Cloning and function exploration comparison of Grik1 in Antarctic crocodile icefish (Chionodmco hamatus) and Nile tilapia(Oreochromis niloticus) exposed to cold stress
ZHANG Xiaowen, ZHANG Yanjie, LI Wei, ZHONG Wenqian, CHEN Liangbiao*
1.International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China; 2.Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China; 3.National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
Abstract: To investigate the effect of Grik1 (glutamate ionotropic receptor kainate type subunit 1) on cell apoptosis in Antarctic crocodile icefish (Chionodracohamatus) and Nile tilapia (Oreochromisniloticus) with body weight of some 500 g under cold stress, the coding sequence (CDS) of their Grik1 was cloned by RT-PCR, and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct the overexpression plasmids pcDNA3.1-ChGrik1 and pcDNA3.1-OnGrik1. The 293T cell line was transfected using the overexpression plasmids for 24 h and then exposed to different treatments at temperature of 37, 28, 18 and 8 ℃ for 48 h, and the apoptosis of cells was detected using flow cytometry. It was found that the apoptosis of 293T cells was induced by cold stress, with most significant (92.43%±0.46%) under 8 ℃ (P<0.05), and significant decrease in the apoptosis rate by overexpression of ChGrik1 or OnGrik1 by 2% to 5%, compared with the pcDNA3.1(+) group under cold stress (P<0.05). Further transcriptome sequencing analysis of cells under 8 ℃ for 48 h showed that overexpression of ChGrik1 or OnGrik1 led to reduce the number of DGEs under cold stress, with 1 346 (differentialy expressed genes) in pcDNA3.1(+)group, 722 in ChGrik1 group and 1 053 DGEs in OnGrik1 group. The expression patterns of genes related to apoptosis and stress response were shown to be changed under cold stress, with up-regulated from down-regulated in NUPR1, STC2 and CHAC1 and up-regulated from down-regulated in HSP. The findings indicated that in Antarctic crocodile icefish and Nile tilapia inhibited in different temperature Grik1 played a similar role in responding to cold stress by reducing cell apoptosis, which provides foundation for further research on the response mechanism of fish to cold stress.
2023, Vol. 38 
