日本沼虾MT基因克隆、组织差异性表达及与饲料铜、锌含量的相关性

摘要

为了更全面理解日本沼虾(Macrobrachium nipponense)的铜/锌营养生理作用,研究利用RACE技术从日本沼虾肝胰腺中克隆获得一金属硫蛋白基因cDNA全长(mn-MT),并对该基因分子特征、组织表达谱和饲料铜/锌水平对其表达的影响进行分析。结果显示：(1)mn-MT cDNA全长665 bp,含编码59个氨基酸的180 bp的开放阅读框,预测该多肽的理论分子量为6.085 kD,等电点为7.73。该蛋白中半胱氨酸含量最高(30.5%),其次是赖氨酸(16.95%)和丝氨酸(10.17%)。相似性分析显示 mn-MT 氨基酸序列与美洲海螯虾、斑节对虾和中华绒螯蟹MT的相似性分别达到78%、75%和75%。(2)qRT-PCR分析显示,mn-MT mRNA在肝胰腺、血细胞、鳃、胃、卵巢、肠和肌肉中都有表达,其中肝胰腺中表达量最高。(3)用4组铜添加量分别为0、20、40及160 mg/kg的饲料和3组锌添加水平分别为0、35和210 mg/kg的饲料饲喂初重为(0.101±0.002) g日本沼虾56d后,分析各组虾肝胰腺的mn-MT mRNA表达。mn-MT mRNA表达随饲料铜水平的提高而升高,到40 mg/kg组达到最高(P<0.05),而后开始下降；饲料中高锌(210 mg/kg)显著提高mn-MT表达(P<0.05),0和35 mg/kg组间差异不显著(P>0.05)。结果表明饲料中铜/锌均可影响mn-MT表达,且呈现不同的剂量依赖效应。%Metallothionein (MT) is a metal binding protein with low molecular weight. The expression of MT can be induced by minerals such as copper or zinc, and it is involved in the metabolism of trace elements. To better understand the physiological and nutritional effects of copper and zinc onMacrobrachium nipponense, we clonedmn-MT from the oriental river prawn using rapid amplification of the cDNA ends (RACE), and evaluated the effects of dietary copper and zinc on the expression ofMT. The full length ofmn-MT cDNA was 665 bp, and it had a 180 bp open reading frame (ORF) encoding a 59aa peptide. The calculated molecular weight of the peptide was 6.085 kD and the predicted isoelectric point was 7.73. The most abundant amino acid in this protein was cysteine residues (30.5%), followed by lysine (16.95%) and serine (10.17%). The similarity analysis showed thatmn-MT shared the similarities of 78%, 75% and 75% with the counterparts in theHomarus americanus,Penaeus monodon andEriocheir sinensisrespectively. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that themn-MT mRNA was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary, muscles and stomach, with the highest level in the hepatopancreas.M. nipponense were fed for 56 days with seven different diets supplemented with Cu at 0, 20, 40 and 160 mg/kgdiet and Zn at 0, 35, 210 mg/kgdiet, and then we analyzed the expression ofmn-MT mRNA in the hepatopancreas. The expression ofmn-MT mRNA was first elevated along with the increase in dietary Cu level, reached to the maximum at 40 mg/kg diet, and then dropped as the Cu concentration was further increased. The expression ofmn-MT mRNA was higher in prawns fed with high Zn (210 mg/kg diet) than that in prawns fed with 35 mg Zn/kg dietor 0 mg Zn/kg diet(P<0.05). There was no sig-nificant difference between the 0 mg Zn/kg groupand the 35 mg Zn/kg group (P>0.05). These results suggested that the expression ofmn-MT mRNA could be affected by dietary copper and zinc with different dose-dependent response curves.