木薯栽培种ZM-Seaside和花叶变种块根蛋白组学分析

doi:10.13560/j.cnki.biotech.bull.1985.2017.03.012

摘要/Abstract

摘要： 为研究木薯栽培种ZM-Seaside（高产种质）和花叶变种（低产种质）块根产量差异的原因,从农艺性状和蛋白质组学角度对以上2个种质进行分析,为选育高产木薯品种提供理论依据。试验中采用旋光法测定淀粉含量;硝酸银滴定法测定氢氰酸含量;苯酚抽提法提取蛋白质;双向电泳技术分离蛋白质;Delta2D软件确定差异蛋白质点;质谱技术鉴定差异蛋白质点,结合KEGG数据库将其功能分类;利用Western blot技术对部分差异蛋白质进行验证;String在线软件构建蛋白质互作调控网络。结果显示,ZM-Seaside块根淀粉含量为29.18%,显著高于花叶变种的25.83%;两种木薯鲜薯薯肉氢氰酸含量均低于50 mg/kg,属可食用木薯种质。ZM-Seaside干物率为40.28%,显著高于花叶变种的37.16%。以花叶变种块根的全蛋白质为对照,ZM-Seaside的块根存在39个差异蛋白质点,其中上调表达23个,下调表达16个;经质谱技术成功鉴定到其中28个,其功能涉及到碳水化合物和能量代谢（7个）、分子伴侣（8个）、解毒和抗氧化（2个）、蛋白质合成（1个）、结构蛋白（3个）及未知功能蛋白质（7个）。STRING代谢网络显示：热激蛋白Heat shock protein和分子伴侣Molecular chaperone Hsp90-1互作关系最多,是整个互作调控网络的枢纽。推测这2个蛋白质是影响ZM-Seaside和花叶变种块根产量差异的关键蛋白质,这些蛋白质有可能成为选育高产木薯种质的标记蛋白质。

关键词: 木薯栽培种, 花叶变种, 块根, 农艺性状, 蛋白质组

Abstract: In order to study yield differences between cassava cultivar ZM-Seaside（high yield）and mosaic-leaf mutation（low yield）,the tuberous roots were analyzed from the aspects of agronomic traits and proteomics in the present study. The results will provide a theoretical basis for the selection of high yield cassava varieties. Starch content was determined by polarimetry,and the hydrocyanic acid concentration was measured by silver nitrate titration method. Protein extract were performed by phenol extraction and protein was separated using two-dimensional electrophoresis. Delta 2D software was used to determine the differentially expressed proteins. The differentially expressed proteins were identified using mass spectrometry in combination with the KEGG database to classify proteins according to their functions. Western blot was used to analyze the protein expression level. String online software was used to construct protein-protein interaction network. Results showed that the starch content of ZM-Seaside（29.18%）was significantly higher than that of mosaic-leaf mutation（25.83%）. Hydrocyanic acid contents of two cassava genotypes in flesh were less than 50 mg/kg,and both of them are in the range of edible cassava. The dry matter rate of ZM-Seaside was 40.28%,significant higher that of mosaic-leaf mutation（37.16%）. 39 differentially expressed protein spots were detected in the tuberous root of ZM-Seaside compared with mosaic-leaf mutation,of which 23 were up-regulated,16 were down-regulated. 28 protein spots were successfully identified,in which their functions related to carbohydrate and energy metabolism（7）,chaperones（8）,detoxifying and antioxidant（2）,protein biosynthesis（1）,structure（3）and function unknown proteins（7）. STRING metabolic network showed that Heat shock protein and Molecular chaperone Hsp90-1 were the hub proteins,which probably are the key of the whole regulatory network. They would be the key proteins affecting the yields between mosaic-leaf mutation and ZM-Seaside,suggesting they may use as the marked proteins to select high-yield cassava varieties.

Key words: Cassava cultivar, mosaic-leaf mutation, tuberous root, agronomic traits, proteomics

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和花叶变种块根蛋白组学分析[J]. 生物技术通报, 2017, 33(3): 78-85.

SONG Yan-chao, An Fei-fei, Xue Jing-jing, Qin Yu-ling, Li Kai-mian, CHEN Song-bi. Proteomic Analysis on Tuberous Roots of Cassava Cultivar ZM-Seaside and Mosaic-leaf Mutation[J]. Biotechnology Bulletin, 2017, 33(3): 78-85.

