草鱼TP53INP1基因的克隆及微囊藻毒素对其表达的影响

MOLECULAR CLONING OF TP53INP1 IN GRASS CARP AND ITS EXPRESSION IN RESPONSE TO MICROCYSTINS

为深入研究肿瘤蛋白p53诱导核蛋白1(Tumor protein 53-induced nuclear protein 1,TP53INP1)的结构及其在微囊藻毒素-LR(MC-LR)胁迫下的表达变化,以MC-LR诱导的草鱼(Ctenopharygodon idella)肝脏转录组测序获得的unigenes序列为基础,扩增获得了TP53INP1基因的cDNA序列(GenBank登录号:MG797689),其中开放阅读框(Open reading frame,ORF)为759 bp,编码252个氨基酸,属于β类型,具有4个PEST结构。氨基酸同源性分析结果表明,TP53INP1具有较高的保守性,其中与鱇浪白鱼(Anabarilius grahami)相似性最高。系统进化分析结果表明其与鱇浪白鱼(Anabarilius grahami)、斑马鱼(Danio rerio)等鱼类聚为一大支。采用荧光定量PCR分析发现TP53INP1基因在草鱼各组织中广泛分布,其中,在肝脏和血液等组织中表达丰富,显著高于在头肾组织中的表达(P<0.05)。采用Western blot检测分析不同剂量(25、75和100μg MC-LR/kg BW)MC-LR染毒96h后对草鱼肝脏TP53INP1蛋白的影响,结果发现草鱼在100μg MC-LR/kg BW剂量组中染毒96h后表达显著下降(P<0.05),暗示TP53INP1可能在响应MC-LR胁迫中起着重要作用。研究为进一步了解TP53INP1的功能及为深入探讨TP53INP1在鱼类响应MC-LR胁迫后的作用机制提供了基础资料。

In order to investigate its structure and expression changes induced by microcystin-LR(MC-LR),the cDNA sequence of TP53INP1 was amplified by using rapid-amplification of cDNA ends(RACE)(GenBank accession number:MG797689).TP53INP1 was 1154 bp in length,containing a 216 bp 3’untranslated region(UTR)and a 759 bp open reading frame that encodes a protein of 252 amino acids.TP53INP1 of grass carp belongs to the typeβsubgroup and possesses four PEST motifs.The maximum identity between the derived TP53INP1 of grass carp and Anabarilius graham TP53INP1 was 98%,and the maximum identity with Homo sapiens TP53INP1αand TP53INP1βwere 58%and 55%,respectively.Phylogenetic analyses showed that TP53INP1 fell into well-supported clades with Anabarilius graham,Danio rerio and other teleost.The results of qRT-PCR revealed that TP53INP1 gene was ubiquitously expressed in all examined tissues of healthy grass carp,and it was highly expressed in liver,blood and other tissues,and its relative expression was significantly higher than that in the head kidney(P<0.05).The livers of grass carp injected with the dose of 25μg MC-LR/kg BW,75μg MC-LR/kg BW and 100μg MC-LR/kg BW were analyzed by Western blot.TP53INP1 protein was significantly down-regulated in the 100μg MC-LR/kg BW group at 96h(P<0.05)compared with control group with a fold change of 0.07.These results provide a basis for further research on the function of TP53INP1 and its mechanism in response to MC-LR.