抗Bt棉铃虫种群的交互抗性研究

分别用对CyrlAc抗性达11．6倍的Bt棉叶和Bt粉剂（Btk)汰选棉铃虫（Heliocoverpa armigera)种群（对Btk的抗性指数为5．2倍）,测定了其对不同Bt制剂,Bt毒素,化学农药的交互抗性,结果表明：停止汰选后,Bt棉叶汰选种群对CrylAc的抗性指数为5．9倍,对CrylAc 1C的交互抗性为3．7倍,对Btk的敏感性降低,对Bta,Cry2A无交互抗性,Btk汰选种群对Btk的抗性指数为4．1倍,对Bta,CrylAc,CrylAc 1C的交互抗性为2．3,3．1,2．3倍,对Cry2A无交互抗性,两汰选种群对化学农药灭多威,辛硫磷,氯氰菊酯的敏感性均有所增加。     

棉铃虫田间种群对Bt制剂的敏感性差异

本研究采用室内毒力测定和诊断剂量两种方法测定了华北地区３省５县棉铃虫对Ｂｔ制剂敏感性的地区间差异。以最敏感的河北固安种群ＬＣ５０和ＬＣ９０值为基线计算其它种群的抗性指数，其最大值分别为１．８０（河南西华）和５．１３（山东高密）倍，证明不同地区的棉铃虫对Ｂｔ制剂的敏感性有显著差异。     

棉铃虫Bt毒素受体蛋白—氨肽酶N与抗性的关系

随着我国转基因棉花种植面积的日益增加,主要目标害虫—棉铃虫的抗性问题越来越被大家所关注。氨肽酶N(aminopeptidase N,APN)是一类昆虫中肠内Bt毒素的受体蛋白,其结构、结合位点的改变或基因突变可能是昆虫对Bt毒素产生抗性的重要原因。本文通过分析Bt毒素的作用方式,从生化、生理、分子生物学等方面探讨了棉铃虫Bt毒素受体蛋白APN与抗性的关系。     

新疆地区棉铃虫自然种群对Bt棉的抗性频率监测

为监测新疆棉区棉铃虫Helicoverpa armigera(Hübner)田间种群对Bt棉的抗性频率,在2010年和2011年分别采集石河子和喀什地区莎车的棉铃虫单雌系,以Cry1Ac毒蛋白作为人工饲料,用单雌系F1/F2代法进行棉铃虫种群抗性个体检测。2010年筛选了123个石河子的棉铃虫单雌系,2 011年筛选了152个莎车的棉铃虫单雌系。两地的棉铃虫种群均没有筛选到相对平均发育级别≥0.8的抗Bt棉个体,估算出石河子和莎车的棉铃虫种群的抗性频率低于10-3。莎车F2单雌系与其对应的F1单雌系相对平均发育级别有明显差异。研究表明新疆石河子地区田间棉铃虫种群仍保持敏感状态,喀什地区田间棉铃虫种群对Bt棉的耐受性增高。     

棉铃虫Bt抗感种群间数种解毒酶和中肠蛋白酶活性的比较

比较Bt抗性种群和敏感种群棉铃虫解毒酶和中肠蛋白酶的变化，测定两种群的α-乙酸萘酯酶、乙酰胆碱酯酶、谷胱甘肽-S-转移酶的酶活力，比较α-乙酸萘酯酶和乙酰胆碱酯酶的Km和Vmax值，并采用特异性酶抑制剂进行鉴定，结果发现，抗性种群棉铃虫的乙酰胆碱酯酶活力、Km和Vmax值都 显著升高，甲基对氧磷对抗性种群棉铃虫乙酰胆碱酯酶的抑制程度比敏感种群明显霸产加，抗生种群5和敏感种群总蛋白酶活力差异不显著，弱碱性类胰蛋白酶和类凝乳蛋白酶活力差异也不显著，但抗性种群3龄幼虫的强碱性类胰蛋白酶活图形显著高于敏感种群。     

