【作者】 饶桂荣；

【导师】 杨继华； 薛妙男；

【作者基本信息】 广西师范大学 ， 有机化学， 2000， 硕士

【摘要】 我们选取经过人工自花授粉3天后的沙田柚(Citrus grandis VarShatirtyu Hort)花柱为实验材料，切取1/2部位处花柱组织，研磨成匀浆后，进行抽提和硫酸铵盐析，得到35％级分的蛋白质粗提液，此液经聚丙烯酰胺凝胶电泳检测，显示出9条蛋白带，其中靠近正极端的两条蛋白带占较大优势。此液经CoA—Sepharose4B亲和柱层析，特异峰(S~#)仅显示一条蛋白带，恰好是35％级分中近正极端的两种蛋白中的一种最优势蛋白。对其部分生化性质测定定结果为：该蛋白为碱性糖蛋白，糖含量为92％,SDS—PAGE的实验结果表明它由两个亚基组成，分子量分别为38.0kD、32.0kD，等电点分别约为7.2、6.9。此外，作者还对其进行了生物学活性测定，该糖蛋白能抑制离体(in vitro)萌发白花花粉的花粉管的生长，并在一定范围内，加入的蛋白量越多，抑制作用也越强。以酵母rRNA为底物，测其核酸酶活性，酶活为118.3u／mg，这说明该糖蛋白有强烈降解RNA的能力。以上实验结果与目前国内外大量资料报道的控制自交不亲和相关蛋白(S—蛋白)的性质极为吻合，我们是在充分了解沙田柚配子体自交不亲和的结构基础和生化基础上着手此实验的。因此，我们可以准确的推测该糖蛋白为控制沙田柚自交不亲和的S—(糖)蛋白，根据此实验结果，作者还详细讨论了在沙田柚自交不亲和过程中S—蛋白的的重要作用机制。这极可能是：沙田柚自花授粉3天后，花柱S—蛋白大量被表达，此时，萌发花粉的花粉管正在不断向前伸长，但S—蛋白能选择地进入花粉管的内部降解其rRNA，从而使得管内蛋白质合成受阻而停止生长，最终导致不亲和反应。 另外，作者采用了SDS—PAGE技术对沙田柚的茎、叶、花萼、花瓣、花药、花柱和子房的可溶性蛋白进行比较分析，检测到茎中的31.0kD，叶中56.0kD，花药中的59.0kD、58.0kD、44.0kD、37.2kD、36.0kD、35.5kD、33.0kD、21.0kD、15.4kD，花柱中的43.0kD、38.5kD，子房中的33.5kD分别为各器官的特异蛋白质。对茎、叶、花药、花柱的全蛋白进行IEF—SDS—PAGE双向电泳分析结果也确定了相应于各自SDS—PAGE上特异蛋白带的特异斑点，而且，SDS—PAGE图谱上的一条蛋白带在IEF—SDS—PAGE双向图谱上呈现等电点不同而分子量相同的几个斑点，如花药的SDS—PAGE上的21.0kD和15.4kD蛋白带在IEF—SDS—PAGE分别为三个斑点、两个斑点。各器官的蛋白质含量以花药的最高，花萼的为最低。此实验结果，将为建立沙田柚特异蛋白的识别和鉴定体系，并进而对其特异蛋白基因的克隆和表达，提供较好的实验依据。更多还原

【Abstract】 S~es of do-us grardic r.shatitry~i Hoit ~e choosed as experhnattal material, which wew in only three days tlmgh artificial pollination of their own. About b2 style tisst~ ~ cut, and iriturated as homogeneity. 35% protein soluticm was gained by use of abstraction mid salting out. The solution showed nine protein brands through PAGE, but it was ob~iow that there was two dominative protein brands near positive anode. The solution showed only one protein brand (S~ peak) through ConA-Sepharose 4B affinitive chromotography column, which ~ one of die two exceptional proteins. We did experiments in order to determine die S~ peak protein’s part biochemical natures. The results: S~ peak protein ~ basic glycoprotein,sugar accouting fcr 924~and it ~ made ~ of two subunits through SDS-PAGE. whose molecular weight were 38.0k!). 32.OkD and iso-electric point 72. 6.9. In addition, S~ peak protein’s biochemical activity was experimented S~ protein could restrain the gro~1h of pollen tubes which germinated in vitro. The restrainarrt role was stronger v?th more S~ protein in certain extent We took for Yeast’s rRNA as substrate to determine S~ protein’s nuclease activity which was 11 8.3u/mg.The final fact was diat S~ protein could degtadate RNA strongly. Above experimental resuhs coincided v~idi reported S-glycoproteins associated whh selfirrcompan’oility. After we enough understood Citrus grc~iis Var.shaiinyu Ho~t gametophytic self incompatibility smicttne and biochemical bases did we begin doing experimentSo we could deduce exactly diat die S~ glycopn)tein we ptnified is S-protein associated ~viui Citrus gram~lis \~shc~j I-bit gametophytic self-incompatibility From what we had done,we dieussed minutely S~ protein’s important role dtning the reaction of Citrus grandis Var.shatinyu Hort gametophytic self incompanl,ilitymar was, afcer artificial pollination to Citrus grcn~dic Var.shat~i Hort in three days, S-protein of style was translated and expressed in magnitxle. In the same time, pollen tubes of germinated pollen were grov~ing foreheacL But S-protein could enter speflcally into pollen tubes and degrariate its rRNA. Finally, pollen tubes stoped growing because its proteins were not expressed,.and die reaction of Citrus grandis Var.shatfrzyu Hort gametophytic selfincompaffbility occineiMorever, Soluble proteins were analyzed by use of the techr4e of SDS-PAGE, which were extracted fiom various organs (stem. leafs sepia]. petaL stamen.. style. ovaiy) of Citrus ra’idis \~rShaz~i Hort.The majorities of the proteins were expressed in these organs. But there are some proteins specific to one organ,They were 31.0W in stem,56.OkD in leaf,59.OkD, 58.OkD.. 44.OkD..37.0W.36.0W. 35.5k!). 33.0k!), 21.0k!), 15.4k!) in stamen,43.OkD, 38.5k!) in style,33.SkD in ovaxylEF-SDS two-dimensional electrophoresis showed the total proteins of die stem. leaf.stamen. style. Many 51)5-PAGE proteins bands represented several proteins dots in LEF-SDS-PAGE.Proteins ananvsis of the various organs showed that stamen contained the gtealest amount and septa] the least amount更多还原