欢迎访问作物学报,今天是

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).

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

[1] 张振文, 李开绵, 叶剑秋, 许瑞丽. 木薯光合作用特性研究 云南大学学报自然科学版, 2007, 29: 628–632
Zhang Z W, Li K M, Ye J Q, Xu R L. The study on photosynthetic characteristic of cassava. J Yunnan Univ (Nat Sci), 2007, 29: 628–632 (in Chinese with English abstract)
[2] 陈冠喜, 李开绵, 叶剑秋, 许瑞丽. 6个木薯品种生长发育及产量性状的初步研究. 热带农业科学, 2009, 29: 26–29
Chen G X, Li K M, Ye J Q, Xu R L. Growth and yield of 6 cassava varieties. J Trop Agric , 2009, 29: 26–29 (in Chinese with English abstract)
[3] Li K, Zhu W, Zeng K, Zhang Z, Zhang Z W, Ye J Q, Ou W J, Rehman S, Heuer B, Chen S B. Proteome Characterization of cassava (Manihot esculenta Crantz) somatic embryos, plantlets and tuberous roots. Proteome Sci, 2010, 8: 10
[4] Nassar N M A, Junior O P, Sousa M V, Ortiz R. Improving carotenoids and amino-acids in cassava. Nutr Agric, 2009, 1: 32–38
[5] Cazzonelli C, Pogson B. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci, 2010, 15: 266–274
[6] Kris G. Joel V. Protein identification methods in proteomics. Electrophoresis, 2000, 21: 1145–1154
[7] Thierry R. Two-dimensional gel electrophoresis in proteomics: old. old fashioned, but it still climbs up the mountains. Proteomics, 2002, 2: 3–10
[8] Fan P X, Wang X C, Kuang T Y, Li Y X. An efficient method for the extraction of chloroplast proteins compatible for 2-DE and MS analysis. Electrophoresis, 2009, 30: 3024–3033
[9] 王金辉. 水稻、玉米叶绿体蛋白质组学的研究和玉米叶绿体转化体系的建立. 中国农业科学院硕士学位论文, 北京, 2011
Wang J H. Proteomic Analysis of Rice and Maize Chloroplast and Construction of Maize Chloroplast Transformation System. Chin Acad Agri Sci, 2011 (in Chinese with English abstract)
[10] THIELLEMENT H. Plant proteomics methods and protocols. Totowa: Humana Press Inc, 2007. pp 43–48
[11] Tanka N, Fujita M, Handa H, Murayama S, Uemura M, Kawamura Y, Mitsui T, Mikami S. Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments. Mol Genet Genom, 2004, 271: 566–576
[12] Huang S B, Nicolas L T, Reena N, Holger E, James W, Harvey M. Experimental analysis of the rice mitochondrial proteome, its biogenesis, and heterogeneity. Plant Physiol, 2009, 149:719–734
[13] Taise S, Miwa O, Takashi S, Naoto M. Ikuko H, Kenichior S, Masayoshi M, Akiho Y, Kenichi T, Tetsuro M. Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension- cultured cells of Arabidopsis thaliana. Plant Cell Physiol, 2004, 45: 672–683
[14] Barsan C, Sanchez-Bel P, Rombaldi C, Egea I, Rossignol M, Kuntz M, Zouine M, Latche A, Bouzayen M, Pech J C. Characteristics of the tomato chromoplast revealed by proteomic analysis. J Exp Bot, 2010, 61: 2413–2431
[15] Siddique M A, Grossmann J, Gruissem W, Baginsky S. Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts. Plant Cell Physiol, 2006, 47: 1663–1673
[16] Zeng Y, Pan Z, Ding Y, Zhu A, Cao H, Xu Q, Deng X X. A proteomic analysis of the chromoplasts isolated from sweet orange fruits (Citrus sinensis L. Osbeck). J Exp Bot, 2011, 62, 5297–5309
[17] Wang Y Q, Yong Y, Fei Z J, Hui Y, Tara F, Theodre W, Michael M, Leao V, Wang X W, Li L. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplasts function and development. J Exp Bot, 2013, 64, 949–961
