用于植物基因研究的mCherry表达载体构建及定位表达

doi:10.7525/j.issn.1673-5102.2017.02.006

摘要/Abstract

摘要： 荧光蛋白在生物学研究中具有广泛的应用和重要的作用，其中红色荧光蛋白mCherry因其颜色和良好的特性，对于植物基因研究具有重要的使用价值，本研究将mCherry基因构建到pBI121植物表达载体系统中，构建了pBI121MCS-mCherry载体。利用基因枪转化法转入洋葱表皮进行表达验证，显微镜观察结果显示整个洋葱细胞具有红色荧光，证明该载体能够在植物细胞中表达红色荧光蛋白。利用双酶切连接法将转录因子BpMYB4基因构建到该载体上，得到融合表达载体pBI121MCS-mCherry-BpMYB4，在洋葱表皮中表达，结果显示细胞核具有红色荧光，证明该载体能够准确表达融合蛋白，进行亚细胞定位。同时融合基因时不再需要中间载体，构建简便，引入的KpnⅠ酶切位点，增加了可选择性。因此该载体可用于植物基因表达定位及转基因植株筛选研究中，为今后的白桦基因组学研究提供了材料。

关键词: mCherry, 红色荧光蛋白, 植物表达载体构建, 细胞定位

Abstract: Fluorescent proteins were widely used and played important roles in the biological research. The red fluorescent protein mCherry is valuable to the plant research because of its color and stability. The mCherry gene was inserted into pBI121 plant expression vector, and the pBI121MCS-mCherry vector was constructed. The vector were transferred into onion epidermis by particle bombardment, and the whole onion cells showed red fluorescence under the microscope, which suggested that the vector could express red fluorescent protein in plant cells. The transcription factor BpMYB4 gene was constructed in this vector using double enzyme digestion, and the fusion expression vector pBI121MCS-mCherry-BpMYB4 was obtained and expressed in onion epidermis. Only the nucleus showed red fluorescence, which indicated that the vector can be used to express the fusion protein and used in the subcellular localization accurately. While the fusion expression vector can be constructed simply, and the intermediate vectors are no longer required. The KpnⅠ sites was introduced which can help the fusion of more genes. Therefore, mCherry vector can be used in cell localization of plant genes and screening of transgenic plants. This study would provide materials for genomics research of birch in future.

Key words: mCherry, red fluorescent protein, plant expression vector construction, cell localization

中图分类号:

于颖, 刘佳欣, 周美琪, 赵磊飞, 王超. 用于植物基因研究的mCherry表达载体构建及定位表达[J]. 植物研究, 2017, 37(2): 194-199.

YU Ying, LIU Jia-Xin, ZHOU Mei-Qi, ZHAO Lei-Fei, WANG Chao. Construction and Expression of mCherry Expression Vector Used in Plant[J]. Bulletin of Botanical Research, 2017, 37(2): 194-199.

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