GFP与水稻条纹病毒病害特异蛋白的融合基因在sf9昆虫细胞中的表达

 用重叠延伸PCR(overlap Polymerase Chain Reaction,overlap-PCR)方法获得了含有绿色荧光蛋白(Green fluorescence protein,GFP)和水稻条纹病毒(Rice stripe virus,RSV)病害特异性蛋白SP(Disease-specific protein)两者的融合基因(GFP-SP),并将其克隆至载体pMD18-T,得到的重组质粒pMD18-T-GFP-SP经Xba Ⅰ/Hind Ⅲ双酶切后与经相同方法处理过的杆状病毒转移载体pFastBacHTb相连接构建成重组转移质粒pFastBacHTb-GFP-SP。酶切和测序鉴定证明了其序列的正确性并且无移码现象发生。将此质粒转化入含穿梭载体Bacmid的感受态细胞DH10Bac,得到含有目的基因片段的重组杆状病毒穿梭质粒rb-GFP-SP。以之转染草地贪夜蛾(Spodoptera frugiperda,sf9)细胞,24~48h后在显微镜200倍可见光视野下观察到被感染细胞发生了细胞和细胞核变大、细胞内颗粒物增多、细胞脱落甚至裂解等一系列与正常的st9昆虫细胞形态有明显区别的变化。荧光显微镜下可见部分细胞发出清晰的绿色荧光,且大部分荧光集中在细胞质部分。这些结果表明,GFP-SP融合蛋白在st9昆虫细胞内成功表达。     

两种夜蛾科昆虫卵的超微形态特征对比研究

[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。     

甜菜夜蛾对白粉菌侵染的中国月季的嗅觉行为研究

采用Y型嗅觉仪测试了白粉菌侵染中国月季对甜菜夜蛾成虫嗅觉行为的影响及这种影响的空间效应。根据反应虫数分析,健康叶片挥发性成分对甜菜夜蛾成虫有显著的引诱作用（P=0.002）,同时感病叶片挥发性成分对雌蛾有显著的驱避作用（P=0.006）,直接比较2种叶片时差异更显著（P=0.000）。通过植物介导,中国月季感染白粉菌对甜菜夜蛾成虫产卵地的选择有明显抑制作用。进一步的空间效应研究结果显示,这种抑制作用存在局部效应和系统效应,能在同小叶内传递,并能向上或向下传递至同叶轴的其他相邻小叶。该研究为甜菜夜蛾成虫产卵地选择机制的深入研究提供了重要依据。     

4%高氯·甲维盐微乳剂防治甜菜夜蛾田间药效试验研究

[目的]明确4%高氯.甲维盐微乳剂对甜菜夜蛾的田间防治效果。[方法]在苞菜田中,于甜菜夜蛾发生初期,按照不同施药量兑水进行叶面喷雾,计算各处理防效。[结果]该药剂有效成分用量21~27 g/hm2,药后2 d防效为91%~99%,药后5 d防效为92%~99%,药后10天防效为92%~98%,显著优于对照药剂4.5%高效氯氰菊酯乳油和1%甲氨基阿维菌素苯甲酸盐乳油。[结论]该药剂速效性好,持效性显著,可作为防治十字花科蔬菜甜菜夜蛾的有效药剂和替代乳油的环保剂型进行推广使用,推荐用药量为有效成分用量24~27 g/hm2。     

Comparative effectiveness and field persistence of insect growth regulators on a field strain of the cotton leafworm, Spodoptera littoralis, Boisd (Lepidoptera: Noctuidae)

Toxicity effects and field persistence of the insect growth regulators lufenuron, flufenoxuron and triflumuron were assessed in the laboratory using second and fourth larval instars of Spodoptera littoralis. Laboratory bioassays indicated that lufenuron was more effective on both 2nd and 4th larval instars, as well as killing both larval instars faster than flufenoxuron or triflumuron. Field-laboratory experiments were conducted to show direct and residual effects of the tested IGRs in terms of toxicity and stability. They indicated that all the tested insecticides were stable under field conditions and give high percentages of mortality. Overall, lufenuron was more efficient than the other tested insecticides. In addition, it gave a faster kill in some testing periods. Data presented in this work show greater efficiency of lufenuron in controlling S. littoralis than flufenoxuron or triflumuron. Using this insecticide for cotton leafworm control in cotton fields may give better results under field condition.     

牛耳枫提取物对甜菜夜蛾酚氧化酶的抑制作用

 【目的】筛选对甜菜夜蛾（Spodoptera exigua（Hübner)）酚氧化酶具有高抑制活性的化合物,寻找新型害虫控制剂的线索。【方法】以牛耳枫（Daphniphyllum calycinum Benth.）提取物Deoxycalyciphylline B和Methyl homosecodaphniphyllate为抑制剂,采用酶标仪微量法研究了其对甜菜夜蛾酚氧化酶的抑制活性及抑制类型。【结果】Deoxycalyciphylline B和Methyl homosecodaphniphyllate对甜菜夜蛾酚氧化酶的抑制中浓度(IC50)分别为2.439 mmol?L-1和0.879 mmol?L-1,2种化合物均为典型的可逆竞争型抑制剂,抑制常数（Ki）分别为2.051 mmol?L-1和1.269 mmol?L-1。【结论】2种生物碱对甜菜夜蛾酚氧化酶具有较好的抑制活性,可以以这2种化合物或其类似物为模板指导酶抑制剂的分子设计,是寻找新型害虫控制剂的重要途径。     

