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作物学报 ›› 2017, Vol. 43 ›› Issue (09): 1290-1299.doi: 10.3724/SP.J.1006.2017.01290

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

木薯块根有色体分离及其蛋白质组学的研究

邓昌哲1,2,姚慧1,安飞飞1,李开绵1,陈松笔1,*   

  1. 1中国热带农业科学院热带作物品种资源研究所 / 农业部木薯种质资源保护与利用重点实验室，海南儋州 571737; 2海南大学农学院, 海南海口 5702281
  • 收稿日期:2016-11-24 修回日期:2017-04-19 出版日期:2017-09-12 网络出版日期:2017-05-22
  • 通讯作者: 陈松笔，E-mail: songbichen@catas.cn
  • 基金资助:

    本研究由国家自然科学基金项目(31271776)和海南省高层次创新创业人才基金项目(2012-2016)资助。

Chromoplast Isolation and Its Proteomic Analysis from Cassava Storage Roots

DENG Chang-Zhe1,2,YAO Hui1,AN Fei-Fei1,LI Kai-Mian1,CHEN Song-Bi1,*   

  1. 1 Tropical Crops Genetic resources Institute, Chinese Academy of Tropical Agricultural Sciences / Key Laboratory of Ministry of Agriculture for Germplasm Resources Conservation and Utilization of Cassava, Danzhou 571737, China; 2 College of Agriculture, Hainan University, Haikou 570228; China
  • Received:2016-11-24 Revised:2017-04-19 Published:2017-09-12 Published online:2017-05-22
  • Contact: Chen Songbi，E-mail: songbichen@catas.cn
  • Supported by:

    The work was supported by the National Natural Science Foundation of China (31271776) and The Initial Fund of High-level Creative Talents in Hainan Province (2012–2016).

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摘要：

木薯(Manihot esculenta Crantz)块根有色体是类胡萝卜素贮藏和调控多种生理生化过程的场所。本研究发现Percoll密度梯度离心法最适合于木薯块根有色体的提取，利用光学显微镜观察发现40%~50% Percoll梯度层富含完整有色体，Western blot分析显示该层的线粒体标志酶Vdac1杂交信号较低，而质体标志酶RbcL杂交信号最高，并以此确定木薯块根有色体的分离方法。利用蛋白质组学方法显示SC9块根有色体存在34个差异蛋白质，其中上调表达17个，下调表达17个；涉及碳代谢及能量代谢相关蛋白所占比例最高。STRING蛋白质互作网络显示，Enolase 2与Elongation Factor互作关系最多，是整个互作网络的核心蛋白质。qRT-PCR定量分析显示Enolase 2在高类胡萝卜素的蛋黄木薯SC9的表达水平显著高于低类胡萝卜素品种SC6068。推测其可能是影响SC9与SC6068类胡萝卜素差异的主要蛋白质之一。

关键词: 木薯块根, 有色体蛋白质, 分离, 蛋白质组

Abstract:

Chromoplasts are the sites to store carotenoids and regulate a variety of physiological and biochemical process in the storage roots of cassava (Manihot esculenta Crantz). In the present study, it was found that Percoll density gradient centrifugation was suitable for isolating the chromoplasts from cassava storage roots. Rich and intact chromoplasts were located in 40% to 50% layer of Percoll and the expression level of Vdac1, a mitochondrial marker, was the lowest and the expression level of RbcL, a plastid marker, was the highest compared with other layers using Western blot. Thirty-four differentially expressed proteins were detected in SC9, in which 17 were up-regulated, and the others were down-regulated. The differential proteins related to carbohydrate and energy metabolism accounted for the highest proportion. The STRING protein-protein interaction network showed that Enolase 2 and Elongation Factor were the hub proteins, which play the key roles in the whole regulatory network. Quantitative analysis by qRT-PCR confirmed the Enolase 2 expression was more significantly up-regulated in the high carotenoid cassava variety than in the low carotenoid cassava SC6068. These two proteins may be the key points for affecting the carotenoid content between SC9 and SC6068.

Key words: Cassava storage root, Chromoplast protein, Isolation method, Proteomics

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