桃果实生长素结合蛋白ABP1的组织定位及蛋白表达分析

doi:10.3864/j.issn.0578-1752.2015.05.10

摘要/Abstract

摘要： 【目的】生长素几乎参与调控植物发育的各个方面。生长素信号转导存在由ABP1（auxin binding protein 1）介导的途径，ABP1作为一种生长素快速响应蛋白参与调控果实发育等生理过程，其主要存在于正在发育的胚及胚的周围组织中。本文旨在研究ABP1是否参与桃果实发育过程，探讨其分布与表达特点，为进一步研究桃果实发育机制奠定理论基础。【方法】以‘24号’桃果实为研究对象，测定其生长曲线以确定果实发育的3个典型时期。分离不同发育时期的中果皮、内果皮和种子，切块后，一部分样品放入EDAC溶液抽真空后进行戊二醛和多聚甲醛固定，固定后的样品经脱水和浸蜡处理后用于石蜡切片；另一部分样品液氮速冻后-80℃保存提取蛋白。制备效价较高的ABP1兔源多克隆抗体，应用免疫组织化学定位技术对自然发育状态下不同发育时期桃果实种子和中果皮中ABP1进行定位分析；应用Western blot技术分析桃果实中果皮、内果皮和种子中ABP1的表达情况。【结果】根据桃果实生长曲线，将发育时期分为3个时期，即第一次快速生长期、硬核期和第二次快速生长期。免疫组织化学定位结果表明，同一发育时期ABP1在种子的不同部位都有分布，且分布信号差异不明显。在种子发育的各时期，ABP1在种子的不同部位都有分布，主要分布在种子的内外种皮细胞以及种皮内维管组织周围的细胞中，且在观测的发育时期内，ABP1的信号强弱没有明显变化。在种子内、外种皮之间的细胞层中会出现零散的、呈带状分布的ABP1信号。而在中果皮发育的整个时期，ABP1只在硬核期维管组织周围的细胞中有明显分布。Western blot结果表明，中果皮中ABP1的表达在果实第一次快速生长期开始（盛花后41 d）及第二次快速生长期开始（盛花后76 d）时的表达量最高，在盛花后39 d的表达量最低。种子中ABP1的表达量要高于中果皮与内果皮。【结论】ABP1在果实内的分布与表达水平存在组织和发育时期的差异性，ABP1因子参与了生长素对桃果实发育的调控过程。

关键词: 桃果实, ABP1, 免疫组化, 表达分析

Abstract: 【Objective】Auxin is almost involved in all aspects of plant development regulation, and ABP1 (Auxin binding protein 1) is established as a crucial component of auxin signaling．ABP1 as a kind of auxin fast response protein participates in a series of physiological processes such as regulation of fruit development. It is distributed mainly in the developing embryo and surrounding tissue of embryo. The purpose of this paper is to study whether ABP1 plays a role in peach fruit development and explore its expression characteristics and hence to provide a theoretical foundation for the further research．【Method】The peach ‘No.24’ was used as experimental material. Three periods of development were divided through the assay of growth curve. The fruits from different stages were separated into mesocarp, endocarp and seeds, small tissue was cut from seed and mesocarp, then put them into the EDAC solution, fixed by glutaraldehyde and paraformaldehyde after vacuumizing. The fixed samples continued dehydrated, paraffin embeded, and then used in paraffin section. The other samples which was used for extracting protein immediately froze in liquid nitrogen, then stored at -80℃. ABP1 rabbit polyclonal antibody was made successfully. The immunohistochemical localization technology was used to analyze the ABP1 distribution in seed and mesocarp at different development stages of peach fruit．The protein expression in mesocarp, endocarp and seed was analyzed by Western blot．【Result】According to peach fruit growth curve, it could be divided into three periods that are the first fast growing period, the hardcore period, and the second fast growing period. The results of immunohistochemical localization showed that ABP1 was distributed in both seed and mesocarp at all stages. ABP1 was evenly distributed in different parts of the seeds of all stages, the signal had no difference among different parts of seed at the same development stage. The stronger signals were detected in cells of outer and inner integument and around vascular tissue of seed coat. In the whole growth period, ABP1 signal did not significantly change. Between the outer and inner integuments, there were sporadic, zonal signals. The expression of ABP1 was around vascular tissue in mesocarp during the hardcore period of fruit development. The level of ABP1 expression was higher at the first rapid growth period（46DAFB）and the start of second rapid growth period (76DAFB) according to Western blot result．Its expression level was the lowest at 39 DAFB. In different tissues, the expression of ABP1 in the mesocarp and endocarp were lower than that in seed．【Conclusion】The distribution and expression level of ABP1 have a difference in tissue and at developmental stage in peach fruit, ABP1 plays an important role in peach fruit development.

Key words: peach fruit, ABP1, immunohistochemistry, expression analysis

余佳，李阳，龚硕，关伟，刘悦萍. 桃果实生长素结合蛋白ABP1的组织定位及蛋白表达分析[J]. 中国农业科学, 2015, 48(5): 921-930.

YU Jia, LI Yang, GONG Shuo, GUAN Wei, LIU Yue-ping. Tissue Location and Protein Expression Analysis of Auxin Binding Protein ABP1 in Peach Fruit (Prunus persica L.)[J]. Scientia Agricultura Sinica, 2015, 48(5): 921-930.

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