水稻花药中Ca~（2+）的定位与花粉细胞骨架荧光标记方法的探索
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摘要
光敏雄性核不育水稻是培育杂交稻的重要种质资源,其雄性不育的细胞学机制与很多方面有关,包括绒毡层发育、ATP酶、Ca~(2+)浓度、细胞骨架和细胞程序性死亡等。Ca~(2+)作为植物的第二信使广泛参与并调节着植物体内的生理生化反应,其在花药发育过程中对花粉形成和发育起重要作用;细胞骨架紊乱导致细胞死亡的现象比较普遍。因此,研究不育水稻花药发育过程中Ca~(2+)分布以及细胞骨架的变化与花粉败育的关系,有助于揭示光敏核不育水稻雄性不育的机理。
本文采用焦锑酸钾沉淀法,观察了光敏核不育水稻(PGMR)农垦58S和正常可育品种农垦58N花药发育过程中药隔、药壁和花粉细胞中Ca~(2+)的分布差异,分析不育花药中Ca~(2+)分布与花粉败育的关系;以水稻为研究材料,采用几种不同的方法获得花粉细胞,分别进行微丝和微管的荧光标记和观察。
主要研究结果如下:
1.花药发育过程中Ca~(2+)异常分布及其与雄性不育的关系
(1)可育花粉单核晚期细胞表面聚集丰富的Ca~(2+)沉淀,花粉壁发育完全,细胞质中Ca~(2+)颗粒很少;而不育花粉细胞表面Ca~(2+)较少,花粉外壁发育异常,不形成花粉内壁,细胞质中积累丰富的Ca~(2+)沉淀。
(2)可育花药绒毡层细胞在单核早期开始解体,单核晚期迅速解体;而不育花药在花粉母细胞时期启动解体,解体过程十分缓慢,一直持续至花粉败育。不育花药较可育花药提前形成乌氏体,但其表面无Ca~(2+)沉淀分布,直到单核晚期才有明显分布,数量少于同期可育花药。除乌氏体以外,不育花药各壁层细胞中的Ca~(2+)沉淀多于可育花药。
(3)花药发育过程中,不育花药和可育花药药隔组织中的Ca~(2+)沉淀都呈逐渐增多的趋势,同期相比,不育花药多于可育花药。
结果表明,光敏核不育水稻绒毡层细胞提早解体及乌氏体功能异常,导致Ca~(2+)向药室的运输发生障碍,致使花粉表面缺乏Ca~(2+)而引起花粉外壁形成不正常;花粉发育后期细胞质内积累大量Ca~(2+)是花粉败育的主要因素之一。
2.花粉细胞骨架荧光标记方法
(1)多聚赖氨酸粘片法:固定花药后压片,挤出花粉于涂有多聚赖氨酸的盖玻片上,对黏附在盖玻片上的花粉进行酶解和标记。结果表明,实验过程中花粉较易脱落,且一些组织碎片影响荧光观察,不易进行后期花粉标记,也不易进行微管荧光标记。
(2)研磨—离心法:固定小花后,研磨压出花粉,过滤,离心收集花粉,在离心管中对花粉进行酶解和标记。结果表明该法与粘片法相比,花粉细胞损失很少,制片上的花粉细胞分布均匀,无背景杂质影响荧光观察。只是此法不易获得早期花粉细胞。采用此法,分别设酶解时间梯度和标记液浓度梯度来摸索最佳标记条件,结果表明:标记微丝以酶解15～30min为宜,标记微管以酶解0.5～1h为宜;适当降低标记液浓度可使标记效果更好。
(3)冰冻切片法:固定小花后,进行冰冻包埋,冷冻后切片,真空粘片后脱包埋剂再进行标记。此技术克服了以上两种方法存在的问题,可以方便快捷地获得各时期的花药结构,DAPI染核效果很好,但是荧光标记效果不太理想,药壁组织细胞荧光信号很强,无法分辨花粉中微丝和微管的特异性荧光。
结果表明,粘片法和研磨—离心法虽各有优缺点,但可以用于微丝和微管的荧光标记,观察花粉发育中细胞骨架的动态变化,可以将两种方法结合起来使用,采用粘片法标记早期花粉细胞,采用研磨—离心法标记后期花粉细胞。
Photo-sensitive Genic Male-sterile Rice(PGMR) is a very important germplasm resource for cross-breeding of rice. Its cytological mechanism of male sterility including many aspects: the development of tapetum, ATPase, concentration of Ca~(2+), cytoskeleton and programmed cell death, etc. Ca~(2+)widely participate and regulate physiological and biochemical response in plants as second messengers, its very important for the formation and development of pollen during the development of anther; disorder of cytoskeleton lead to cell death is general. Therefore, researching the relationship between pollen abortion both distribution of Ca~(2+) and variation of cytoskeleton during the development of anther of rice, helpful to reveals the mechanism of male sterility for PGMR.
