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

doi:10.3864/j.issn.0578-1752.2015.13.017

摘要/Abstract

摘要： 【目的】通过对华南8号木薯及其四倍体块根淀粉含量与结构及蛋白质组学分析，揭示染色体加倍对木薯块根淀粉含量与结构的影响及其蛋白质调控机制。【方法】植后10个月收获木薯块根，采用空、水重测定块根鲜薯的淀粉含量，分光光度法分别测定淀粉中支链淀粉与直链淀粉的比例，采用Excel 2013和DPS v7.05统计软件对数据进行分析，差异显著性标准采用新复极差法，通过扫描电镜对块根淀粉体的大小、形态及数量进行显微结构观察，利用蛋白质印迹（Western blot）技术对参与淀粉合成与降解的酶表达水平进行验证，采用双向电泳技术分离块根蛋白质，Delta 2D软件分析差异倍数在2.0以上的差异蛋白质点，并通过基质辅助激光解析电离飞行时间质谱技术（MALDI-TOF-TOF-MS/MS）鉴定差异蛋白质，结合KEGG数据库将其按照功能进行分类。【结果】木薯染色体加倍后，块根干物率、淀粉含量及单株鲜薯重均显著下降，分别比二倍体降低了12.18%、11.41%和35.34%；而淀粉中直链淀粉和支链淀粉比例无显著变化；淀粉体形态无明显差异，主要为球形，不规则球体、椭球体也有存在，大小较均一，淀粉体排列较为疏松，空间间隙较大，视野内淀粉体数量减少；蔗糖磷酸合成酶（SPS）表达水平降低，β-淀粉酶表达水平升高，而颗粒结合型淀粉合成酶I（GBSSI）表达水平无显著变化；经软件分析得到的20个表达差异显著的蛋白质点中上调表达的有2个，下调表达的有18个；经质谱技术成功鉴定到其中的19个，1个下调表达的蛋白质点16未得到匹配，17个下调表达的蛋白质其功能涉及到碳代谢及能量代谢（4个）、结构蛋白（3个）、DNA和RNA代谢（2个）、分子伴侣（2个）、HCN代谢（2个）、抗氧化与解毒（1个）、蛋白质合成（1个）及未知功能（2个）；2个上调表达蛋白质为茎特异性蛋白TSJT1。【结论】参与碳代谢及能量代谢、核酸代谢、分子伴侣等7个代谢途径相关蛋白质表达水平下调；淀粉体疏松排列，数量减少；参与淀粉代谢合成过程的SPS表达水平的下降，参与淀粉降解过程的β-淀粉酶表达水平的升高。表明四倍体块根淀粉合成能力降低，分解能力提高，从而降低了块根的淀粉含量，而直链淀粉与支链淀粉比例无显著变化。

关键词: 木薯, 四倍体, 块根, 淀粉, 蛋白质

Abstract: 【Objective】The aim of the study was to better understand the change of starch content, starch structure and its proteins regulatory mechanisms of cassava tuberous roots of diploid and its tetraploid.【Method】Tuberous roots of 10 months after planting were used as the materials, starch content was measured by weight in air and water, the proportion of amylose and amylopectin was determined by spectrophotometry, Excel 2013 and DPS v7.05 statistical software were used to analyze the data, the significant difference was determined by using Duncan method, the size, shape and number of amyloplast were observed by scanning electron microscopy, Western blot was used to verify some enzymes involved in starch synthesis and degradation, roots proteins of cassava tetraploid and diploid genotypes were excised and purified from 2D gels, Delta 2D software were used to analyze the different proteins with more than 2.0 fold changes and assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF-MS/MS) were used to identify them, and the proteins were classified according to their functions combined with KEGG database.【Result】After chromosome doubling in tetraploid cassava, dry matter content, starch content and fresh roots weight were significantly decreased by 12.18%, 11.41%, and 35.34%, respectively. No significant difference was observed in amylase and amylopectin proportion. No significant difference in amyloplast morphology, mainly spherical, irregular sphere and ellipsoid also exist, the size was uniform, while the loose degree of amyloplast and space gap were increased, the number was decreased. The level of sucrose phosphate synthase (SPS) significantly decreased, β-amylase expression was increased, while the level of granule bound starch synthase I (GBSSI) was not changed. Twenty differential expressed protein spots were showed and analyzed by software, including 2 up-regulated spots and 18 down-regulated spots, 19 were successfully matched in database, while spot 16, which was down-regulated, was no match. The functions of the 17 down regulated proteins were associated with carbohydrate and energy metabolism (4), structure (3), DNA and RNA metabolism (2), chaperones (2), HCN metabolism (2), antioxidant (1), protein synthesis (1) and unknown function (2), 2 up-regulated proteins were stem-specific proteins (TSJT1).【Conclusion】Protein related with carbohydrate and energy metabolism, nucleic acid metabolism and chaperones were down-regulated, the amyloplast loosely arranged and number decreased, the expression level of SPS decreased, β-amylase expression increased, indicating the low starch synthesis ability and high starch decomposition ability in tetraploid cassava, then decreased starch content, while no significant changes in amylose and amylopection proportion was found.

Key words: cassava, tetraploid, tuberous roots, starch, proteins

安飞飞，陈松笔，李庚虎，周锴，李开绵. 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.

AN Fei-fei, CHEN Song-bi, LI Geng-hu, ZHOU Kai, LI Kai-mian. Comparison Analysis of Starch and Protein Expression Profiles on Cassava Tuberous Roots cv. SC8 and Its Tetraploid[J]. Scientia Agricultura Sinica, 2015, 48(13): 2656-2665.

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