He-Ne激光对增强UV-B辐射小麦微丝骨架的影响
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摘要
本研究以‘晋麦8号’(Triticum aestivum)小麦为研究对象,采用He-Ne激光(5mW·mm-2)辐照和增强UV-B(10.08 kJ·m-2·d-1)辐射处理小麦,以异硫氰酸荧光素标记的鬼笔环肽(FITC-Ph)为探针,利用激光共聚焦扫描显微镜,研究了小麦叶肉细胞原生质体及根尖细胞微丝骨架分布格局与变化,以揭示分束分裂与细胞骨架的关系,为进一步阐明细胞的抗性机理、细胞分裂的生物学机制等奠定理论基础。主要研究结果如下:
1、采用适当的酶的种类及较低的酶浓度(纤维素酶R-10 1.5 %、果胶酶Y-23 0.1% ),在适宜的温度(26℃)和酶解时间(3h)内,可以获得大量的原生质体。
2、利用SDS-PAGE电泳技术分析和检测小麦幼苗肌动蛋白,结果表明,增强UV-B辐射在一定程度上使肌动蛋白含量受到影响,而He-Ne激光可降低UV-B辐射造成的损伤。
3、以小麦叶肉细胞原生质体为材料,在激光共焦扫描镜下,可观察到小麦叶肉细胞原生质体内精细的微丝骨架。对照组(CK)中叶肉细胞原生质体微丝呈现网状或平行状的随机排列。UV-B辐射处理(B)后微丝网络结构遭到破坏,部分聚集成束,部分或呈点状或碎片状分布于原生质体中。UV-B复合处理(BL)后,只有部分微丝网络被打乱,部分聚集成点状分布于胞质中。He-Ne激光组(L)微丝束有些变得较短且分支明显,呈随机排列的交叉网络。
4、以小麦根尖细胞为材料,激光共焦扫描镜下可清晰地观察到小麦根尖细胞微丝骨架的有丝分裂时相。UV-B辐射处理后,间期细胞肌动蛋白纤丝排列紊乱;前期细胞周质中肌动蛋白纤丝变为短的片段,可见点状荧光随机分布在周质;中期细胞荧光强度明显减弱,明亮的的微丝片段消失,点状荧光也消失;有丝分裂后期到末期,成膜体区域肌动蛋白纤丝或成弥散状或完全消失,且出现落后染色体,染色体桥,不均等分裂及三束分裂等染色体畸变类型和异常分裂现象。
The seedlings of wheat(Jin mai 8)were exposed to He-Ne laser irradiation with 5mW·mm-2 Power density after enhanced UV-B radiated(10.08kJ·m-2·d-1). Microfilaments (MFs) were observed by confocal laser scanning microscopy (CLSM) in the wheat mesophyll protoplast and wheat root-tip cells treated with FITC- Phalloidin . The results were showed as follows:
1. We can gain a large number of protoplasts using appropriate types of enzymes and low enzyme concentrations (cellulase R-10 1.5% and pectinase Y-23 0.1%), at the appropriate temperature (26℃), and digestion time (3h ).
2. The actin in wheat mesophyll protoplast was studied by SDS-PAGE electrophoresis analysis .The results showed that the contents of actin were affected in a certain extent after UV-B radiation; but the He-Ne laser can reduce the UV-B radiation damage.
3. Microfilaments (MFs) were observed by confocal laser scanning microscopy (CLSM) in the wheat mesophyll protoplast treated with FITC- Phalloidin .Two types of MFs bundles could be recognized(CK): those beneath the plasma membrane running parallel to each other; those surrounding the chloroplast. When the wheats were treated by enhanced ultraviolet-B radiation(B), the distribution pattern of MFs was changed into bundles or granules. Part of the actin network is disrupted treated with compound treatment (BL). The distribution of MFs have become shorter and arrange crossing networks by He-Ne laser irradiation(L).
4. The distribution of microfilaments (MFs) were examined by confocal laser scanning microscopy (CLSM) in the wheat root-tip cells under the enhanced ultraviolet-B (10. 08 kJ·m - 2·d- 1 ) radiation treated with FITC- Phalloidin .The results showed that under the condition of the enhanced ultraviolet-B radiation, distribution of actin MFs were disordered in interphase cells; The arrays of MFs disintegrated into short fragments of random during prophase; Fluorescence intensity significantly decreased and bright fragments of microfilaments disappeared in Metaphase cells; MFs disappeared in the phragmoplast regions when cells were in anaphase and telophase time . And some types of chromosome aberration were induced ,including Chromosomal logging , chromosomal bridge and partition-bundle division .
