决明子胰蛋白酶抑制剂COTI对菜青虫生长抑制作用的研究

从决明子中分离得到胰蛋白酶抑制剂COTI,利用其缓冲液浸泡油菜叶片饲喂菜青虫(Pieris rapae L)幼虫后,幼虫体重明显下降,分析显示菜青虫中肠提取物中蛋白酶活性受到COTI的抑制,表明决明子胰蛋白酶抑制剂对菜青虫的生长具有明显的抑制作用。     

Dietary proteinase inhibitors alter complement of midgut proteases

Plant serine proteinase inhibitors (Pls) have the potential to restrict the growth and/or development of herbivorous insects. However, there are limitations to the efficacy of these Pls. An insect's susceptibility to a Pl is determined, at least in part, by the relative proportion of proteolytic enzyme activity in the midgut that is suppressed by that inhibitor. Insects adapt to dietary trypsin inhibitor in their host plant by secreting “inhibitor-resistant” trypsin(s). These “inhibitor-resistant” enzyme(s) may be the standard proteinase(s) secreted into the midgut (e.g., Pieris rapae), or may be enhanced following ingestion of proteinase inhibitor (e.g., Helicoverpa zea). In addition, insects may be pre-adapted to specific Pl(s), following adaptation to a Pl from the same family. For example, Pieris rapae is a crucifer specialist that is resistant to cabbage Pl, but is also resistant to Kunitz soybean trypsin inhibitor, a Pl in the same family as cabbage Pl, but from a non-host plant. The ultimate value of this pre-adaptation to herbivory by a species of insect will be determined by the number of different families of Pl in host plant(s) to which the species has adapted, and the distribution of those families among other species of plants. Thus, it is possible that the presence of a plant Pl limits herbivory by insect(s). However, multiple inhibitors, matched to the complement of enzymes in the insect's midgut, may be required to enhance this resistance of plants to herbivorous insects. © 1996 Wiley-Liss, Inc.     

The use of oviposition‐induced plant cues by Trichogramma egg parasitoids

1. Female parasitoids have evolved various foraging strategies in order to find suitable hosts. Egg parasitoids have been shown to exploit plant cues induced by the deposition of host eggs. 2. The tiny wasp Trichogramma brassicae uses oviposition‐induced cues from Brussels sprouts to locate eggs of the cabbage white butterflies Pieris brassicae and Pieris rapae that differ in their egg‐laying behaviour. These plant cues are elicited by male‐derived anti‐aphrodisiac pheromones in the accessory reproductive gland (ARG) secretions of mated female butterflies. However, the closely related generalist species Trichogramma evanescens does not respond to Brussels sprout cues induced by the deposition of P. brassicae egg clutches. 3. Here we showed in two‐choice bioassays that T. evanescens wasps respond to Brussels sprout cues induced by (i) the deposition of single eggs by P. rapae, and (ii) the application of ARG secretions from either mated P. rapae females, or from virgin female butterflies in combination with P. rapae's anti‐aphrodisiac compound indole. The wasps only associatively learned to respond to Brussels sprout cues after applying indole alone by linking those cues with the presence of P. rapae eggs. 4. Our results indicate that Trichogramma wasps more commonly exploit oviposition‐induced plant cues to locate their host eggs. Generalist wasps show less specificity in their response than specialists and employ associative learning.     

Molecular cloning and expression patterns of FK506‐binding protein 12, an immunophilin from the cabbage butterfly,Pieris rapae

FK506‐binding protein (FK506BP) class belonging to immunophilin protein family has been known to play key roles in modulating T‐cell activation, regulation of cell cycle and protein folding. However, little is known about the involvement of FK506BP during viral pathogenesis in insect host. In this study, an attempt has been made to focus on the involvement of FK506BP in antiviral innate immunity, by cloning the full‐length cDNA of FK506BP12 (PrFK506BP12) from the cabbage butterfly, Pieris rapae. It comprised of 532 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 327 bp encoding 108 amino acids. In silico analysis of PrFK506BP12 ORF revealed a highly conserved FK506‐binding domain (FKBD). As expected, it showed high homology to other FK506BPs identified from Bombyx mori (92%), Manduca sexta (91%), Suberites domuncula (82%), Tribolium castaneum (81%) and Aedes aegypti (74%) . Expression of PrFK506BP12 was observed during developmental stages of P. rapae, but was pronounced in late pupal and adult stage. In addition, spatial expression pattern analysis indicated its high expression in the head and fat body. Furthermore, PrFK506BP12 mRNA was induced 12 h after LTA, Poly I:C treatment and 3h after Pieris rapae granulovirus (PrGV) treatment in carcass. It suggests that PrFK506BP12 appears to be involved in immune responses and also play an important role in the fat body, although it remains to be clarified about their precise role in response to granulovirus.     

Kunitz-type trypsin inhibitor with high stability from <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. seeds

The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography. This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation of stable SOTI-trypsin complex (K _i = 2.3·10⁻⁶ M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 1, pp. 131–140.     

A trypsin inhibitor from <Emphasis Type="Italic">Cassia obtusifolia</Emphasis> seeds: isolation,characterization and activity against <Emphasis Type="Italic">Pieris rapae</Emphasis>

A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70°C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 μM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.     

