银鲳3个营养代谢相关基因的克隆、表达及摄食水母对其表达模式的影响

Cloning and Expression of Three Pampus argenteus Genes and the Effects of Jellyfish Feeding on Their Expression Patterns

本研究基于转录组学的分析结果,克隆分析了银鲳(Pampus argenteus)二肽酶(dehydropeptidase,dp1)、羧肽酶A(carboxypeptidases,cpa2l)和磺基转移酶(sulfotransferase,sult2)3个营养代谢相关基因,并探讨了其在消化吸收水母(Scyphozoa)过程中发挥的功能。通过RACE技术克隆获得了银鲳dp1、cpa2l和sult2基因的c DNA全长序列。dp1基因全长2522 bp,包含1个1272 bp的开放阅读框(ORF),含有1个由23个氨基酸组成的信号蛋白肽和1个典型的酰胺水解酶超家族的结构域。cpa2l基因全长1421 bp,ORF长1260 bp,含有1个由16个氨基酸组成的信号蛋白肽和1个典型的M14金属羧肽酶家族的结构域。sult2基因全长1834 bp,ORF长714 bp,含有1个典型的磺基转移酶家族的结构域。银鲳Dp1、Cpa2l和Sult2与蓝鳍金枪鱼(Thunnusmaccoyii)同源性最高,系统进化树结果也显示,银鲳dp1、cpa2l和sult2与蓝鳍金枪鱼对应基因聚在一个分支上,亲缘关系最近。运用实时荧光定量PCR(RT-q PCR)检测了dp1、cpa2l和sult2基因在不同组织中的表达水平及摄食水母对3个基因不同组织中表达规律的影响。结果显示,银鲳dp1基因在肝脏中表达量最高(P<0.05),与未摄食水母组相比,dp1基因在摄食水母组的脑和鳃中表达量均极显著增加(P<0.01),在中肠和肾脏中显著增加(P<0.05),而在肌肉中显著下降(P<0.05)。cpa2l基因在未摄食水母组的中肠中表达量最高,而在摄食水母组的肾脏中表达量最高(P<0.05),与未摄食水母组相比,摄食水母组cpa2l基因在肝脏中的表达量极显著增加(P<0.01),而在中肠和肌肉中却极显著下降(P<0.01)。sult2基因在肝脏中表达量最高(P<0.05),与未摄食水母组相比,sult2基因在摄食水母组的中肠、脑、鳃、肝脏和肾脏中表达量显著增加(P<0.05)。研究表明,dp1、cpa2l和sult2基因在银鲳摄食水母后机体营养物质的消化吸收和代谢过程中起重要的调节作用,基于摄食水母后3个基因组织表达模式的比较分析,推测dp1主要参与调控消化吸收和组织中营养素沉积的过程,cpa2l主要参与调控肝脏中营养代谢的过程,而sult2可能在整个消化吸收和代谢过程中均起到重要的调控作用。

In this study,three genes associated with the metabolism of Pampus argenteus,namely dehydropeptidase(dp),carboxypeptidase A(cpa),and sulfotransferase(sult),were cloned and evaluated using transcriptomics analysis,and their functions in the digestion and absorption of jellyfish were discussed.First,full-length cDNA sequences for dp1,cpa2l,and sult2 were cloned using RACE and confirmed using sequencing.The total length of the dp1 was 2522 bp,with this transcript including an open reading frame(ORF)of 1272 bp,a signal peptide composed of 23 amino acids,and an amide hydrolase superfamily domain.The cpa2l transcript was 1421 bp long and encoded a 1260 bp ORF.This protein consisted of a signal peptide of 16 amino acids and was characterized by a typical M14 metal carboxypeptidase family domain.sult2 was 1834 bp in length,producing an ORF of 714 bp,which included a typical sulfatase domain.In addition,homology evaluations of these three P.argenteus genes revealed that they were most similar to the bluefin tuna(Thunnus maccoyii),and phylogenetic tree analysis showed that dp1,cpa2l,and sult2 were all closely related to their corresponding genes from T.maccoyii.RT-qPCR was then used to evaluate the expression levels of these three genes in different tissues and the changes in expression patterns in response to jellyfish ingestion.These experiments revealed that dp1 expression was naturally the highest in the liver(P<0.05)and significantly increased in the brain,gills(P<0.01),midgut,and kidney(P<0.05),but significantly decreased in the muscle(P<0.05)when compared to that in the non-feeding control.cpa2l expression was highest in the middle intestine in the non-jellyfish feeding group and the kidney in the jellyfish feeding group(P<0.05)and was shown to be significantly increased in the liver(P<0.01)but significantly decreased in the midgut and muscle(P<0.01)when compared to that in the non-feeding group.sult2 expression was highest in the liver(P<0.05)and significantly increased in the midgut,brain,gill,liver,and kidney in the feeding group compared to that in the non-feeding group(P<0.05).Taken together,these results suggest that dp1,cpa2l,and sult2 play important roles in nutrient digestion,absorption,and metabolism in P.argenteus,especially in response to jellyfish feeding.In addition,the results of the tissue expression patterns for each of these genes following jellyfish ingestion suggest that dp1 is primarily involved in the regulation of digestion,absorption,and nutrient deposition;cpa2l is involved in the regulation of nutrient metabolism in the liver;and sult2 may play an important role in the overall process of digestion,absorption,and metabolism.