Identification And Functional Studies Of Cytochrome P450 And Carboxylesterase Family Genes From The Silkworm, Bombyx Mori

Many members of lepidopteran insects are agricultural and forest pests, which cause immeasurable losses every year all over the world. Wide use of insecticides has resulted in increased insecticide resistance in many pests, and has then caused great difficulties to control these pests.Cytochrome P450 is a superfamily of heme proteins, which partipicates in oxidative metabolism of endogenous and exogenous compounds. In insects, a substantial amount of research has indicated that cytochrome P450 enzyme system plays pretty important roles in detoxifying metabolism of pyrethroid insecticides and plant toxins. Carboxylesterase belongs toα/βhydrolase fold protein family, which comprise of a multigene family and can effectively hydrolyze the endogenous and exogenous compounds containing carboxylester, amide, and thioester bonds. In insects, the research about carboxylesterase is mainly focused on its involvement in insecticide resistance. Moreover, many studies have demonstrated that many metabolic enzymes such as cytothrome P450 and carboxylesterase are involved in the degradation of odorants.Bombyx mori is another insect whose whole genome sequences have been fully completed after Drosophila melanogaster and Anopheles gambiae. It has been considered as the lepidopteran model insect and is also the only whole genome sequence available lepidopteran insect. A great number of genome sequences and EST data have provided great convenience to study new genes and their corresponding functions. Bombyx mori and Bombyx mandarina originated from the same ancestor, and belong to family Bombycidae, order Lepidoptera. There have been great differences in insecticide resistance between the two species due to different selective pressures of insecticides. To research detoxifying metabolism and olfactory detection related cytochrome P450 and carboxylesterase family genes will provide clues to the control of pests such as lepidopteran insects, and facilitate the research about the physiological functions of the two family genes and evolutionary differences of species under domestication. In this research, multiple cytochrome P450 and carboxylesterase genes were identified by using bioinformatic and molecular approaches. The evolutionary relationships between these genes were analyzed at the molecular level. Tissue expression and developmental expression profiles were studied, as well as the inductive effects of xenobiotics on these genes. Some genes were expressed by using insect baculovirus express system, and the enzymatic activity of a carboxylesterase gene product was further assayed. We first compared the differences of esterases between Bombyx mori and Bombyx mandarina at the biochemical and molecular levels. A first carboxylesterase gene was also cloned from the agricultural pest, Helicoverpa armigera.1. The research of cytochrome P450 genes from Bombyx moriIn this study, we cloned two CYP4 and four CYP6 genes using bioinformatics and RT-PCR approaches. Sequence analysis showed that these genes contained conserved P450 gene sequence regions and one conserved intron. CYP4M5 and CYP4M9 were clustered together in a head-to-tail arrangement possibly due to gene duplication. Blast analysis showed that these P450 genes shared significant similarity with CYP4 and CYP6 families involved in the metabolism and detoxification of xenobiotics in other insects. RT-PCR analysis showed that these P450 genes were all highly expressed in the midgut or fat body of silkworms. Some of the P450 gene expression was increased while expression of other P450 genes was decreased as the instar age increased. When the larvae were exposed to 1.75×10-5 % of cypermethrin, 3.5×10-6 % of cypermethrin and 0.1 % of rutin, expression of CYP6AB5 was increased by 2.3-fold, 2.2-fold and 1.9-fold, respectively, while exposure to 0.1 % quercetin did not influence the expression of CYP6AB5. In contrast, expression of the other five P450 genes was suppressed after exposure to these compounds.CYP6AB5 gene was expressed by using insect Bac-to-Bac baculovirus expression system. The expression of this gene lay the foundation for characterization of substrate specificity of this enzyme by further constructing in vitro metabolizing enzymatic system.Signal inactivation is a critical step in the olfactory dynamic process, which involves various odorant-degrading enzymes. In this study, a cytochrome P450 cDNA CYP6AE21 was cloned from the moth antenna of Bombyx mori by RT-PCR method. CYP6AE21 contains a 1 572 bp ORF, which encodes a putative protein of 523 amino acids. This putative protein have a calculated molecular mass of 60.5 kD, pI of 8.4, and a P450 characteristic structure heme-binding region. CYP6AE21 and Bombyx mori CYP6AE2 both contain only one intron which exists at the same site, and their relative two exons have exactly the same size. The two genes share 94.5% nucleotide similarity, and cluster together on the same chromosome in a head-to-tail arrangement separated by a approx. 