凡纳滨对虾水孔蛋白-4的cDNA克隆及盐度胁迫对其肝胰腺mRNA表达水平的影响

Molecular cloning of aquaporin-4(AQP4) gene in the Pacific white shrimp(Litopenaeus vannamei) and the effect of salinity stress on its expression in hepatopancreas

为了探索水孔蛋白(Aquaporin,AQP)在凡纳滨对虾(Litopenaeus vannamei)渗透压调节过程中作用,本研究通过RACE方法获得克隆的凡纳滨对虾AQP(命名为LvAQP4)的c DNA全长序列,并分析了盐度胁迫对其肝胰腺m RNA表达的影响。结果发现:LvAQP4 c DNA序列全长为1 048 bp,其中包括75 bp的5￠UTR,187 bp的3￠UTR和786 bp的ORF。根据ORF序列推导出LvAQP4编码261个氨基酸,预测其分子质量为27.85 k Da,等电点为8.11。推导的氨基酸序列与其他甲壳物种的AQP相似度为48.8%到97.3%。进化分析显示LvAQP4属于AQP1-like亚族、AQP4类。定量PCR(q PCR)检测到LvAQP4在不同组织中均有表达,其中鳃的表达量最高,肝胰腺、肌肉、脑和眼柄中也较高,而血淋巴、肠、胃和胸神经节中表达量则较低。在高盐(盐度40)刺激下,凡纳滨对虾的肝胰腺LvAQP4 m RNA表达量会随着时间推移而上升,到6 h达到最高,而后逐步下降。而在低盐(盐度4)刺激下,凡纳滨对虾的肝胰腺LvAQP4 m RNA表达量并无明显变化。在原代分离和培养的肝胰腺细胞中,培养液中加入额外的Na Cl会剂量依赖地提高LvAQP4 m RNA表达水平。同样,通过在培养液中额外加入蔗糖提高培养液渗透压,也会剂量依赖地提高LvAQP4 m RNA表达水平,但作用不如Na Cl明显。这些结果表明盐度与肝胰腺LvAQP4的表达量有关,LvAQP4对凡纳滨对虾的渗透压调节具有非常重要的作用。

In this study,to explore the function of aquaporin(AQP)in the osmoregulation of Litopenaeus vannamei,the full-length cDNA of L.vannamei aquaporin-4(termed as LvAQP4)was cloned by RACE method,and the effect of salinity stress on the transcriptional expression of LvAQP4in the hepatopancreas was examined.The results showed that the full length cDNA of LvAQP4was1048bp in length,containing a75bp5?UTR,a187bp3?UTR,and a786bp ORF that encode for a deduced protein of261amino acids with the estimated molecular mass of27.85kDa and an isoelectric point of8.11.The LvAQP4protein shared identities from48.8%to97.3%with its counterparts in other decapod species.Additionally,the phylogenetic analysis suggested that LvAQP4belonged to AQP1-like subfamily and clustered with other AQP4s.Quantitative real-time PCR(qPCR)showed that the expression of LvAQP4was ubiquitous,with the highest expression in the gills.The expression levels of LvAQP4were also high in the hepatopancreas,muscle,brain,and eyestalk but low in hemocytes,intestine,stomach,and thoracic ganglion.Under hypersalinity stress(40),the expression levels of LvAQP4mRNA in the hepatopancreas increased consistently from the beginning to6h,reaching the highest level at6h,and then decreased after that.Under hypersalinity stress(4),on the contrary,the mRNA levels of LvAQP4showed no significant change.In the hepatopancreatic primary cells,the expression levels of LvAQP4mRNA exhibited a dose-dependent upregulation when increased dosage of NaCl was added.Similar results were observed with addition of sucrose in the culture medium to increase the osmotic pressure,but the effect of sucrose on LvAQP4transcripts was weaker than that with NaCl.These results suggested that the expression of LvAQP4in the hepatopancreas was affected by salinity and LvAQP4may play important roles in the osmoregulation of L.vannamei.