一种监测棉铃虫对Bt杀虫晶体蛋白抗性的技术

研究了以诊断试剂盒监测棉铃虫对苏云金杆菌(Bt)杀虫晶体蛋白(ICP)抗性的技术。将1日龄幼虫在含诊断剂量的饲料中(每毫升饲料含Bt ICP0.01mg)取食7天,以生长至3龄以上的个体作为抗性个体的判断指标;筛选出人工饲料中防腐剂剂量的最佳值为每350毫升饲料中含丙酸2.5ml。经对多个棉铃虫不同抗性种群的验证试验,证明诊断试剂盒检测抗性个体频率较为精确且应用简便。     

棉铃虫对Bt杀虫蛋白抗性测定方法的研究

对饲料混合法 ,体重抑制率法 ,浸叶接虫法进行比较研究 ,结果表明 ,饲料混合法比较稳定 ,比较适宜作为抗性测定的方法。用饲料混合法测定比较不同龄期幼虫的测定结果 ,确定 2龄作供试幼虫为宜。比较不同处理天数下的生测结果 ,确定 Bt粉剂对 2龄幼虫的适宜处理时间为 5d,Cry1Ac为 14d。用改进稀饲料隔离饲养供试幼虫的方法后 ,得到理想的生测数据 ,生测时间延长到 14d,对照死亡率也能控制在 2 0 %以下。从我们 1998年对棉铃虫的抗性监测结果说明 ,尽管田间棉铃虫对 Bt ICP未达抗性水平 ,但对 Cry1Ac型蛋白敏感性已有降低 ,值得引起重视。     

转Bt基因植物对不同抗性棉铃虫的生长抑制作用

采用转Bt基因的烟草和棉花 ,对经转Bt单基因、转Bt与CpTI双基因烟草汰选 1 8代和常规烟草处理的棉铃虫种群进行生长抑制作用的比较测定。结果表明 ,转Bt烟草对以汰选后的抗Bt单基因汰选棉铃虫种群的体重抑制作用显著低于转双基因烟草汰选种群和对照种群 ;转Bt棉花对单基因种群的杀虫活性、体重、龄期发育以及化蛹率的抑制作用亦显著低于双基因种群和对照种群     

转Bt基因玉米对棉铃虫的抗性评价

棉铃虫Helicoverpa armigem（Htlbner）是常年危害玉米的重要害虫。转Bt基因抗虫玉米为害虫的防治提供了新的途径。采用室内离体生物测定方法测定了孟山都转Bt基因抗虫玉米Yield—Gard不同组织对棉铃虫初孵幼虫的杀虫活性以及田间人工接虫抗性鉴定。Bt玉米花丝、苞叶和幼嫩雌穗对棉铃虫具有较高的杀虫活性，取食Bt玉米花丝和幼嫩雌穗（籽粒和穗轴）的棉铃虫初孵幼虫不能存活和完成幼虫期发育，而对照分别有51．0％和22．0％个体完成幼虫期发育。以Bt玉米雌穗苞叶饲养棉铃虫初孵幼虫，5天后幼虫全部死亡，而非Bt玉米对照仅为16．0％，差异显著。田间花丝期人工接种棉铃虫抗性鉴定试验结果表明，Bt玉米雌穗被害率为3．5％，危害级别为0．007，百株存活幼虫14．3头。而非Bt玉米雌穗被害率达到82．5％，危害级别为2．51，平均百株存活幼虫79．6头，显著高于Bt玉米。这些结果说明转Bt基因玉米穗期对棉铃虫的防治效果明理．     

转Bt 基因抗虫玉米对棉铃虫杀虫活性的初步评价

国内外在转 Bt基因抗虫作物的研究和应用上已取得重大进展 ,其中 Bt抗虫棉已在美国、澳大利亚、中国等进入商品化应用阶段 ,近年来种植面积增长很快 ,并取得了显著的经济、生态和社会效益。 1 997年下半年 ,我国农业部分别批准了孟山都公司申报的保铃棉○R在河北省商业化生产和 Bt玉米在我国多省份的田间种植。其中保铃棉 ○R已由河北省定名为新棉 33B,1 998年在全省实际种植了近 1 0万 hm2 ,1 999年的种植面积已达 1 5万 hm2 。我国自主研制的 Bt抗虫棉也于 1 998年开始商品化应用 ,当年种植面积即达 1 0万 hm2以上 ,1 999年推广总面积…     

