杨树叶片蛋白质双向电泳图谱的建立  

Two-dimensional Electrophoresis Profiles for Proteins of Poplar Leaf

作　　者：索慧英 郑密 卢晗 曲冠证[3] 李莹 SUO Hui-ying;ZHENG Mi;LU Han;QU Guan-zheng;LI Ying(College of Life Science,Northeast Forestry University,Harbin 150040,Heilongjiang,China;Harbin Sport University,Harbin 150008,Heilongjiang,China;State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University Harbin 150040,Heilongjiang,China)

机构地区：[1]东北林业大学生命科学学院,黑龙江哈尔滨150040 [2]哈尔滨体育学院,黑龙江哈尔滨150008 [3]东北林业大学林木育种国家重点实验室,黑龙江哈尔滨150040

出　　处：《林业科学研究》2020年第2期128-137,共10页Forest Research

基　　金：国家自然科学基金(31300554);中央高校基本科研业务费专项资金项目(2572018BW04);东北林业大学林木遗传育种国家重点实验室创新项目(2013B08)。

摘　　要：[目的]探究适用于杨树叶片蛋白质组学研究的双向电泳体系,建立杨树叶片蛋白质的双向电泳图谱。[方法]本研究以小黑杨(Populus simonii×P. nigra)叶片为材料,对小黑杨叶片双向电泳体系的2D裂解液、蛋白的纯化、IPG胶条的pH范围、蛋白上样量、等电聚焦时间和SDS(Sodium Dodecyl sulfate)平衡时间进行优化。利用优化后的双向电泳体系分别对小黑杨、大青杨和84 K杨叶片蛋白质进行双向电泳实验。[结果]采用2D裂解液Ⅱ对小黑杨叶片蛋白质进行溶解,可显著提高蛋白的溶解性,双向电泳图谱中可分辨出326个蛋白质,比采用2D裂解液Ⅰ溶解的蛋白样品多209个蛋白质。通过蛋白裂解液提取小黑杨叶片蛋白质,利用2D clean-up试剂盒法对蛋白样品进行纯化,所得的双向电泳图谱背景清晰、蛋白质的分离效果较好。小黑杨叶片蛋白质主要集中在pH 4~7内,选用24 cm、pH 4~7的线性IPG胶条进行双向电泳,可获得分离效果较好的393个蛋白质。蛋白上样量提高至1 mg时,可分辨的蛋白质的个数明显增加,由326个增加至454个。10 000V的等电聚焦时间为13 h,SDS平衡时间为40 min时可得到背景清晰、蛋白质个数多、分辨率较高的双向电泳图谱;采用优化后的双向电泳体系对小黑杨、大青杨(Populus ussuriensis Kom.)和84 K杨(Populus alba×P.glandulosa)叶片蛋白质进行双向电泳实验,分别可检测到531、828、525个蛋白质,且蛋白质分离效果好、图谱分辨率高。[结论]优化后的双向电泳体系提高了小黑杨叶片蛋白质双向电泳图谱的分辨率和重复性,采用优化后的双向电泳体系成功的建立了小黑杨、大青杨和84 K杨叶片蛋白质的双向电泳图谱,该体系适用于小黑杨、大青杨和84 K杨叶片蛋白质组学的分析,为杨树叶片蛋白质组学的研究奠定了基础。[Objective] To explore a two-dimensional electrophoresis(2-DE) system suitable for proteomics research of poplar leaf and to establish 2-DE profiles for proteins of poplar leaf. [Method] 2-DE was used to conduct the optimization of 2 D lysis buffer, the purification of protein, the immobilized pH gradient(IPG), sample loading amount, isoelectric focusing(IEF) time and the equilibration time of the 2-DE system of Populus simonii×P. nigra leaves. 2-DE experiments for proteins of P. simonii×P. nigra, P. ussuriensis and P. alba×P. glandulosa leaves were carried out using optimized 2-DE system. [Result] The solubility of proteins of P. simonii×P. nigra leaves could be significantly improved by using 2 D lysis buffer II. There were 326 proteins detected by using 2 D lysis buffer II,which were 209 more than that detected by using 2 D lysis buffer I. The proteins of P. simonii×P. nigra leaves were extracted by plant protein lysis buffer and purified by 2 D clean-up kit. The background of the 2-DE profile was clear and the protein separation effect was better. The proteins of P. simonii×P. nigra leaves was mainly distributed in the range of pH 4~7, and 393 proteins could be obtained with good separation using IPG strips of 24 cm in length with the pH ranges from 4 to 7. The proteins detected increased from 326 to 454 when the proteins loading amount was up to 1 mg. With the condition of 6 h IEF time of 10 000 V and 40 minutes of Sodium Dodecyl Sulfate equilibration time, the background of 2-DE profile was more clear, more proteins were detected and the resolution was higher.There were 531, 828 and 525 proteins, which were detected from P. simonii×P. nigra, P. ussuriensis and P. alba×P.glandulosa leaves separately by using optimized 2-DE system. The separation of the protein was clear and the resolution was high. [Conclusion] This study optimized the key steps of 2-DE system of protein of P. simonii×P. nigra leaves and establish 2-DE profiles for proteins of P. simonii×P. nigra, P. ussuriensis and P. alba×P. gla

关 键 词：小黑杨 蛋白质 双向电泳 优化 

分 类 号：S722[农业科学—林木遗传育种]