昆虫对苏云金芽孢杆菌杀虫晶体蛋白的交叉抗性

昆虫对苏云金芽孢杆菌杀虫晶体蛋白的交叉抗性是昆虫的一个重要抗性特征,它对于抗性管理具有重要意义,本文综述了近年来昆虫交叉抗性的分类、遗传基础、抗性机制等方面的研究进展.     

淡色库蚊对溴氰菊酯抗性的遗传分析

目的：对室内选育的淡色库蚊抗溴氰菊酯品系（抗性系数５１８）进行遗传分析。方法：采用ＬＣ－Ｐ值分析法研究淡色库蚊溴氰菊酯抗性的形式遗传。采用抑制剂增效试验探讨抗性机制。结果：正交和反交Ｆ１代的抗性系数分别为１５４．８和１５９．２。Ｆ１代的ＬＣ－Ｐ线靠近抗性亲本一方，Ｄ＝０．６２５；ＢＣ代在死亡率５０％处、Ｆ２代在死亡率２５％和７５％处未出现明显平坡，ＢＣ和Ｆ２的实测曲线和理论曲线均存在较大差异，Ｐ均＜０．０１；ＰＢ、ＤＥＦ和ＴＰＰ对抗性品系的增效比分别为２７．１３、１．２２和１．０５。结论：淡色库蚊对溴氰菊酯的抗性为多基因遗传，其主要基因为不完全显性，多功能氧化酶是抗性的主要因子。     

嗜人按蚊对溴氰菊酯抗性的遗传分析

目的 对实验室选育的嗜人按蚊抗溴氰菊酯品系进行抗性遗传特性分析。方法 采用LC-P线法研究嗜人按蚊对溴氰菊酯抗性的遗传方式。结果 正交和反交F_1代的抗性系数分别为15.19、16.66,接近1∶1。D-0.651,F_1代的LC-P线靠近抗性亲本一方。BC代在死亡率50%处与F_2代死亡率在25%—75%处未出现明显平坡,BC和F_2的实际死亡率和理论死亡率曲线的适合性检验差异具有显著性,P均<0.01。结论 嗜人按蚊对溴氰菊酯的抗性是由常染色体遗传所致的多基因遗传,抗性遗传表现为不完全显性。     

苏云金杆菌以色列亚种晶体蛋白CryIVD基因的克隆及表达产物的效果测定

目的　　克隆、表达苏云金杆菌以色列亚种晶体蛋白CryIVD基因并测定其杀蚊毒效。　 方法　采用PCR技术 ,扩增得到CryIVD基因片段 ;通过双酶切及连接反应 ,将该基因克隆入大肠杆菌质粒 pUC18构建重组克隆及表达载体 ;转化E .coliDH5α,提取重组质粒进行酶切鉴定及DNA序列测定 ;以IPTG诱导表达CryIVD蛋白 ,SDS PAGE分析表达产物并进行杀蚊毒效测定。　结果　CryIVD基因成功克隆并在宿主菌中正确表达 ,表达产物对蚊幼的杀灭毒效测定显示其对淡色库蚊Ⅱ～Ⅲ龄健康幼虫的LC50 为 2 .3 8× 10 6 cells ml,对白纹伊蚊健康幼虫的LC50 为 1.6× 10 7cells ml。　结论　CryIVD基因被成功克隆和表达 ,且其表达产物有明显杀蚊幼活性。     