Potassium antimontate was adopted to investigate Ca~(2+) distribution in the anthers of photo-sensitive genic male-sterile line of rice Nongken 58S and fertile line Nongken 58N during their development, to study the relationship between Ca~(2+) distribution and male-sterile of rice. In order to investigate the relationship between the dynamic distribution of cytoskeleton and male-sterile of rice, a feasible immunofluorescence labeling method must be established firstly to observe the framework of cytoskeleton of pollen. We attempted several ways to gain pollen and made great efforts to improve experimental method, finally, a mature labeling system was established in our laboratory, by which we can label both microfilament and microtubule easily.
The main results are listed as bellows:
1. To research the relationship between Ca~(2+) distribution and male-sterile of rice
(1) In the locule of fertile anther, the calcium precipitates gradually accumulated on the surface of uninucleate pollen until pollen mature, but its lack in the cytoplasm, the pollen wall formed completely composing of exine and intine. The calcium precipitates on the surface of sterile pollen were obviously fewer than fertile pollen, but they were abundant in the degradated cytoplasm of abortive pollen, the exine formed singularly and the pollen wall composed without intine.
(2) The tapetum cells of the fertile anther began to degenerate at early microspore stage and degraded rapidly at late microspore stage, but that of sterile anther startuped degenerate at microsporocyte stage, and went on to pollen abortion. Ubish bodies began to form earlier in sterile anther than fertile anther, but with no calcium granules on them, till late microspore stage, many calcium granules appeared on the surface of them, but obviously less than in fertile anther. In sum, there were much more calcium granules in anther wall of sterile anther than in fertile anther, but the calcium granules on the surface of ubish bodies in sterile anther were much less than in fertile anther.
(3) The calcium precipitates gradually increase in parechymatous cells of both sterile and fertile anther, but there were more in sterile anther than in fertile anther.
Due to the tapetum cells degrading ahead and the abnormal function of ubish bodies, the transport of Ca~(2+) to locule occurred hindrance, induced to form abnormal exine. Abundent Ca~(2+) in the cytoplasm was one of the most factors of the pollen abortion.
2. A labeling system was established to observe the cytoskeleton of pollen.
(1) Pollen were extruded out from anther and were allowed to settle on the coverslip. The anther wall debris was removed, but difficult to clean out fully. The pollen lose easily during the process of experiment. The anther wall debris disturbed the backdrop of fluorescence. The method was not adapt to labell microtubule easily.
(2) Gain pollen through grinding and centrifugation. This was used successfully to labell both the microfilament and microtubule. During the process of experiment, only a few pollen lost, and the backdrop of fluorescence was very clean. But it was difficult to gain the early pollen.
(3) Used freezing apparatus to slice anther to flake could obtain pollen of each stage. its was easily to slice but was difficult to labell the cytoskeleton of pollen.
(4) The pollen under label were photographed under both fluorescence microscope and Confocal microscope. The results showed that the cytoskeleton under the Confocal microscope was clearer and more bright than under the fluorescence microscope.
It was obvious that these methods have virtue as well as defect. We could used two methods simultaneously to labell the pollen during its development. But it was a pity that we fail to label pollen by slicing.
本文采用焦锑酸钾沉淀法,观察了光敏核不育水稻(PGMR)农垦58S和正常可育品种农垦58N花药发育过程中药隔、药壁和花粉细胞中Ca~(2+)的分布差异,分析不育花药中Ca~(2+)分布与花粉败育的关系;以水稻为研究材料,采用几种不同的方法获得花粉细胞,分别进行微丝和微管的荧光标记和观察。
主要研究结果如下:
1.花药发育过程中Ca~(2+)异常分布及其与雄性不育的关系
(1)可育花粉单核晚期细胞表面聚集丰富的Ca~(2+)沉淀,花粉壁发育完全,细胞质中Ca~(2+)颗粒很少;而不育花粉细胞表面Ca~(2+)较少,花粉外壁发育异常,不形成花粉内壁,细胞质中积累丰富的Ca~(2+)沉淀。
(2)可育花药绒毡层细胞在单核早期开始解体,单核晚期迅速解体;而不育花药在花粉母细胞时期启动解体,解体过程十分缓慢,一直持续至花粉败育。不育花药较可育花药提前形成乌氏体,但其表面无Ca~(2+)沉淀分布,直到单核晚期才有明显分布,数量少于同期可育花药。除乌氏体以外,不育花药各壁层细胞中的Ca~(2+)沉淀多于可育花药。
(3)花药发育过程中,不育花药和可育花药药隔组织中的Ca~(2+)沉淀都呈逐渐增多的趋势,同期相比,不育花药多于可育花药。
结果表明,光敏核不育水稻绒毡层细胞提早解体及乌氏体功能异常,导致Ca~(2+)向药室的运输发生障碍,致使花粉表面缺乏Ca~(2+)而引起花粉外壁形成不正常;花粉发育后期细胞质内积累大量Ca~(2+)是花粉败育的主要因素之一。
2.花粉细胞骨架荧光标记方法
(1)多聚赖氨酸粘片法:固定花药后压片,挤出花粉于涂有多聚赖氨酸的盖玻片上,对黏附在盖玻片上的花粉进行酶解和标记。结果表明,实验过程中花粉较易脱落,且一些组织碎片影响荧光观察,不易进行后期花粉标记,也不易进行微管荧光标记。
(2)研磨—离心法:固定小花后,研磨压出花粉,过滤,离心收集花粉,在离心管中对花粉进行酶解和标记。结果表明该法与粘片法相比,花粉细胞损失很少,制片上的花粉细胞分布均匀,无背景杂质影响荧光观察。只是此法不易获得早期花粉细胞。采用此法,分别设酶解时间梯度和标记液浓度梯度来摸索最佳标记条件,结果表明:标记微丝以酶解15～30min为宜,标记微管以酶解0.5～1h为宜;适当降低标记液浓度可使标记效果更好。
