[Show abstract][Hide abstract] ABSTRACT: The ladybird Propylaea japonica (Thunberg) is one of most important natural enemies of aphids in China. This species is threatened by the extensive use of insecticides but genomics-based information on the molecular mechanisms underlying insecticide resistance is limited. Hence, we analyzed the transcriptome and expression profile data of P. japonica in order to gain a deeper understanding of insecticide resistance in ladybirds. We performed de novo assembly of a transcriptome using Illumina's Solexa sequencing technology and short reads. A total of 27,243,552 reads were generated. These were assembled into 81,458 contigs and 33,647 unigenes (6,862 clusters and 26,785 singletons). Of the unigenes, 23,965 (71.22%) have putative homologues in the non-redundant (nr) protein database from NCBI, using BLASTX, with a cut-off E-value of 10-5. We examined COG, GO and KEGG annotations to better understand the functions of these unigenes. Digital gene expression (DGE) libraries showed differences in gene expression profiles between two insecticide resistant strains. When compared with an insecticide susceptible profile, a total of 4,692 genes were significantly up- or down- regulated in a moderately resistant strain. Among these genes, 125 putative insecticide resistance genes were identified. To confirm the DGE results, 16 selected genes were validated using quantitative real time PCR (qRT-PCR). This study is the first to report genetic information on P. japonica and has greatly enriched the sequence data for ladybirds. The large number of gene sequences produced from the transcriptome and DGE sequencing will greatly improve our understanding of this important insect, at the molecular level, and could contribute to the in-depth research into insecticide resistance mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Peptidoglycan recognition proteins (PGRPs) are non-specific immune molecules of insects, and vertebrates etc., but are not present in plants and nematodes. In the current experiment, a PGRP DNA sequence (2,910 bp containing four exons) was identified from genomic DNA library of Asian corn borer, Ostrinia furnacalis, and a full-length cDNA programming PGRP was cloned (designed as OfPGRP-S) with an open reading frame of 579 bp, having 192 amino acid. This inferred amino acid sequence showed maximum similarity to known lepidopteran PGRPs. Quantitative real-time PCR investigation disclosed the level of mRNA of OfPGRP-S to be constitutively expressed in the whole developmental stages and with higher expression in the mature larvae. Even more the OfPGRP-S was mainly expressed in immune capable organs i.e., fat body and midgut, and was strongly induced by injecting gram-positive bacteria i.e., Staphylococus aureus. Recombinant protein OfPGRP-S could bind to S. aureus and Bacillus thuringiensis which enhance proPO activation in the presence of these microbes. The results indicated that OfPGRP-S is an inducible protein acting as a receptor-type PGRP for enhancing the proPO activation on exposure to bacteria.
No preview · Article · Aug 2013 · World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology)
[Show abstract][Hide abstract] ABSTRACT: The entomopathogenic fungus Metarhizium anisopliae is currently used as an efficient biological control agent against different insects. The prevalence and genetic variability
of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae in Asian countries (China, Laos, Singapore and South Korea) and a European country (The Netherlands) was examined. The fungus
was found to be widespread in agricultural and forest soils throughout China especially in the south and south western regions,
with a maximum recovery percentage of 81.6, while in Laos it was found to be abundant in the forest soils only, the soil of
Netherlands also seemed to be an excellent source of entomopathogens with a significant recovery of insect pathogenic fungi.
Simple sequence repeats (SSR) and ITS-rDNA sequence data showed that the isolates are closely linked to each other. The gene
diversity (He) and polymorphic information content values of sixty two isolates showed mean values of 0.37 and 0.63. The majority
of the isolates belonged to one of the closely related genotypes and these were found to be dominant in the agricultural as
well as forest ecosystem. Genetic distances among the isolates according to locations and different sources showed minimal
variation ranging from 0.23 to 0.34%, with maximum genetic distance of 0.34% among the isolates from Laos and The Netherlands.
The reason for the limited variation is uncertain, however even the similarity index among the isolates can endow with sufficient
knowledge for the implications of the methods to develop this fungus as a microbial control agent.
–Genetic diversity–Simple sequence repeats–ITS-rDNA–Biocontrol–Phylogenetic analysis
No preview · Article · Feb 2011 · World Journal of Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Hybrid antibacterial peptide CecropinAD (CAD) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach to express the hybrid peptide CAD in the methylotrophic yeast Pichia pastoris, the cDNA sequence encoding CAD was obtained by recursive PCR (rPCR) and cloned into the vector pPICZα-A. The Sac I-linearized recombinant plasmid pPICZα-CAD was transformed into P. pastoris GS115 by electroporation. Expression of recombinant CAD was induced for 96 h with 1.0% methanol at 28 °C, pH 5.0. The recombinant CAD was purified by two steps of reversed-phase HPLC and 1.8 mg pure active CAD was obtained from 100 ml culture. Tricine-SDS-PAGE and mass spectrometry analyses demonstrated that the molecular weight of the purified CAD was 3.8 kDa. Analysis of circular dichroism (CD) revealed that CAD mainly has α-helixes in the presence of 10 mM phosphate buffer (pH 7.2), 50% TFE/water solution (pH 2.0), or 30 mM SDS (pH 10.8). FACScan analysis showed that the antibacterial mechanism of CAD is to act on the cell membrane to disrupt bacterial cell structure. Antimicrobial assays demonstrated that recombinant CAD has a broad spectrum of anti-microbial property against fungi, as well as Gram-positive and Gram-negative bacteria, but does not have hemolytic activity against human erythrocytes. Our results suggest that recombinant antimicrobial peptide CAD may serve as an attractive candidate for the development of therapeutic antimicrobial drugs.
No preview · Article · Jan 2009 · PROCESS BIOCHEMISTRY
[Show abstract][Hide abstract] ABSTRACT: The bioactivities of destruxins against whitefly, Bemisia tabaci and its natural enemy, ladybird beetle Serangium japonicum were evaluated. Destruxins A and B (DA and DB) showed insignificant ovicidal, oviposition deterrent and systemic insecticidal activities to B. tabaci; however, DA and DB had certain contact virulence to its nymphs. The LC(50) values of DA at 120h to 2nd, 3rd and 4th instars were 89.8 (95% confidence interval as 85.4-94.4), 199.3 (187.7-211.5) and 270.7 (251.5-291.5)mg/L, while the LC(50)s of DB at 120h were 96.5 (92.0-101.2), 216.7 (203.0-231.2), 359.4 (326.6-395.4)mg/L, respectively. In addition, DA exhibited moderate acute contact toxicities towards S. japonicum, the LC(50)s at 48h were 165.4 (132.3-229.4) and 192.5 (148.1-289.2)mg/L for 4th instar larvae and adults. Furthermore, the results from experiments of residual toxicities of DA towards mortalities of 4th instar larvae and adults, pupation rate, emergence rate, average number of egg/female and hatching rate suggested that DA had minimal effects to the ladybird beetle. Generally, the toxicity decreased about 50% from 1st to 3rd-5th day of post-treatment. Specially, the residual toxicity at 50mg/L and the 7th day post-treatment was down to a value not differing significantly from the control.
[Show abstract][Hide abstract] ABSTRACT: Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.
No preview · Article · Oct 2008 · Protein Expression and Purification