病原体重组枯草芽孢杆菌疫苗的研制现状

枯草芽孢杆菌是一种新型疫苗载体,它可以表达细菌、病毒和吸虫等病原体的多种蛋白,本文拟就这方面的研究现状进行综述。     

云南省埃及伊蚊对拟除虫菊酯类抗性群体的击倒抗性基因突变分析

目的 检测云南省登革热重点地区对氯菊酯和高效氯氟氰菊酯杀虫剂已产生抗性的埃及伊蚊的击倒抗性(knockdown resistance,kdr)基因,进行基因突变分析,阐明抗性表型与kdr基因突变的关系。方法 收集云南省景洪市、勐腊县、勐海县、瑞丽市和耿马县经过氯菊酯和高效氯氟氰菊酯杀虫剂测定的埃及伊蚊成蚊样本,分别提取单蚊基因组DNA,Allele-specific PCR(AS-PCR)扩增和分析其kdr部分基因片段,统计抗性与敏感表型中kdr突变的基因型和基因频率。结果 共580份雌性埃及伊蚊样本被用于V1016G和F1534C击倒抗性突变的检测。V1016G的突变频率为99.83%(579/580),F1534C的突变频率为46.38%(269/580)。V1016G和F1534C突变频率差异存在统计学意义(χ²=421.338,P=0.000)。5个埃及伊蚊种群中,V1016G突变率除了瑞丽市外,其他区域均达到100.00%。F1534C突变率景洪市最高为84.16%,耿马县最低为5.66%。抗性表型与敏感表型的V1016G和F1534C突变率差异均存在统计学意义(χ²=7.298,P=0.007;χ²=14.010,P=0.000)。580份样本中,有65份样本同时存在V1016G和F1534C位点突变,突变率为11.21%;不同地区同时突变率范围为0~33.66%,景洪市同时突变率最高为33.66%。结论 云南省登革热重点地区埃及伊蚊种群存在V1016G和F1534C突变,V1016G突变率高且分布广,F1534C突变以景洪市埃及伊蚊种群为最高。敏感表型与抗性表型V1016G和F1534C突变存在差异,提示抗性产生与突变存在一定的关系。     

对溴氰菊酯抗性和敏感淡色库蚊不同发育阶段的抗性水平

对室内选育的淡色库蚊（对溴氰菊酯抗性品系和敏感品系）用浸渍法测定了Ⅱ龄、Ⅲ龄和Ⅳ龄幼虫，用接触筒法测定了早期、中期和晚期成虫；并观察了增效醚（ＰＢ）、磷酸三苯酯（ＴＰＰ）和杀虫脒与溴氰菊酯混用后的增效效果。结果表明：（１）淡色库蚊各发育阶段对溴氰菊酯的抗性水平，在敏感品系由高至低依次为中期、晚期、早期成虫、Ⅳ龄、Ⅲ龄和Ⅱ龄幼虫；在抗性品系则为早期、中期、晚期成虫、Ⅳ龄、Ⅲ龄和Ⅱ龄幼虫。（２）ＰＢ和杀虫脒均有明显增效作用，提示抗性可能与氧化代谢增强和靶标部位不敏感有关。其中似乎幼虫阶段以氧化代谢增强为主要抗性机制；在成虫阶段，氧化代谢增强和靶标部位不敏感性均在抗性中起重要作用     

球形芽孢杆菌1593与苏云金杆菌H—14对蚊幼虫的毒效的比较研究

由于化学杀虫剂引起的蚊虫抗性及环境污染等问题,蚊虫生物防治日益受到重视。苏云金杆菌H-14(Bacillus thurtugiensis H-14)(简称BTH-14)和球形芽孢杆菌(Bacillus sphaericus     

吡喹酮衍生物DW-3-15对日本血吸虫PZQ抗性虫体的生物学效应观察北大核心CSCD

目的通过体外观察吡喹酮(PZQ)衍生物DW-3-15对日本血吸虫PZQ抗性虫体的生物学效应,探讨该衍生物作为抗日本血吸虫候选新药的潜在价值。方法单性日本血吸虫尾蚴(70±5条)感染小鼠,21d后,用PZQ的半数有效剂量(ED50,25.98mg/kg)对小鼠进行灌胃给药,每天一次,连续30d,停药21d后,再给予小鼠治疗剂量PZQ(200mg/kg),连续5d,停药2周后肝门静脉灌注收集虫体,置DMEM培养液中培养,分别加入不同浓度PZQ和DW-3-15,作用16h后换新鲜培养液,继续培养72h,每隔24h体视显微镜下观察、记录一次虫体活力和形态变化,评价诱导虫体对PZQ及DW-3-15的敏感性。结果 PZQ和DW-3-15体外作用于未诱导日本血吸虫成虫的临界致死浓度(作用虫体72h活力降低率达90%的最低浓度)分别为14μmol/L和45μmol/L;诱导虫体对PZQ的敏感性较未诱导虫体显著下降,是其临界致死浓度的8倍(112μmol/L),而诱导虫体对DW-3-15的敏感性与未诱导虫体相比没有明显差异,临界致死浓度仍为45μmol/L。结论经诱导的日本血吸虫PZQ抗性虫体对DW-3-15没有交叉抗性;提示DW-3-15抗日本血吸虫的靶点可能与PZQ不同,具有作为抗日本血吸虫候选新药的潜在价值,值得进一步研究。     

