虾夷马粪海胆溶菌酶基因全长cDNA的克隆与表达分析

Cloning and expression analysis of a sea urchin(Strongylocentrotus intermedius) lysozyme gene

本实验采用RT-PCR和cDNA末端快速扩增(RACE)技术克隆得到了虾夷马粪海胆(Strongylocentrotus intermedius)溶菌酶(LYZ)基因的全长cDNA序列。结果表明,虾夷马粪海胆LYZ基因全长为912 bp,含有1个480 bp的开放阅读框(ORF),编码159个氨基酸,其中第1 20个氨基酸为信号肽,蛋白计算分子量为17.69 kD,等电点为7.75。氨基酸比对分析表明,虾夷马粪海胆LYZ基因与紫球海胆(Strongylocentrotus purpuratus)和刺参(Apostichopus japonicus)的i型LYZ基因相似百分比分别为91.4%和59.3%,并且含有i型LYZ基因的保守序列DVGSLSCGP(Y)Y(F)QIK,所以推断本实验克隆的溶菌酶为i型。采用实时定量PCR方法,以β-actin为内标,对其在虾夷马粪海胆各组织中的表达进行研究,发现LYZ基因在围口膜中表达量最高,其次是齿间肌、管足、肠、体腔液、雄性性腺和雌性性腺。利用脂多糖(LPS)刺激虾夷马粪海胆,取刺激后不同时间的海胆体腔液,对该基因的表达差异进行分析。结果表明,虾夷马粪海胆的LYZ基因在LPS刺激后8 h时表达量最高,12 h时开始逐步回落,至36 h时回落至对照组相近水平。本结果可为虾夷马粪海胆免疫学研究及抗病相关分子标记的开发提供参考依据。

A full-length cDNA coding lysozyme （LYZ） was cloned from sea urchin Strongylocentrotus intermedius by reverse transcriptase-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends（RACE） methods. The full-length cDNA of LYZ was 912 bp with a 480 bp open reading frame（ORF） encoding 159 amino acids, which included a signal peptide of 20 amino acids at the N-terminus and a mature peptide of 139 amino acids. The deduced amino acid sequence had a putative size of 17.69 kD and the theoretical isoelectric point was 7.75. The multiple alignments revealed identity of 91.4% between S. intermedius and S. purpuratus and 59.3% identity between S. intermedius and Apostichopus japonicus in the i-type LYZ amino acid sequence. The i-type lysozyme conserved sequence DVGSLSCGP（Y）Y（F）QIK was detected in the S. intermedius amino acid sequence. These results indicate that the cDNA sequence cloned from S. intermedius is a member of the i-type lysozyme family. Real-time quantitative PCR was carried out to measure LYZ mRNA expression in different tissues and monitor LYZ mRNA expression patterns in the coelomic fluid after a lipopolysaccharide （LPS） challenge. LYZ mRNA expression levels in peristome membrane were significantly higher than those of muscle in the Aristotle＇s lantern, tube-foot, intestines, coelomic fluid, male gonad, and female gonad （P〈0.05）. LYZ expression in the coelomic fluid was up-regulated after the LPS challenge and reached its maximum level at 8 h post-stimulation, and decreased gradually thereafter. Thirty-six hours after the LPS challenge, LYZ levels in the coelomic fluid were similar to those of the control.