印度芥菜细胞中Hg~(2+)分布的可视化研究
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摘要
汞对动物和植物均会产生损害。印度芥菜是目前发现的较好的能够超累积汞的植物,能够高富集包括Hg2+在内的多种二价重金属离子,是比较理想的研究超富集Hg2+的分子机制和解毒机制的植物。本研究通过愈伤组织诱导获得悬浮细胞,寻找到最适宜的印度芥菜愈伤组织诱导的最优条件。在印度芥菜愈伤组织诱导试验中,选择子叶为外植体诱导愈伤组织速度较快,明显快于下胚轴和幼叶,同时愈伤组织较为疏松,呈浅黄色,形态较好。筛选出的最适诱导愈伤组织的条件为MS+2,4-D(1.0mg·L-1)+NAA (1.0mg-L"1)+6-BA(3.0mg·L-1),诱导出的愈伤组织呈现浅绿色疏松水渍状,增殖很快,数量最多,质量也最好。
本研究中使用EPNP作为对Hg2+高度敏感的探针,观察Hg2+在印度芥菜植物体的分布。结果表明,EPNP能够透过细胞壁和细胞膜,分布到细胞内部。随着培养基中Hg2+浓度从10μM升高到100μM,可以明显观察到荧光信号明显增强。时间依赖实验表明,经过3小时的培养,EPNP与汞离子结合后产生的荧光并没有淬灭。同时,通过共聚焦显微镜的观察,可以判断Hg2+主要富集在溶酶体,而不是细胞核或者线粒体中。这些观察为Hg2+在印度芥菜活体细胞中的分布提供了实验证据。
利用分子成像仪,观察印度芥菜外植体中EPNP对汞离子响应的浓度和时间依赖。随着汞离子浓度的升高,荧光的亮度明显增加。说明在活体植物中,EPNP对植物体本身无毒无害,而且随着Hg2+浓度的增加,EPNP的荧光亮度也明显增强。经过1h、2h、5h、6h培养后,印度芥菜下胚轴均呈现明显的荧光。随着培养时间的增长,可以明显看到汞离子荧光的运输情况。本研究实现了活体、实时和动态地观察研究Hg2+在印度芥菜根、茎等部位的吸收、转移和运输的过程。利用荧光分子成像技术,研究Hg2+在活体内的迁移,并以此初步开始分子影像学的研究。这是第一次使用Hg2+的荧光探针观察活体植物细胞。将EPNP应用于其他受到Hg2+污染的环境和植物,实时观察汞离子的分布和运输,进一步研究与植物修复和解毒相关的基因和蛋白,可能会对植物解毒的研究有所帮助。
Exposure to mercury causes severe damage to plants, animals and even humans. Brassica juncea, as a plant species that has been reported as a new heavy metal hyper-accumulator recently, is used in this research. Brassica juncea can accumulate many divalent metals including Hg2+due to its particular transmembrane proteins. Calli of Brassica juncea were induced from cotyledons, stems and young leaves explants under the effect of hormonal supplementation (NAA,6-BA,2,4-D). The results showed that the stem is the best organ to obtain loose callus material. The MS medium containing1.0mg·L-1NAA+1.0mg·L-1-BA+2.0mg·L-12,4-D was found to be the most effective in callus induction. Suspension cells were established by transferring calli to a liquid MS medium in triangular flasks containing the same composition and same hormonal supplementation and were shaken for21days.
By using EPNP as a highly selective and sensitive probe for Hg2+, the distribution of Hg2+in Brassica juncea can be detected. Our results show that EPNP can penetrate the cell wall and cell membrane, and is distributed inside the cell. With the increase of HgCl2concentrations from10μM to100μM in the cultured medium, a gradual enhanced fluorescence can be observed. A time-dependent experiment showed the fluorescence of the EPNP-Hg complex was still not quenched after3hours of exposure. It was also found that Hg2+mostly accumulated in lysosomes and not in the nucleus or mitochondria. These observations provide direct experimental evidence for the localization of Hg2+in Brassica juncea suspension cells in vivo.
In vivo fluroseence imaging to visualize the transportation of Hg2+, the progress can be detected. Our results show that with the increase of HgCl2from10μM to100μM in the cultured medium, gradually enhanced fluorescence intensity can be observed. A time-dependent experiment showed the fluorescence of the EPNP-Hg complex in the plant was still accumulated. To the best of our knowledge, this is the first example of a probe-based methodology for imaging Hg2+in living plants and suspension cells. More applications of EPNP for fluorescence imaging of other plants grown in Hg2+polluted environment may be helpful to better understand plant poisoning.