淡色库蚊敌敌畏抗性和氯氰菊酯抗性品系对常用化学杀虫剂的交互抗性

目的了解淡色库蚊对6种常用化学杀虫剂的交互抗性,为科学使用化学杀虫剂提供依据。方法采用WHO生物测定方法检测淡色库蚊敏感品系、敌敌畏抗性品系和氯氰菊酯抗性品系Ⅳ龄幼虫对三氯杀虫酯、敌敌畏、马拉硫磷、残杀威、氯氰菊酯和溴氰菊酯等6种常用化学杀虫剂的敏感性,每次测定设置7个浓度,每个浓度设2组,每组25个幼虫,24h后统计幼虫死亡数据,计算致死中浓度(median lethal concentration,LC_(50))、回归方程和抗性指数。结果淡色库蚊敌敌畏抗性品系对三氯杀虫酯、敌敌畏、马拉硫磷、残杀威、氯氰菊酯和溴氰菊醑的LC_(50)分别为1.9623mg/L、1.1607mg/L、0.7359mmg/L、0.9002mg/L、0.0220mg/L和0.000 1 mg/L,回归方程分别为Y=3.5287+5.0254X、Y=4.6962+4.693 7X、Y=5.5051+3.853 6X、Y=5.2350+5.1476X、Y=10.4995+3.3184X和Y=13.2977+2.1683X,抗性指数分别为9.25、12.17、9.14、7.93、183.47和0.71;淡色库蚊氯氰菊酯抗性品系对三氯杀虫酯、敌敌畏、马拉硫磷、残杀威、氯氰菊酯和溴氰菊酯的LC_(50)分别为5.572 8mg/L、0.2464mg/L、0.0892mg/L、0.202 7mg/L、0.064 1 mg/L和0.008 5mg/L,回归方程分别为Y=2.7728+2.9852X、Y=7.2054+3.6260X、Y=9.475 1+4.2635X、Y=6.8106+2.6125X、Y=8.7404+3.1352X和Y=14.6951+4.6853X,抗性指数分别为26.27、2.58、1.10、1.79、534.31和40.60。结论长期使用一种化学杀虫剂会导致蚊虫产生抗药性,并对其他化学杀虫剂产生不同的交互抗性,应采取合理选择杀虫剂品种和确定使用剂量等有效措施以避免蚊虫抗药性的产生。     

Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.

A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from Bacillus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in approximately 15% of Bacillus strains analyzed. The sequence of the vip3A(b) gene, a homologue of vip3A(a) isolated from B. thuringiensis strain AB424 is also reported. Vip3A(a) and (b) proteins confer upon Escherichia coli insecticidal activity against the lepidopteran insect larvae mentioned above. The sequence of the gene predicts a 791-amino acid (88.5 kDa) protein that contains no homology with known proteins. Vip3A insecticidal proteins are secreted without N-terminal processing. Unlike the B. thuringiensis 5-endotoxins, whose expression is restricted to sporulation, Vip3A insecticidal proteins are expressed in the vegetative stage of growth starting at mid-log phase as well as during sporulation. Vip3A represents a novel class of proteins insecticidal to lepidopteran insect larvae.     

苏云金芽孢杆菌以色列亚种毒性蛋白基因的克隆及其原核表达

目的从苏云金芽孢杆菌（Bacillus thuringiensis,Bti）以色列亚种中获得cry4B、cry11A和cyt1A3种主要杀蚊幼虫蛋白的基因,并进行克隆和原核表达。方法根据GenBank上3种基因的已知序列设计合成引物,应用PCR技术扩增目的基因,克隆入pUC19质粒,阳性克隆质粒经酶切和测序鉴定。将测序鉴定的3种蛋白基因克隆入原核表达载体（pET32c）进行原核表达。结果PCR扩增获得特异性的基因片段,重组质粒经酶切后获得预计大小的基因片段,并测序鉴定。序列结果与已知序列一致。原核表达载体经ITPG诱导,获得预计大小的目的蛋白。结论成功克隆了苏云金芽孢杆菌以色列亚种的3个主要杀蚊幼虫蛋白的基因,并进行原核表达。     

Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5, 000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.     

Reversal of resistance to Bacillus thuringiensis in Plutella xylostella.

Continued success of the most widely used biopesticide, Bacillus thuringiensis, is threatened by development of resistance in pests. Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B. thuringiensis, revealed factors that promote reversal of resistance. In strains of P. xylostella with 25- to 2800-fold resistance to B. thuringiensis compared with unselected strains, reversal of resistance occurred when exposure to B. thuringiensis was stopped for many generations. Reversal of resistance was associated with restoration of binding of B. thuringiensis toxin CryIA(c) to brush-border membrane vesicles and with increased biotic fitness. Compared with susceptible colonies, revertant colonies had a higher proportion of extremely resistant individuals. Revertant colonies responded rapidly to reselection for resistance. Understanding reversal of resistance will help to design strategies for extending the usefulness of this environmentally benign insecticide.     

Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli.

Sau 3A1 partial digestion fragments from Bacillus thuringiensis var. kurstaki HD-1 plasmid DNA were ligated into the BamHI site of the cloning vector pBR322 and transformed into Escherichia coli strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with antibody made against B. thuringiensis crystals. One strain, ES12, was isolated by using this procedure. ES12 contains a plasmid of Mr 11 X 10(6) that has DNA sequence homology with pBR322 as well as with Mr 30 X 10(6) and Mr 47 X 10(6) plasmids of B. thuringiensis. It makes a protein antigen, detected by antibodies to crystal, which has the same electrophoretic mobility as the B. thuringiensis crystal protein. Protein extracts of ES12 are toxic to larvae of the tobacco hornworm Manduca sexta.     

From the Cover: Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize

The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.The global area devoted to transgenic crops producing insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) has increased rapidly over the past 15 y, with Bt crops covering more than 69 million hectares in 2012 (1). Most of this area was planted in Bt cotton and Bt maize (1). Benefits of Bt crops include effective management of target pests, decreased use of conventional insecticides, and reduced harm to nontarget organisms (2–5). However, the evolution of resistance could diminish these benefits. The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of maize, with larval feeding on maize roots and associated management costs causing economic losses in excess of $1 billion per year (6). Through 2013, three Bt toxins have been used in transgenic maize for management of western corn rootworm: Cry3Bb1, mCry3A, and Cry34/35Ab1 (7).In the United States and elsewhere, commercial registration of a Bt crop is accompanied by a resistance-management plan to delay the onset of pest resistance. Resistance management for Bt crops has focused on the refuge strategy, in which refuges of non-Bt crops allow the survival of Bt-susceptible insects, which may mate with resistant insects that survive on the Bt crop (8). To the extent that the heterozygous progeny from these matings have lower fitness on a Bt crop than their Bt-resistant parent, delays in resistance may be achieved, and these delays in resistance increase with the quantity of refuge (9). Additionally, refuges are far more effective in delaying resistance when Bt crops achieve a high dose of toxin against a target pest. High-dose Bt crops kill more than 99.99% of susceptible insects and render resistance a functionally recessive trait (9, 10). None of the currently commercialized Bt maize targeting the western corn rootworm is high dose, so the risk of resistance is increased (11, 12).In 2003, Cry3Bb1 maize was registered by the United States Environmental Protection Agency (US EPA) for management of western corn rootworm larvae (7). In 2009, farmers in Iowa observed severe injury to Cry3Bb1 maize by larval western corn rootworm in the field, and subsequent laboratory assays revealed that this injury was associated with Cry3Bb1 resistance (13). More fields with Cry3Bb1 resistance were identified in 2010 (14), and research in fields identified in 2009 as harboring Cry3Bb1-resistant western corn rootworm found no difference in survival for this pest between non-Bt maize and Cry3Bb1 maize (11). Current threats to Bt maize include the spread of Bt-resistant western corn rootworm and the loss of additional Bt toxins through the presence of cross-resistance. In this paper we report that injury to Cry3Bb1 maize in the field has persisted through 2011 and expanded to include mCry3A maize. Analysis of western corn rootworm collected in 2011 revealed that (i) severe injury to Cry3Bb1 maize and mCry3A maize in the field was associated with resistance, and (ii) cross-resistance between Cry3Bb1 and mCry3A was present. These results demonstrate that insects can evolve resistance rapidly to Bt crops that are not high dose and raise concerns about the adequacy of current resistance-management strategies.     

淋病奈瑟菌常见抗生素耐药机制的研究进展

淋病奈瑟菌是淋病的病原菌。淋病目前在我国性传播疾病中感染率较高,有效的抗生素治疗是防治淋病的主要方法,但淋病奈瑟菌可通过改变作用靶点、改变对细胞膜的通透性、产生抗生素灭活酶以及药物外排机制等方式降低对抗生素的易感性。为了更好地指导临床用药、对淋病进行预防和防治,需要严密监测淋病奈瑟菌的耐药情况。本文针对淋病奈瑟菌常见抗生素的耐药机制进行综述。