番荔枝花发育不同阶段的差异蛋白质组分析

doi:10.3864/j.issn.0578-1752.2018.01.014

摘要/Abstract

摘要： 【目的】从蛋白质组学的角度研究番荔枝花发育不同阶段差异表达的蛋白质，分析相关信号通路，为揭示番荔枝花发育的分子机制提供理论基础。【方法】以番荔枝不同发育阶段的花为材料，采用双向电泳技术（Two-dimensional gel electrophoresis，2-DE）分离获得花芽（1 mm＜花芽＜2 mm）、花蕾（5 mm＜花蕾＜6 mm）、开花前期（花瓣轻微张开）及开花期（花瓣张开）等不同阶段表达丰度不同的蛋白点，利用MALDI-TOF-MS质谱鉴定技术分析相关差异蛋白质，并对其进行功能注释。【结果】通过蛋白图谱比较分析发现，番荔枝花双向电泳图谱中的每张凝胶图谱中都可以检测到800多个重复蛋白点，其中在不同花发育阶段存在表达差异的有50个蛋白点。经质谱鉴定分析，并采用MASCOT软件在线检索，共检测到36个表达量相差2倍以上的蛋白点。36个蛋白质可分为7个主要的功能类别，这些功能类别包括：呼吸与能量代谢（11个蛋白质），蛋白合成与代谢（8个蛋白质），转录与翻译（3个蛋白质），胁迫与防御（2个蛋白质），细胞分化与增殖（3个蛋白质），次生物质代谢（8个蛋白质）和未知功能蛋白（1个蛋白质）。通过GO分析，发现36个已鉴定的蛋白，分别归属于20个分类（term）。通过对差异蛋白进行定量分析，发现呼吸与能量代谢相关的蛋白中，3个蛋白（A07、C28、C42）随着花发育表达量不断下降，4个蛋白（A36、A37、B13、B29）表达则显著上升。在蛋白合成与代谢相关蛋白中，2个蛋白（A13和A22）在花发育的4个阶段都处于低水平表达，而另5个蛋白（B17、B20、A19、A21和C21）表达量显著上升。一个胁迫与防御相关蛋白（B32）在开花期，表达量达到最大值。2个细胞分化与增殖相关蛋白（B27和C57）表达量也呈逐渐升高的趋势。5个次生物质代谢相关蛋白（A06、A29、A34、C100和C110）随着花发育，表达量逐渐降低，而蛋白质C79和D38在花蕾阶段表达量达到最大值后，又逐渐下降。【结论】通过比较番荔枝花发育4个阶段的蛋白图谱，发现36个蛋白具有不同的差异表达谱。其中呼吸与能量代谢相关蛋白质占最大比例，诸如两个ATP合酶α亚基和丙酮酸脱羧化同工酶，都在番荔枝花发育过程呈现出显著的差异表达，这可能与花发育过程需要耗费大量的能量有关。此外，发掘了两个含有三角状五肽重复蛋白，其表达随着番荔枝花发育阶段的变化而改变，这些蛋白的功能还有待进一步研究。能量代谢、次生代谢、蛋白质合成等生物过程可能都参与了对番荔枝开花过程的调控。

关键词: 番荔枝, 花发育, 差异表达蛋白质, 蛋白功能

Abstract: 【Objective】In order to reveal the molecular mechanism of sugar-apple flower development, the function of differential expression proteins among four flower developmental stages were studied by using an approach of plant proteomics. 【Method】Flower samples were collected at four flower developmental stages: floral bud stage, bud stage, the mature flowers with partially opened petals stage and mature flowers with opened petals stage. The expressed proteins of flowers in four stages were detected using two-dimensional gel electrophoresis (2-DE) technology. All expressed proteins and their functional annotation were analyzed by MALDI-TOF-MS mass spectrometry .【Result】The results of protein profiling showed that there was more than 800 protein spots in each gel electrophoresis and 50 of them showed differences in different stages of sugar apple flowers. 36 proteins were identified by spectrum analysis as well as MASCOT online retrieval. These proteins could be divided into 7 functional categories, including respiration and energy metabolism (11 proteins), protein synthesis and metabolism (8 proteins), transcription and translation (3 proteins), stress and defense (2 proteins), cell proliferation and differentiation (3 proteins), secondary metabolites (8 proteins) and unknown proteins (1 protein). Furthermore, the 36 identified proteins could be grouped into 20 GO classifications. Among 11 respiration and energy metabolism related proteins, three proteins (A07, C28, C42) were continuously decreased with flower development while the other four proteins (A36, A37, B13, B29) were significantly increased; among the proteins related synthesis and metabolism, two proteins (A13 and A22) expressed at low level for all four flower developmental stages, while other five proteins (B17, B20, A19, A21 and C21) expression increased significantly with the flower development. A stress and defense related protein (B32) showed highest expression in last flower developmental stage. The expression of two cell differentiation and proliferation related proteins (B27 and C57) expression was gradually increased. Five secondary metabolism related proteins expression (A06, A29, A34, C100 and C110) gradually decreased with flower development, while proteins C79 and D38 reached the maximum value in the bud stage, and then gradually decreased. 【Conclusion】Comparing the protein spectrum of the sugar-apple at four different flower developmental stages, 36 proteins were identified as differentially expressed proteins. The respiration and energy metabolism related proteins accounted as the largest proportion, such as two ATP synthase alpha subunits and pyruvate decarboxylation isozyme. Differential expression of these proteins suggested the flower development process requires a lot of energy. In addition, our study first reported two pentatricopeptide repeat-containing proteins, and the expression of them changed with the flower development process. However, the function of these proteins remains to be further study. The results suggest that energy metabolism, secondary metabolism, protein synthesis and other biological processes may be involved in the process of flowering regulation of sugar-apple.

Key words: sugar apple, flower development, differential expressed protein, protein function

刘锴栋，莫亿伟,冯少娴，吴婉仪，黎海利，钟军弟，袁长春. 番荔枝花发育不同阶段的差异蛋白质组分析[J]. 中国农业科学, 2018, 51(1): 149-159.

LIU KaiDong, MO YiWei, FENG ShaoXian, WU WanYi, LI HaiLi, ZHONG JunDi, YUAN ChangChun. Comparative Proteomic Analysis in Different Developmental Stages of Sugar-Apple (Annona squamosa L.) Flowers[J]. Scientia Agricultura Sinica, 2018, 51(1): 149-159.

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