棉铃虫磷脂酰乙醇胺结合蛋白的克隆及表达分析   总被引：1，自引：0，他引：1  

赵洁  任苏伟  刘宁  艾新宇  马纪  刘小宁 《中国农业科学》2018,51(8):1493-1503

【目的】克隆获得棉铃虫（Helicoverpa armigera）磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine binding protein,PEBP)的cDNA序列,分析HaPEBP在棉铃虫体内的表达规律,检测2-十三烷酮处理条件下棉铃虫体内HaPEBP的变化情况,为进一步明确棉铃虫PEBP的功能和选择该基因作为调控棉铃虫种群数量的分子靶标提供依据。【方法】利用RACE技术从棉铃虫6龄幼虫的中肠组织中扩增得到HaPEBP的cDNA序列,并对其氨基酸序列和蛋白结构进行分析。将HaPEBP的ORF序列连接至pET32a载体并转化大肠杆菌BL21(DE3),IPTG诱导后检测目的蛋白的表达形式,并通过镍柱的亲和层析纯化融合蛋白。最后通过实时荧光定量PCR检测棉铃虫幼虫不同时期和6龄幼虫不同组织中HaPEBP的表达规律,以及2-十三烷酮处理棉铃虫6龄幼虫后中肠内HaPEBP的变化规律。【结果】获得的HaPEBP cDNA序列长760 bp,其中ORF为588 bp,编码195个氨基酸,蛋白质分子量和等电点分别为21.76 kD和5.93。氨基酸序列分析表明HaPEBP无信号肽、跨膜结构域和二硫键,是一个由4个α螺旋和9个β折叠片组成的胞质单体蛋白。将BL21(DE3)-pET32a-HaPEBP重组菌用1 mmol·L^-1的IPTG在37℃条件下诱导4 h,约40 kD的融合蛋白His-HaPEBP能以可溶的形式存在于重组菌中。按照同样的方法诱导1 L的重组菌,将超声处理后的上清液与Ni-NTA柱结合,进行咪唑缓冲液梯度洗涤,200 mmol·L^-1的咪唑洗涤两次后得到约40 kD的单一蛋白,与融合蛋白的大小一致。将此流出液超滤浓缩,获得183.3 ng·μl^-1的融合蛋白,能被抗His-Tag的单克隆抗体识别发生免疫反应。HaPEBP在棉铃虫幼虫的1-6龄期和预蛹期均表达,且6龄幼虫的表达量最高。该基因在6龄幼虫的脂肪体、中肠、头部和体壁中也均有表达,且脂肪体内的表达量最高。不同浓度的2-十三烷酮处理后,棉铃虫6龄幼虫中肠内HaPEBP的表达量均有所降低;低浓度(2.5和5 mg·g^-1)在6 h就能降低HaPEBP的表达量,其mRNA含量随着时间的延长逐渐增加;而高浓度(10和15 mg·g^-1)在12 h才会降低HaPEBP的表达量,其mRNA含量随着时间的延长先下降后上升。【结论】克隆分析了HaPEBP,利用大肠杆菌系统表达出可溶的融合蛋白His-HaPEBP,并通过镍柱-咪唑纯化法得到了高纯度的融合蛋白,时空表达分析显示HaPEBP在棉铃虫6龄幼虫和脂肪体中表达量最高,2-十三烷酮处理6龄幼虫后中肠内HaPEBP的表达量显著降低。  相似文献   

丙磺舒在阿维菌素作用棉铃虫细胞中的增效机制研究     

王赞永  周勇  马海昊  刘佳  朱航  邓希乐  谭辉华  周小毛 《湖南农业科学》2017,(5)

丙磺舒是ABCC转运蛋白抑制剂,用系列浓度阿维菌素和200μmol/L丙磺舒的食物饲喂棉铃虫,检测中肠组织中阿维菌素的含量。结果表明:与单独使用阿维菌素相比,混合使用丙磺舒后阿维菌素在中肠中的平均驻留时间(MRT)增加了13%,消除半衰期(t1/2z)增加了27%,药物峰值提高了2.8倍。细胞活力测定发现,亚致死剂量阿维菌素与400μmol/L丙磺舒混用后,棉铃虫细胞的活力降低了67.1%。表明抑制ABCC转运蛋白可以提高阿维菌素在棉铃虫中肠的驻留能力,提高药物对棉铃虫细胞的毒力。  相似文献   

A perspective on management of Helicoverpa armigera: transgenic Bt cotton,IPM, and landscapes          下载免费PDF全文

Sharon Downes  Darren Kriticos  Hazel Parry  Cate Paull  Nancy Schellhorn  Myron P Zalucki 《Pest management science》2017,73(3):485-492

Helicoverpa armigera is a major pest of agriculture, horticulture and floriculture throughout the Old World and recently invaded parts of the New World. We overview of the evolution in thinking about the application of area‐wide approaches to assist with its control by the Australian Cotton Industry to highlight important lessons and future challenges to achieving the same in the New World. An over‐reliance of broad‐spectrum insecticides led to Helicoverpa spp. in Australian cotton rapidly became resistant to DDT, synthetic pyrethroids, organophosphates, carbamates and endosulfan. Voluntary strategies were developed to slow the development of insecticide resistance, which included rotating chemistries and basing spray decisions on thresholds. Despite adoption of these practices, insecticide resistance continued to develop until the introduction of genetically modified cotton provided a platform for augmenting Integrated Pest Management in the Australian cotton industry. Compliance with mandatory resistance management plans for Bt cotton necessitated a shift from pest control at the level of individual fields or farms towards a coordinated area‐wide landscape approach. Our take‐home message for control of H. armigera is that resistance management is essential in genetically modified crops and must be season long and area‐wide to be effective. © 2016 Society of Chemical Industry  相似文献   

