| 摘要: |
| 为探究龙须菜(Gracilariopsis lemaneiformis)磷脂酶D (PLD)在高温响应中的作用,以龙须菜的全长转录组数据为基础,克隆获得了龙须菜GlPLD1、GlPLD2和GlPLD3基因,采用生物信息学、原核表达及酶活测定等方法对其功能进行初步分析。结果表明,GlPLD1、GlPLD2和GlPLD3基因cDNA序列长度分别为2 667 bp、2 661 bp和2 511 bp,编码898、888和886个氨基酸。序列比对分析表明,GlPLD1、GlPLD2和GlPLD3均具有保守的C2结构域和HKD结构域。通过原核表达分别获得了龙须菜GlPLD1、GlPLD2和GlPLD3重组蛋白,经体外酶促反应证明GlPLD1、GlPLD2和GlPLD3均具有PLD功能,且GlPLD2活性最高;并且,当pH为8.0,温度为33℃时,GlPLD2的比活力最大。另外,高温胁迫会上调GlPLD1、GlPLD2和GlPLD3的基因表达水平和PLD酶活,推测其在龙须菜高温胁迫应答中发挥功能。因此,本研究可为进一步研究龙须菜PLD在高温胁迫应答中的作用奠定基础。 |
| 关键词: 龙须菜 磷脂酶D 原核表达 酶学特征 高温胁迫 |
| DOI：10.11759/hykx20240319001 |
| 分类号:S917.3 |
| 基金项目:国家自然科学基金项目(32002376);浙江省自然科学基金项目(LY24C190002) |
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| Prokaryotic expression, enzymatic characterization, and high temperature response analysis of phospholipase D from Gracilariopsis lemaneiformis |
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CUI Zhenhao, TANG Wenting, ZHANG Xiaoqian, SUN Xue, XU Nianjun
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School of Marine Sciences, Ningbo University, Ningbo 315832, China
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| Abstract: |
| To investigate the role of phospholipase D (PLD) in high temperature stress response in Gracilariopsis lemaneiformis. Based on full-length transcriptome data of G. lemaneiformis, GlPLD1, GlPLD2, and GlPLD3 were cloned, and their functions were preliminarily analyzed through bioinformatics, prokaryotic expression, and enzyme activity assay. Results revealed that cDNA sequences of GlPLD1, GlPLD2, and GlPLD3 were 2, 667 bp, 2, 661 bp, and 2, 511 bp in length, encoding 898, 888, and 886 amino acids, respectively. Furthermore, sequence alignment analysis demonstrated that all three genes had conserved C2 and HKD domains. Recombinant proteins of GlPLD1, GlPLD2, and GlPLD3 were obtained by prokaryotic expression, and in vitro enzymatic reactions confirmed that all of them had PLD function, with the highest activity being observed in PLD2. Moreover, when pH was 8.0 and temperature was 33℃, the specific activity of GlPLD2 was the highest. Additionally, high temperature stress upregulated GlPLD1, GlPLD2, and GlPLD3 expression levels and PLD enzyme activity, suggesting that PLD played a role in the high temperature stress response of G. lemaneiformis. Thus, this study can provide valuable insights for further research on the role of PLD in high temperature stress response of G. lemaneiformis. |
| Key words: Gracilariopsis lemaneiformis phospholipase D prokaryotic expression enzymatic characterization heat stress |
