坛紫菜谷胱甘肽过氧化物酶基因的克隆及表达特征

谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)是植物活性氧(ROS)清除酶促系统的重要成员之一,在植物逆境胁迫应答中发挥着重要作用。本研究以坛紫菜(Pyropia haitanensis)为研究材料采用RACE技术克隆获得了一条坛紫菜的GPX全长基因序列,命名为PhGPX(Gen Bank收录号:JX673908)。该基因序列全长1027 bp,包含555 bp的开放阅读框,所编码的多肽包含184个氨基酸,分子量为19.9 k D,等电点为8.76。多序列比对和系统进化树分析结果表明Ph GPX属于植物GPX基因家族成员。基因表达水平的q PCR分析结果表明Ph GPX基因在坛紫菜叶状体和丝状体世代中的表达水平没有显著差异;高温胁迫不同时间水平下,Ph GPX基因的表达水平呈现为先上调后下调的趋势;不同失水胁迫条件下,Ph GPX基因的表达不受低水平的失水胁迫影响,但可被高水平(40%)的失水胁迫所抑制,且在复水30 min后仍然无法恢复失水胁迫前的水平。由此推测坛紫菜体内的GPX也可能存在多种家族成员,不同的逆境胁迫条件、甚至不同的逆境胁迫水平可能需要不同家族成员的GPX参与ROS的清除和防御。

Cloning and expression analysis of the glutathione peroxidase gene from Pyropia haitanensis

At normal situation, algae maintain a balance between reactive oxygen species (ROS) production and scavenging states, and a little amount of ROS molecules can be used as intracellular signal transduction molecules involved in biochemical metabolic processes. But under abiotic stress conditions, excessive accumulation of ROS will cause oxidative damage to cell membrane, then resulting in irreparable loss of metabolic function and eventu-ally leading to cell death. Algae evolved a complex antioxidant defense mechanism (including non-enzymatic system of antioxidant enzymes and antioxidant enzymatic systems) to resist the damages of ROS. Glutathione peroxidase (GPX) is an important member of the ROS scavenging enzymatic system, which can clean the lipid peroxide and H2O2, its mechanism is to reduce ROS through catalytic the reactions between glutathione (GSH) and lipid peroxides (ROOH) or free H2O2, thereby blocking the production of free radicals, inhibiting the peroxidation processes of cells and protecting the enzyme activity of some sulfhydryl. Previous studies have concluded that the level of GPX activity has a positive correlation with the ability of plants to adapt to adversity. So GPX synthe-sis-related gene has been considered as an important stress resistance-related gene.Pyropia haitanensis, naturally growth in coastal intertidal zone, is an important economic macroalgae in Fujian, Zhejiang, Guangdong provinces. With tidal changes, the thallus required to undergo periodic dehydration and rehydration process, therefore it should has a very strong adaptability to adversity. Simultaneously, with global warming, the temperature of sea water increases, which causes damages to seedling ofP. haitanensisand lead to big economic loss for aquaculture industry. As for the physiological and molecular response of P. haitanensis to high temperature and dehydration stress conditions, our previous studies found GPX plays an important role in stress resistance. In this study, the full-length ofGPX gene fromPyropia haitanensis was cloned by using rapid amplification of cDNA ends (RACE) technology, which namedPhGPX (GenBank accession: JX673908). The nucleotide sequence ofPhGPX consists of 1027 bp, including an open reading frame (ORF) of 555 bp, which can be translate into a 184-amino-acid putative peptides with a molecular mass of 19.91 kD and a theoretical PI of 8.76. Multiple sequence alignment and Phy-logenetic analysis indicated that the protein belong to the plant GPX enzyme family. Expression patterns analyzed by qPCR revealed that the expression ofPhGPX gene between the thallus phage and conchocelis phage was not significantly different; during high-temperature stress, the expression levels ofPhGPX significantly upregulated firstly and then decreased; However, during desiccation, the expression levels ofPhGPX had no significantly change when the water loss was≤40%, but as the desiccation continue, the expression levels ofPhGPX were significantly downregulated. These results suggested that the expression ofPhGPX was induced by different stress at different levels.