Serine proteinase over-expression in relation to deltamethrin resistance in Culex pipiens pallens

Two serine proteinase genes were isolated from Culex pipiens pallens as significantly up-regulated genes in a deltamethrin-resistant strain through a combination of suppression substractive hybridization and gene expression profiling by macroarrays. These two genes were found to be expressed at least threefold higher in the resistant strain than in the susceptible one. By using rapid amplification of cDNA ends to screen the constructed cDNA library, we cloned these two sequences. There were 909 bp with an open reading frame of 786 bp in the sequence of trypsin cDNA (GenBank/NCBI AF468495), the deduced protein had 261 amino acids, which was most similar to the trypsin gene of Anopheles gambiae. There were 992 bp with an open reading frame of 816 bp in the chymotrypsin cDNA (GenBank/NCBI AY034060), and its deduced amino acid sequence had 271 amino acids, which was most similar to the chymotrypsin-like protein from Aedes aegypti. The two genes were stably expressed in mosquito C6/36 cells, and the expected 29 and 30 kDa bands were shown with Western blot, respectively. In these cells, after deltamethrin treatment, they had protective effects on the viability. The results indicate that trypsin and chymotrypsin were more highly expressed in the deltamethrin-resistant strain, and was related to insecticide resistance in mosquitoes, Cx. pipiens pallens.     

三疣梭子蟹Profilin基因全长cDNA的克隆与表达分析

三疣梭子蟹（Portunus trituberculatus）是我国沿海重要养殖品种之一,近年来养殖病害呈逐年上升趋势,制约了三疣梭子蟹养殖产业的健康可持续发展。克隆三疣梭子蟹免疫相关基因,研究免疫基因的功能和作用机制,可为三疣梭子蟹养殖病害的防治奠定基础。本研究从三疣梭子蟹血细胞全长cDNA文库中克隆了742 bp 的profilin基因全长cDNA。Profilin全长cDNA中开放阅读框长375 bp,编码125 aa。推导的三疣梭子蟹profilin理论等电点pI 5.87,氨基酸序列与冈比亚按蚊（Anopheles gambiae）profilin同源性最高,序列一致性为42.9%。荧光定量RT-PCR分析结果显示在正常的三疣梭子蟹机体中,血细胞profilin表达水平最高,其次为肝胰脏;在致病菌副溶血弧菌（Vibrio parahaemolyticus）诱导后,血细胞中profilin表达量显著上升（P<0.01）,表明profilin可能参与了三疣梭子蟹的免疫防御反应,是一个免疫相关因子。     

Identification and classification of detoxification enzymes from Culex quinquefasciatus (Diptera: Culicidae)

Molecular characterization of the insecticide resistance has become a hot research topic ever since the first disease transmitting arthropod (Anopheles gambiae) genome sequence has unveiled in 2002. A recent publication of the Culex quinquefasciatus genome sequence has opened up new opportunities for molecular and comparative genomic analysis of multiple mosquito genomes to characterize the insecticide resistance. Here, we utilized a whole genome sequence of Cx. quinquefasciatus to identify putatively active members of the detoxification supergene families, namely cytochrome P450s (P450s), glutathione-S-transferases (GSTs), and choline/carboxylesterases (CCEs). The Culex genome analysis revealed 166 P450s, 40 GSTs, and 62 CCEs. Further, the comparative genomic analysis shows that these numbers are considerably higher than the other dipteran mosquitoes. These observed speciesspecific expansions of the detoxification super gene family members endorse the popular understanding of the involvement of these gene families in protecting the organism against multitudinous classes of toxic substances during its complex (aquatic and terrestrial) life cycle. Thus, the generated data set may provide an initial point to start with to characterize the insecticide resistance at a molecular level which could then lead the development of an easy to use molecular marker to monitor the incipient insecticide resistance in field environs.     

Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: Members of PAS transcriptional factor family

The Methoprene-tolerant (Met) gene in Drosophila melanogaster has been shown to function in juvenile hormone (JH) action. Met homologs were isolated from three mosquito species, Culex pipiens, Aedes aegypti and Anopheles gambiae. Sequence similarity was found to be high in bHLH and PAS conserved domains, and the majority of the 7-9 introns in AaMet and AgMet are located in either identical or similar positions, indicating evolutionary relatedness. Sequence comparison with Met and the similar germ-cell expressed (gce) gene in D. melanogaster showed that the mosquito genes are more similar to gce than to Met. Moreover, the multiple introns in AgMet and AaMet are more similar in number with the 7 introns in Dmgce than to the single intron in DmMet; in fact, six intron positions in AaMet and AgMet are similar to those in Dmgce. Efforts to identify a second homologous gene in mosquitoes were unsuccessful, suggesting a single gene in lower Diptera, consistent with the single gene uncovered in genomic sequencing of Ae. aegypti and An. gambiae. These results suggest that a gene duplication occurred during the evolution of higher Diptera, resulting in Met and gce.     