棉铃虫入土化蛹的生物学习性

室内模拟试验表明 ,棉铃虫蛹道形状为“U”型 ,蛹室一般距土表 3～5cm ;入土孔道和羽化出土孔道不同 ,羽化出土孔道在筑蛹室之前已事先做好 ,且孔道上端距土表0.5～1.0cm。棉铃虫入土第 3天处于预蛹和蛹初期 ,入土第 10天为羽化前期 ;入土第 12天时 ,已有部分成虫羽化出土 ,其他个体正处于羽化期     

Selection and heritability of resistance to Bacillus thuringiensis subsp kurstaki and transgenic cotton in Helicoverpa armigera (Lepidoptera: Noctuidae)

Compared with an unselected susceptible population, a cotton bollworm, Helicoverpa armigera (Hübner), population selected for 22 generations with transgenic cotton leaves (modified Cry1A) in the laboratory developed 11.0-fold resistance to Cry1Ac (one single-protein product MVPII). Resistance to Bacillus thuringiensis Berliner subsp kurstaki (Btk) was selected for 22 generations with a 5.2-fold increase in LC50. The estimated realized heritabilities (h2) of resistance for transgenic-cotton- and Btk-selected populations were 0.1008 and 0.2341, respectively. This reflects the higher phenotypic variation in response to Cry1Ac in the transgenic-cotton-selected population. This variation may have been caused by differences in protein toxin levels expressed in different growth stages of the transgenic cotton. Because of the different slopes of the probit regression lines between Cry1Ac and Btk, the estimated realized h2 cannot be used visually to compare resistance development to Cry1Ac and Btk in H armigera. Thus, the response quotient (Q) of resistance was also estimated. The Q values of resistance for transgenic-cotton- and Btk-selected populations were 0.0763 and 0.0836, respectively. This showed that the rate of resistance development would be similar in both selection populations. This result indicates that the selection of resistance using transgenic cotton is different from that selected using the single toxin. Resistance risk to transgenic cotton and Btk in field populations was assessed assuming different pressures of selection by using the estimated h2. Assuming the h2 of resistance in a field population was half of the estimated h2, and the population received prolonged and uniform exposure to transgenic cotton or Btk causing >70% mortality in each generation, we predicted that resistance would increase 10-fold after <23 generations for Cry1Ac in transgenic cotton-selected-populations and after <21 generations for Btk in Btk-selected populations. Cross-resistance would be expected after <48 generations for Btk in transgenic-cotton-selected populations and after <21 generations for Cry1Ac in Btk-selected population. The results show that the potential to evolve resistance is similar in both transgenic-cotton- and Btk-selected populations, but that cross-resistance development to Btk is slower in transgenic-cotton-selected populations than cross-resistance development to Cry1Ac in Btk-selected populations.     

Toxicity of Mixtures of Bacillus thuringiensis with Endosulfan and Other Insecticides to the Cotton Boll Worm Helicoverpa armigera

In the laboratory, low concentrations of Bacillus thuringiensis Berliner (B.t.), as ‘Dipel’ 2X applied (at about the LC₅) to cotton leaves enhanced the activity of endosulfan and reduced resistance to endosulfan from approximately seven fold to two fold in neonate larvae of Helicoverpa armigera (Hubner). Low (LC₅) concentrations of endosulfan also increased the toxicity of B.t. Both endo-sulfan and B.t. increased the toxicity of the stomach poison cryolite to H. armigera. This increased toxicity was not observed where B.t./endosulfan combinations were applied topically to larvae or where B.t. was combined with organophosphorus or pyrethroid insecticides which are considered primarily to be contact toxicants. Mixtures of B.t. and endosulfan applied at equitoxic concentrations were less toxic than similar concentrations applied alone.     

