【作者】 王春雷；

【导师】 张绍铃；

【作者基本信息】 南京农业大学 ， 果树学， 2010， 博士

【摘要】 以砂梨(Pyrus pyrifolia)品种‘幸水’(S4S5)、‘今村秋’(S1S6)为试材,利用微量花粉管线粒体分离技术、线粒体膜电位测定技术,以及透射电镜、蛋白免疫印迹、荧光标记等方法研究了离体条件下自花授粉后花粉管生长抑制和死亡的特点及相关信号传递,并在活体条件下验证了部分实验结果,主要结论如下：1.建立了一套有效的微量花粉管线粒体分离纯化技术。首先利用细胞筛研磨花粉管,破碎花粉管的同时过滤细胞溶液,避免因研磨过度导致线粒体被破坏,再综合前人分离纯化线粒体的经验,通过差速离心和密度离心,成功从微量梨花粉管中分离纯化出大量线粒体。经透射电镜、流式细胞仪检测显示,从花粉管分离得到的线粒体纯度较高,并且结构完整,同时具有很好生理活性,能够满足后续实验的需要。2.确定了活体和离体条件下自花授粉花粉管停止生长的时间。利用MTT染色法和伊文思蓝染色法分别研究了S-RNase作用下,自花花粉管丧失活力和死亡的时间,结果表明在处理30 min后,经S-RNase处理的自花花粉管具有活力和正常花粉管所占比率明显减少,随着时间的推移,这种趋势有所加强；而用苯胺蓝染色法观察活体条件下自花授粉后花粉管生长情况,结果表明花粉管生长发生抑制主要在授粉后9 h。这些数据为后面研究提供了很好的依据。3.确定了自花花粉管死亡具有程序性死亡(programmed cell death,PCD)特点。为了研究梨自花授粉不亲和性反应中自花授粉花粉管死亡是否属于PCD,利用荧光标记、透射电镜和蛋白免疫印迹等技术研究了S-RNase对自花花粉管线粒体和核DNA的影响,并且研究了类Caspase是否在S-RNase抑制自花花粉管生长反应中被激活。结果表明,在S-RNase处理30 min后,自花花粉管线粒体膜电位特异性降低,而异花花粉管和对照却没有明显变化。此外,在处理120 min,发现自花花粉管线粒体细胞色素c泄漏到细胞质,并且线粒体发生明显的溶胀现象,电子透射密度降低,脊消失,并伴随大量空泡出现。更为重要的是,不论是在离体系统中,还是在活体授粉中,自花授粉花粉管核DNA都发生降解,并且降解顺序是先营养核,后生殖核。此外,我们的研究结果表明,在S-RNase抑制自花花粉管死亡的过程中,并没有类Caspase酶被激活。据此推断自花授粉花粉管死亡可能属于PCD,但引起核降解的核酸酶性质还有待进一步研究。4.确定了S-RNase能够特异性破坏自花花粉管顶端活性氧(reactive oxygen species,ROS)梯度,发现了一条引起自花授粉花粉管核DNA降解的信号传递路径。利用荧光标记、组织定位和亚细胞定位等方法,研究了S-RNase对自花花粉管顶端ROS梯度的影响,以及ROS梯度消失引起花粉管生理代谢变化。结果表明：S-RNase通过破坏线粒体结构和降低花粉管NADPH含量,减少ROS在线粒体和细胞壁上的积累,破坏自花花粉管顶端ROS梯度,最终抑制自花花粉管生长；花粉管顶端活性氧梯度被破坏后,能够抑制花粉管钙离子通道开放、引起微丝骨架解聚,并最终引起核DNA降解。这是到目前为止,首次报道一条梨自花花粉管死亡的信号传递路径。更多还原

【Abstract】 Using Pyrus pyrifolia cultivars ’Kosui’(S4S5) and’Imamuraaki’(S1S6) as materials, and a new method of isolation mitochondria or a method of mitochondrial membrane potential stain in intact pollen tube as procedure, the features of incompatible pollen tube death and the signal transduction mechanisms of self-incompatibility were studied in vitro, and some experimental results were validated in vivo. The main results as follows:1. A new method was developed for isolation mitochondria from a few amounts of pollen tubes. The method of homogenization method usually represents the critical step affecting the yield of mitochondria isolated from plant cells. The problem of balancing total disruption of cell wall structures with need to maximize preservation of intact mitochondria places crucial emphasis on the optimization of both the intensity and the duration of homogenization. We developed a method for cell homogenization, which homogenize the pollen tube on cell sieve and purify the mitochondria by differential density centrifugation. The isolated mitochondria were tested with transmission electron microscope, flow cytometry. The results showed that the isolated mitochondria were intact and with physiological activity. What’s more important, the isolated mitochondria are of high purity. This new method was easy, fast, reliable and efficient.2. The influence of SI in incompatible pollen tubes growing was evaluated. The MTT stain and Evarisblue stain were used to evaluate the time of incompatible pollen tube losing viability and death under S-RNase challenge in vitro, respectively. The results showed that the frequencies of viable pollen tube and live pollen tube were less changed at 30 min; at 60 min, the frequencies decreased obviously, and at 120 min, the values were less than half of control. Furthermre, the pollen tubes were stained with anilin blue, the result showed that incompatible pollen tubes were arrested in style at 9 h after pollination. Those data were helpful for the following experiments.3. It was demonstrated that death of incompatible pollen tubes shared some feature of programmed cell death (PCD). To investigate whether the death of incompatible pollen tube belongs to PCD, the fluorescence stain, transmission electron microscope, western blotting were used to observe the effect of S-RNase on mitochondria and nuclear DNA, and the caspase-like activity was tested in incompatible pollen tube at the same time. The results showed that the mitochondrial membrane potential (ΔΨmit) of incompatible pollen tube was specifically collapsed by S-RNase at 30 min after SI challenge in vitro. Moreover, it was also confirmed by western blotting at 2 h after SI challenge that ΔΨmit collapse induced cytochrome c leakage into the cytosol. Swollen mitochondria had been detected by transmission electron microscopy as early as 1 h after SI challenge, the swollen mitochondria accompany with reduction of crista, reducing of electron density and large bleb. The most important, the nuclear DNA degradation was detecteded both in vitro and in vivo, and the vegetative nucleus was degraded in advance of the generative nucleus. Otherwise, the data showed that the caspase-like activity was not triggered in incompatible pollen tube under S-RNase challenge. These results suggested that the death of incompatible pollen tube might belong to PCD and caspase-like activity was not involved in nuclear DNA degradation.4. The effect of tip-localized reactive oxygen species (ROS) on SI was evaluated, and a cascade signal pathway of nuclear DNA degradation was uncovered. The results showed that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest the ROS formation in mitochondria and cell walls. The mitochondria ROS disruption was related to the mitochondria alteration, while cell walls ROS disruption was related to quantity of NADPH decrease. Tip-localized ROS disruption not only decreased the Ca2+ current, depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. This was the first time to report a cascade signal pathway of self-incompatibility in P. pyrifolia L.更多还原