(3)冰冻切片法:固定小花后,进行冰冻包埋,冷冻后切片,真空粘片后脱包埋剂再进行标记。此技术克服了以上两种方法存在的问题,可以方便快捷地获得各时期的花药结构,DAPI染核效果很好,但是荧光标记效果不太理想,药壁组织细胞荧光信号很强,无法分辨花粉中微丝和微管的特异性荧光。
结果表明,粘片法和研磨—离心法虽各有优缺点,但可以用于微丝和微管的荧光标记,观察花粉发育中细胞骨架的动态变化,可以将两种方法结合起来使用,采用粘片法标记早期花粉细胞,采用研磨—离心法标记后期花粉细胞。
Photo-sensitive Genic Male-sterile Rice(PGMR) is a very important germplasm resource for cross-breeding of rice. Its cytological mechanism of male sterility including many aspects: the development of tapetum, ATPase, concentration of Ca~(2+), cytoskeleton and programmed cell death, etc. Ca~(2+)widely participate and regulate physiological and biochemical response in plants as second messengers, its very important for the formation and development of pollen during the development of anther; disorder of cytoskeleton lead to cell death is general. Therefore, researching the relationship between pollen abortion both distribution of Ca~(2+) and variation of cytoskeleton during the development of anther of rice, helpful to reveals the mechanism of male sterility for PGMR.
Potassium antimontate was adopted to investigate Ca~(2+) distribution in the anthers of photo-sensitive genic male-sterile line of rice Nongken 58S and fertile line Nongken 58N during their development, to study the relationship between Ca~(2+) distribution and male-sterile of rice. In order to investigate the relationship between the dynamic distribution of cytoskeleton and male-sterile of rice, a feasible immunofluorescence labeling method must be established firstly to observe the framework of cytoskeleton of pollen. We attempted several ways to gain pollen and made great efforts to improve experimental method, finally, a mature labeling system was established in our laboratory, by which we can label both microfilament and microtubule easily.
The main results are listed as bellows:
1. To research the relationship between Ca~(2+) distribution and male-sterile of rice
(1) In the locule of fertile anther, the calcium precipitates gradually accumulated on the surface of uninucleate pollen until pollen mature, but its lack in the cytoplasm, the pollen wall formed completely composing of exine and intine. The calcium precipitates on the surface of sterile pollen were obviously fewer than fertile pollen, but they were abundant in the degradated cytoplasm of abortive pollen, the exine formed singularly and the pollen wall composed without intine.
(2) The tapetum cells of the fertile anther began to degenerate at early microspore stage and degraded rapidly at late microspore stage, but that of sterile anther startuped degenerate at microsporocyte stage, and went on to pollen abortion. Ubish bodies began to form earlier in sterile anther than fertile anther, but with no calcium granules on them, till late microspore stage, many calcium granules appeared on the surface of them, but obviously less than in fertile anther. In sum, there were much more calcium granules in anther wall of sterile anther than in fertile anther, but the calcium granules on the surface of ubish bodies in sterile anther were much less than in fertile anther.
(3) The calcium precipitates gradually increase in parechymatous cells of both sterile and fertile anther, but there were more in sterile anther than in fertile anther.
Due to the tapetum cells degrading ahead and the abnormal function of ubish bodies, the transport of Ca~(2+) to locule occurred hindrance, induced to form abnormal exine. Abundent Ca~(2+) in the cytoplasm was one of the most factors of the pollen abortion.
2. A labeling system was established to observe the cytoskeleton of pollen.
(1) Pollen were extruded out from anther and were allowed to settle on the coverslip. The anther wall debris was removed, but difficult to clean out fully. The pollen lose easily during the process of experiment. The anther wall debris disturbed the backdrop of fluorescence. The method was not adapt to labell microtubule easily.
(2) Gain pollen through grinding and centrifugation. This was used successfully to labell both the microfilament and microtubule. During the process of experiment, only a few pollen lost, and the backdrop of fluorescence was very clean. But it was difficult to gain the early pollen.
(3) Used freezing apparatus to slice anther to flake could obtain pollen of each stage. its was easily to slice but was difficult to labell the cytoskeleton of pollen.
(4) The pollen under label were photographed under both fluorescence microscope and Confocal microscope. The results showed that the cytoskeleton under the Confocal microscope was clearer and more bright than under the fluorescence microscope.
It was obvious that these methods have virtue as well as defect. We could used two methods simultaneously to labell the pollen during its development. But it was a pity that we fail to label pollen by slicing.
引文
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