Identification, expression pattern, cellular location and potential role of the caveolin-1 gene from Artemia sinica

详细信息    查看全文

• 作者：Xuejie Li^a ; Feng Yao^a ; Wei zhang^a ; Cheng Cheng^a ; Bing Chu^a ; Yan Liu^a ; Yanli Mei^a ; Yang Wu^a ; Xiangyang Zou^b ; ^{zouxiangyang@126.com" class="auth_mail} ; Lin Hou^a ; ^{houlin@lnnu.edu.cn" class="auth_mail}
• 关键词：As-CAV-1 ; caveolin-1 protein from Artemia sinica ; As-cav-1 ; caveolin-1 gene from Artemia sinica ; ORF ; open reading frame ; CSD ; caveolin scaffolding domain ; PCR ; Polymerase Chain Reaction ; UTR ; untranslated region
• 刊名：Gene
• 出版年：1 May, 2014
• 年：2014
• 卷：540
• 期：2
• 页码：161-170
• 全文大小：2209 K

文摘

Caveolins are integral membrane proteins that serve as scaffolds to recruit numerous signaling molecules. Caveolins play an important role in membrane trafficking, signal transduction, substrate transport and endocytosis in differentiated cells. In this study, a caveolin-1 gene from Artemia sinica (As-cav-1) was successfully cloned for the first time. The full-length cDNA of As-cav-1 comprises 974 bp, with a 675 bp open reading frame (ORF) that encodes a polypeptide of 224 amino acids with a caveolin scaffolding domain (CSD) and two transmembrane domains. Multiple sequence alignment revealed that the putative As-CAV-1 protein sequence was relatively conserved across species, especially in the CSD domain. Real-time PCR revealed high levels of the As-cav-1 transcript at 0 h of embryo development. Furthermore, As-cav-1 transcripts were highly upregulated under high salinity (200鈥? and low temperature stresses (15 掳C). To further characterize As-cav-1, recombinant pET30a-cav-1 protein was expressed using a prokaryotic expression system. The recombinant protein comprised 290 amino acids with a theoretical molecular weight of 32 kDa, and a predicted isoelectric point of 5.6. Western blotting of the expression levels of As-CAV-1 during different embryo development stages revealed that As-CAV-1 levels decreased gradually during development stages from 0 h to 40 h, and increased at 3 d. Furthermore, western blotting showed that As-CAV-1 was upregulated to its highest expression level by low temperature stress (15 掳C) and high salinity. Confocal laser microscopy analysis, using antibodies generated against the recombinant As-CAV-1 protein, showed that As-CAV-1 was mostly located in the cell membrane. Our results suggested that As-cav-1 plays a vital role in protecting embryos from high salt damage and low temperature stress, especially during post-diapause embryonic development.