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引用本文:林丽春,吕燕,孙鹏,孙雪,徐年军.龙须菜(Gracilariopsis lemaneiformis)中两种分支酸代谢酶对温度和水杨酸的响应及其原核表达研究.海洋与湖沼,2019,50(1):220-227.
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龙须菜(Gracilariopsis lemaneiformis)中两种分支酸代谢酶对温度和水杨酸的响应及其原核表达研究
林丽春, 吕燕, 孙鹏, 孙雪, 徐年军
宁波大学海洋学院 宁波 315211
摘要:
龙须菜(Gracilariopsis lemaneiformis)在我国主要用作鲍饵料和琼胶生产原料,但是南方夏季高温限制了龙须菜的栽培和产业化。抗逆植物激素水杨酸(salicylic acid,SA)与分支酸代谢之间存在着密切的联系。本文以两种分支酸代谢酶——异分支酸合成酶(isochorismate synthase,ICS)和分支酸变位酶(chorismate mutase,CM)为研究对象,分析了23℃(常温)和33℃(高温)条件下添加SA对龙须菜中两种分支酸代谢酶编码基因转录水平的影响。结果表明高温和SA不同程度地刺激了两种分支酸代谢酶转录水平的表达,如在3-12h,SA添加后ics表达量分别提高到常温对照组的2.84-4.76倍和高温对照组的1.25-1.62倍;在24h时,cm表达量分别提高到常温对照组的2.73倍和高温对照组的1.82倍。然后,我们将两种酶的编码基因分别转化到原核表达载体pET28a中,结果得到了多以包涵体形式大量表达的重组ICS和CM蛋白,其最佳诱导条件为0.1mmol/L IPTG在16℃诱导24h,并用镍柱初步纯化了重组蛋白。该研究为阐明温度和外源SA对龙须菜中两种分支酸代谢酶的影响规律,以及为从蛋白水平研究这两种代谢酶提供了资料。
关键词:  龙须菜  水杨酸  异分支酸合成酶  分支酸变位酶  原核表达
DOI:10.11693/hyhz20180800200
分类号:S968.43
基金项目:国家自然科学基金项目,31672674号,41376151号。
RESPONSES OF TWO CHOTISMATE METABOLIC ENZYMES TO TEMPERATURE AND SALICYLIC ACID IN GRACILARIOPSIS LEMANEIFORMIS AND THEIR PROKARYOTIC EXPRESSION ANALYSIS
LIN Li-Chun, LÜ Yan, SUN Peng, SUN Xue, XU Nian-Jun
School of Marine Sciences, Ningbo University, Ningbo 315211, China
Abstract:
The economic seaweed Gracilariopsis lemaneiformis is widely cultivated for feeding abalone and producing agar in China. However, high temperatures have limited its growth and industrialization in the southern coast. The stress-resistant phytohormone salicylic acid (SA) is intimately related to chorismate metabolism. In this study, the transcriptional level of two chorismate metabolic enzymes, isochorismate synthase (ICS) and chorismate mutase (CM) from G. lemaneiformis, were used to analyze the effects of the addition of SA under two temperature conditions. Results showed that high temperature (33℃) and SA stimulated the transcriptional expression of the two enzymes, e.g., compared to the corresponding control, ics expression level ranged from 2.84 to 4.76 times at 23℃ and from 1.25 to 1.62 times at 33℃ after addition of SA within 3-12h; cm expression level increased to 2.73 times at 23℃ and 1.82 times at 33℃ at 24h. Then, the encoding genes of the two enzymes were transferred into pET28a vector, respectively, and the recombinant ICS and CM proteins were expressed largely in the inclusion bodies. The optimal induction conditions of the recombinant proteins were at 16℃ for 24h by 0.1mmol/L IPTG. Finally, the recombinant proteins were preliminarily purified with a nickel column. This work provides the base for the research on the regulation of temperature and exogenous SA on the chorismate metabolism enzymes, and for the further research on the two enzymes at the protein levels in G. lemaneiformis.
Key words:  Gracilariopsis lemaneiformis  salicylic acid  isochorismate synthase  chorismate mutase  prokaryotic expression
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