Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Gene Prediction and Sequence Analysis of Insect OBP CSP and sid-1

Author XuYaLong
Tutor LiFei
School Nanjing Agricultural College
Course Agricultural Entomology and Pest Control
Keywords Insect expressed sequence tag (EST) Odorant binding protein (OBP) Chemosensory proteins (CSP) sid-1 Bioinformatics RT-PCR Evolution
Type Master's thesis
Year 2010
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Gene cloning is a time-consuming and laborious work. In recent years, expressed sequence tags (ESTs) of many insect species have been sequenced, providing a good and useful resource for gene discovery. Insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) play an important role in chemical communication of insects, The researching of OBP and CSP can help us understand the coevolution and interaction of insects and their environment; The sid-1 gene encodes a transmembrane protein which is an important participator in systemic RNAi pathway, exploring the distribution and evolution of sid-1 in insects can help us know which insects could occur systemic RNAi, and then the RNAi technology can be applicated in the agriculture pest control.In this paper, we developed a computational pipline to discover insect genes from ESTs. In total,752,841 insect ESTs were collected from 54 species covering eight Orders of Insecta. From these ESTs,142 OBPs and 177 CSPs were identified, of which 117 OBPs and 129 CSPs are new. The intact open reading frames (ORFs) of 88 OBPs and 123 CSPs were obtained by electronic elongation. Together with all family members obtained from the NCBI (OBPs) or the UniProtKB (CSPs),850 OBPs and 237 CSPs were analyzed for their motif, C-pattern structural characteristics and evolutionary relationship. Generally, there were no major differences between different Orders, except for the presence of a subclass of OBPs, C-plus OBPs, in Diptera containing eight conserved cysteines. In the typical C-pattern, there were three amino acids between the second and third cysteines in all OBPs, while eight residues were present between the fifth and sixth cysteines in most insect OBPs. The numbers of amino acids between the other three neighboring cysteines were rather variable. In most insects, the distance between the fourth and the fifth cysteines was the most variable. However, in Hymenoptera, the distance between the first and the second cysteines was the most variable with a coefficient of variation of 11.66. The highest variations were found in the OBPs of Diptera. By contrast, C-patterns of CSPs were much more conserved. In the evolutionary tree for GOBPs and PBPs, these two subfamilies were mainly clustered by Orders, indicating that most genes appeared after diversification of different Orders. However, the situation is different for CSPs. Although lepidopteran CSPs were mainly clustered as an independent group, some of their CSPs are in the same clade with other Orders, suggesting that some CSPs are ancient, whereas others appeared after the diversification of Orders.In addition,42 non-redudant sid-1 genes were found in 18 specieces, of which 16 sepecieces were first report. The evolution of sid-1 homologues in different species and their similarities are highly relevant to evolutional status of the species. There is still no report about sid-1 homologue gene in dipter untill now. We deduced that that the sid-1 homologue gene might be lost in the evolution of Diptera. The sid-1 gene structure analysis shows that the lengths of vertebrate sid-1 genes were longer than their invertebrate counterparts. The exon numbers of vertebrate sid-1 are from 21 to 33 but only 7-13 in invertebrate. This implies that verterbrate sid-1 genes might have fine-tuned expression patterns and complex functions.Caenorhabditis elegans sid-2 is required for environmental RNA interference. We found no sid-2 homologues when searching of sid-2 in insects, and there is still no report about sid-2 homologue gene in insects untill now. It is interesting that many insects can accur environmental RNAi, so we can get a conclution that Caenorhabditis elegans and insects evolued different environmental RNAi mechanism respectively.

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