参考文献

[1] Nassar NMA. Wild and indigenous cassava, Manihot esculenta Crantz diversity：An untapped genetic resource[J]. Genetic Resources and Crop Evolution, 2007, 54（7）：1523-1530.
[2] An F, Chen T, Stéphanie DMA, et al. Domestication syndrome is investigated by proteomic analysis between cultivated cassava（Manihot esculenta Crantz）and its wild relatives[J]. PLoS One, 2016, 11（3）：e0152154 .
[3] Gomes PTC, Nassar NMA. Cassava interspecific hybrids with increased protein content and improved amino acid profiles[J]. Genetics and Molecular Research, 2013, 12（2）：1214-1222.
[4] 陈松笔, 安飞飞, 朱文丽, 等. 蛋白质组学在木薯育种中的应用[J]. 生物技术通报, 2015, 31（11）：18-26.
[5] Wang W, Feng B, Xiao J, et al. Cassava genome from a wild ancestor to cultivated varieties[J]. Nat Commun, 2014, 5：5110
[6] 徐娟, 黄洁. 自然低温条件下木薯种质出苗率和株高的调查评价[J]. 广东农业科学, 2013, 40（1）：16-18.
[7] 陈旭红. 旋光法测定木薯粗淀粉的含量[J]. 食品工业科技, 2000, 21（2）：66-67.
[8] 蒋治国, 何飞燕, 李兴芳. 饲料中氢氰酸含量测定方法的研究[J]. 饲料工业, 2005, 26（22）：47-48.
[9] 安飞飞, 陈松笔, 李庚虎, 等. 华南8号木薯及其四倍体块根淀粉及蛋白表达谱的差异分析[J]. 中国农业科学, 2015, 48（13）：2656-2665.
[10] Li K, Zhu W, Zeng K, et al. Proteome characterization of cassava（Manihot esculenta Crantz）somatic embryos, plantlets and tuberous roots[J]. Proteome Science, 2010, 8：10.
[11] Chen S, Glazer I, Gollop N, et al. Proteomic analysis of the entomopathogenic nematode steinernema feltiae IS-6 IJs under evaporative and osmotic stresses[J]. Molecular and Biochemical Parasitology, 2006, 145（2）：195-204.
[12] 安飞飞, 凡杰, 李庚虎, 等. 华南8号木薯及其四倍体诱导株系叶片蛋白质组及叶绿素荧光差异分析[J]. 中国农业科学, 2013, 46（19）：3978-3987.
[13] Khan S, Ahmad K, Alshammari EMA. Implication of caspase-3 as a common therapeut target for multineurode generative disorders and its inhibition using nonpeptidyl natural compounds[J]. BioMed Research International, 2015, 175（2）：235-244.
[14] 李永宏, 黄清臻. 新复极差法在生物统计中的应用[J]. 医学动物防制, 2002, 18（5）：270-272.
[15] 关海宁, 刁小琴, 徐桂花, 等. Duncar新复极差法优化特色“明目”花草茶工艺的研究[J]. 饮料工业, 2010, 13（6）：30-33.
[16] 宋雁超, 姚惠, 吕亚, 等. 花叶木薯变种和木薯栽培种ZM-Seaside叶片光合参数及蛋白组学分析[J]. 植物遗传资源学报, 2016, 17（5）：935-941.
[17] 李健, 古碧, 龙罡, 等. 广西食用木薯淀粉卫生质量检测结果分析[J]. 农业研究与应用, 2014, 3：41-46.
[18] Munch-Petersen A, Kalckar HM, Cutolo E, et al. Uridyl transferases and the formation of uridine triphosphate;enzymic production of uridine triphosphate：uridine diphosphoglucose pyrophosphorolysis[J]. Nature, 1953, 172（4388）：1036-1037.
[19] Daran JM, Dallies N, Thines-Sempoux D, et al. Genetic andbiochemical characterizarion of the UGP1 gene encoding theUDP-glucose pyrophosphorylase from Saccharomyces cerevisiae[J]. European of Journal Biochemistry, 1995, 233（2）：520- 530.
[20] 吴晓俊, 刘涤, 胡之璧. 尿苷二磷酸葡萄糖焦磷酸化酶[J]. 植物生理学通讯, 2000, 36（3）：193-200.
[21] Eimert K, Villand P, Kilian A, Kleczkowski LA . Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley（Hordeum vulgare）tissues[J]. Gene, 1996, 170（2）：227- 232.
[22] Ap Rees T, Leja M, Macdonald FD, Green JH . Nucleotide sugars and starch synthesis in spadix of Arum maculatum and suspension cultures of Glycine max[J]. Phytochemistry, 1984, 23（11）：2463- 2468.
[23] 姚庆荣. 木薯遗传转化体系的建立与优化及转AGPase基因的研究[D]. 海口：华南热带农业大学, 2007.
[24] Sweetlove LJ, Müller-Röber B, Willmitzer L, et al. The contribution of adenosine 5'-diphosphoglucose pyrophosphorylase to the control of starch synthesis in potato tubers[J]. Planta, 1999, 209（3）：330-337.
[25] Leloir LF, Cardini CE. The biosynthesis of sucrose phosphate[J]. Journal of Biological Chemistry, 1955, 214（1）：157-165.
[26] 刘凌霄, 沈法富, 卢合全, 等. 蔗糖代谢中蔗糖磷酸合成酶（SPS）的研究进展[J]. 分子植物育种, 2005, 3（2）：275-281.
[27] 李永庚, 于振文, 姜东, 等. 冬小麦旗叶蔗糖和籽粒淀粉合成动态及与其有关的酶活性的研究[J]. 作物学报, 2001, 27（5）：658-664.
[28] Farrar J, Pollock C, Gallagher J. Sucrose and the integration of metabolism in vascular plants[J]. Plant Science, 2000, 154（1）：1-11.
[29] Hubbard NL, Huber SC, Pharr DM. Sucrose phosphate synthase and acid invertase as determinants of sucrose concentration in developing muskmelon（Cucumis melo L. ）fruits[J]. Plant Physiol, 1989, 91（4）：1527-1534.
[30] Carvalho LJ, Lippolis J, Chen S, et al. Characterization of carotenoid-protein complexes and gene expression analysis associated with carotenoid sequestration in pigmented cassava（Manihot Esculenta Crantz）storage root[J]. Open Biochem J, 2012, 6：116-130.
[31] 吕亚, 安飞飞, 宋雁超, 等. 木薯叶片光合作用日变化的差异蛋白分析[J]. 湖南农业大学学报：自然科学版, 2016, 42（3）：256-261.