凡纳滨对虾ANT2 基因的克隆及低温表达谱分析

为研究凡纳滨对虾适应低温的分子机理, 实验对从低温处理凡纳滨对虾抑制性消减文库中筛选出的一个360 bp EST 序列进行了研究。首先, 同源比对显示该EST 片段与其他物种的ANT2 基因高度同源, 命名为凡纳滨对虾ANT2 基因(LVANT2); 其次, 通过构建凡纳滨对虾肝胰腺全长cDNA 文库, PCR 扩增获得LVANT2 基因的全长cDNA1540 bp, 其中包括1011 bp 的完整开放阅读框, 编码336 个氨基酸残基。然后, 对基因进行了不同组织和低温处理的表达谱分析: (1)组织表达谱的结果显示, 该基因在凡纳滨对虾肌肉组织中表达量最高; (2)在不同低温处理下的表达结果显示, 该基因在15℃处理下基因表达量发生显著变化, 13℃开始呈下调表达, 11℃时表达量又升高; 13℃低温处理不同时间发现该基因在12h 内表达量发生显著变化,48h 后表达量最高。LVANT2 基因的低温诱导表达模式说明其可能在凡纳滨对虾低温适应中发挥作用  

MOLECULAR CLONING OF LVANT2 GENE AND ITS EXPRESSION PATTERN BY COLD INDUCTION

To study the molecular mechanism of cold tolerance of Litopenaeus`vannamei,a 360 bp EST was isolated from suppression subtractive hybridization library of hepatopancreas which treated by low temperature.The 360 bp EST shared highly homology with ANT2 gene of other species,so we named it Litopenaeus vannamei ANT2 gene(LVANT2).Then we cloned whole cDNA sequence of PVANT2 from a hepatopancreas cDNA library.The LVANT2 gene was 1540 bp with a 1011 bp open reading frame encoding a peptide of 309aa.Tissue expression pattern by real-time PCR revealed that LVANT2 mRNA existed in heart,hepatopancreas,muscle,stomach,intestinal and eyestalk but mainly existed in muscle tissue.Cold induction by different temperatures revealed that LVANT2 mRNA expression started to increase after 15℃ treatment for 36 hours,and then decreased after 13℃ treatment for 36 hours,but increased again after 11℃ treatment.As for 13℃ treatment for different times,the expression pattern changed during the first 12 hours,but the expression with low temperature treatment for 24 hours and 36 hours was almost the same with the room temperature.The low temperature induction expression pattern demonstrated that LVANT2 may function in cold tolerance of Litopenaeus vannamei.