脊尾白虾蜕皮抑制激素基因全长cDNA的克隆及其对环境胁迫的响应

CLONING AND EXPRESSION OF MOLT-INHIBITING HORMONE GENE FROM EXOPALAEMON CARINICAUDA UNDER ENVIRONMENTAL STRESSES

为研究脊尾白虾蜕皮抑制激素基因(MIH)在环境适应中的作用,本文采用RACE技术从蜕皮间期脊尾白虾眼柄中克隆获得MIH-like基因全长c DNA序列,命名为Ec MIH。该基因全长1218bp,包括360bp的开放阅读框,420bp的5'端非编码区和394bp的3'端非编码区;开放阅读框编码120个氨基酸,包括41个氨基酸组成的信号肽和79个氨基酸组成的成熟肽,预测蛋白分子量为13.71k Da,理论等电点p I为8.02。同源性比较分析发现,脊尾白虾MIH-like基因与日本沼虾和罗氏沼虾同源性最高,分别为87%和86%。荧光定量PCR分析结果表明,脊尾白虾MIH-like基因在眼柄中的表达量最高,在卵巢中的表达量最少,而在肝脏、肌肉、鳃和血细胞中不表达。环境胁迫后该基因具有明显的时序性。p H6.5胁迫后24h和48h,眼柄和腹神经节中MIH-like基因的表达量较对照组均显著增加(P<0.05);4mg/L氨氮浓度组胁迫后12h和24h,眼柄和腹神经节中MIH-like基因的表达量较对照组均显著增加(P<0.05);盐度39胁迫后12h和24h,眼柄和腹神经节中MIH-like基因的表达量较对照组均显著增加(P<0.05)。本研究结果表明脊尾白虾mih基因参与了环境胁迫后的机体应激反应。

We cloned the full-length MIH（molt-inhibiting hormone）-like c DNA from the eyestalk ganglia of ridgetail white prawn Exopalaemon carinicauda, and named as Ec MIH by rapid amplification of c DNA ends（RACE）. The full-length of MIH c DNA was 1218 bp containing a 420 bp 5＇ untranslated region（5＇UTR）, a 438 bp 3＇ untranslated region（3＇UTR） and a 360 bp open reading frame（ORF） encoding 120 amino acids consisting of a 41-residue signal peptide and a 79-residue mature peptide; the molecular mass was 13.71 k Da and the p I（isoelectric point） was 8.02. Sequence analysis indicated that the Ec MIH was in homology with those of Marsupenaeus japonicas and Macrobrachium rosenbergii, in 87% and 86% identity, respectively. Tissue expression analysis showed that the Ec MIH m RNA expressed at the highest level in eyestalk while extremely low in ovary, and not expressed in liver, muscle, gill, and blood cells. Under different environmental stresses, the MIH-like gene expression level in eyestalk and ventral ganglion significantly increased（P〈0.05） compared to the control（i.e., in p H 6.5 group for 24 h and 48 h, in 4mg/L NH4 Cl group for 12 h and 24 h, and in salinity 39 group for 12 h and 24h）. Therefore, the Ec MIH is involved in response against environmental stresses.