《植物生理学报》 2014, 50(8): 1259-1264

莱茵衣藻和斜生栅藻蛋白核的染色方法优化

吕学兰^1,2, 陆贻超¹, 葛亚明¹, 张亚杰^1,*

¹中国科学院宁波材料技术与工程研究所, 浙江宁波315201; ²中国科学院大学, 北京100049

通信作者：张亚杰；E-mail: zhangyj@nimte.ac.cn；Tel: 0574-8669-0283

摘　要：

蛋白核(pyrenoid)作为真核藻类重要的、相对独立的固碳结构, 在CO₂高效浓缩和固定过程中发挥中重要作用, 可为开发高效生物固碳技术提供理论支持。染色法是快速观察蛋白核的最佳方法之一，有助于深入研究蛋白核的结构和功能，但目前对染色法仍缺乏系统研究。本文以较易染色的莱茵衣藻(Chlamydomonas reinhardtii)和较难染色的斜生栅藻(Scenedesmus obliquus)为材料, 探讨了碘染和溴酚蓝(BPB)染色观察蛋白核的方法。结果表明, 碘染法最适用于蛋白核外周淀粉观察, 最适染色液浓度和时间分别是0.1%~0.2% (W/V)和5 min。热激预处理和溴酚蓝染色相结合的蛋白核染色效果更好, 优化条件为: 70 ℃水浴热激40 s, 0.05% (W/V) BPB染色15 min。本方法为研究微藻蛋白核提供了一种快速有效的直接观察方法。

关键词：真核微藻; 蛋白核; 碘染色; 溴酚蓝染色

收稿：2014-03-27   修定：2014-05-26

资助：国家重点基础研究发展计划(2011CBA00905)和国家自然科学基金青年基金(31100183)。

Optimization of Method for Staining Pyrenoid of Chlamydomonas reinhardtii and Scenedesmus obliquus

LÜ Xue-Lan^1,2, LU Yi-Chao¹, GE Ya-Ming¹, ZHANG Ya-Jie^1,*

¹Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, Zhejiang 315201, China; ²University of Chinese Academy of Science, Beijing 100049, China

Corresponding author: ZHANG Ya-Jie; E-mail: zhangyj@nimte.ac.cn; Tel: 0574-8669-0283

Abstract:

Pyrenoid, as an important and relatively independent structure of carbon fixation in eukaryotic microalgae, plays a crucial role in CO₂ concentration and fixation. Study of pyrenoid could support the development of efficient bio-sequestration technologies. However, quick characterization of pyrenoid in algae cell was imperfect, which to some extent limited the in-depth study of its structure and function. In this study, two typical microalgae strains, Chlamydomonas reinhardtii and Scenedesmus obliquus, representing easily and difficult staining species, and two dyes, iodine and bromophenol blue (BPB), were used to optimize methods for staining pyrenoid. Results demonstrated that iodine could simutaneously stain stereoscopical pyrenoid and its starch sheath, and the optimal staining concentration and time were 0.1%–0.2% (W/V) and 5 min, respectively. In comparison to iodine staining, the combination of BPB staining and heat shocking showed a better result than iodine staining, which could clearly staining pyrenoid without staining starch sheath. The optimal staining condition was at 70 ℃ heat shock for 40 s, and 0.05% (W/V) BPB staining for 15 min. Our result provided a practical method that could characterize pyrenoid of microalgae in vivo fast and effectively.

Key words: eukaryotic microalgae; pyrenoid; iodine staining; bromophenol blue staining