斜纹夜蛾普通气味结合蛋白ⅠcDNA的克隆及在原核细胞中的表达

 【目的】克隆、序列分析和原核表达编码斜纹夜蛾（Spodoptera litura）GOBPⅠ（SlGOBPⅠ）的cDNA。【方法】采用同源克隆结合RACE的方法克隆SlGOBPⅠ基因的cDNA序列,并在 pET-32a/BL21（DE3）系统中进行原核表达。【结果】从斜纹夜蛾触角中克隆了GOBPⅠ的cDNA序列（GenBank登录号为EU086372）,序列分析表明,SlGOBPⅠ开放阅读框序列为495 bp,编码164个氨基酸,分子量为19.3 kD,等电点为5.54。SlGOBPⅠ具有昆虫气味结合蛋白的典型特征,即氨基酸序列中具有 6个半胱氨酸残基,呈酸性。SlGOBPⅠ氨基酸序列与鳞翅目昆虫的气味结合蛋白氨基酸序列具有较高的相似性,表明SlGOBPⅠ属于GOBP家族。将SlGOBPⅠ编码序列,克隆到表达载体pET-32 a上,构建原核表达载体pET-SlGOBPⅠ,在表达宿主菌BL21（DE3）中,经IPTG诱导成功表达了相对分子质量为32.0 kD的可溶性融合蛋白,利用Ni2+-NTA亲和柱一步纯化了SlGOBPⅠ融合蛋白,以此融合蛋白免疫新西兰大白兔制备了SlGOBPⅠ的抗血清,ELISA滴度为1﹕5 000,Western印迹检测结果显示,SlGOBPⅠ抗血清与表达的融合蛋白质呈阳性反应,表明所表达的融合蛋白保持原有的免疫原性。【结论】克隆、分析和表达了编码斜纹夜蛾气味结合蛋白GOBPⅠcDNA 序列,为今后深入研究斜纹夜蛾GOBPⅠ基因的结构和功能奠定了基础。     

抗氰戊菊酯甜菜夜蛾品系的交互抗性及相对适合度

用氰戊菊酯对采自福建福州的甜菜夜蛾[Spodoptera exigua (Hubner)]进行抗性品系的选育.经连续10代的室内抗性选育,获得抗性倍数达698.5倍的抗性品系(Fen-R),与起始种群相比抗性提高了27.6倍.Fen-R抗性品系对溴氰菊酯具有7.3倍的交互抗性,对三氟氯氰菊酯、毒死蜱、灭多威、氟啶脲和虫螨腈不存在交互抗性.通过构建Fen-R抗性品系、敏感品系(S)的实验种群生命表,比较了一系列生长发育和繁殖特征,并采用净增殖率(R0)来确定其相对适合度.结果表明,Fen-R抗性品系与S品系相比,存在明显的生长发育和繁殖不利性,表现为幼虫发育历期延长、蛹重下降、产卵量和卵孵化率降低,Fen-R抗性品系的适合度为0.53.     

匍匐翦股颖害虫禾灰翅夜蛾生物学习性研究

禾灰翅夜蛾是一种多食性害虫,可危害匍匐翦股颖草坪草。本试验结合室内饲养法,对禾灰翅夜蛾在匍匐翦股颖草上的发生危害和生长发育情况进行了系统的观察研究。结果表明, 禾灰翅夜蛾幼虫取食匍匐翦股颖草的叶片及嫩茎,可对匍匐翦股颖草坪草造成毁灭性的损害;雌蛹的体重明显大于雄蛹的体重。在广州,禾灰翅夜蛾的最高产卵量为251粒/雌蛾,平均产卵量为54.6粒/雌蛾,卵块产。     

Efficacy of insecticide mixtures against pyrethroid‐ and organophosphate‐resistant populations of Spodoptera litura (Lepidoptera: Noctuidae)

BACKGROUND: Spodoptera litura (F.) is an important pest worldwide, with over 112 host plants, and is exposed to insecticides throughout the year, resulting in the rapid development of resistance. Insecticide mixtures can delay the development of resistance more effectively than sequences or rotations. Cypermethrin, deltamethrin, profenofos, chlorpyrifos and fipronil were assessed separately and in mixtures against laboratory susceptible S. litura and two field‐collected populations. RESULTS: The field‐collected population from Khanewal (KWL) was significantly more resistant to cypermethrin, deltamethrin, chlorpyrifos and profenofos than one collected from Muzaffar Garh (MGH). Mixtures of cypermethrin + chlorpyrifos or profenofos and of deltamethrin + chlorpyrifos or profenofos at 1:1, 1:10 and 1:20 ratios significantly increased (P < 0.01) toxicity to cypermethrin and deltamethrin in field populations. The combination indices of cypermethrin + chlorpyrifos at 1:1 and 1:10 ratios and cypermethrin + fipronil at 1:1, 1:10 and 1:20 ratios for the KWL strain and of cypermethrin + profenofos or fipronil at 1:1, 1:10 and 1:20 ratios for MGH were significantly below 1, suggesting synergistic interactions. The inhibitors DEF and PBO largely overcame resistance to deltamethrin, cypermethrin and profenofos, suggesting that resistance to the insecticides was associated with esterase and monooxygenase detoxification respectively. CONCLUSION: Chlorpyrifos, profenofos and fipronil could be used in mixtures to restore cypermethrin and deltamethrin susceptibility. These findings may have considerable practical implications for S. litura resistance management. Copyright © 2008 Society of Chemical Industry