雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

doi:10.11983/CBB16171

植物学报 ›› 2017, Vol. 52 ›› Issue (6): 774-782.DOI: 10.11983/CBB16171

雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

胡添源¹, 王睿¹, 陈上¹, 马宝伟¹, 高伟^1,^2,^*(), 黄璐琦³

¹首都医科大学中医药学院, 北京 100069
²中医络病研究北京市重点实验室, 北京 100069
³中国中医科学院中药资源中心, 道地药材国家重点实验室培育基地, 北京 100700

收稿日期:2016-08-21 接受日期:2017-01-10 出版日期:2017-11-01 发布日期:2018-02-22
通讯作者: 高伟
基金资助:
国家自然科学基金优秀青年科学基金(No.81422053)、国家杰出青年科学基金(No.81325023)和“十三五”时期北京市属高校高水平教师队伍建设支持计划(No.CIT&TCD20170324)

Protoplast Isolation and Establishment of Transient Expression System of Tripterygium wilfordii Suspension Culture Cells

Hu Tianyuan¹, Wang Rui¹, Chen Shang¹, Ma Baowei¹, Gao Wei^1,^2,^*(), Huang Luqi³

¹School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China
²Key Laboratory of Collateral Diseases, Beijing 100069, China
³Key Laboratory of Dao-di Herbs, National Resources Center for Chinese Materia Medica, China Academy of Chinese Medicinal Sciences, Beijing 100700, China

Received:2016-08-21 Accepted:2017-01-10 Online:2017-11-01 Published:2018-02-22
Contact: Gao Wei

摘要/Abstract

摘要： 为探索药用植物雷公藤(Tripterygium wilfordii)悬浮细胞原生质体提取的最优条件, 并建立雷公藤原生质体瞬时转化体系, 以雷公藤悬浮细胞为材料, 对酶解液配比、酶解时间、甘露醇浓度及处理转速进行考察。用PEG介导的瞬时转化法将外源基因转化到雷公藤原生质体中。结果表明, 以雷公藤悬浮细胞为材料提取原生质体的最佳条件是酶液配比为2.0%纤维素酶+0.5%果胶酶+0.5%离析酶, 甘露醇浓度为0.6 mol?L^-1, 酶解10小时, 处理转速为67×g; 用PEG介导法将含有编码GFP的植物表达载体转化雷公藤悬浮细胞原生质体, 激光共聚焦扫描显微镜下细胞显示绿色荧光。通过实验筛选得到雷公藤悬浮细胞原生质体的最佳提取条件, 建立了雷公藤悬浮细胞原生质体的瞬时转化体系, 为进一步开展雷公藤功能基因及合成生物学研究奠定了基础。

关键词: 原生质体, 悬浮细胞, 瞬时转化, 雷公藤

Abstract: We aimed to find the best protoplast extraction conditions with Tripterygium wilfordii suspension culture cells and establish an efficient transient expression system. Key factors in protoplast isolation, such as concentration of enzymes, time of enzymolysis, osmotic pressure and centrifugation speed, were studied. PEG mediation was used to transfer the GFP gene into protoplasts. The optimal extraction system was with cellulose R-10 2.0%, pectinase Y-23 0.5%, macerozyme R-10 0.5%, 0.6 mol?L^-1mannitol. The suitable enzymolysis time was 10 h, and the optimal centrifugation speed was 67 ×g. Under LSCM, the protoplast emitted green fluorescence after transforming the plasmid encoding green fluorescent protein into it. We revealed the optional conditions to isolate protoplast of T. wilfordii suspension cells and established the transient transformation system; it laid a foundation for further study on the functional genes and syn- thetic biology of T. wilfordii.

Key words: protoplast, suspension culture cells, transient transformation, Tripterygium wilfordii

胡添源, 王睿, 陈上, 马宝伟, 高伟, 黄璐琦. 雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立. 植物学报, 2017, 52(6): 774-782.

Hu Tianyuan, Wang Rui, Chen Shang, Ma Baowei, Gao Wei, Huang Luqi. Protoplast Isolation and Establishment of Transient Expression System of Tripterygium wilfordii Suspension Culture Cells. Chinese Bulletin of Botany, 2017, 52(6): 774-782.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB16171

https://www.chinbullbotany.com/CN/Y2017/V52/I6/774

图/表 7

表1 雷公藤悬浮细胞细胞壁酶解液配比

Table 1 Proportion of enzymic digestion to remove Triptery- gium wilfordii suspension cells cytoderm

Group	Cellulase (%)	Pectinase (%)	Macerozyme (%)
1	1.5	0.3	0.5
2	2.0	0.3	0.5
3	2.0	0.5	0.5
4	2.0	0.7	0.5
5	2.5	0.3	0.5

图1 酶解液浓度对雷公藤悬浮细胞原生质体产量(A)和活力(B)的影响不同小写字母表示在P<0.05水平上差异显著。

Figure 1 The influence of enzyme concentration on Tripterygium wilfordii suspension cells protoplast yield (A) and activity (B) Different lowercase letters indicate significant differences at P<0.05.

图2 不同酶解时间对雷公藤悬浮细胞原生质体产量(A)及活力(B)的影响不同小写字母表示在P<0.05水平上差异显著。

Figure 2 The influence of enzymatic hydrolysis time on Trip- terygium wilfordii suspension cells protoplast yield (A) and activity (B)Different lowercase letters indicate significant differences at P<0.05.

图3 不同甘露醇浓度对雷公藤悬浮细胞原生质体产量(A)和活力(B)的影响不同小写字母表示在P<0.05水平上差异显著。

Figure 3 The influence of mannitol concentration on Tripterygium wilfordii suspension cells protoplast yield (A) and activity (B)Different lowercase letters indicate significant differences at P<0.05.

图4 不同转速对雷公藤悬浮细胞原生质体的产量(A)及活力(B)的影响不同小写字母表示在P<0.05水平上差异显著。

Figure 4 The influence of centrifugal speed on Tripterygium wilfordii suspension cells protoplast yield (A) and activity (B)Different lowercase letters indicate significant differences at P<0.05.

图5 光学显微镜下雷公藤悬浮细胞原生质体形态(A) 台盼蓝染色前的原生质体; (B) 台盼蓝染色后的原生质体(红框标出的为死细胞)。Bars=100 μm

Figure 5 Morphology of Tripterygium wilfordii suspension cells protoplast under optical microscope(A) Morphology of protoplast before trypan blue staining; (B) Morphology of protoplast after trypan blue staining, and the blue cell in red box was dead. Bars=100 μm

图6 激光共聚焦扫描显微镜下观察雷公藤悬浮细胞原生质体(A) 明场下的原生质体; (B) 488 nm激发光下GFP荧光; (C) 原生质体无自发荧光; (D) 叠加效果图。Bars=1 μm

Figure 6 Protoplast of Tripterygium wilfordii suspension cells under laser scanning confocal microscop(A) Protoplasts under the laser confocal microscope in bright field; (B) GFP fluorescence of protoplast under the laser confocal microscope in excitation 488 nm; (C) There is no red chloroplast spontaneous fluorescence of protoplast under the laser confocal microscope in excitation 488 nm; (D) Red chlorophyll fluorescence signals and GFP signals from protoplast. Bars=1 μm

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雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

Protoplast Isolation and Establishment of Transient Expression System of Tripterygium wilfordii Suspension Culture Cells

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