雪莲愈伤组织蛋白质提取及双向电泳分析  

Analysis of Proteome Changes in Cold-acclimated Saussurea involucrata Callus by an Improved Efficient Protein Extraction Method

作　　者：金德善[1] 卢存福[1] 兰小中[2] 刘雪萍[1] 乔佩[1] 李红英[1] 陈玉珍[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京林业大学林木育种国家工程实验室,林木花卉遗传育种教育部重点实验室,北京100083 [2]西藏大学,西藏农牧学院,西藏林芝860000

出　　处：《西北植物学报》2012年第9期1903-1909,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家科技支撑计划项目(2011BAI13B06);北京市自然科学基金(6112016)

摘　　要：以产自西藏绵头雪莲为材料,分别采用TCA/丙酮法、尿素法和酚法,提取雪莲愈伤组织蛋白质并进行双向电泳,对蛋白产量和纯度以及电泳图谱进行比较。结果表明:(1)酚法较TCA/丙酮法和尿素法获得的蛋白质更纯,杂质少,蛋白点多且分辨率较高,在二维电泳图谱中背景清晰,横纹和纵纹较少。(2)对酚法提取的蛋白质样品进行上样量的比较结果显示,17cm胶条500μg上样量二维图谱的背景和蛋白点分布较好。(3)用0℃处理雪莲愈伤组织12h,利用建立的双向电泳体系分析结果发现,有33个蛋白点比常温(23℃)上调1.5倍表达,有5个蛋白点的表达下调2倍。(4)质谱鉴定结果表明,低温诱导的蛋白包括NADP-依赖型异柠檬酸脱氢酶、腺苷高半胱氨酸酶、抗性RPP8类蛋白、蛋白点gi|13129470、α-微管蛋白,分别与新陈代谢、植物防御、能量代谢、细胞的结构蛋白相关。Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore,the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. To understand mechanisms in cold resistance of alpine plants, a proteomic analysis of callus from a Tibet alpine plant Saussurea involucrata grown at 0℃ was performed. Three improved protein extraction methods, i. e. TCA/aceton extraction, urea/thiourea extraction and phenol extraction, were adopted to extract proteins from samples. The prepared protein samples were separated by two-dimensional electrophoresis, followed by colloidal Coomassie Blue G-250 staining. The three methods were compared in protein yield,protein purity,and electrophoregram. The results showed that. （1）Most proteins can be extracted by the phenol protein extraction. In addition, the background is more clear, and less longitudinal and transverse stripes exist. （2）About 500 μg protein sample was the most appropriate for 17 cm IPG strip. （3） 2D-MALDI-TOF analysis of the callus samples resulted in the identification of 38 proteins that were differentially accumulated during cold acclimation （0℃ for 12 h）. 33 proteins were up-regulated by 1.5 fold and 5 proteins were down-regulated by 2 fold. （4）NADP-dependent isocitrate dehydrogenase,adenosylhomocys-teinase, resistance protein RPPS-like protein, hypothetical protein gi113129470, and alpha-tubulin were induced in cold-acclimated callus. The proteins are related to metabolism, plant defense, energy metabolism, and cell structure, respectively.

关 键 词：雪莲 双向电泳 样品制备 蛋白质组 

分 类 号：Q946.1[生物学—植物学]