7.6 kb intergenic region. CYP6AE21 was highly expressed in larval head and fat body, and moth antenna. It was also detected in many other larval and moth tissues. NADPH P450 reductase (CPR), a very important component of monooxygenase system, was also highly expressed in the moth antenna, and detected in other moth tissues with low levels. Subcellular localization analysis showed that the expression product of CYP6AE21 is localized in cytoplasm, which is consistent with the classical localization of microsomal P450s. It was postulated that this P450 gene might participate in the degradation of odorants after their internalization into cells.2. The research of carboxylesterase genes from Bombyx mori and Bombyx mandarinaCarboxylesterase belongs toα/βhydrolase fold protein family, which can effectively catalyze the hydrolysis of endogenous and exogenous compounds containing carboxylester, amide and thioester bonds. In insects, the research of carboxylesterase is mainly focused on its involvement in insecticide resistance. Whereas, there is few study related to its physiological functions. Bioinformatics method was used to screen the genome sequence of Bombyx mori, and 12 candidate carboxylesterase genes were identified through molecular approaches. Two of the 12 candidate genes can generate various variants through alternative splicing. Some of these identified genes are clustered together on the chromosomes, while others are distributed among different chromosomes. These carboxylesterase genes can be divided into two groups according to the number of their introns. The 1 group contains conservative corresponding intron sites; the 2 group contains introns with great varieties. Homology comparison and phylogenetic analysis showed that there are great differences among these sequences, suggesting that they must have undergone great evolutionary divergences. Expression analysis indicated that some of these carboxylesterase genes were dominantly expressed in midgut or silk gland, while some others were detected at high levels in moth antenna. Moreover, BmCarE-12 and BmCarE-15 transcripts were increased in fat body after the induction by lipopolysaccharide (LPS). The putative functions of these carboxylesterase genes were discussed with regard to their respective expression patterns and phylogenetic relationships.BmCarE-5 was expressed at high levels in larval midgut, and moth antenna. Promoter analysis showed that the expression of this gene might be regulated by endogenous hormones. In order to research its potential function, we expressed this gene by using insect baculovirus expression system. SDS-PAGE analysis showed that there is a specific band of about 76 kD at the lane of BmCarE-5 recombinant bacmid infected BmN cells compared with control group. Western blot analysis showed that there is also a specific band at the same position, indicating that BmCarE-5 was successfully expressed. Enzymatic activity of this enzyme was assayed after purification of its expression product. The results showed that the optimum temperature for BmCarE-5 is 40℃, and Km and Vmax for this enzyme are 2.653 mM and 178 OD/min·mg protein, respectively. This research will facilitate the characterization of the physiological function of carboxylesterase genes.In order to research the realtionship between esterases and the insecticide resistance differences between Bombyx mandarina M. and Bombyx mori L., and the evolutionary difference between esterases from the two species, esterase activities at different larval stage and different tissues of day 3 fifth-instar larvae of Bombyx mori and Bombyx mandarina were detected using the kinetic method. The esterase activities of Bombyx mori and Bombyx mandarina were both higher at late larval stage and had similar tissue distributions with highest values in midguts and less in silk glands, suggesting that the esterases of Bombyx mori and Bombyx mandarina were relatively conserved during the course of evolution. The esterase activity in the midgut of Bombyx mori was 1.9-fold higher than that in Bombyx mandarina. According to the mutant ali-esterase hypothesis, the insecticide resistance of Bombyx mandarina was possibly not caused by overexpression of esterases, but probably through point mutations or other detoxifying enzymes. BmmCarE-4 and BmmCarE-5 had 7 variants caused by splicing differently at 5′terminal. The two genes were located on the same transcription unit, and shared over 52% amino acid identity. BmmCarE-4 and BmmCarE-5 originated from the duplication of 3′terminal sequence of one of the two genes in a pattern similar to the two orthologous genes in Bombyx mori, which further demonstrates the high conservation of esterases at the molecular level between the two species. The esterase activity in the silk gland of Bombyx mori was 4.49-fold higher than that in Bombyx mandarina. The expression level of CarE-9 in silk gland was nearly identical between Bombyx mori and Bombyx mandarina. Whereas, CarE-7 was expressed highly in silk gland of Bombyx mori, but nearly not detected in Bombyx mandarina. The expression variance of CarE-7 in silk glands of Bombyx mori and Bombyx mandarina was one of the main reasons for the difference in enzymatic activities in these tissues, and this gene might play a role in the growth and development of silk gland or the synthesis of silk protein. Moreover, BmmCarE-2 shared highest amino acid similarity (98.9%) with BmCarE-2 from Bombyx mori (Genbank accession number: DQ311250). Tissue expression analysis demonstrated that BmmCarE-2 was expressed highly in head and fat body, slightly lower in silk gland and midgut, and extremely low in hemolymph.3. The research of carboxylesterase gene from Helicoverpa armigeraIn order to study the molecular mechanisms of insecticide resistance in Helicoverpa armigera, a first full-length carboxylesterase cDNA was cloned from Helicoverpa armigera through RT-PCR and rapid amplification of cDNA ends (RACE) strategies. Sequence analysis revealed that this gene contains a 1 794 bp ORF, a 129 bp 5′UTR and 139 bp 3′UTR, which encodes a 597 amino acid protein. The predicted molecular weight and isoelectric point of this carboxylesterase were 67.1 kD and 4.92, respectively. This gene was deposited in GenBank under the accession number EF547544. Homology analysis showed that this carboxylesterase is most similar to the carboxylesterase from Spodoptera litura with 60﹪amino acid identity. Semi-quantitative RT-PCR showed that this gene was highly expressed in midgut, low-expressed in fat body and gonad, and not expressed in head. It was postulated that this carboxylesterase gene may be involved in the detoxification of xenobiotics.