双齿围沙蚕金属结合蛋白(MPⅡ)基因克隆与表达

Cloning and expression of the metalloprotein(MPⅡ) gene from poly-chaetes Perinereis aibuhitensis(Annelida: Polychaeta)

根据已知杂色沙蚕(Nereis diversicolor)金属结合蛋白Ⅱ(MPⅡ)部分基因序列设计引物,应用RACE技术从双齿围沙蚕(Perinereis aibuhitensis)中克隆得到MPⅡcDNA序列全长为904 bp,其中,开放阅读框为357 bp,编码119个氨基酸。Blast比对结果显示,由双齿围沙蚕MPⅡcDNA序列推导的氨基酸序列与多毛类Periserrula leucophryna蚯蚓血红蛋白(Hr)、杂色沙蚕(Nereis diversicolor)蚯蚓肌血红蛋白(MHr)及星虫类Themiste zostericola Hr氨基酸序列的同源性分别为81.51%、77.12%和61.02%,由此推断该cDNA序列可能属于血红蛋白家族。应用Real-time PCR方法,分别研究了沙蚕暴露于Cd2+质量浓度为40 mg/L的环境中12、24、48和72 h后MPⅡ基因表达水平的变化,以及3种质量浓度5、10和20 mg/L Cd2+暴露15 d后MPⅡ基因的表达水平。实验结果表明:(1)沙蚕暴露于40 mg/LCd2+72 h后,MPⅡmRNA表达量显著增加,为空白对照组的12.6倍,表现出极显著差异(P<0.01);(2)不同质量浓度Cd2+诱导15 d后,各组MPⅡmRNA表达量均呈显著升高(P<0.05),达到空白对照组的4.8倍以上,且随Cd2+浓度增大,MPⅡmRNA表达量增加。据此认为,MPⅡ对Cd2+的应激调控发生于转录水平。本研究为海洋沉积环境早期污染的生态风险预测提供了分子生物学依据。

In the present study,the partial cDNA of metalloproteinⅡ（MPⅡ） was amplified from the total RNA of epidermis muscle of the polychaete,Perinereis aibuhitensis,by reverse transcription polymerase chain reaction with the primers designed based upon the homologous cDNA sequence of MPII from Nereis diversicolor.Other primers were designed according to the partial sequence of Perinereis aibuhitensis MPII and two fragments of approximately 600 bp and 250 bp were obtained using 3′-and 5′-RACE,and sequenced.Sequence analysis re-vealed a 904 bp cDNA containing an 81 bp 5′-untranslated region,463 bp 3′-untranslated regions,and 357 bp open reading frame putatively encoding a protein of 119 amino acids.The deduced amino acid sequence of MPII was subjected to BLAST search for similarity with published sequences and it was found that MPII shared high iden-tity（81.51%） with Periserrula leucophryna hemerythrin,also with myohemerythrin of Nereis diversicolor（77.12%）,and with the hemerythrin of Themiste zostericola（61.02%）.Furthermore,the deduced amino acid se-quence of MPII contained the conserved motif（HX29HX3EX16HX3HX23/28HX4D） of the hemerythrin family of proteins,suggesting that MPII belongs to the hemerythrin family.The expression levels of Perinereis aibuhitensis MPII were detected after exposure to 40 mg/L Cd2＋ for 12,24,48,and 72 h using quantitative PCR.The expres-sion levels of MPII mRNA during 15 d were also detected under three concentrations of Cd2＋.The current study showed that the expression levels of the MPII were approximately 12.6-fold higher（P〈0.01） than control group at 40 mg/L Cd2＋ after 72 h.The level of MPII mRNA produced in the muscle tissue of sandworm increased signifi-cantly following exposure to Cd2＋ of 5,10,and 20 mg/L after 15 d,and were above 4.8-fold than control group（P〈0.05）.Therefore,the expression level of the MPII increased with increasing concentrations of Cd2＋.We sug-gest that heavy metal Cd2＋ is associated with the induction of MPII expression,and the present study provided molecular support for the assessment of early ecological risks for monitoring the marine sediment environment based on the health of clamworm.