LR型微囊藻毒素对鲢鱼PEPCK基因表达的影响

MC-LR effects on the expression of PEPCK genes in the silver carp

在前期经LR型微囊藻毒素(MC-LR)处理后建立鲢鱼肝脏组织抑制差减杂交文库的基础上,利用RACE方法克隆了经MC-LR处理后差异表达明显的鲢鱼磷酸烯醇式丙酮酸羧激酶PEPCK基因cDNA全长,并对其进行了序列分析,然后通过半定量分析了经MC-LR投与1、5、10 h鲢鱼肝脏组织PEPCK表达水平。结果显示:鲢鱼PEPCK基因cDNA全长2 605 bp,包括101 bp的5'端非翻译区、564 bp的3'端非翻译区、1 911 bp的开放阅读框,编码636个氨基酸。将鲢鱼PEPCK基因cDNA核酸及氨基酸序列与GenBank中已发表的其他几种鱼类的相应序列进行比对,表明鲢鱼PEPCK与翘嘴红鲌的同源性最高,其核酸及氨基酸序列的相似性分别达到97%和99%;其次为斑马鱼,同源性均达到90%以上;与星斑川鲽的同源性最差,但核酸及氨基酸序列的相似性也分别达到77%和84%,说明鱼类PEPCK在长期的进化过程中具有很高的保守性。鲢鱼PEPCK具有与草酰乙酯结合的特有结构域以及与GTP三磷酸链结合的激酶1和激酶2基序。另外,鲢鱼投予MC-LR后,肝脏组织PEPCK经过1、5、10 h的表达量均有显著升高,说明鲢鱼投予MC-LR 1 h内该基因即被诱导表达,且在10 h内皆维持较高的表达水平。这与微囊藻毒素作用有关,推测与微囊藻毒素解毒过程相关。

In the present study, RACE was performed to clone the silver carp PEPCK cDNA and the sequence was analyzed. Under the previous work of construction of the silver carp, liver cDNA suppression subtractive hybridization libraries exposed to MC-LR. Then, semi-quantitative RT-RCR was used to analyze the transcriptional expression of the silver carp liver PEPCK after 1, 5, 10 h exposed to MC-LR. The results showed that silver carp possessed 2 605 base pair （bp） nucleotide that comprisesd of a 5＇-UTR （un-translated region） of 101 bp, a 3＇-UTR of 564 bp and a 1 911 bp open reading frame （ORF） which encoded 636 amino acids. Sequence analysis showed that the nucleotides and amino acids sequence identity of silver carp PEPCK were highest （97% and 99% respectively） with Erythroculter ilishaeformis, secondly with Danio rerio （more than 90%）, and lowest with Platichthys stellatus （77% and 84% respectively）, implying that PEPCK was highly conserved during the long course of evolution. Silver carp PEPCK has combined with oxalyl ethyl specific domain, as well as three chain phosphate of GTP-binding kinase 1 and kinase 2 motifs. On the other hand, significant increases in PEPCK transcriptional expression were observed in the livers of silver carp at 1, 5, 10 h after exposed to MC- LR, this indicated PEPCK transcription was induced by exposing MC-LR at 1 h. These results were related to microcystin effects, and might be related with microcystin detoxification process.