry1AbCry1FCry2AbCry1Ac,对草地贪夜蛾抑制生长发育的顺序为Cry1AbCry1FVip3ACry1AcCry2Ab。此外,与美国相对敏感种群比较,云南瑞丽草地贪夜蛾种群对Cry1Ab、Cry1Ac、Cry1F、Cry2Ab和Vip3A的敏感性指标在0.28～3.76之间,表明该入侵种群对此5种Bt蛋白均未产生抗性。此研究可为将来建立以转Bt基因玉米作为防控草地贪夜蛾的技术体系提供依据。     

Does cotton bollworm show cross-resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab? A mini review

Since 1996, transgenic Bacillus thuringiensis(Bt) cotton has been commercially grown in numerous countries in an effort to stem the losses caused by key lepidopteran pests. However, the development of pest resistance to Bt toxins has jeopardized the continued utilization of Bt cotton. As a strategy designed to circumvent the development of resistance, Bt cotton varieties expressing two or more toxins targeting the same pest have been introduced. Nevertheless, from the perspective of long-term planting of Bt cotton, the potential risk of cross-resistance to these Bt toxins is a threat that cannot be ignored. In this paper, we review current research(including that based on the analysis of protein binding sites and resistance genes) on the resistance of cotton bollworm(Helicoverpa armigera) to the Bt toxins Cry1 Ac and Cry2 Ab and the interrelationship between these toxins. On the basis of existing evidence, we assume that the actions of Cry1 Ac and Cry2 Ab against cotton bollworm are not completely independent, and then propose the "resistance-associated gene mutation potential hypothesis". Although the mechanisms underlying the resistance of pests to Bt toxins are yet to be comprehensively elucidated, this hypothesis could undoubtedly have important implications for adopting "pyramid" strategy in the future. Further research is recommended to devise strategies to retard the development of H. armigera resistance to Bt cotton, either using different Bt toxins or their various combinations.     

Microarray analysis of global gene regulation in the Cry1Ab-resistant and Cry1Ab-susceptible strains of Diatraea saccharalis

BACKGROUND: Extensive adoption of transgenic Bt corn in recent years for stalk borer control has increased risk of resistance evolution in the target pest populations. A Bt‐resistant strain of the sugarcane borer, Diatraea saccharalis, was approximately 100‐fold more tolerant to Cry1Ab toxin than the susceptible counterpart. To gain a better understanding of the molecular mechanisms of Bt resistance, the Cry1Ab‐susceptible (Cry1Ab‐SS) and Cry1Ab‐resistant (Cry1Ab‐RR) strains of D. saccharalis were subjected to a microarray analysis. RESULTS: Results showed that the expression levels of many genes were significantly different between the Cry1Ab‐RR and Cry1Ab‐SS strains. Microarray analysis of 7145 cDNAs revealed 384 differentially expressed genes. A total of 273 genes were significantly upregulated 2–51.6‐fold, and 111 genes were significantly downregulated 2–22.6‐fold in the Cry1Ab‐RR strain. The upregulation of three potential resistance‐related genes, coding for a glutathione S‐transferase (GST), a chymotrypsin‐like protease (CHY) and a lipase (LP), was confirmed using real‐time PCR, indicating a reproducibility of the microarray data. Ontology analysis revealed that more than twice the number of metabolic‐related genes were upregulated compared with downregulated genes with the same biological function. Up to 35.2% of the upregulated genes in the resistant strain were associated with catalytic activity, while only 9.5% of the downregulated genes were related to the same catalytic molecular function. CONCLUSION: The large portion of metabolic‐ or catalytic‐related genes with significant upregulations indicated a potential large increase in metabolic or catalytic activities in the Cry1Ab‐RR strain. This cDNA microarray gene expression data could be used to characterize and identify new genes that may be associated with Bt resistance in D. saccharalis. Copyright © 2012 Society of Chemical Industry