[18] 安飞飞, 凡杰, 李庚虎, 间纯平, 李开绵. 华南8号木薯及其四倍体诱导株系叶片蛋白质组叶绿素荧光差异分析. 中国农业科学, 2013, 46: 3978–3987
An F F, Fan J, Li G H, Jian C P, Li K M. Comparison of leaves proteome and chlorophyll fluorescence of cassva cv. SC8 and Its tetraploid mutants. Sci Agric Sin, 2013, 46: 3978–3987 (in Chineses with English abstract)
[19] 宋雁超, 安飞飞, 薛晶晶, 秦于玲, 李开绵, 陈松笔. 木薯栽培种ZM-Seaside和花叶变种木薯块根蛋白质组学分析. 生物技术通报, 2017, 33(3): 78–85
Song Y C, An F F, Xue J J, Qin Y L, Li K M, Chen S B. Protemic analysis on tuberous roots of cassava cultivar ZM-Seaside and Mosaic-leaf mutantion. Boll Biol, 2017, 33(3): 78–85 (in Chineses with English abstract)
[20] Sánchez T, Salcedo E, Ceballos H, Dufour D, Mafla G, Morante N, Calle F, Pérez J C, Debouck D, Jaramillo G, Moreno I X. Screening of starch quality traits in cassava (Manihot esculenta Crantz). Starch/St?rke, 2009, 61, 12–19
[21] An F F, Jie F, Jun L, Li K M, Zhu W L, Wen F, Luzi J C B, Songbi C. Comparison of leaf proteomes of cassava (Manihot esculenta Crantz) cultivar NZ199 diploid and autotetraploid genotypes. PLoS One, 2014, 9(4): e85991
[22] Cristina M S, Petersen M, Mundy J. Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol, 2010, 61: 621–649
[23] Neuhaus H E, Emes M J. Nonphotosynthetic metabolism in plastid. Plant Physiol, 2000, 51: 111–140
[24] Eage I, Brasan C, Bian W, Purgatto E, Purgatto E, Latche A, Chervin C, Bouzayen M, Pech J C. Chromoplast differentiation: current status and perspectives. Plant Cell Physiol, 2010, 51: 1601–1611
[25] Reiser J, Linka N, Lemke L, Jeblick W, Neuhaus H E. Molecular physiological analysis of the two plastidic ATP/ADP transporters from Arabidopsis. Plant Physiol, 2004, 136: 3524–3536
[26] Li L, Van Eck J. Metabolic engineering of carotenoid accumulation by creating a metabolic sink. Transgen Res, 2007, 16: 581–585
[27] Pojidaeva E, Zinchenko V, Shestakov S, Sokolenko A. Involvement of the SppAl peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol, 2004. 186: 3991–3999
[28] Jarvis P. Targeting of nucleus-encoded proteins to chloroplasts in plants. New Phytol, 2008, 179: 257–285
[29] Sun W, Montagu M W, Verbruggen N. Small heat shock protein and stress tolerance in plants. Biochim Biophys Acta 2002, 1577: 1–9
[30] Wang W X, Vinocur B, Shoseyov O. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Altman, Trends Plant Sci, 2004, 9: 244–252
[31] Sakamoto W. Protein degradation machineries in plastids. Annu Rev Plant Biol, 2006, 57: 599–621
[32] Peltier J-B, Ripon D R, Friso G, Rudella A, Cai Y, Ytterberg J, Giacomelli L, Pillardy J, van Wijk K J. Clp protease complexes from photosynthetic and non-photosynthetic plastids and mitochondria of plants, their predicted three-dimensional structures, and functional implications. J Biol Chem, 2004, 279: 4768–4781
[33] Veronica A, Ingenfeld A, Klaus A. Characterization of the snow cotyledon 1mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality. Plant Mol Biol, 2006, 60: 507–518

[1] 王靖天, 张亚雯, 杜应雯, 任文龙, 李宏福, 孙文献, 葛超, 章元明. 数量性状主基因+多基因混合遗传分析R软件包SEA v2.0[J]. 作物学报, 2022, 48(6): 1416-1424.
