拟穴青蟹(Scylla paramamosain)蛋白二硫键异构酶基因的分子克隆与表达分析

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF PROTEIN DISULFIDE ISOMERASE GENE IN THE MUD CRAB(SCYLLA PARAMAMOSAIN)

蛋白二硫键异构酶(PDI)是内质网中的关键酶,参与蛋白合成过程中二硫键的形成、还原和异构。本文首次从拟穴青蟹(Scylla paramamosain)克隆获得了PDI基因cDNA全长,该序列长度为2015bp,开放阅读框为1452bp,编码483个氨基酸。荧光定量PCR检测发现PDI存在于拟穴青蟹的多个组织中;在拟穴青蟹卵巢发育过程中,PDI基因的表达量在前4个时期逐渐上升,到了第5期(成熟期)表达量则下降,表明PDI参与了卵巢发育的蛋白合成过程。免疫组化表明,拟穴青蟹卵母细胞存在PDI阳性反应,进一步为该分子参与卵巢发育提供了形态学证据。

Protein disulfide isomerase （PDI） is a multifunctional protein in all eukaryotic cells for assisting many protein maturation processes within the endoplasmic reticulum （ER）. We cloned and characterized PDI gene from mud crab Scylla paramamosain, and studied its localization and developmental expression. The full length of cDNA was 2016bp long with an ORF of 1452bp encoding a putative peptide of 483 amino acids （aa）. The deduced aa sequence of Sp-PDI showed high similarity to related sequences of other species. Molecular analysis was carried out by examining gene expression via real-time quantitative RT-PCR. In the crabs of same developmental stage and size, Sp-PDI transcripts occurred in all the various tissues, including ovary, hepatopancreas, hemocyte, muscle, heart, thoracic ganglia, cerebral ganglia, stomach, gill, eyestalk, and epidermis. In the ovaries of different stages, Sp-PDI expression increased gradually until it become matured and then decreased at last. By means of immunochemistry, PDI reactivity was found in the oocyte of S. paramamosain. These results suggest that Sp-PDI is implicated during the growth and ovarian development.