南极独角雪冰鱼与尼罗罗非鱼Grik1基因的克隆及其在低温胁迫下的作用比较

doi:10.16535/j.cnki.dlhyxb.2022-144

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大连海洋大学学报 ›› 2023, Vol. 38 ›› Issue (1) : 68-75. DOI: 10.16535/j.cnki.dlhyxb.2022-144

南极独角雪冰鱼与尼罗罗非鱼Grik1基因的克隆及其在低温胁迫下的作用比较

张晓雯,张艳杰,李玮,钟文倩,陈良标*

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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*

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摘要

为了探究南极独角雪冰鱼(Chionodmco hamatus)和尼罗罗非鱼(Oreochromis niloticus)离子型谷氨酸受体海人藻酸亚型1基因(glutamate ionotropic receptor kainate type subunit 1, Grik1)在低温胁迫下对细胞凋亡的影响，采用RT-PCR法克隆得到体质量约为500 g的南极独角雪冰鱼和尼罗罗非鱼Grik1基因，分别命名为ChGrik1和OnGrik1，并将其插入到真核表达载体pcDNA3.1(+)上构建pcDNA3.1-ChGrik1和pcDNA3.1-OnGrik1过表达载体，对293T细胞系进行转染，24 h后在不同温度(37、28、18、8 ℃)下对细胞处理48 h，然后用流式细胞仪检测不同温度下转染不同质粒的细胞凋亡情况。结果表明：低温胁迫诱导293T细胞凋亡，8 ℃时最显著，凋亡率达到92.43%±0.46%(P<0.05)；过表达ChGrik1或OnGrik1基因相比空载组均能显著降低细胞凋亡率2%～5%(P<0.05)；进一步对 8 ℃低温胁迫48 h的细胞进行转录组测序分析显示，空载组有1 346个差异表达基因，过表达ChGrik1和OnGrik1基因组均会减少受低温胁迫的差异表达基因数目，分别为722个和1 053个，同时还改变了与细胞凋亡相关基因的表达趋势，如NUPR1、STC2和CHAC1等基因由下调变为上调，HSP等基因由上调变为下调。研究表明，南极独角雪冰鱼和尼罗罗非鱼的生存温度差异较大，但两者的谷氨酸受体基因Grik1功能类似，均能通过减少细胞凋亡来应对低温胁迫。

Abstract

To investigate the effect of Grik1 (glutamate ionotropic receptor kainate type subunit 1) on cell apoptosis in Antarctic crocodile icefish (Chionodraco hamatus) and Nile tilapia (Oreochromis niloticus) 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.

导出引用

张晓雯, 张艳杰, 李玮, 钟文倩, 陈良标. 南极独角雪冰鱼与尼罗罗非鱼Grik1基因的克隆及其在低温胁迫下的作用比较[J]. 大连海洋大学学报, 2023, 38(1): 68-75 https://doi.org/10.16535/j.cnki.dlhyxb.2022-144

ZHANG Xiaowen, ZHANG Yanjie, LI Wei, ZHONG Wenqian, CHEN Liangbiao. Cloning and function exploration comparison of Grik1 in Antarctic crocodile icefish (Chionodmco hamatus) and Nile tilapia(Oreochromis niloticus) exposed to cold stress[J]. Journal of Dalian Fisheries University, 2023, 38(1): 68-75 https://doi.org/10.16535/j.cnki.dlhyxb.2022-144

中图分类号： S 961.6