Dissertation
Dissertation > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant pest and its control > Lepidoptera pests

Molecular Chracterization and Tissue Expression of Cytochrome P450 and Esterase Gene Fragemets in the Antennae of Spodoptera Exigua (H(?)bner) and Spodoptera Litura (Fabricius)

Author ShaoGangFeng
Tutor DongShuangLin
School Nanjing Agricultural College
Course Agricultural Entomology and Pest Control
Keywords Spodoptera exigua S. litura Odorant degrading enzyme (ODE) Cytochrome P450 Antennal esterase Tissue expression
CLC S433.4
Type Master's thesis
Year 2009
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In insects, the olfactory systems have evolved to be highly sensitive, enabling detection and discrimination among a diverse array of volatile chemicals, and thus leading to various behavioral responses such as mate-seeking, location of food and habitat, oviposition. Therefore, olfactory systems play crucial roles in insect survival and reproductive success. The olfactory-based control strategies have developed as an important means for pest control. The beet armyworm, Spodoptera exigua (Hiibner) and common cutworm, S. litura (Fabricius) (Lepidoptera: noctuidae), are severe pests of various agricultural crops. Their sex pheromone of female moths have been identified as complex blends, and successfully used in pest population monitoring. However, it is not satisfactory to use it in pest control neither by means of mass-trapping nor mating-disruption. Therefore, it is very important to gain insight on the molecular mechanism of the perception of female sex pheromone by the male. The insights into the molecular mechanism will facilitate molecular designing of specific regulator to olfactory related proteins, and for further development of more effective pest behaviorally interfering techniques. Here, we report the molecular identification and sequence analysis of P450 and antennal esterase genes from S. exigua and S. litura, and further tissure expression patterns of these genes. The main results are as follows.1. Molecular characterization and Tissue expression analysis of P450 genes from S. exiguaPCR was performed with a pair of degenerate primers designed on the conserved amino acid regions in P450 of other insects, afforded two cDNA products of 444 and 441 bp, respectively. They were named CYP4L15 and CYP4L16, respectively. Alignment with other p450 genes putatively involved in odorant degradation, high identities were found among these proteins. The identity between gene CYP4L15 and CYP4L4 from M. brassicae was 94%; the identity between CYP4L16 and CYP4L4 was 92%. Two novel genes had a high identity of 96%.To investigate the expression of P450 in different tissues of S. exigua, reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed using specific primers. CYP4L15 and CYP4L16 were expressed in male and female antennae of S. exigua, and got equal expression levels. CYP4L15 expressed in all tissues tested (antennae, head without antennae, thorax, abdomen, feet, wing) in male moth. CYP4L16 expressed in all tissues tested except for the abdomen in male moth. Both CYP4L15 and CYP4L16 were lowerly expressed in wings. Based on these results,2 genes were putatively proposed to involve in chemoreception, with CYP4L15 also in the metabolism of toxical chemicals in the alimentary canal.2. Molecular characterization and Tissue expression analysis of P450 genes from S. lituraSimilarly as in S. exigua, with RT-PCR technique, three cDNA fragments (444bp) were cloned. They were named CYP4L19, CYP4L20 and CYP4L21, respectively. Alignment with the other p450s putatively involved in odorant degradation, high identities were found among these proteins. The identity with CYP4L19 and CYP4L4 was 94%; both CYP4L20 and CYP4L21 have identity with CYP4L4 of 92%. CYP4L19 has identity of 97% and 95% with two P450 genes cloned from S. exigua CYP4L15 and CYP4L16; CYP4L20 has identity of 95% and 93% with CYP4L15 and CYP4L16; CYP4L21 has identity of 96% and 93% with CYP4L15 and CYP4L16. Three novel genes have high identity of 97%.To investigate expression of P450 in different tissues of S. litura, RT-PCR experiments were performed using specific primers. RT-PCR experiments show CYP4L19, CYP4L20 and CYP4L21 were expressed with equal level in male and female S. litura antennae. Six tissues were selected for expression comparison in the male moth, including antennae, head with antennae deduced, thorax, abdomen, feet and wing. CYP4L19 and CYP4L20 were expressed in all tested tissues, with higher amount in antennae, abdomen and feet. CYP4L21 expressed in all tested tissues except for in wing. Three cloned novel genes were proposed to play roles in metabolism of odorant moleculars, toxical chemicals and pesticide entered into alimentary canal with food. 3. Molecular cloning, Characterization and Tissue expression analysis of esterase genes from S. exigua and S. lituraBy RT-PCR method with a pair of degenerate primers designed against an alignment of several known insect odorant degrading esterase animo acid sequences. Two cDNA fragments of antennal esterase gene, SexiAE and SlitAE were cloned and sequenced from the antennae of male S. exigua and S. litura, respectively. Alignment with the other esterase genes that putatively involved in odorant degradation, identities were analysised. SexiAE shared the highet identity of 54% with odorant degrading esterase gene of MbraODE, and SlitAE shared the highest identity of 51% with esterase of MbraODE and SlitEST. Two novel genes shared an identity of 67%.To investigate the expression of SexiAE and SlitAE in different tissues of S.exigua and S. litura, RT-PCR experiments were performed using specific primers. SexiAE and SlitAE were both expressed in male and female antennae of S. exigua and S. litura, respectively. Six tissues were selected for the expression study in the male moth, and these tissues were male antennae, head with antennae deduced, thorax, abdomen, feet and wing. SexiAE was expressed in all tested tissues, but the expression leve in wing was very low. SlitAE was expressed with higher level in antennae and abdomen than that in feet. There was no expression in other tissues tested. The expression pattern of these two esterase genes indicated that 2 esterases may play multiple roles besides odorant metabolism in chemoreception, and that. SexiAE comparing to SlitAE, may has wider substrate spectrum and thus undertake more physiological functions.In conclusion, we described the identification and characterization of odorant reception related enzymes from the male antennae of S. exigua and S. litura, and reported their tissue expression pattern in S. exigua or S. litura. The results could be helpful for both understanding of the molecular mechanism of odorant reception in S. exigua and S. litura, and providing theoretical bases in designing and developing new tactics in pest control.

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