| 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。本研究以大型经济海藻—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1035 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,是不稳定的疏水蛋白,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,发现低盐和高光条件下会促进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酶活性,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。本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。 |
| 关键词: 海带 血红素加氧酶 SjHO1基因 表达分析 功能鉴定 |
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| IDENTIFICATION, EXPRESSION AND FUNCTIONAL ANALYSISI OF A HEME OXYGENASE1 GENE FROM SACCHARINA JAPONICA |
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Tang Liuqing, Xu Mengxue, Li Le, Lv Fang, Zhan Dongmei, Wu Haiyi
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Marine Science Research Institute of Shandong Province (National Oceanographic Center, Qingdao ）
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| Abstract: |
| 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 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. 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 low-salt and high-light 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. |
| Key words: Saccharina japonica heme oxygenase SjHO1 expression analysis functional identification |