无核荔枝胚败育相关蛋白质的分离鉴定及cDNA克隆
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摘要
关于荔枝胚胎发育的研究,我国学者的研究较为深入,主要集中于形态解剖学和生理生化研究水平。形态学和解剖学的研究表明,胚胎败育型荔枝在授粉后10d左右,其胚胎发育受阻,花后15d,胚珠已明显萎缩导致果实无核。荔枝胚胎发育过程中内源激素变化表明,败育胚胎中IAA、GA3、CTK等促进型激素低于正常发育胚胎,而抑制型激素ABA则高于正常胚胎。败育胚胎内源激素与正常胚胎的差异,可能造成生理代谢紊乱及物质能量的亏损而导致果实无核。目前关于荔枝胚胎发育的基因调控及蛋白质组研究较少,仅陈伟及张以顺等采用双向电泳技术进行了荔枝胚胎发育后期总蛋白质的分离,还没有进一步的蛋白质组分析及鉴定研究。张以顺等克隆到一个与荔枝胚败育相关的S-腺苷甲硫氨酸合成酶基因。目前尚无更多的相关研究。
海南无核荔枝是荔枝中的珍稀品种,其无核率高达95%,但其无核率极易受花期温度的影响,无核率的不稳定性严重影响了该品种的推广种植和经济效益。形态学观察表明,海南无核荔枝无核果实的形成是由于授粉后低温的影响导致胚胎败育所引起。
海南无核荔枝经中国热带农业科学院和华南热带农业大学国家重点实验室鉴定,认为无核荔枝是一个突变体,可能由于某些基因发生突变,使胚胎发育早期的生化过程发生紊乱或受到抑制,导致胚胎败育。目前已对海南无核荔枝进行了部分应用基础方面的研究,包括胚退化的生物学,胚内源激素动力学,与无核荔枝花、果、胚发育相关基因的克隆等,为无核荔枝的产业化提供了有力的科技支撑。
蛋白质作为基因功能的体现者和执行者,与生命体的生物功能和生理活动密切相关。机体所处的不同环境和本身的生理状态差异,会导致基因转录产物有不同的剪切和转译成不同的蛋白。因而蛋白质组学已成为当今研究热点。采用蛋白质组学技术及方法获得野生型与突变体间的差异蛋白质,对于目的蛋白质的功能及其编码这些蛋白的基因的功能研究,具有重要的意义,也为进一步探讨目的蛋白质与生物功能及生理活动的关系奠定了基础。
本研究从差异蛋白质组学入手,分别提取无核荔枝开花后20d和开花后40d正常发育胚和败育胚的总蛋白质,采用二维聚丙烯酰胺凝胶电泳技术,得到无核荔枝
Litchi chinensis Sonn is an important agricultural product of subtropical Southern China. Hainan seedless Litchi is an excellent variety with a high seedless rate of 95%. Unfortunately, the seedless rate is unstable and dependent on the temperature during flowering stage. The unstable seedless rate seriously impacts the seedless Litchi's planting area and its commercial value. Although there are many morphological biochemical studied during embryo developing of litchi, there are few studies on the proteins and proteomatic relating to the embryonic development of litchi.
The purpose of this research is to isolate the proteins of seedless Litchi at the different embryo development stages by using the two-dimensional polyacrylamide gel electrophoresis (2DE) in order to isolate the differential proteins between normal embryo and aborted embryo. The next step is to identify these proteins by using the MALDI-TOF MS search homologous protein and obtain their sequence through the protein data bank.
The major results of the study are as follows:
1. From 2DE electrophoresis maps, we compared the global protein patterns between normal and aborted embryo of litchi at the 20 DAA (day after anthesis) through computer image analysis. In total, 129 and 130 protein spots were obtained in the normal and aborted embryo maps, respectively. Of these, 24 spots were not significantly different, while 14 spots increased and 21 spots decreased in quantity. Furthermore, some spots were missing and some new spots emerged in aborted embryo compared with the normal embryo. The differential proteins between normal and aborted embryo of litchi are useful for regulating and controlling embryo abortion.
2. We compared the global protein patterns between normal and aborted embryo of litchi at the 40 DAA. The result shows, the rate of the differential proteins were increased compared with the 20 DAA. 388 and 482 protein spots were obtained in normal and aborted embryo map, respectively, of which 60 spots were not significantly different, while 46 spots increased and 54 spots decreased in quantity. Furthermore, some spots
海南无核荔枝是荔枝中的珍稀品种,其无核率高达95%,但其无核率极易受花期温度的影响,无核率的不稳定性严重影响了该品种的推广种植和经济效益。形态学观察表明,海南无核荔枝无核果实的形成是由于授粉后低温的影响导致胚胎败育所引起。
海南无核荔枝经中国热带农业科学院和华南热带农业大学国家重点实验室鉴定,认为无核荔枝是一个突变体,可能由于某些基因发生突变,使胚胎发育早期的生化过程发生紊乱或受到抑制,导致胚胎败育。目前已对海南无核荔枝进行了部分应用基础方面的研究,包括胚退化的生物学,胚内源激素动力学,与无核荔枝花、果、胚发育相关基因的克隆等,为无核荔枝的产业化提供了有力的科技支撑。
蛋白质作为基因功能的体现者和执行者,与生命体的生物功能和生理活动密切相关。机体所处的不同环境和本身的生理状态差异,会导致基因转录产物有不同的剪切和转译成不同的蛋白。因而蛋白质组学已成为当今研究热点。采用蛋白质组学技术及方法获得野生型与突变体间的差异蛋白质,对于目的蛋白质的功能及其编码这些蛋白的基因的功能研究,具有重要的意义,也为进一步探讨目的蛋白质与生物功能及生理活动的关系奠定了基础。
本研究从差异蛋白质组学入手,分别提取无核荔枝开花后20d和开花后40d正常发育胚和败育胚的总蛋白质,采用二维聚丙烯酰胺凝胶电泳技术,得到无核荔枝
Litchi chinensis Sonn is an important agricultural product of subtropical Southern China. Hainan seedless Litchi is an excellent variety with a high seedless rate of 95%. Unfortunately, the seedless rate is unstable and dependent on the temperature during flowering stage. The unstable seedless rate seriously impacts the seedless Litchi's planting area and its commercial value. Although there are many morphological biochemical studied during embryo developing of litchi, there are few studies on the proteins and proteomatic relating to the embryonic development of litchi.
The purpose of this research is to isolate the proteins of seedless Litchi at the different embryo development stages by using the two-dimensional polyacrylamide gel electrophoresis (2DE) in order to isolate the differential proteins between normal embryo and aborted embryo. The next step is to identify these proteins by using the MALDI-TOF MS search homologous protein and obtain their sequence through the protein data bank.
The major results of the study are as follows:
1. From 2DE electrophoresis maps, we compared the global protein patterns between normal and aborted embryo of litchi at the 20 DAA (day after anthesis) through computer image analysis. In total, 129 and 130 protein spots were obtained in the normal and aborted embryo maps, respectively. Of these, 24 spots were not significantly different, while 14 spots increased and 21 spots decreased in quantity. Furthermore, some spots were missing and some new spots emerged in aborted embryo compared with the normal embryo. The differential proteins between normal and aborted embryo of litchi are useful for regulating and controlling embryo abortion.
2. We compared the global protein patterns between normal and aborted embryo of litchi at the 40 DAA. The result shows, the rate of the differential proteins were increased compared with the 20 DAA. 388 and 482 protein spots were obtained in normal and aborted embryo map, respectively, of which 60 spots were not significantly different, while 46 spots increased and 54 spots decreased in quantity. Furthermore, some spots
引文
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