凡纳滨对虾碱性磷酸酶和酸性磷酸酶基因的克隆、表达及盐度应答效应

cDNA cloning and gene expressionin response to salinity of alkaline phosphatase and acid phosphatase from Litopenaeus vannamei

本研究通过c DNA末端快速扩增法(RACE)首次克隆得到凡纳滨对虾(Litopenaeus vannamei)碱性磷酸酶(alkaline phosphatase,ALP)和酸性磷酸酶(acid phosphatase,ACP)基因的c DNA全长序列,其中ALP基因全长序列1 910 bp,编码536个氨基酸;ACP基因全长序列1 544 bp,编码439个氨基酸。盐度调控设3个盐度组:31(对照)、16和3。养殖45 d后,荧光定量PCR测定对虾肝胰腺、肌肉、胃、肠和鳃5个组织中的ALP和ACP基因m RNA的表达情况。RT-PCR显示,ALP基因在5个组织中普遍表达,在肝胰腺中表达量最高(P<0.05),肠道中的表达量最低(P<0.05);ACP基因在5个组织中也均表达,在肝胰腺和鳃中表达量明显高于其他3个组织(P<0.05)。盐度应答实验表明,不同盐度条件下,不同组织中,ALP和ACP基因存在不同的表达模式。在胃和鳃组织中,ALP基因在31盐度组的表达量显著高于其他两组(P<0.05);在肝胰腺和肠道中,ALP基因在16盐度下的表达量明显低于其他两组(P<0.05);在肌肉中,ALP基因的表达与其他组织均不同,31盐度组表达量最高,而16盐度组最低(P<0.05);在肝胰腺中,ACP基因在31盐度组的表达量明显低于其他盐度组(P<0.05);在肌肉和鳃两个组织中,ACP基因在31盐度下的表达量明显高于其余两个盐度组(P<0.05);在肠道中,ACP基因的表达量在3盐度组明显高于其他两组(P<0.05);而在胃中,ACP基因在3个盐度下的表达量没有显著性差异。上述结果为研究低盐条件下凡纳滨对虾的磷酸酶基因的变化机制提供了参考。

In this study,alkaline phosphatase(ALP)and acid phosphatase(ACP)genes were cloned from Litopenaeus vannamei by the rapid amplification of cDNA endsmethod.The full-length cDNA sequence of ALP was1910bp,encoding536amino acids,whereas the full-length cDNA sequence of ACP was1544bp,encoding439amino acids.A salinity regulation experiment was performed with three treatments:31‰(control),16‰,and3‰salt water.ALP and ACP gene expression was determined in tissues of the hepatopancreas,muscle,stomach,intestine and gills with real-time PCR after45days in saline.RT-PCR reveals that the ALP gene was detected in five tissues with the highest expression in the hepatopancreas(P<0.05)and the lowestin theintestine(P<0.05).The ACP gene was also detected in five tissues,and the gene expression in the hepatopancreas and gills was higher than that in the other tissues(P<0.05).After the salinity regulation experiment,the ALP and ACP genes presented different expression patterns among the various tissues.In the stomach and gill tissues,the higher expression level of the ALP gene occurred in the31‰saline group compared with the other two salinity groups(P<0.05);in the hepatopancreas and intestinal tissues,the expression level of the ALP gene in the16‰saline group was significantly lower than that in the other two salinity groups(P<0.05);in muscle tissue,the expression level of the ALP gene was the highest in the31‰saline group(P<0.05)and the lowest in the16‰salinegroup(P<0.05).Whereas in the hepatopancreas,the expression level of the ACP gene in the31‰salinegroup was significantly lower than that in the other two salinity groups(P<0.05);in muscle and gill tissues,the expression level of the ACP gene in the31‰salinegroup was significantly higher than that in the other two salinity groups(P<0.05);in the intestines,the expression of the ACP gene was the highest in the3‰salinegroupand was significantly higher than that in the other two groups(P<0.05);in the stomach,the expression level of the ACP gene was not significantly different among the three groups.These results provide useful data for the molecular mechanism of Litopenaeus vannamei phosphatase metabolism under conditions of low salinity.