【作者】 李惠敏；

【导师】 秦新民；

【作者基本信息】 广西师范大学 ， 生态学， 2004， 硕士

【摘要】 本研究探讨了沙田柚(Citrus grandis var.Shatinyu Hort.)花粉离体(in vitro)萌发的优化条件；对超声波破碎花粉管提取其可溶性蛋白的方法也进行了研究；并且利用了灵敏度高、样品需求量少的双向电泳结合银染技术，对沙田柚花粉自交、异交条件下离体萌发的花粉管可溶性蛋白的电泳格局的差异进行了分析；另外，还分析了沙田柚成熟花粉萌发前后三种同工酶（即过氧化物酶、酯酶、淀粉酶）的类型和活力变化的情况。为沙田柚配子体自交不亲和性在雄蕊方面的研究提供了一些新的实验证据，并且为最终分离、鉴定沙田柚花粉S蛋白打下了基础。研究结果发现贮藏时花粉的含水量、撒播密度对花粉萌发率和萌发速度的影响至关重要。所贮藏花药的含水量保持在11.0~12.5%范围内，每个加有1.5ml培养液的凹面皿(?5㎝)中，一般花粉的撒播量为8~12个花药，或者每片滴加0.5ml培养液的载玻片上，花粉的撒播量为6~8个花药；培养温度28~30℃，这种情况下培养的花粉，其萌发率最高，可达到80~90%的萌发率。另外，蔗糖的含量在10~20%之间条件下，花粉的萌发均较好。其中以12%蔗糖浓度的BKS培养液作为花粉管培养基，花粉不仅有较好的萌发率，花粉管也长得较整齐。SDS-PAGE检测利用超声波破碎花粉管提取其可溶性蛋白的效果，结果表明：与常规研磨法相比，超声波提取法结果稳定、可靠性强、操作简便、大大缩短提取时间。经过比较，以选用600W功率，超声时间3s/次，间歇时间6s/次，分三个回合共工作120次，每回合之间相隔10分钟的组合效果最好。采用这一组参数进行超声波破碎花粉管以提取其可溶性蛋白时，镜检发现90%的花粉管断裂成碎片，但花粉粒几乎没有被破碎。对沙田柚自交、异交条件下花粉管可溶性蛋白的电泳结果进行分析和比较，结果发现，在SDS-PAGE单向电泳图谱上检测到自交条件下相对分子量为22.0ku和32.5ku的两条带不同于异交条件下蛋白图谱；而异交下条件下出现了分子量为44.8ku的差异蛋白。在IEF-PAGE的图谱上，检测到pI为5.85和6.20的条带为自交花粉管的特异蛋白谱带。对自交、异交条件下的花粉管可溶性蛋白的IEF/SDS-PAGE双向电泳图谱分析也确定了相似于单向电泳上的特异蛋白带的蛋白斑点。比较分析沙田柚自交、异交花粉管蛋白的双向电泳图谱，两者的蛋白分布格局相似，具有重叠性，可分辨出200多个蛋白点。在已知花柱与自交不亲和有关的蛋白处于的pI范围5.00~7.50之间，考察该范围内自交、异交条件下的蛋白点的变化或差异情况，多次重复实验的结果发现：在异交花粉管电泳图谱中出现了1种特异蛋白（A），A蛋白（MrA=58.2ku，pI=5.90）；在自交花粉管电泳图谱中出现了2种特异蛋白（B、C），B蛋白（MrB=26.4ku，pI=6.10）,C蛋白（MrC=28.0ku，pI=6.30），这些蛋白可能是沙田柚花粉管中与自交不亲和有关的蛋白。鉴于现有的关于配子体型自交不亲和性的两种模型学说，我们的结果从一个方面支持了“核酸酶抑制剂”模型。沙田柚花粉萌发前后同工酶变化分析结果显示：萌发前后花粉的过氧化物酶、淀粉酶同工酶种类变化不大，但萌发后花粉的酶活力比未萌发前高；萌发前后酯酶同工酶类型差异较大，但均表现较高的酶活性，这说明萌发过程中，代谢活动比较活跃，酶的活性也增强。更多还原

【Abstract】 In this study we discussed the better conditions for the pollen culture of Citrus grandis var. Shatinyu Hort. in vitro and made some research on the method of ultrasonic waves on extracting proteins from the pollen tube．In addition，we established the two-dimensional electrophoresis analysis technology that is highly sensitive and demands less loading．Using the two-dimensional electrophoresis together with silver staining, we compared and analyzed the proteins extracted from self and non-self pollinated pollen tube．Moreover，the types and activity of three kinds of isoenzymes of Shatinyu pollen，i.e. peroxidase(POD)，amylase(Amyl)and esterase(Est)were analyzed with vertical slab PAGE and by specific cytochemical staining during the process of pollen germination．So here we provided some new data for GSI(Gametophytic Self Incompatibility)about pollen and have done some basic work to finally identify the male determinate of GSI in Shatinyu．Pollen that has been stored at -70℃ was used in the experiments to determine the time course of pollen tube growth，the effects of pollen density on germination rate of pollen and the effects of osmotic potential of the medium on pollen tube growth．Finding that it is most important considering the moisture content of pollen，the temperature under storing and the density of pollen when sowed in the culture medium．We got the highest germination rate about 80％ to 90％ when meeting the needs such as the moisture content of pollen ranges from 11.0% to 12.5% during the culture of pollen tube．To maximize pollen tube growth，we also optimized pollen density and the concentration of sucrose．The medium contained 12% sucrose is better though the pollen is to be well germinated under 10% to 20% sucrose．The density of pollen required to achieve the highest growth rate was 8 to 12 anthers’ pollen in concave utensil(?5㎝) with 1.5ml culture medium or 6 to 8 anthers’ pollen in microscope slides with 0.5ml culture medium．Both the concave utensil and the microscope were incubated in the dark in the plate with wet paper at 28℃ to 30℃．Measured the extracts by SDS-PAGE after the pollen tubes were processed by ultrasonic waves．Compared with the routine method，the efficiency of extracting proteins from pollen tubes was greatly enhanced and the result displays more stable and dependable by ultrasonic process．So proteins extracted by ultrasonic process is significantly higher than that extracted by the routine method．We did the experiment with a series of parameters to find out which were better for the result．After having done 120 times as ultrasonic waves lasts 3 seconds every 9 seconds under 600W power，90 percent <WP=4>pollen tubes were crashed into shatters but the microspores were almost integrated．Two specific proteins with molecular weights of 22.0ku and 32.5ku were detected in self-pollinated pollen tube while another specific protein about 44.8ku was less than the protein owing the same Mr in cross-pollinated pollen tube by SDS-PAGE．In addition，there were two bands whose pI are 5.85 and 6.20 found in self-pollinated pollen tube by the method of IEF-PAGE．Compared and analyzed the patterns of pollen tube proteins between self and non-self pollinated in the two-dimensional electrophoresis，their proteins’ distribution is similar and shows overlapping．More than 200 spots are differentiated on the gel．Two special proteins spots were detected in self-pollinated pollen tube electrophoresis．Their molecular weights are 28.0ku and 26.4ku，respectively．Their pIs are 6.30 and 6.10 approximately．However，a 58.2ku protein whose pI is about 5.90 was different from self-pollinated pollen tube in the two-dimensional electrophoresis of cross-pollinated．These three kinds of proteins from pollen tube of Shatinyu maybe interrelate to self-incompatibility．Herein the two hypothesis about GSI，our result supports the RNase inhibitor model rather than the receptor or gatekeeper model on the one hand．Compared the types and the activity of three kinds of isoenzymes of Shtinyu germ更多还原