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

Cloning and function exploration comparison of Grik1 in Antarctic crocodile icefish(Chionodmco hamatus)and Nile tilapia(Oreochromis niloticus)exposed to cold stress

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1346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1053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。

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 1346(differentialy expressed genes)in pcDNA3.1(+)group,722 in ChGrik1 group and 1053 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.