A b-actin gene,Libb-actin1,from the psocid,Liposcelis bostrychophila,was isolated,sequenced,and expressed in Escherichia coli.The cDNA sequence was 1 281 bp in length and contained an open reading frame of 1 131 bp encoding 376 amino acids with a predicted molecular weight of 41.82 kDa.According to a BlastN search,the coding region shared the highest identity(97%) with Pediculus humanus actin 5C,while the deduced amino acid sequence was completely identical to a mutant of Drosophila melanogaster actin 5C.Comparison of the nucleotide and deduced amino acid sequences confirmed the high similarity between Libb-actin1 and homologs in other insect species.The 3' untranslated region(3' UTR) of the Libb-actin1 mRNA had a high A+U content(approximately 75%) and contained three repeats of the AUUUUUA and AUUUA motifs,which may play a role in regulating mRNA decay.The expression of Libb-actin1 was further analyzed in insecticide induced and control psocids.The results indicated that there was no significant difference in expression of Libb-actin1 between the induced and control groups,suggesting that Libb-actin1 may be an appropriate internal control for the gene expression profiling in this insect.Furthermore,Libb-actin1 was also heterologously expressed in Escherichia coli,which provided a basis to investigate the physiological functions of actin genes in the psocid.
An allele of CYP6BQ13, named CYP6BQ13v2 (GenBank accession no. FJ209361), was isolated from the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) by RT-PCR. The cDNA sequence of CYP6BQ13v2, 1 563 bp in length, contains an open reading frame of 1 554 nucleotides encoding a putative protein of 518 amino acid residues with a predicted molecular weight of 59.92 kDa and a theoretical pI of 7.60. The putative protein contains the classic hemebinding sequence motif F××G×××C×G (residues 456-465) conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 98% identity with the previously published sequence of CYP6BQ13 (GenBank accession no. XP_967146) from the T. castaneum genome project. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that there was closer phylogenetic relationship of CYP6BQ13v2 with CYP302A1 and CYP307A1 mediating synthesis of the insect molting hormone, distant relationship with CYP6B1 metabolizing plant allelochemicals, CYP6D1 linking to pyrethroid resistance and other members of CYP6 family. Expression test of the gene in the adults and immature stages of T. castaneum by quantitative real-time PCR revealed that CYP6BQ13v2 is expressed in all life stages investigated. The mRNA expression level in 1st instar larvae was 14.9-and 3.86-fold higher than those in pupae and adults, respectively. The CYP6BQ13v2 expression levels appeared in the order of 1st instar larvae, followed by 4th instar larvae, 7th instar larvae, adult, and pupae from high to low. The more bioinformation of CYP6BQ13v2 was also analyzed.
XU Yong-qiang WANG Jin-jun JIANG Hong-bo DOU Wei TANG Pei-an AN Feng-ming
Glutathione S-transferases(GSTs) from Liposcelis bostrychophila Badonnel and L.entomophila(Enderlein)(Psocoptera:Liposcelididae) were purified by glutathione-agarose affinity chromatography,and characterized subsequently by their Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2,4-dinitrobenzene(CDNB) and reduced glutathione(GSH),respectively.The specific activity of the purified GST toward CDNB was 2.3-fold higher in L.bostrychophila than in L.entomophila.Though the specific activities of purified enzymes varied between the two species,the purification yields were similar.SDS-PAGE revealed one band at 23 kDa for both the species.GSTs of L.entomophila exhibited higher Michaelis-Menten constants(Km) but lower maximal velocity(Vmax) values than those of L.bostrychophila.The optimum pH for CDNB conjugation of L.bostrychophila and L.entomophila GSTs was 7.0 and 7.5,and optimum temperature was 35 and 40°C,respectively.Inhibition kinetics showed that cibacron blue,curcumin,bromosulfalein,ethacrynic acid,and carbosulfan had excellent inhibitory effects on GSTs in both species,but the inhibitory effects of beta-cypermethrin,fenpropathrin,tetraethylthiuram disulfide,and diethyl maleate were not significant.
DOU Wei XIAO Li-sha NIU Jin-zhi JIANG Hong-bo WANG Jin-jun