丹参咖啡酸-O-甲基转移酶基因(SmCOMT1)的克隆及其分析

依据丹参转录组数据库得到的咖啡酸-O-甲基转移酶基因序列设计特异性引物,采用RT-PCR方法从丹参分离得到一个新的COMT基因,命名为SmCOMT1(GenBank注册号为JF693491）。该基因cDNA全长1 158 bp,包含一个长为1 095 bp的开放阅读框,编码364个氨基酸。SmCOMT1 gDNA序列长2 275 bp,包含4个外显子和3个内含子。序列分析结果表明,SmCOMT1编码的多肽具有COMT的序列保守元件,与同科植物罗勒COMT编码的多肽高度同源,同源性达到89%。系统进化树分析表明,SmCOMT1与双子叶植物的COMT亲缘关系较近。qRT-PCR结果表明,SmCOMT1基因在丹参不同组织器官中差异表达,其中茎中的表达量最高,并且其表达受茉莉酸甲酯和病原菌的诱导,显示SmCOMT1基因可能在植物防御反应中发挥作用。     

The histone-like C-terminal extension in ribosomal protein S6 in Aedes and Anopheles mosquitoes is encoded within the distal portion of exon 3

In eukaryotic cells, ribosomal protein S6 (RPS6) is the major phosphorylated protein on the small ribosomal subunit. In the mosquitoes Aedes aegypti and Aedes albopictus, the cDNA encoding RPS6 contains 300 additional nucleotides, relative to the Drosophila homolog. The additional sequence encodes a 100-amino acid, lysine-rich C-terminal extension of the RPS6 protein with 42-49% identity to histone H1 proteins from the chicken and other multicellular organisms. Using mass spectrometry we now show that the C-terminal extension predicted by the cDNA is present on RPS6 protein isolated from ribosomal subunits purified from Ae. albopictus cells. To expand our analysis beyond the genus Aedes, we cloned the rpS6 cDNA from an Anopheles stephensi mosquito cell line. The cDNA also encoded a lysine-rich C-terminal extension. However, in An. stephensi rpS6 the extension was approximately 70 amino acids longer than that in Ae. albopictus, and at the nucleotide level, it most closely resembled histone H1 proteins from the unicellular eukaryotes Leishmania and Chlamydomonas, and the bacterium Bordetella pertussis. To examine how the histone-like C-terminal extension is encoded in the genome, we used PCR-based approaches to obtain the genomic DNA sequence encoding Ae. aegypti and Ae. albopictus rpS6. The sequence encoding the histone-like C-terminal extension was contiguous with upstream coding sequence within a single open reading frame in Exon 3, indicating that the lysine-rich extension in mosquito RPS6 is not the result of an aberrant splicing event. An in silico investigation of the Anopheles gambiae genome based on the cDNA sequence from An. stephensi allowed us to map the An. gambiae gene to chromosome 2R, to deduce its exon-intron organization, and to confirm that Exon 3 encodes a C-terminal histone-like extension. Because the C-terminal extension is absent from Drosophila melanogaster, we examined a partial cDNA clone from a Psychodid fly, which shares a relatively recent common ancestor with the mosquitoes. The absence of the C-terminal extension in the Psychodid rpS6 cDNA suggests that the unusual RPS6 structure is restricted to a relatively small group of flies in the Nematocera.     

Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae

The binary toxin (Bin) from Bacillus sphaericus exhibits a highly insecticidal activity against Culex and Anopheles mosquitoes. The cytotoxicity of Bin requires an interaction with a specific receptor present on the membrane of midgut epithelial cells in larvae. A direct correlation exists between binding affinity and toxicity. The toxin binds with high affinity to its receptor in its primary target, Culex pipiens, and displays a lower affinity to the receptor in Anopheles gambiae, which is less sensitive to Bin. Although the Bin receptor has previously been identified and named Cpm1 in C. pipiens, its structure in Anopheles remains unknown. In this study, we hypothesize that the Anopheles Bin receptor is an ortholog of Cpm1. By screening the Anopheles genomic database, we identified a candidate gene (Agm3) which is expressed primarily on the surface of midgut cells in larvae and which functions as a receptor for Bin. A Cpm1-like gene is also present in the Bin-refractory species Aedes aegypti. Overall, our results indicate that the three mosquito genes examined share a very similar organization and are strongly conserved at the amino acid level, in particular in the NH(2)-terminus, a region believed to contain the ligand binding site, suggesting that relatively few amino acids residues are critical for high affinity binding of the toxin.     

Molecular cloning and expression of the gene encoding a phospholipase A1 from Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.     

