The cDNA cloning and functional verification of the PhCUL1 gene from Pyropia haitanensis

Abstract:

Heat stress affects Pyropia haitanensis yield and quality, and is the main factor restricting the development of the Pyropia industry. Previous studies found that the genes related to the ubiquitin proteasome system (UPS) of P. haitanensis were significantly up-regulated under heat stress; however, its molecular function in response to high temperatures is unknown. Therefore, in this study, the function of the cullin E3 ligase gene (PhCUL1) was studied using molecular biology and genetics. The results showed that the full-length cDNA of PhCUL1 was 2,500 bp, including an open reading frame (ORF) of 2,481 bp. This gene has a Cullin domain (407~618 aa) and a Cullin Nedd8 domain (754~821 aa). The Cullin Nedd8 domain is a protein fusion site that confers PhCUL1 with E3 ligase activity. Phylogenetic analysis showed that PhCUL1 was clustered with P. umbilicalis. qRT-PCR results showed that the PhCUL1 gene was significantly induced by high temperature. In addition, a transgenic expression system of Chlamydomonas reinhardtii was constructed to verify the function of the PhCUL1 gene. Transgenic C. reinhardtii plants overexpressing PhCUL1 exhibited obvious tolerance to heat stress. The relative PhCUL1 expression in C. reinhardtii exhibited upregulation within 6 hours. These results indicated that PhCUL1 plays a crucial role in response to heat stress in P. haitanensis and its specific regulatory mechanism needs further study. This study provided a better understanding of the mechanisms of UPS in P. haitanensis in response to high temperature and a theoretical basis for the selection of new varieties with high temperature resistance.