Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica

Nov 10, 2020Molecular biology reports

Gene and muscle protein levels in three types of mud crab

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Abstract

EF1α was identified as the most suitable reference gene for measuring tropomyosin expression in mud crabs.

Tropomyosin is a major allergen found in mud crab species.
Expression of candidate reference genes was evaluated in different tissues: abdomen, walking leg, and cheliped.
The expression stability of reference genes was analyzed using multiple methods, including comparative delta-Ct and RefFinder.
Tropomyosin expression levels were highest in Scylla tranquebarica and least in Scylla paramamosain.
All mud crab species showed the highest tropomyosin expression in the abdomen.

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Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is the most sensitive approach to produce accurate relative gene expression that has yet to be demonstrated for allergenic tropomyosin in mud crab species. This study was conducted to identify the suitable reference gene and tropomyosin expression in different body parts of local mud crabs, Scylla olivacea, Scylla paramamosain and Scylla tranquebarica. Myosin, 18S rRNA, GADPH and EF1α were selected as candidate reference genes and their expression was measured in the abdomen, walking leg and cheliped tissues of local Scylla spp. The expression stability was analyzed using the comparative delta-Ct method, BestKeeper, NormFinder and geNorm then comprehensively ranked by RefFinder algorithm. Findings showed that EF1α was the most suitable reference gene across three mud crab species. Meanwhile, the abdomen, walking leg and cheliped selected their own suitable reference gene either Myosin, 18S rRNA, EF1α or GADPH. Overall, tropomyosin was the highest in S. tranquebarica, whereas the least was in S. paramamosain. Interestingly, tropomyosin was the highest in the abdomen of all mud crab species. This is the first analysis on reference genes selection for qRT-PCR data normalization of tropomyosin expression in mud crab. These results will provide more accurate findings for further gene expression and allergen analysis in Scylla spp.

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Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica

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