Characterization of the nicotinic acetylcholine receptor subunit gene Mdalpha2 from the house fly, Musca domestica

A nicotinic acetylcholine receptor (nAChR) subunit gene, Mdalpha2, was isolated and characterized from the house fly, Musca domestica. This is the first nAChR family member cloned from house flies. Mdalpha2 had a cDNA of 2,607 bp, which included a 696 bp 5'-untranslated region (UTR), an open reading frame of 1,692 bp, and a 219 bp 3'-UTR. Its deduced amino acid sequence possesses the typical characteristics of nAChRs. Mdalpha2 genomic sequence was 11.2 kb in length in the aabys strain and 10.9 kb in the OCR strain, including eight exons and seven introns. Based on the deduced amino acid sequence, Mdalpha2 had the closest phylogenetic relationship to the Drosophila melanogaster Dalpha2 and Anopheles gambiae Agamalpha2, and a similar genomic structure to Dalpha2. Quantitative real-time PCR analysis showed that Mdalpha2 is expressed in the head and the thorax at 150- and 8.5-fold higher levels than in the abdomen. Linkage analysis of a Mdalpha2 polymorphism indicates this gene is on autosome 2. The importance of these results in understanding the diversity and phylogenetic relationships of insect nAChRs, the physiology of nAChRs in the house fly, and the utility of nAChR sequences in resistance detection/monitoring is discussed.     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

Sodium channel genes and their differential genotypes at the L-to-F kdr locus in the mosquito Culex quinquefasciatus

The para-type sodium channel in insects is the primary target of pyrethroid and DDT insecticides. However, modifications in the target protein structure such as point mutations or substitutions, resulting from single nucleotide polymorphisms (SNP), cause insensitivity of the insect’s nervous system to pyrethroids and DDT and, in turn, result in insecticide resistance. Among these mutations, substitution of leucine to phenylalanine (L to F) in the 6th segment of domain II (IIS6) has been clearly associated with pyrethroid and DDT resistance in many insect species, including mosquitoes. Here, multiple copies of the sodium channel gene were identified in the mosquito Culex quinquefasciatus by Southern blot analysis and polymerase chain reaction (PCR) analysis. Two genomic DNA fragments of the mosquito sodium channel gene (509 and 181 bp) were detected by a single PCR primer pair. Sequence analysis indicated the lack of an intron sequence in the 181 bp sodium channel fragment. Single nucleotide polymorphism (SNP) analysis revealed a strong correlation among the frequencies of L-to-F allelic (T) expression at the RNA level, the frequencies and resistance allele (T) at the L-to-F site of the 509 bp genomic DNA fragment, which did include an intron sequence, and the levels of insecticide resistance. Taking together, this study, for the first time, not only revealed multiple copies of the sodium channel gene presented in the Culex mosquito genome but also suggested that the one with the intro sequence may be a functional copy of the sodium channel gene in the Culex mosquitoes.     

Assessment of esterase gene expression as a risk marker for insecticide resistance in Florida Culex nigripalpus (Diptera: Culicidae)

Esterases are enzymatic proteins known to play a role in insecticide resistance formation. To further our understanding of the development of insecticide resistance, we tested the gene expression level of a gene implicated in insecticide resistance (Temsha est‐1) from Culex nigripalpus Theobald (Diptera: Culicidae) in field mosquitoes. We found that the level of expression of TE‐1 differed depending on the frequency of exposure to organophosphate insecticide through expression studies. Temsha est‐1 cDNA is 1,808 base pairs and fully sequenced with up to 96% nucleotide sequence identity to esterase B genes of other mosquito species. The genes from five different species, including TE‐1, were closely related by genetic distance and phylogenetic analysis. Differential expression of this gene that is correlated to differences in susceptibility towards organophosphate would provide the ability to use Temsha est‐1 as an indicator of the formation of tolerance/resistance. This would greatly enhance mosquito control efforts by allowing targeted application of insecticides to mosquito populations that are most susceptible. Also, it would provide resistance information so that a rational design could be used for insecticide rotation schedules.     

Cloning and characterization of a gene encoding HMG-CoA reductase from Ganoderma lucidum and its functional identification in yeast

A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) was isolated from a triterpene-producing fungus, Ganoderma lucidum (Reishi or Lingzhi). This report provides the complete nucleotide sequence of the full-length cDNA encoding HMGR and its genomic DNA sequence. The cDNA of the HMGR (GenBank Accession no., EU263989) was found to contain an open reading frame (ORF) of 3,681 bp encoding a 1,226-amino-acid polypeptide, whereas the HMGR genomic DNA sequence (GenBank Accession no., EU263990) consisted of 4,262 bp and contained seven exons and six introns. The deduced amino acid sequence of G. lucidum HMGR showed significant homology to the known HMGRs from Ustilago maydis and Cryptococcus neoformans, and contained four conserved domains. Gene expression analysis showed that the expression level was relatively low in mycelia incubated for 10, 12, and 14 d, and reached the highest level in the primordia. Functional complementation of Gl-HMGR in a HMGR-deficient mutant yeast strain indicated that the cloned cDNA encoded a HMG-CoA reductase.     

Isolation and sequence analysis of P450 genes from a pyrethroid resistant colony of the major malaria vector Anopheles funestus.

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.     

Cloning of CYP9G2 from the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae).

Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%).