Genomic Localization and Nucleotide Sequence of a lef-8 Gene of the Spodoptera Littoralis Nucleopolyhedrovirus

A gene similar to lef-8 of the Autographa californica (Ac) nucleopolyhedrovirus (MNPV) was identified in the Spodoptera littoralis (Spli) MNPV. The SpliMNPV lef-8-like gene was localized on the genomic map between 26.9 and 29 map units and is flanked by a chitinase gene and p47 gene. This gene arrangement differs from that of similar genes in the AcMNPV genome, where the lef-8 gene is located about 62 kbp from the chitinase gene and about 7 kbp from the p47 gene. Sequence analyses of the lef-8 gene revealed an ORF of 2730 nucleotides, predicted to encode a protein with M _r 104876. The putative protein is 60.9% identical to the AcMNPV LEF-8 and contains an identical sequence of a conserved motif of DNA-dependent RNA polymerases. Sequences downstream of the lef-8 gene contain two sequence repeats which resemble a repeated motif of the SpliMNPV enhancer element and other repetitive sequences from the viral genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification of DNA for the transfection of a Spodoptera frugiperda cell line

Spodoptera frugiperda (Sf-9) cells have been widely used in baculovirus expression systems, transient gene expression studies and transgenic cell lines. These applications commonly require the transfection of bacterial plasmid DNA. One of the most reliable methods of preparing transfection-quality plasmid DNA is cesium chloride (CsCl) density gradient centrifugation. However, the traditional CsCl DNA purification is a long and laborious process. We have made a series of modifications to the traditional method that makes it faster, safer and easier. In the current study we demonstrate that DNA prepared by our modified CsCl method was also better for the transfection of Sf-9 cells than DNA prepared by the traditional CsCl method.     

胡椒碱对SF-21细胞株的杀伤作用及对其形态影响的研究

目的检测胡椒碱对草地贪夜蛾SF-21细胞的毒性作用和生长抑制作用,以便进一步研究其对昆虫细胞离子通道的影响。方法胡椒碱处理SF-21细胞后,用MTT微量酶反应比色法、改良微波Giemsa染色法检测细胞死亡率及形态学改变,用冲击式测试法检测细胞生长率及受损细胞的恢复。结果胡椒碱处理24h后,对SF-21细胞的半数毒性浓度(IC50)为95.37μg/ml,且毒性作用强度随着药物浓度增加而增强。胡椒碱浓度在10μg/ml以上时可诱发SF-21细胞形态学改变,表现为细胞呈多形性、细胞间有间隙、胞质内充满颗粒,以后随药物浓度的升高,胞质出现空泡、染色质凝成粗大颗粒或无结构大块、大片细胞脱落、崩解、死亡。低浓度胡椒碱(5μg/ml)对SF-21细胞增殖没有影响,较高浓度胡椒碱(10、20、40μg/ml)则对SF-21细胞增殖有明显的抑制作用,抑制作用强度随药物浓度的增加而增加,去除药物作用后,受抑制的细胞又重新恢复生长、分裂。结论胡椒碱对昆虫细胞有较强的毒性作用和生长抑制作用,可作为新型杀虫剂的功能物质。     

Nuclear factor erythroid‐derived 2–related factor 2 activates glutathione S‐transferase expression in the midgut of Spodoptera litura (Lepidoptera: Noctuidae) in response to phytochemicals and insecticides

Detoxication enzymes play an important role in insect resistance to xenobiotics such as insecticides and phytochemicals. We studied the pathway for activating the expression of glutathione S‐transferases (GSTs) in response to selected xenobiotics. An assay of the promoter activity of GST epsilon 1 (Slgste1) of Spodoptera litura led to the discovery of a cis‐regulating element. An antioxidant response element was activated in response to indole‐3‐carbinol (I3C) and chlorpyrifos (CPF) and was able to bind with the xenobiotic sensor protein nuclear factor erythroid‐derived 2–related factor 2 (SlNrf2). SlNrf2 and Slgste1 were responsive to reactive oxygen species induced by I3C and CPF in a S. litura cell line, as well as in S. litura midguts. SlNrf2 RNA interference (RNAi) reduced the message RNA levels of Slgste1 and the peroxidase activity of GSTs in response to I3C, xanthotoxin, CPF and deltamethrin. SlNrf2 RNAi and inhibitor treatment of GST activity decreased the viability of I3C‐treated cells. These results indicate that SlNrf2 activates the expression of GSTs in response to oxidative stresses caused by exposure to xenobiotics.     

Antifeedant Activity of Ursolic Acid Isolated from Duboisia Myoporoides

Ursolic acid isolated from the leaves and stems of Duboisia myoporoides (Solanaceae) was bioassayed by leaf disc method for feeding deterrence using Spilosoma obliqua and Spodoptera litura as test insects. This compound was proved to be a potent antifeedant under laboratory conditions. Azadirachtin was used as standard. Ursolic acid produced 90.12% and 91.96% inhibition at 5000 ppm concentration, respectively, against S. obliqua and S. litura .