1、采用适当的酶的种类及较低的酶浓度(纤维素酶R-10 1.5 %、果胶酶Y-23 0.1% ),在适宜的温度(26℃)和酶解时间(3h)内,可以获得大量的原生质体。
2、利用SDS-PAGE电泳技术分析和检测小麦幼苗肌动蛋白,结果表明,增强UV-B辐射在一定程度上使肌动蛋白含量受到影响,而He-Ne激光可降低UV-B辐射造成的损伤。
3、以小麦叶肉细胞原生质体为材料,在激光共焦扫描镜下,可观察到小麦叶肉细胞原生质体内精细的微丝骨架。对照组(CK)中叶肉细胞原生质体微丝呈现网状或平行状的随机排列。UV-B辐射处理(B)后微丝网络结构遭到破坏,部分聚集成束,部分或呈点状或碎片状分布于原生质体中。UV-B复合处理(BL)后,只有部分微丝网络被打乱,部分聚集成点状分布于胞质中。He-Ne激光组(L)微丝束有些变得较短且分支明显,呈随机排列的交叉网络。
4、以小麦根尖细胞为材料,激光共焦扫描镜下可清晰地观察到小麦根尖细胞微丝骨架的有丝分裂时相。UV-B辐射处理后,间期细胞肌动蛋白纤丝排列紊乱;前期细胞周质中肌动蛋白纤丝变为短的片段,可见点状荧光随机分布在周质;中期细胞荧光强度明显减弱,明亮的的微丝片段消失,点状荧光也消失;有丝分裂后期到末期,成膜体区域肌动蛋白纤丝或成弥散状或完全消失,且出现落后染色体,染色体桥,不均等分裂及三束分裂等染色体畸变类型和异常分裂现象。
The seedlings of wheat(Jin mai 8)were exposed to He-Ne laser irradiation with 5mW·mm-2 Power density after enhanced UV-B radiated(10.08kJ·m-2·d-1). Microfilaments (MFs) were observed by confocal laser scanning microscopy (CLSM) in the wheat mesophyll protoplast and wheat root-tip cells treated with FITC- Phalloidin . The results were showed as follows:
1. We can gain a large number of protoplasts using appropriate types of enzymes and low enzyme concentrations (cellulase R-10 1.5% and pectinase Y-23 0.1%), at the appropriate temperature (26℃), and digestion time (3h ).
2. The actin in wheat mesophyll protoplast was studied by SDS-PAGE electrophoresis analysis .The results showed that the contents of actin were affected in a certain extent after UV-B radiation; but the He-Ne laser can reduce the UV-B radiation damage.
3. Microfilaments (MFs) were observed by confocal laser scanning microscopy (CLSM) in the wheat mesophyll protoplast treated with FITC- Phalloidin .Two types of MFs bundles could be recognized(CK): those beneath the plasma membrane running parallel to each other; those surrounding the chloroplast. When the wheats were treated by enhanced ultraviolet-B radiation(B), the distribution pattern of MFs was changed into bundles or granules. Part of the actin network is disrupted treated with compound treatment (BL). The distribution of MFs have become shorter and arrange crossing networks by He-Ne laser irradiation(L).
4. The distribution of microfilaments (MFs) were examined by confocal laser scanning microscopy (CLSM) in the wheat root-tip cells under the enhanced ultraviolet-B (10. 08 kJ·m - 2·d- 1 ) radiation treated with FITC- Phalloidin .The results showed that under the condition of the enhanced ultraviolet-B radiation, distribution of actin MFs were disordered in interphase cells; The arrays of MFs disintegrated into short fragments of random during prophase; Fluorescence intensity significantly decreased and bright fragments of microfilaments disappeared in Metaphase cells; MFs disappeared in the phragmoplast regions when cells were in anaphase and telophase time . And some types of chromosome aberration were induced ,including Chromosomal logging , chromosomal bridge and partition-bundle division .
引文
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