EFFECT OF PARASITIZATION BY THE PUPAL ENDOPARISITOID,PTEROMALUS PUPARUM (HYMENOPTERA: PTEROMALIDAE) ON HUMORAL IMMUNE REACTIONS OF PIERIS RAPAE (LEPIDOPETERA: PIERIDAE)*

Abstract Studies on the effect of parasitization by the endoparasitoid on host humoral immune reactions are carried out with the pupal endoparisitic wasp, Pteromalus puparum, and its host, Pieris rapae. Phenoloxidase (PO) activity of parasitized hosts hemolymph increased significantly at 12 h, day four and day five after parasitization. Hem‐agglutination activity of parasitized hosts hemolymph was always higher than that of wounded and unparasitized ones. Moreover, antibacterial activity of parasitized hosts hemolymph became more and more stronger, whilst wounded and unparasitized pupae only owned a weak antibacterial activity. It suggested that activities of humoral immune factors of Pieris rapae could be influenced to some degrees by P. puparum.     

The molecular bases of plant resistance and defense responses to aphid feeding: current status

Plant genes participating in the recognition of aphid herbivory in concert with plant genes involved in defense against herbivores mediate plant resistance to aphids. Several such genes involved in plant disease and nematode resistance have been characterized in detail, but their existence has only recently begun to be determined for arthropod resistance. Hundreds of different genes are typically involved and the disruption of plant cell wall tissues during aphid feeding has been shown to induce defense responses in Arabidopsis, Triticum, Sorghum, and Nicotiana species. Mi‐1.2, a tomato gene for resistance to the potato aphid, Macrosiphum euphorbiae (Thomas), is a member of the nucleotide‐binding site and leucine‐rich region Class II family of disease, nematode, and arthropod resistance genes. Recent studies into the differential expression of Pto‐ and Pti1‐like kinase genes in wheat plants resistant to the Russian wheat aphid, Diuraphis noxia (Mordvilko), provide evidence of the involvement of the Pto class of resistance genes in arthropod resistance. An analysis of available data suggests that aphid feeding may trigger multiple signaling pathways in plants. Early signaling includes gene‐for‐gene recognition and defense signaling in aphid‐resistant plants, and recognition of aphid‐inflicted cell damage in both resistant and susceptible plants. Furthermore, signaling is mediated by several compounds, including jasmonic acid, salicylic acid, ethylene, abscisic acid, giberellic acid, nitric oxide, and auxin. These signals lead to the development of direct chemical defenses against aphids and general stress‐related responses that are well characterized for a number of abiotic and biotic stresses. In spite of major plant taxonomic differences, similarities exist in the types of plant genes expressed in response to feeding by different species of aphids. However, numerous differences in plant signaling and defense responses unique to specific aphid–plant interactions have been identified and warrant further investigation.     

Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.     

Cloning and expression profiles of tumor suppressor QM homologue in response to granulovirus in Pieris rapae

The tumor suppressor, QM, has been cloned and characterized from various model organisms such as human, plant and invertebrates. Yet, it has not been seriously investigated for its role in conjunction with antiviral mechanisms involving innate insect immunity. From the expressed sequence tag (ESTs) project, conducted with larval cDNA library of cabbage butterfly, Pieris rapae, a partial fragment (718 bp) of QM homologue, termed PrQM containing 660 bp long open reading frame (ORF) encoding protein of 219 amino acids was identified. In silico analysis of PrQM ORF revealed the presence of ribosomal protein L10a/L10e type domain. Phylogenetic analysis of the P. rapae QM‐like protein showed high amino acid sequence similarity with other PrQM polypeptides identified from Heliothis virescenes (95%), Plutella rapae (92%), Bombyx mori (92%), Drosophila melanogaster (89%), and Polyrhachis vicina (85%). The butterfly QM has the closest phylogenetic relationship to a moth (Hv) QM homologue. Further investigations revealed the expression of PrQM at all developmental stages, with pronounced presence at the egg stage. In addition, spatial pattern analysis indicated its high expression in the head, salivary gland, integument and fat body with visible presence in Malpighian tubule and gut. Time course expression studies conducted after immune‐challenge with lipoteichoic acid (LTA) showed the induction of PrQM mRNA at 12 h and 24 h after challenge and also in response to granulovirus (GV). Results of this investigation therefore suggest possible role of QM‐like proteins from Pieris rapae to be involved in innate antiviral immune responses. Further elucidation on the precise function of PrQM during antiviral immune responses by using RNA interference remains a viable research front.     

Transcriptional analysis of Pieris rapae in response to P. rapae granulovirus

Pieris rapae granulovirus (PrGV) is an important pathogen that has been exploited as a microbial insecticide to control agriculture pests. They can specifically infect cabbage butterfly (Pieris rapae), causing a series of pathological symptoms. In this infected P. rapae at 6?h and 72?h. As a result, a series of host genes were significantly modulated following PrGV infection, including those correlated with exoskeleton, ribosome, heat shock protein (HSP), proteasome, oxidation-reduction and apoptosis. Taken together, our study unveiled the P. rapae response to PrGV at different time point and provided a potential strategy for pest management.