Gene Cloning Of Esterases And MRNA Expression Level In Three Psocid Species

Psocids,belonging to Liposcelididae,Psocoptera,are worldwide and commonly found in various processed and unprocessed dry foods.Outbreaks of psocids could pose an alarming threat to stored product.Routine fumigations of warehouses and storage facilities with methyl bromide and PH₃ have failed to control these pests.In addition,the rapid development of resistance to chemical insecticide by psocids has also been reported.In China,the commonly occurred psocid species are Liposcelis bostrychophila,L.entomophila,L.decolor,and L.paeta.In this dissertation,the molecular characteristics of acetylcholinesterases(ACHE),carboxylesterase(CarE),and nicotinic acetylcholine receptor(nAChR) in L.bostrychophila,L.entomophila,and L.decolor were investigated.The project is supported by National Natural Science Foundation of China(30570231) and the Program for New Century Excellent Talents in University(NCET-04-0854).The major results are summarized as follows:1 Gene cloning of esterases and mRNA expression level in L. bostrychophila1.1 Gene cloning and sequence analysis of AChE in L.bostrychophilaThe second full length cDNA encoding AChE(GenBank Accession No.:FJ647185) was cloned from L.bosrtychophila by the methods of reverse transcriptase PCR(RT-PCR) and rapid amplification of cDNA ends(RACE).The complete cDNA of this gene consists of 3316 bp with an open reading frame(ORF) of 2814 bp,encoding a protein of 937 amino acids residues with the putative signal peptide of 23 residues.The mature protein has a molecular weight of 104.8 kDa with an isoelectric point of 6.63.This gene is paralogous to AChE of Drosophila melanogaster by sequence homology analysis,and thus named Lb acel.Although a low identity between two AChEs of L.bostrychophila(38.75%),both of them share high identity to the same type of AChEs from other insect species.ACHE1 from L.bostrychophila possesses 92%and 90%amino acid reside identities to those of L.entomophila and L.decolor.Also,the identities to ACHE1 from Spodoptera exigua,Nephotettix cincticeps and Helicoverpa assulta are higher than 70%.According to sequence alignment,the predicted amino acids contained all typical residues of AChE such as the catalytic triad and the oxy-anion hole.Homology modeling of 3-D structure of ACHE1 from L. bostrychophila is constructed using Homo sapiens(1p0i:A) native BuChE structure as template by SWISS-MODEL.The catalytic triad were found and denoted in the 3-D structure of AChE1 from L.bostrychophila referring to Torpedo californica. 1.2 mRNA expression levels of two AChE genes from L.bostrychophilaThe mRNA expression levels of two ace genes from L.bostrychophila in different strains, development stages,and insecticide treatments were studied using Real Time PCR.The results showed that the expression levels of two ace genes in resistant strains(DDVP_R and PH_3R) were significantly higher than those of susceptible strain(P＜0.05).After treated by dichlorvos(DDVP) or phosphine(PH₃),the expression levels of two ace genes were all significantly increased than control.The highest expression level of two ace genes was detected at the second stadium and the lowest was at the first stadium and adult stage.1.3 Gene cloning and sequence analysis of CarE in L.bostrychophilaTwo full length cDNA encoding Care were cloned from L.bostrychophila by the methods of RT-PCR and RACE,named Lb est1(GenBank Accession No.:EU854151) and Lb est2(GenBank Accession No.:EU854152).The complete cDNA of Lb est1 consists of 2049 bp with an ORF of 1713 bp,encoding a protein of 570 amino acids residues with the putative signal peptide of 19 residues.Lb est2 consists of 2525 bp encoding a protein of 617 amino acids residues with the putative signal peptide of 17 residues.Based on the online software ScanProsite,two conserved regions of Care were founded:serine active site(FGGDPNKVTIFGESAG for Lb est1; FGGDPNRITLFGESAG for Lb est2) and the conserved cysteine that compose disulfide bridge (EDCLFLNVFTP for Lb est1;EDCLYLNIYSP for Lb est2).The sequence homology analysis showed that the two Care genes cloned from L.bostrychophila share low identities to other insect species,but the sequences is highly conserved in active site.1.4 mRNA expression levels of two CarE genes from L.bostrychophilaReal Time PCR analysis showed that the expression level of Lb est2 in DDVP_R and PH_3R strains were 1.91 and 1.42 fold- higher than that of susceptible strain,respectively,and they were significantly different by one way ANOVA(P＜0.05).The expression levels of two Care genes after treated by DDVP or PH₃ were also significantly higher than control(P＜0.05).The expression level of Lb est1 gradually decreased with the growth of the psocid,and lowest level was detected at adult stage.In contrast,the expression levels of Lb est2 in nymphal stages are lower than that in adult stage.2 Gene cloning of AChE and mRNA expression level in L.entomophila2.1 Gene cloning and sequence analysis of AChE in L.entomophilaTwo full length cDNA encoding AChE were cloned from L.entomophila by the methods of RT-PCR and RACE,named Le ace1(GenBank Accession No.:EU854149) and Le ace2(GenBank Accession No.:EU854150).The complete cDNA of Le ace1 consists of 1958 bp with an ORF of 1890 nucleotides,encoding a protein of 629 amino acids residues.Le ace2 consists of 2171 bp with an ORF of 1914 bp,encoding a protein of 637 amino acids residues with the putative signal peptide of 20 residues.The identity of two AChEs is low(35.73%).The predicted amino acids of two genes from L.entomophila both contained all typical residues of AChE family by sequence alignment, such as the catalytic triad and the oxy-anion hole.Homology modeling of 3-D structure of two AChE from L.entomophila were constructed using H.sapiens(1p0i:A) native