甘蔗叶绿体分离及其蛋白质提取方法研究  

Isolation of chloroplasts from leaf tissue of sugarcane and extraction of their protein

作　　者：孙富[1,2] 黄巧玲[1,2] 黄杏[1,2] 张保青[1,2] 陈明辉[1,2] 杨丽涛[1,2] 李杨瑞[2,3,4,5] 

机构地区：[1]广西大学农学院,南宁530005 [2]亚热带农业生物资源保护与利用国家重点实验室,南宁530005 [3]广西农业科学院,南宁530007 [4]广西作物遗传改良生物技术重点开放实验室,南宁530007 [5]广西甘蔗遗传改良重点实验室中国农业科学院甘蔗研究中心,南宁530007

出　　处：《南方农业学报》2011年第5期463-467,共5页Journal of Southern Agriculture

基　　金：国家科技支撑计划项目(2007BAD30B00;2008BADB8B00);科技部国际合作项目(2008DFA30600;2009DFA30820);农业部"948"计划项目(2009-Z8);广西自然科学基金创新研究团队项目(2011GXNSFF018002);广西科技攻关项目(桂科能0815011)

摘　　要：【目的】探索甘蔗幼苗叶片完整叶绿体的分离纯化及其叶绿体蛋白质提取方法,为深入研究甘蔗叶绿体蛋白质组学奠定基础。【方法】以甘蔗品种ROC22幼苗叶片为材料,通过差速离心法制备粗叶绿体制品,分别利用蔗糖密度梯度离心和Percoll梯度离心进一步分离纯化完整叶绿体;提取叶绿体蛋白质,并进行电泳分析。【结果】两种密度梯度离心法均可获得纯度和完整率较高的叶绿体,但蔗糖密度梯度离心法具有分离时间较短、成本较低,分离的叶绿体完整率、纯度和含量高等特点,并且提取的叶绿体蛋白质电泳条带清晰,蛋白质数量多且较全。【结论】与Pecoll梯度离心法相比,蔗糖密度梯度离心法更适宜于在亚细胞水平上进行甘蔗蛋白质组学研究。[Objective]In order to study sugarcane chloroplasts proteomics, the methods for isolation and purification of intact chloroplast and its protein extraction from leaves of sugarcane seedling were compared. [Method]The leaves of young seedling of sugarcane variety ROC22 were sampled to isolate the chloroplasts by differential centrifugation. The obtained intact chloroplasts were isolated and purified using sucrose density gradient centrifugation and Percoll gradient centrifugation. The proteins in chloroplast were extracted and analyzed by SDS-PAGE electrophoretic. [Result]The results indicated that both density gradient centrifugation methods could obtain high rate and high purity of intact chloroplasts, but sucrose density gradient centrifugation saved the time and cost, and resulted in high purity and integrity of chloroplasts. The SDS-PAGE profiles of proteins, separated from the intact chloroplasts, were rich and clear, with high quantity and quality of proteins.[Conclusion]Compared with Percoll gradient centrifugation method, sucrose density gradient centrifugation method was suitable for proteomics studies of sugarcane at subcellular level.

关 键 词：甘蔗 叶绿体 蛋白质 密度梯度离心 

分 类 号：S566.1[农业科学—作物学]