海带(Saccharina japonica)SjHO1基因的鉴定、表达与功能分析  

IDENTIFICATION,EXPRESSION AND FUNCTIONAL ANALYSIS OF A HEME OXYGENASE-1 GENE FROM SACCHARINA JAPONICA

作　　者：唐柳青 许梦雪 李乐 吕芳 詹冬梅 吴海一 TANG Liu-Qing;XU Meng-Xue;LI Le;LYU Fang;ZHAN Dong-Mei;WU Hai-Yi(Shandong Province Macroalgae Resources Conservation and Application Engineering Research Center,Marine Science Research Institute of Shandong Province,Qingdao 266104,China;Nearshore Seaweed Farm Construction Engineering Research Center of Shandong Province,Qingdao 266104,China;Shandong Key Laboratory of Intelligent Marine Ranch(under preparation),Qingdao 266104,China)

机构地区：[1]山东省海洋科学研究院,山东省大型海藻资源保护与应用工程技术研究中心,山东青岛266104 [2]近岸海藻场建设山东省工程研究中心,山东青岛266104 [3]山东省智慧海洋牧场重点实验室(筹),山东青岛266104

出　　处：《海洋与湖沼》2025年第1期186-195,共10页Oceanologia Et Limnologia Sinica

基　　金：山东省重点研发计划,2022LZGC004号;山东省现代农业产业技术体系创新团队项目,SDAIT26号。

摘　　要：血红素加氧酶(heme oxygenase, HO)是生物体内催化血红素分解代谢的限速酶,在生物的生长发育中起着重要的作用。以大型经济海藻海带(Saccharina japonica)为材料,利用基因组数据库,以分子生物学手段和生物信息学工具鉴定获得一个HO1同源基因(SjHO1)。SjHO1基因由核基因组编码,开放阅读框为1 035 bp,编码344个氨基酸。预测编码的蛋白SjHO1分子量为36.75 kDa,等电点为8.71,是不稳定的亲水蛋白,不含信号肽和跨膜结构,具有HemeO保守结构域,亚细胞定位预测其主要定位在叶绿体。通过氨基酸序列比对和系统进化树分析发现,SjHO1蛋白与同属于褐藻的两种水云以及鼠尾藻的HO1蛋白序列相似性较高。采用实时荧光定量PCR分析海带SjHO1基因响应非生物胁迫的表达模式,发现高光和低盐条件下会促进SjHO1的表达,而高温、干燥和黑暗条件下SjHO1的表达量则没有显著变化。为进一步鉴定其功能,将SjHO1连接到表达载体pET-28a(+)上,转化获得SjHO1编码基因的大肠杆菌BL21(DE3)工程菌,通过IPTG诱导表达,SDS-PAGE和Western blot分析表达产物发现成功表达目的蛋白。进而测定HO活性,发现其具有结合血红素降解的特征峰,但未检测到反应产物BV的相应吸收峰。研究结果为进一步分析SjHO1基因功能及海带抗逆分子育种奠定了基础。Heme oxygenase(HO)is the rate-limiting enzyme that catalyzes the catabolism of heme in organisms and plays a crucial role in their growth and development.In this study,a homologous gene of HO1(SjHO1)was identified using the genome database of the seaweed Saccharina japonica,employing molecular biology and bioinformatics tools.The SjHO1 gene is encoded by the nuclear genome and the open reading frame of the SjHO1 gene is 1035 bp,encoding a protein of 344 amino acids.The predicted protein,SjHO1,has a molecular mass of 36.75 kDa and an isoelectric point of 8.71.It is characterized as an unstable hydrophobic protein without signal peptide and transmembrane domains,and it contains a conserved HemeO domain,subcellular localization predicts that it is mainly localized in chloroplasts.Comparative analysis of the amino acid sequence and phylogenetic analysis indicate that the SjHO1 protein is homologous to HO1 proteins from two species of Ectocarpus and Sargassum thunbergii,all of which are members of marine brown algae.Real-time quantitative analysis of SjHO1 gene expression patterns in response to abiotic stress revealed that high-light and low-salt conditions significantly enhanced SjHO1 expression,while no significant changes were observed under high-temperature,desiccation,or darkness conditions.Additionally,the SjHO1 gene was ligated into the expression vector pET-28a(+)and transformed into E.coli BL21(DE3),where it was induced with IPTG.Successful expression of the target protein was confirmed by SDS-PAGE and Western blot analyses.Furthermore,the enzyme activity of HO was measured,revealing a characteristic peak associated with heme degradation;however,the corresponding absorption peak of the reaction product,biliverdin(BV),was not detected.These findings provide a foundational reference for further research on the function of SjHO1 gene and its potential applications in molecular breeding.

关 键 词：海带 血红素加氧酶 SjHO1基因 表达分析 功能鉴定 

分 类 号：S968[农业科学—水产养殖] Q786[农业科学—水产科学]