中间球海胆乳酸脱氢酶基因克隆及其对海水酸化的响应

Identification and characterization of LDH gene and its response to seawater acidification in the sea urchin(Strongylocentrotus intermedius)

为明确中间球海胆乳酸脱氢酶(LDH)基因序列及表达特征,研究其对海水酸化胁迫的响应,实验利用cDNA末端快速扩增(RACE)技术获得中间球海胆LDH基因(命名为SiLDH)的全长cDNA,并且比较了海水酸化胁迫下,中间球海胆不同组织SiLDH基因表达及总LDH活性的变化情况。结果显示:①SiLDH基因的cDNA全长为1 499 bp,包含133 bp的5′非编码区,349 bp的3′非编码区和1 017 bp编码338个氨基酸的开放阅读框(ORF)。SiLDH编码蛋白的相对分子量为36.40 ku,理论等电点为6.55,属于无信号肽的非跨膜、温和疏水蛋白。②生物信息学分析显示,SiLDH蛋白序列与紫球海胆LDH X4蛋白序列一致性最高(90.88%)。③实时荧光定量PCR(qRT-PCR)发现,SiLDH基因在检测的4种组织中均有表达,相对表达量从高到低为性腺>管足>肠>体腔液;总LDH活性检测显示,中间球海胆总LDH活性从高到低为性腺>管足>体腔液>肠;④海水酸化处理60 d后发现,与自然海水组(pH 8.06±0.01)相比,SiLDH基因在中间球海胆性腺、管足和肠道中呈现总体降低趋势,在体腔液中则呈现随海水pH降低先降低后显著升高的趋势;总LDH活性在性腺、管足和体腔液中呈现随海水pH降低而降低的趋势,在肠道中则呈现随海水pH降低先降低后显著升高的趋势。研究表明,中间球海胆面对海水酸化时可能通过调节SiLDH基因的表达和LDH活性来缓解海水酸化带来的负面影响。

In order to identify and characterize the lactate dehydrogenase(LDH)gene and investigate its expression and activity alterations in response to seawater acidification stress in sea urchins,we used database data mining and rapid amplification of cDNA ends(RACE)to identify a novel LDH gene in the sea urchin Strongylocentrotus intermedius(here designated SiLDH).The full-length cDNA of SiLDH was 1 499 bp,including a 1 017 bp open reading frame encoding 338 amino acid residues.Sequence analysis indicated that the predicted SiLDH protein was similar to the LDH homolog in the sea urchin S.purpuratus.Quantitative real-time PCR analysis showed that SiLDH was ubiquitously expressed in coelomic fluid,gonad,tube foot and intestine of healthy adult S.intermedius,with the highest level of expression identified in the gonad.Total LDH enzyme activity in different tissues of S.intermedius from high to low was as gonad>tubefoot>coelomic fluid>intestine.Both SiLDH mRNA expression profiles and total LDH enzyme activity were altered in the coelomic fluid,gonad,tube foot and intestine after 60-day seawater acidification treatments(Control:natural seawater;OA1:pH=-0.3;OA2:pH=-0.4;OA3:pH=-0.5).Our results provide more information about the characteristics and biological functions of the LDH homolog in sea urchins.