本研究中使用EPNP作为对Hg2+高度敏感的探针,观察Hg2+在印度芥菜植物体的分布。结果表明,EPNP能够透过细胞壁和细胞膜,分布到细胞内部。随着培养基中Hg2+浓度从10μM升高到100μM,可以明显观察到荧光信号明显增强。时间依赖实验表明,经过3小时的培养,EPNP与汞离子结合后产生的荧光并没有淬灭。同时,通过共聚焦显微镜的观察,可以判断Hg2+主要富集在溶酶体,而不是细胞核或者线粒体中。这些观察为Hg2+在印度芥菜活体细胞中的分布提供了实验证据。
利用分子成像仪,观察印度芥菜外植体中EPNP对汞离子响应的浓度和时间依赖。随着汞离子浓度的升高,荧光的亮度明显增加。说明在活体植物中,EPNP对植物体本身无毒无害,而且随着Hg2+浓度的增加,EPNP的荧光亮度也明显增强。经过1h、2h、5h、6h培养后,印度芥菜下胚轴均呈现明显的荧光。随着培养时间的增长,可以明显看到汞离子荧光的运输情况。本研究实现了活体、实时和动态地观察研究Hg2+在印度芥菜根、茎等部位的吸收、转移和运输的过程。利用荧光分子成像技术,研究Hg2+在活体内的迁移,并以此初步开始分子影像学的研究。这是第一次使用Hg2+的荧光探针观察活体植物细胞。将EPNP应用于其他受到Hg2+污染的环境和植物,实时观察汞离子的分布和运输,进一步研究与植物修复和解毒相关的基因和蛋白,可能会对植物解毒的研究有所帮助。
Exposure to mercury causes severe damage to plants, animals and even humans. Brassica juncea, as a plant species that has been reported as a new heavy metal hyper-accumulator recently, is used in this research. Brassica juncea can accumulate many divalent metals including Hg2+due to its particular transmembrane proteins. Calli of Brassica juncea were induced from cotyledons, stems and young leaves explants under the effect of hormonal supplementation (NAA,6-BA,2,4-D). The results showed that the stem is the best organ to obtain loose callus material. The MS medium containing1.0mg·L-1NAA+1.0mg·L-1-BA+2.0mg·L-12,4-D was found to be the most effective in callus induction. Suspension cells were established by transferring calli to a liquid MS medium in triangular flasks containing the same composition and same hormonal supplementation and were shaken for21days.
By using EPNP as a highly selective and sensitive probe for Hg2+, the distribution of Hg2+in Brassica juncea can be detected. Our results show that EPNP can penetrate the cell wall and cell membrane, and is distributed inside the cell. With the increase of HgCl2concentrations from10μM to100μM in the cultured medium, a gradual enhanced fluorescence can be observed. A time-dependent experiment showed the fluorescence of the EPNP-Hg complex was still not quenched after3hours of exposure. It was also found that Hg2+mostly accumulated in lysosomes and not in the nucleus or mitochondria. These observations provide direct experimental evidence for the localization of Hg2+in Brassica juncea suspension cells in vivo.
In vivo fluroseence imaging to visualize the transportation of Hg2+, the progress can be detected. Our results show that with the increase of HgCl2from10μM to100μM in the cultured medium, gradually enhanced fluorescence intensity can be observed. A time-dependent experiment showed the fluorescence of the EPNP-Hg complex in the plant was still accumulated. To the best of our knowledge, this is the first example of a probe-based methodology for imaging Hg2+in living plants and suspension cells. More applications of EPNP for fluorescence imaging of other plants grown in Hg2+polluted environment may be helpful to better understand plant poisoning.
引文
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[2]Liu X, Wu Q, Li P. Phytoremediation of Heavy Metal Contaminated Soil by Hyper-Accumulators:A Review of Researches in China and Abroad [J]. Journal of Agro-Environment Science,2003,22(5):636-640.
[3]Bin Y, He S, Jingjun H, et al. Application of transgenic plants in phytoremediation for contaminated soil by heavy metals and organic pollutants [J]. Scientia Silvae Sinicae,2005,41(4):162-167.
[4]母波,韩善华,张英慧等.汞胁迫对植物细胞结构与功能的影响[J].中国微生态学杂志,2007,19(4)：112-113
[5]张义贤.重金属对大麦毒性的研究[J].环境科学学报,1997,17(2)：199-205
[6]施国新,杜开和,解凯彬.汞、镉污染对黑藻叶细胞伤害的超微结构研究[J].植物学报,2000,42(4)：373-378.
[7]解凯彬,施国新,陈国祥等.Hg2+对萍蓬草光合膜超微结构及功能的影响[J].农村生态环境,2002,18(1)：26-30.
[8]史字,何玉科.重金属污染环境的植物修复及其分子机制[J].植物生理与分子生物学学报,2003，29(4)：267-274.
[9]马剑敏,靳萍,吴振斌.沉水植物对重金属的吸收净化和受害机理研究进展[J].植物学通报,2007,2(1)：12-15
[10]Zhang W H, Tyerman S D. Inhibition of water channels by HgCl2 in intact wheat root cells [J]. Plant Physiology,1999,120(3):849-858.
[11]Dordas C, Chrispeels M J, Brown P H. Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from squash roots [J]. Plant Physiology, 2000,124(3):1349-1362.
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