BuChE structure and Drosophila melanogaster(1d×4:A) native AChE structure as templates,respectively,by SWISS-MODEL.The catalytic triad were found and denoted in the 3-D structure of AChE from L. entomophila referring to T.californica.2.2 Gene cloning ofβ-actin and mRNA expression levels of two AChE genes from L. entomophilaBecause no reference gene has ever been used in Real Time PCR for L.entomophila in GeneBank,a fragment ofβ-actin gene was cloned from L.entomophila(GenBank Accession No.: FJ041117).It consists of 822 bp encoding a protein of 273 amino acids residues.The deduced amino acid sequence possesses a high homology toβ-actin from other species reported in GenBank.Real Time PCR analysis showed that Le ace1 expressed 1.6 fold higher than Le ace2 in L.entomophila. The expression levels of the two ace genes after treated by aldicarb or malathion were significantly higher than control(P＜0.05).3 Gene cloning of AChE and phylogenetic analysis in L.decolorThe full length of Ld ace2(GenBank Accession No.:FJ647187) and fragment of Ld ace1 (GenBank Accession No.:FJ647186) were cloned from L.decolor.The complete cDNA of Ld ace2 consists of 2111 bp with an ORF of 1917 bp,encoding a protein of 638 amino acids residues.The mature protein has a calculated molecular weight of 71.9 kDa with an isoelectric point of 4.7.All the typical residues of AChE family were found in the deduced amino acid sequence of Ld ace2 by sequence alignment.Homology modeling of 3-D structure were constructed using D.melanogaster (1d×4:A) native AChE structure as template.The fragment of Ld ace1 contains 1616 bp encoding 500 amino acid residues.The amino acid sequence has a high identity to ACHE1 from L. bostrychophila and L.entomophila.It includes all the typical residues of AChE family except one conserved cysteine that composes disulfide bridge.The phylogenetic tree of 50 AChE amino acid sequences from 31 species were constructed by MEGA4.1.The results showed that all AChE genes were divided into four distinct main groups: Insect Typeâ… AChE Gene,Vertebrate AChE Gene,Insect Typeâ…¡AChE Gene and Nematode AChE Gene.The 6 AChE Genes from L.bostrychophila,L.entomophila and L.decolor were demarcated into Insect Typeâ… AChE Gene and Insect Typeâ…¡AChE Gene.4 Gene cloning of nAChR and mRNA expression level in L.bostrychophilaTwo nicotinic acetylcholine receptor(nAChR) subunit genes,Lb al and Lb a8,were cloned from the psocid L.bostrychophila.The full length cDNAs of Lb a1(GenBank Accession No.: EU871527) and Lb a8(GenBank Accession No.:EU871526) consist of 2025 and 1763 bp, respectively,and contain ORFs of 1644 and 1608 bp encoding 547 and 535 amino acids, respectively.The two subunits shared high identities to those from other insect species,though they share only 56%identity in amino acid sequence.Both genes have features typical of members of nAChR family,such as the long N-terminal extracellular domain and the four hydrophobic transmembrane domains(TM1-4).The large N-terminal domain of the two nAChR subunits also contains the dicysteine loop(cys-loop) consisting of two disulphide-bond,and the ACh-binding-site-forming regions(loops A-F).They contain both the two adjacent cysteines which was unique toα-type subunit.The dendrogram shows that the proteins deduced by Lbα1 and Lbα8 belong to theα1-type andα8-type subunits,respectively.Lbα1 possesses a total of 15 potential phosphorylation sites and two potential N-glycosylation sites,while Lbα8 shares 9 potential phosphorylation sites and one potential N-glycosylation site.Quantitative real-time PCR analysis showed that Lbα1 expresses 2.03,2.55,4.77,6.54 and 5.28 fold higher than Lbα8 in the first,second,third,fourth stadium and adult,respectively.The highest expression level of both Lbα1 and Lbα8 was detected at adult stage and lowest was at third and fourth stadium,respectively.There was a stable and relatively low expression level for Lbα1 and a progressive expression pattern for Lbα8 among the 1st,2nd,3rd,and 4th nymphal stages.5 Development and reproduction of L.decolor as a function of temperatureThe developmental rate,survival,intrinsic rate of increase(r_m),net reproductive rate(R₀),mean generation time(T),and population doubling time(t) of L.decolor population were evaluated at eight constant temperatures.The female of L.decolor had four stadia in total nymphs,while the male only had three.Between 20 and 37.5℃,the female developmental period from egg to adult varied from 46.2 d at 20℃to 16.1 d at 35℃,and the male varied from 41.8 d at 20℃to 13.6 d at 35℃,but the high temperature(37.5℃) caused a decline in developmental rate of immature stage of L.decolor.The survival rate from egg to adult was highest(57.3%) at 32.5℃,and lowest(19.0%) at 37.5℃.L. decolor reproduced most eggs(130.4) at 32.5℃and the fewest(24.7) at 37.5℃.The populations reared at 32.5℃expressed the largest r_m(0.0609),R_o(16.61) and shortest t(11.39 d).The Weibull frequency distribution suggested a good fit to the data set of age-specific survivorship for all test temperatures.The populations reared at 20-32.5℃have a typeâ… survivorship curve(c＞1.0); whereas populations reared at other temperatures reveal a typeâ…¢survivorship curve(c＜1.0) based on Weibull frequency distribution.In summary,the molecular characteristics of esterases(ACHE and CarE) from three poscid species were studied systematically here.The fact that both two ace genes exist in poscids provides more evidence that two AChE genes are widely distributed in insects.The above results not only fasten the acquaintance of psocid molecular toxicology and enrich the content of evolution and genetic variation,but also contribute to development of molecular diagnostic technique for psocid resistance in field and pave the way for designing new insecticides and develop new strategies for pest management.