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The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses.

ABSTRACT: BACKGROUND: Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated. RESULTS: An SCPx cDNA (SlSCPx) cloned from the common cutworm, Spodoptera litura, was characterized. The SlSCPx cDNA encoded a 535-amino acid protein consisting of a 3-oxoacyl-CoA thiolase (SCPx-t) domain and a SCP-2 (SCPx-2) domain. SlSCPx mRNA was expressed predominately in the midgut, while SlSCPx-2 mRNA was detected in the midgut, fat body and epidermis and no SlSCPx-t mRNA was detected. A 58-kDa full-length SCPx protein and a 44-kDa SCPx-t protein were detected in the midgut of sixth instar larvae when the anti-SlSCPx-t antibody was used in western blotting analysis; a 16-kDa SCP-2 protein was detected when anti-SlSCPx-2 antibody was used. SlSCPx protein was post-translationally cleaved into two smaller proteins, SCPx-t and SCPx-2. The gene appeared to be expressed into two forms of mRNA transcripts, which were translated into the two proteins, respectively. SlSCPx-t and SlSCPx-2 proteins have distinct and different locations in the midgut of sixth instar larvae. SlSCPx and SlSCPx-t proteins were detected predominately in the cytoplasm, whereas SlSCPx-2 protein was detected in the cytoplasm and nuclei in the Spli-221 cells. Over-expression of SlSCPx and SlSCPx-2 proteins enhanced cholesterol uptake into the Spli-221 cells. Knocking-down SlSCPx transcripts by dsRNA interference resulted in a decrease in cholesterol level in the hemolymph and delayed the larval to pupal transition. CONCLUSION: Spatial and temporal expression pattern of this SlSCPx gene during the larval developmental stages of S. litura showed its specific association with the midgut at the feeding stage. Over-expression of this gene increased cholesterol uptake and interference of its transcript decreased cholesterol uptake and delayed the larval to pupal metamorphosis. All of these results taken together suggest that this midgut-specific SlSCPx gene is important for cholesterol uptake and normal development in S. litura.

SUBMITTER: Guo XR

PROVIDER: S-EPMC2779813 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses.

Guo Xing-Rong XR Zheng Si-Chun SC Liu Lin L Feng Qi-Li QL

BMC molecular biology 20091113

<h4>Background</h4>Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated.<h4>Results</h4>An SCPx cDNA (SlSCPx) cloned from the common ...[more]

PMID: 19912624

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Project description:Plants produce secondary metabolites to provide chemical defense against herbivorous insects, whereas insects can induce the expression of detoxification metabolism-related unigenes in counter defense to plant xenobiotics. Tomatine is an important secondary metabolite in tomato (Lycopersicon esculentum L.) that can protect the plant from bacteria and insects. However, the mechanism underlying the adaptation of Spodoptera litura, a major tomato pest, to tomatine in tomato is largely unclear. In this study, we first found that the levels of tomatine in tomatoes subjected to S. litura treatment were significantly increased. Second, we confirmed the inhibitory effect of tomatine on S. litura by adding moderate amounts of commercial tomatine to an artificial diet. Then, we utilized RNA-Seq to compare the differentially expressed genes (DEGs) in the midgut and fat body tissues of S. litura exposed to an artificial diet supplemented with tomatine. In total, upon exposure to tomatine, 134 and 666 genes were upregulated in the S. litura midgut and fat body, respectively. These DEGs comprise a significant number of detoxification-related genes, including 7 P450 family genes, 8 glutathione S-transferases (GSTs) genes, 6 ABC transport enzyme genes, 9 UDP-glucosyltransferases genes and 3 carboxylesterases genes. Moreover, KEGG analysis demonstrated that the upregulated genes were enriched in xenobiotic metabolism by cytochrome P450s, ABC transporters and drug metabolism by other enzymes. Furthermore, as numerous GSTs were induced by tomatine in S. litura, we chose one gene, namely GSTS1, to confirm the detoxification function on tomatine. Expression profiling revealed that GSTS1 transcripts were mainly expressed in larvae, and the levels were the highest in the midgut. Finally, when larvae were injected with double-stranded RNA specific to GSTS1, the transcript levels in the midgut and fat body decreased, and the negative effect of the plant xenobiotic tomatine on larval growth was magnified. These results preliminarily clarified the molecular mechanism underlying the resistance of S. litura to tomatine, establishing a foundation for subsequent pest control.

Dataset Information

The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses.

Publications

The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses.

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