糖对黄瓜子叶节培养物成花的影响及其差异蛋白质组的研究
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摘要
本实验通过黄瓜离体子叶节花芽分化实验体系,初步探索糖种类和浓度对植物成花的影响;并应用蛋白质组学方法和生物信息学技术,在蛋白质水平上探讨植物成花的机理。实验结果如下:
1、组织培养实验结果显示:无糖或高糖处理不利于成花。在添加0.3mg/L PP_(333)的基础上,1.5%蔗糖+1.5%果糖处理的直接花分化率最高;成花培养中蔗糖是基本的因素。果糖的最佳供应时间在开始培养后的48-72h。在供糖基础上,添加GA_3处理对成花的影响不明显;添加ABA处理可提高直接花分化率;适度抑制乙烯有利于成花。
2、建立并优化双向电泳实验条件,结果表明:应采用酚抽提法制备蛋白质干粉,第一向等电聚焦固相pH梯度胶条(24cm)的最适蛋白上样量为1200μg,第二向SDS-PAGE使用11.5%T的分离胶等条件,有助于提高黄瓜离体子叶节总蛋白质双向电泳的分辨率和重复性。
3、通过对PP_(333)诱导的黄瓜离体子叶节花芽分化的差异蛋白研究,经双向电泳检测到蛋白点有1000个左右,质谱分析鉴定出15个差异表达蛋白点,其中可确认有意义的差异蛋白点有5个,分别为14-3-3蛋白(R2)、核酮糖-1,5-二磷酸羧化酶大亚基(R5和R9)、甲硫氨酸合酶(R10)、氨基肽酶(R16)和60S酸性核糖体蛋白(R20)。这些蛋白与光合作用、代谢作用、翻译过程及信号转导等关系密切,可能在黄瓜成花中起着不同的作用。
In this paper, the effect of sugars on flowering was tentatively explored during the course of flower bud differentiation of cucumber cotyledonary nodes cultured in vitro by histological method. The mechanism of flowering was studied by the way of proteomics and biology informatics.
1、The results of tissue culture experiments showed that the flowering efficiency of cucumber cotyledonary nodes treated with sugar-deletion or sugar of high concentration were all unfavourable. By supplement with 0.3mg/L PP_(333), the efficiency of direct floral bud formation was the best when the culture medium was supplied with 1.5% sucrose and 1.5% fructose, sucrose was the basal factor in flowering in vitro. The optimum time for pulse-treatment with fructose was the duration of 48-72 hours after the seedlings were decapitated. On the base of sugar supplement, the treatment of GA_3 didn't obviously change the efficiency of flowering. The treatment of ABA increased the efficiency of direct floral bud formation. Moderate inhibition of ethylene production and/or action could help to flowering.
2、2-DE (two-dimensional eletrophoresis) protocol suited for the separation from cucumber cotyledonary nodes was established. Experiment was showed that: The method of preparing proteins powder was phenol extraction. The optimum loading quantity of sample protein was 1200μg in isoeletric focusing. The optimunm concentration of separation gel was 11.5%T. The optimum protocol of cucumber cotyledonary nodes markddely increased resolution and repeatability of 2-DE of cucumber cotyledonary nodes.
3、Differentially proteomic approaches using 2-DE were adopted to identify differentially expressed proteins from cucumber cotyledonary nodes treated with PP_(333). Nearly 1000 proteins were reproducibly detected. Analysis with MALDI-TOF-TOF/MS showed that 15 differentially expressed proteins including 5 important proteins, 14-3-3 protein (R2), ribulose 1,5-bisphosphate carboxylase large subunit(R5 and R9), methionine synthase(RlO), putative aminopeptidase(R16), 60s acidic ribosomal protein(R20). All of these proteins had a close relation to photosynthesis, metabolism, protein translation process and signal transduction. They were playing different parts in cucumber flowering.
1、组织培养实验结果显示:无糖或高糖处理不利于成花。在添加0.3mg/L PP_(333)的基础上,1.5%蔗糖+1.5%果糖处理的直接花分化率最高;成花培养中蔗糖是基本的因素。果糖的最佳供应时间在开始培养后的48-72h。在供糖基础上,添加GA_3处理对成花的影响不明显;添加ABA处理可提高直接花分化率;适度抑制乙烯有利于成花。
2、建立并优化双向电泳实验条件,结果表明:应采用酚抽提法制备蛋白质干粉,第一向等电聚焦固相pH梯度胶条(24cm)的最适蛋白上样量为1200μg,第二向SDS-PAGE使用11.5%T的分离胶等条件,有助于提高黄瓜离体子叶节总蛋白质双向电泳的分辨率和重复性。
3、通过对PP_(333)诱导的黄瓜离体子叶节花芽分化的差异蛋白研究,经双向电泳检测到蛋白点有1000个左右,质谱分析鉴定出15个差异表达蛋白点,其中可确认有意义的差异蛋白点有5个,分别为14-3-3蛋白(R2)、核酮糖-1,5-二磷酸羧化酶大亚基(R5和R9)、甲硫氨酸合酶(R10)、氨基肽酶(R16)和60S酸性核糖体蛋白(R20)。这些蛋白与光合作用、代谢作用、翻译过程及信号转导等关系密切,可能在黄瓜成花中起着不同的作用。
In this paper, the effect of sugars on flowering was tentatively explored during the course of flower bud differentiation of cucumber cotyledonary nodes cultured in vitro by histological method. The mechanism of flowering was studied by the way of proteomics and biology informatics.
1、The results of tissue culture experiments showed that the flowering efficiency of cucumber cotyledonary nodes treated with sugar-deletion or sugar of high concentration were all unfavourable. By supplement with 0.3mg/L PP_(333), the efficiency of direct floral bud formation was the best when the culture medium was supplied with 1.5% sucrose and 1.5% fructose, sucrose was the basal factor in flowering in vitro. The optimum time for pulse-treatment with fructose was the duration of 48-72 hours after the seedlings were decapitated. On the base of sugar supplement, the treatment of GA_3 didn't obviously change the efficiency of flowering. The treatment of ABA increased the efficiency of direct floral bud formation. Moderate inhibition of ethylene production and/or action could help to flowering.
2、2-DE (two-dimensional eletrophoresis) protocol suited for the separation from cucumber cotyledonary nodes was established. Experiment was showed that: The method of preparing proteins powder was phenol extraction. The optimum loading quantity of sample protein was 1200μg in isoeletric focusing. The optimunm concentration of separation gel was 11.5%T. The optimum protocol of cucumber cotyledonary nodes markddely increased resolution and repeatability of 2-DE of cucumber cotyledonary nodes.
3、Differentially proteomic approaches using 2-DE were adopted to identify differentially expressed proteins from cucumber cotyledonary nodes treated with PP_(333). Nearly 1000 proteins were reproducibly detected. Analysis with MALDI-TOF-TOF/MS showed that 15 differentially expressed proteins including 5 important proteins, 14-3-3 protein (R2), ribulose 1,5-bisphosphate carboxylase large subunit(R5 and R9), methionine synthase(RlO), putative aminopeptidase(R16), 60s acidic ribosomal protein(R20). All of these proteins had a close relation to photosynthesis, metabolism, protein translation process and signal transduction. They were playing different parts in cucumber flowering.
引文
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