摘要
为探究瞬时受体电位锚蛋白 1b(transient receptor potential ankyrin 1b,Trpa1b)在高温条件下对斑马鱼(Danio rerio)的影响,利用CRISPR/Cas9 基因敲除技术构建trpa1b基因缺失的斑马鱼模型,采用组织学和转录组学等方法,研究了高温胁迫下trpa1b基因敲除(trpa1b-/-)斑马鱼和野生型(WT)斑马鱼的存活、组织凋亡及其他相关基因表达的变化.结果表明:通过加热装置使水温以 1℃/h的速度加热,发现相比于野生型斑马鱼,trpa1b-/-纯合子斑马鱼高温下整体存活时间更短;通过对 40℃下不同基因型斑马鱼组织进行取样,TUNEL染色对比其细胞凋亡程度,trpa1b-/-纯合子鳃和脑组织的凋亡程度显著高于野生型斑马鱼(P<0.05);对常温(28℃)和高温(38℃和 40℃)下不同基因型的鳃组织进行转录组测序分析,高温下trpa1b-/-纯合子中hspa1b和hsp90aa1.1 表达量显著性下调(P<0.001),同时细胞内钙离子、泛素-蛋白质转移酶、溶酶体及核糖体相关基因表达发生改变,使其在热刺激下生存能力受损.研究表明,tr-pa1b基因是维持斑马鱼高温耐受所需的重要基因,trpa1b缺失会导致斑马鱼高温不耐受更加明显,本研究结果为后续探究鱼类耐高温机制研究提供了有益参考.
Abstract
To explore transient receptor potential ankyrin 1b(trpa1b)gene on zebrafish(Danio rerio)under high temperature conditions,zebrafish model with trpa1b gene deletion was constructed by CRISPR/Cas9 gene knockout technology.Survival,tissue apoptosis and other related gene expression changes were investigated in Trpa1b knock-out(trpa1b-/-)zebrafish and wild-type(WT)zebrafish exposed to water temperature from 28 ℃ to 40 ℃ at a warming rate of 1 ℃/h per hour by histological and transcriptome methods.Zebrafish tissues of different genotypes were sampled at 40℃.It was found that the overall survival time of trpa1b-/-homozygotes was shorter under high temperature.TUNEL staining showed that there was significantly higher apoptosis in gill and brain of trpa1b-/-ho-mozygotes than that in wild-type zebrafish(P<0.05).Transcriptome sequencing analysis for gill tissues of different genotypes under normal temperature(28℃)and high temperature(38℃and 40℃)revealed that the expression level of hspa1b and hsp90aa1.1in trpa1b-/-homozygotes was significantly down regulated under high temperature(P<0.001),and that the expression level of intracellular calcium ions,ubiquitin protein transferase,lysosome and ribosome related genes were changed,which impaired their viability under heat stimulation.The findings indicate that Trpa1b deletion will lead to more obvious high-temperature intolerance in zebrafish due to the fact that trpa1b gene is an important gene required to maintain high temperature tolerance in zebrafish,which provides reference for further research on the mechanism of high temperature tolerance in fish.
维普
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