养殖水域Cu^(2+)胁迫对虹鳟MT2基因表达模式的影响

Effects of Cu2+Stress on the Expression Pattern of MT2 Gene in Rainbow Trout in Aquaculture Waters

为了探究水体中铜离子胁迫对虹鳟(Oncorhynchus mykiss)MT2基因表达模式的影响,开发Metallothioneins作为检测水域生态中重金属铜污染的分子标志,该研究利用TA克隆法获得了虹鳟MT2基因的CDS序列,检测了MT2基因在虹鳟不同组织中的表达模式,以及Cu^(2+)胁迫(Cu^(2+)浓度75、150和300μg/L)后1、2、3和4 d和胁迫恢复1、2 d时间点的MT2基因表达动态。结果表明,虹鳟MT2基因CDS区长186 bp,编码62个氨基酸,具有典型的半胱氨酸(Cys)-X(1~3个)-半胱氨酸结构,属不稳定亲水性蛋白。虹鳟MT2基因序列与大西洋鲑的同源性最高且在肝脏中表达量最高,其次是脑和肠道。胁迫试验结果显示,MT2基因在肝脏和鳃中呈现先升高后降低的趋势,在头肾、脾脏和脑中呈现逐渐升高的趋势,且呈现一定的剂量依赖性,肠道MT2基因在胁迫2、3和4 d被显著抑制。胁迫恢复后,处理组肝脏、头肾、鳃、脑MT2基因表达量仍显著升高。综上,虹鳟MT2基因能在不同组织中被Cu^(2+)诱导表达,推测MT2基因在虹鳟抗重金属毒性中可能发挥着重要作用,且MT2基因表达量与铜离子的浓度相关,可以作为分子生物标志检测水域生态中的重金属铜污染。

The aim of this study is to investigate the effect of copper ion stress in water on the expression pattern of MT2 gene in rainbow trout(Oncorhynchus mykiss),and to develop Metallothioneins as molecular markers for detecting heavy metal copper pollution in aquatic ecosystems.The CDS sequence of rainbow trout MT2 gene was obtained using TA cloning method,and the expression patterns of MT2 gene in different tissues of rainbow trout were detected.The MT2 gene expression dynamics at 1 d,2 d,3 d,4 d after Cu^(2+)stress(75μg/L,150μg/L,300μg/L)and 1 d and 2 d after stress recovery were detect⁃ed as well.The results showed that the CDS region of rainbow trout MT2 gene was 186 bp,encoding 62 amino acids,with a typical Cys-X(1~3)-Cys structure,which is an unstable hydrophilic protein.The MT2 gene sequence of rainbow trout showed the highest homology with that of Atlantic salmon and the highest expression was found in the liver,followed by the brain.The results of the stress test showed that MT2 gene showed a tendency to increase and then decrease in liver and gill,and a tendency to gradually increase in head kidney,spleen and brain with a certain dose-dependence,and intestinal MT2 gene was significantly suppressed at 2 d,3 d and 4 d of stress.After recovery from stress,the expression of MT2 gene in liver,head kidney,gill and brain of the treated group was still significantly elevated.In summary,rainbow trout MT2 gene can be in⁃duced to be expressed by Cu^(2+)in different tissues,and it is hypothesized that MT2 gene may play an important role in rainbow trout’s resistance to heavy metal toxicity.Moreover,the expression of MT2 gene is correlated with the concentration of copper ions,and it can be used as a molecular biomarker for detecting heavy metal copper contamination in the ecology of waters.