[2] 刘培勋,马小飞,万洪深,郑建敏,罗江陶,蒲宗君. 两个不同籽粒硬度小麦的比较蛋白组学分析[J]. 作物学报, 2020, 46(8): 1275-1282.
[3] 马金姣,兰金苹,张彤,陈悦,郭亚璐,刘玉晴,燕高伟,魏健,窦世娟,杨明,李莉云,刘国振. 过表达OsMPK17激酶蛋白质增强了水稻的耐旱性[J]. 作物学报, 2020, 46(01): 20-30.
[4] 李萍,侯万伟,刘玉皎. 青海高原耐旱蚕豆品种青海13号响应干旱胁迫蛋白质组学分析[J]. 作物学报, 2019, 45(2): 267-275.
[5] 宋奇琦,Pratiksha SINGH,Rajesh Kumar SINGH,宋修鹏,李海碧,农友业,杨丽涛,李杨瑞. 基于iTRAQ技术的甘蔗受黑穗病菌侵染蛋白组分析[J]. 作物学报, 2019, 45(1): 55-69.
[6] 王卫东, 高翔, 赵丹阳. 高分子量麦谷蛋白亚基HPCE高效分离及图谱鉴定[J]. 作物学报, 2018, 44(7): 966-976.
[7] 王道平,徐江,牟永莹,闫文秀,赵梦洁,马博,李群,张丽娜,潘映红. 表油菜素内酯影响水稻幼苗响应低温胁迫的蛋白质组学分析[J]. 作物学报, 2018, 44(6): 897-908.
[8] 彭章, 童华荣, 梁国鲁, 石艺琦, 袁连玉. 茶树叶片和胚根原生质体的分离及PEG诱导融合[J]. 作物学报, 2018, 44(03): 463-470.
[9] 李竹, 许莉萍, 苏亚春, 吴期滨, 成伟, 孙婷婷, 高世武. 基于田间表型和Bru1基因检测分析甘蔗褐锈病抗性遗传[J]. 作物学报, 2018, 44(02): 306-312.
[10] DO Thanh-Trung,李健,张风娟,杨丽涛,李杨瑞,邢永秀. 甘蔗与抗旱性相关差异蛋白质组分析[J]. 作物学报, 2017, 43(09): 1337-1346.
[11] 于涛,李耕,张成芬,刘鹏,董树亭,张吉旺,赵斌. 玉米籽粒早期发育相关蛋白的差异表达特性[J]. 作物学报, 2017, 43(04): 608-619.
[12] 朱亚军,孙强,王金明,陈凯,冯博,方雅洁,林秀云,徐建龙*. 粳稻品种吉粳809的稻瘟病抗性基因分析[J]. 作物学报, 2016, 42(11): 1638-1646.
[13] 刘自刚,袁金海,孙万仓,曾秀存,方彦,王志江,武军艳,方园,李学才,米超. 低温胁迫下白菜型冬油菜差异蛋白质组学及光合特性分析[J]. 作物学报, 2016, 42(10): 1541-1550.
[14] 韩平安,逯晓萍,米福贵,张瑞霞,李美娜,薛春雷,董婧,丛梦露. 基于蛋白质组学的高丹草苗期杂种优势分析[J]. 作物学报, 2016, 42(05): 696-705.
[15] 吴林坤,陈军,吴红淼,王娟英,秦贤金,张重义,林文雄. 地黄连作胁迫响应机制的块根蛋白质组学分析[J]. 作物学报, 2016, 42(02): 243-254.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!