Influence of three diets on susceptibility of selected insecticides and activities of detoxification esterases of Helicoverpa assulta (Lepidoptera: Noctuidae)     

Kai-Yun Wang  Hong-Yan Wang  Tong-Xian Liu 《Pesticide biochemistry and physiology》2010,96(1):51-55

The oriental tobacco worm, Helicoverpa assulta Guenée, is one of the most destructive pests of tobacco and peppers in China. We determined the susceptibility of H. assulta reared on an artificial diet, chili pepper and tobacco to four insecticides (fenvalerate, phoxim, methomyl, indoxacarb) under laboratory conditions associated with the activities of acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione S-transferase (GST) in its larvae. H. assulta larvae that were fed with chili pepper were more susceptible to fenvalerate, indoxacarb, and phoxim than those that were fed with tobacco and the artificial diet, but not to methomyl. The larvae that were fed with chili pepper were 3.65-, 2.49-, 1.92- and 2.44-fold more susceptible to fenvalerate, phoxim, methomyl, and indoxacarb than those fed with tobacco, respectively. The AChE activities of H. assulta larvae that were fed with chili pepper and tobacco were 2.12 and 1.07 μmol mg⁻¹ 15 min⁻¹, respectively, almost 2-fold difference. The CarE activity of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 4.12, 7.40 and 7.12 μmol mg⁻¹ 30 min⁻¹, respectively. Similarly, the GST activities of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 52.02, 79.37 and 80.02 μmol mg⁻¹ min⁻¹, respectively. H. assulta larvae that were fed with chili pepper were more resistance to the tested insecticides. The low activities of AChE and the high activities of CarE and GST lead to H. assulta become more susceptible to the tested insecticides.  相似文献   

Digestive proteolytic and amylolytic activities of Helicoverpa armigera in response to feeding on different soybean cultivars     

Bahram Naseri  Yaghoub Fathipour  Saeid Moharramipour  Vahid Hosseininaveh  Angharad MR Gatehouse 《Pest management science》2010,66(12):1316-1323

  相似文献   

两种夜蛾科昆虫卵的超微形态特征对比研究     

孙会忠  董钧锋  宋月芹  张友军 《农业科学与技术》2011,(5):773-775

[目的]探索甜菜夜蛾和烟青虫卵的形态学特征,为卵的发育及种群鉴定积累资料。[方法]运用扫描电子显微镜对两种昆虫卵的超微结构特征进行观察。[结果]甜菜夜蛾和烟青虫的卵均为球状,前者卵壳表面具网状纹饰,且分布有较多的呼吸空,卵孔外围的梅花状刻纹卵石状;后者卵壳表面隆起贯穿两极,隆起间具梯形纹饰,亚极区的隆起上分布有乳突,未见呼吸孔的分布。[结论]甜菜夜蛾和烟青虫卵的超微结构特征存在种间特异性,超微特征丰富了两种昆虫的个体发育资料,也为两种昆虫的卵及其外类群的准确鉴定提供参考。  相似文献   

人工神经网络在棉铃虫发生等级预报中的应用     

黄健  李晶  张璞  李杨 《安徽农业科学》2010,38(23):12913-12915,12922

国内棉铃虫划分为热带型、亚热带型、温带型和新疆型4个地理型,预测棉铃虫的发生对棉铃虫的防治和安排农事生产有着重大意义。笔者选取新疆麦盖提县植保站棉铃虫发生资料与同期气象资料按月平均处理后,通过相关分析法选出与发生等级相关的因子作为预测因子,分别用逐步回归法和人工神经网络建立该地的预测预报模型。结果表明,BP神经网络预报模型的拟合精度和预报精度高于逐步回归模型,在干旱区,温度对棉铃虫发生的影响强于降雨量的影响。  相似文献   

棉铃虫氨肽酶N基因片段克隆、表达和内源蛋白检测   总被引：1，自引：0，他引：1  

刘京国  赵晓萌  杨爱珍  于同泉  路苹  师光禄 《北京农学院学报》2010,25(1):20-22,26

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。  相似文献   

不同剂量Bt悬浮液对辣椒烟青虫的防治效果     

涂祥敏  刘崇政  杨红  赖卫  詹永发  余文中 《长江蔬菜》2010,(22):69-70

以40%乐果乳油和4.5%高效氯氰菊酯乳油为对照药剂,研究不同剂量的Bt悬浮液对辣椒烟青虫防治效果,结果表明,在辣椒结果盛期（7月25日）,烟青虫1～2龄幼虫期,用Bt悬浮液80 mL/667m2对水50kg进行喷雾,防治效果达83.67%～87.91%,为最佳剂量。  相似文献   

湿度与棉铃虫发生的关系研究     

李胄  郑永善 《湖北农业科学》2016,(12):3084-3088

利用同在半径5 km区域内连续10年的湿度和棉铃虫(Helicoverpa armigera Hübner)百株卵量的调查资料,分析研究了湿度与棉铃虫发生的关系。结果表明,2月平均绝对湿度和最小绝对湿度均与二代棉铃虫百株卵量呈极显著正相关,11月的定时最小相对湿度与次年二代棉铃虫百株卵量呈显著负相关,11月的月平均绝对湿度和月平均相对湿度与次年二代棉铃虫百株卵量呈极显著负相关,10~12月的5种湿度资料均与次年二代棉铃虫百株卵量呈负相关。建立了5个棉铃虫百株卵量与湿度因子呈显著或极显著相关的回归方程。  相似文献