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.

Tropomyosin是一种肌肉组织蛋白，是包括贝蟹在内的大多数贝类的主要过敏原。使用稳定的参考基因进行定量实时PCR（qRT-PCR）是产生准确的相对基因表达的最灵敏方法，对于蟹蟹变应原原肌球蛋白尚待证实。进行了这项研究，以鉴定合适的参考基因和原肌球蛋白在当地泥蟹，Sylla olivacea，Scyla paramamosain和Scylla tranquebarica的不同身体部位的表达。选择肌球蛋白，18S rRNA，GADPH和EF1α作为候选参考基因，并在局部的腹部，步行小腿和唇状组织中检测其表达Scylla spp。使用比较delta-Ct方法，BestKeeper，NormFinder和geNorm分析表达稳定性，然后通过RefFinder算法进行综合排名。研究结果表明，EF1α是三种泥蟹物种中最合适的参考基因。同时，腹部，步行小腿和唇颊动物选择了自己合适的参考基因，即肌球蛋白，18S rRNA，EF1α或GADPH。总体而言，原肌球蛋白在S. tranquebarica中最高，而在S. paramamosain中最低。有趣的是，原肌球蛋白在所有泥蟹物种的腹部中最高。这是针对泥蟹中原肌球蛋白表达的qRT-PCR数据归一化参考基因选择的首次分析。这些结果将为Scylla spp中的进一步基因表达和过敏原分析提供更准确的发现。