Bt对棉铃虫天蚕素抗菌肽基因(HacD)转录的影响

抗菌肽是昆虫体液免疫的重要组成部分, 对抵御外源致病微生物的侵染具有重要作用。明确苏云金芽胞杆菌Bacillus thuringiensis(Bt)诱导下棉铃虫天蚕素抗菌肽基因(HacD)转录的空间特征和时序特征, 有利于进一步了解昆虫体液免疫与棉铃虫抗Bt发展的关联性。本研究测定了HacD cDNA序列, 对序列进行了蛋白质翻译, 并通过RT-qPCR技术研究了棉铃虫HacD基因在Bt诱导下的转录规律。结果表明, 棉铃虫HacD cDNA全长189 bp, 其中GC含量为48.15%, 肽链由63个氨基酸组成。Bt诱导后棉铃虫幼虫HacD相对转录水平整体呈显著上调趋势, 其中脂肪体中HacD的相对转录水平上调最高, 其次为血淋巴。Bt对棉铃虫幼虫进行短期诱导后, 脂肪体和血淋巴中的HacD基因的相对转录水平均呈持续上调趋势, 分别在诱导24 h和18 h时达到最高, 随后均持续下调。对幼虫进行持续诱导后, F0代初孵幼虫、5龄幼虫、蛹和成虫的HacD基因均可以保持上调的转录特性;传代培养获得的F1代初孵幼虫、5龄幼虫、蛹和成虫的HacD基因转录较F0代下调, 但均高于对照组。综上所述, Bt能够诱导棉铃虫HacD基因转录水平上调。     

重组增效蛋白对Bt和氯氰菊酯防治棉铃虫的增效作用

前期在大肠杆菌中表达得到具有显著生物活性的含粉纹夜蛾颗粒体病毒增效蛋白C端818个氨基酸的融合蛋白P96。以毒饲料法研究P96对苏云金芽孢杆菌和氯氰菊酯杀棉铃虫敏感、抗性品系的增效作用。结果表明：P96对Bt杀棉铃虫敏感、低抗、中抗品系的增效比分别为3．34、4．72和9．82；对氯氰菊酯杀棉铃虫敏感、低抗、中抗品系的增效比分别为2．53、3．38和6．24。随棉铃虫抗性升高，P96的增效作用愈为明显。单对汰选是快速选育棉铃虫抗Bt品系的有效方法。     

Genetics of Heliothis and Helicoverpa resistance to chemical insecticides and to Bacillus thuringiensis

Genetic linkage maps of Heliothis virescens and Helicoverpa armigera are being used to identify and characterize resistance-conferring genes. The insensitive acetylcholinesterase conferring resistance to organophosphorus insecticides and the insensitive sodium channel conferring resistance to pyrethroids have both been mapped in H. virescens. The linkage mapping approach permits a genetic dissection of resistance, even when the mode of action and lethal target are not precisely known, such as for the insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). We have identified and mapped a major Bt-resistance locus in a strain of H. virescens exhibiting up to 10000-fold resistance to Cry1Ac toxin and are currently developing a linkage map for H. armigera with a set of ‘anchor’ loci to facilitate comparison with H. virescens. Both species are currently experiencing their first significant selective pressure in the field by transgenic cotton expressing Cry1Ac, and timely identification of resistance mechanisms and their underlying genetic basis will be essential in successfully managing the Bt resistance that will eventually appear. ©1997 SCI     

苏云金芽孢杆菌HBF-1芽孢对棉铃虫幼虫生长发育及酶活的影响

苏云金芽孢杆菌HBF-1菌株的芽孢对初孵棉铃虫幼虫无明显杀伤作用,但可以直接影响其正常的生长发育,使幼虫前期体重增加;而后期发育迟缓,历期延长,成虫产卵量明显减少。通过光学显微镜观察发现,用含有HBF-1芽孢的饲料饲喂棉铃虫幼虫,幼虫中肠肠壁细胞遭到破坏;随着虫龄的增大,微绒毛逐渐脱落,有的部位细胞壁破裂。酶活的测定结果表明:初孵棉铃虫幼虫取食含有HBF-1芽孢(100μg/g)的饲料后,幼虫体内的乙酰胆碱酯酶、羧酸酯酶和谷胱甘肽-S-转移酶的活性发生了变化。     

棉铃虫对常见化学农药的触角电位反应

利用电生理技术测定了棉铃虫对 15种常见农药的触角电位反应 (EAG) ,发现棉铃虫对不同农药品种的EAG反应差异很大 ,并且雌、雄蛾对某些农药品种的EAG反应截然不同 ,存在较大的性别差异。以上结果可为棉田害虫防治有选择地使用化学农药提供部分依据