苹果属植物树皮组织总蛋白提取及分离方法的建立

doi:10.11983/CBB14186

植物学报 ›› 2015, Vol. 50 ›› Issue (6): 739-745.DOI: 10.11983/CBB14186

苹果属植物树皮组织总蛋白提取及分离方法的建立

张彩霞^1,², 田义^1,², 张利义^1,², 肖龙^1,², 丛佩华^1,^2,,A;^*()

¹中国农业科学院果树研究所, 农业部园艺作物种质资源利用重点实验室, 兴城 125100
²中国农业科学院果树研究所, 兴城 125100

收稿日期:2014-10-27 接受日期:2015-01-27 出版日期:2015-11-01 发布日期:2015-09-06
通讯作者: 丛佩华
作者简介:
? 共同第一作者
基金资助:
农业部现代农业产业技术体系建设专项(No;CARS-28)、国家科技支撑计划(No.2013BAD02B01)和国家自然科学基金(No;30900968, No.31201602)

Extraction and Separation Analysis of Total Protein from the Bark of Apple Branches

Caixia Zhang^1,2, Yi Tian^1,2, Liyi Zhang^1,2, Long Xiao^1,2, Peihua Cong^1,2*

¹Key Laboratory of Horticulture Crops Germplasm Resources Utilization, Ministry of Agriculture, Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, China
²Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, China

Received:2014-10-27 Accepted:2015-01-27 Online:2015-11-01 Published:2015-09-06
Contact: Cong Peihua
About author:
? These authors contributed equally to this paper

摘要/Abstract

摘要： 为了建立适于苹果属植物树皮组织总蛋白提取的技术方法, 以8年生华月苹果(Malus domestica)树枝条为试材, 通过比较不同提取方法(TCA-丙酮沉淀法(A)、甲醇/醋酸铵沉淀法(B)和改良的Tris-酚抽提方法(C))并优化提取条件, 确立了最适提取及分离方法为改良的Tris-酚抽提方法。在2-DE分离时, 该方法所获得的样品图谱中蛋白点总数为993个, 明显多于TCA-丙酮沉淀(418个)和甲醇醋酸铵沉淀(674个)方法, 并且与其它两种方法相比, 该方法获得的图谱背景更清晰, 蛋白点聚焦效果更好。此外, 经过3个梯度上样量的图谱分离效果比较, 确定了800 μg为本研究中2-DE分析的理想上样量。另外, 为了验证该提取及分离方法的可行性, 进一步对蛋白质表达谱中的部分蛋白点进行了质谱分析, 且这些蛋白点均得到了成功鉴定。该研究通过优化总蛋白提取方法及样品上样量等条件, 获得了理想的双向电泳分离图谱, 为苹果属植物树皮组织材料的蛋白质组学研究奠定了基础。

Abstract: We aimed to establish a suitable extraction method for analyzing total protein in the bark of apple branches. The branches of an 8-year-old cultivar (Huayue) were used as material. We compared 3 different extraction methods, including TCA-acetone precipitation (A), methanol/ammonium acetate precipitation (B), and improved Tris-phenol extraction (C). We optimized the extraction procedures and established an ideal method for analyzing the total protein in the bark of apple branches. The improved Tris-phenol extraction method could reveal a good map of 993 protein spots, significantly more than the other two methods: 418 and 674 protein spots were obtained with TCA-acetone precipitation and methanol/ammonium acetate precipitation, respectively. Furthermore, the gel background was clear, and the protein spots were better focused than with the two other methods. After comparing sample loading quantity, we selected 800 μg as the ideal loading quantity for our study. Furthermore, we selected several proteins for MALDI-TOF-TOF/MS analysis and database searching, and the protein spots were finally identified. In this study, we optimized the extraction procedures and sample loading quantity for total protein and finally obtained an ideal 2-D electrophoresis map. Our study may provide a good basis for further proteome analysis of apple branches.

张彩霞, 田义, 张利义, 肖龙, 丛佩华. 苹果属植物树皮组织总蛋白提取及分离方法的建立. 植物学报, 2015, 50(6): 739-745.

Caixia Zhang, Yi Tian, Liyi Zhang, Long Xiao, Peihua Cong. Extraction and Separation Analysis of Total Protein from the Bark of Apple Branches. Chinese Bulletin of Botany, 2015, 50(6): 739-745.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB14186

https://www.chinbullbotany.com/CN/Y2015/V50/I6/739

图/表 4

图1 苹果树皮组织分别采用3种方法提取总蛋白后获得的电泳图谱(A) TCA-丙酮沉淀方法; (B) 甲醇醋酸铵沉淀方法; (C) 改良的Tris-酚抽提方法

Figure 1 Representative 2-DE maps of the total protein extracts from the bark of apple branches following 3 proto- cols(A) TCA-acetone precipitation; (B) Methanol/ammonium ace- tate precipitation; (C) Improved Tris-phenol extraction

图2 采用不同上样量的双向电泳图谱比较(A), (D) 600 μg; (B), (E) 800 μg; (C), (F) 1 000 μg

Figure 2 Comparison of 2-DE maps of different sample load(A), (D) 600 μg; (B), (E) 800 μg; (C), (F) 1 000 μg

图3 苹果树皮组织总蛋白表达谱中筛选的蛋白点1-5: 高丰度蛋白点; 6-10: 低丰度蛋白点

Figure 3 Representative protein spots of the total protein extracts from the bark of apple branches 1-5: High-abundance proteins; 6-10: Low-abundance proteins

表1 苹果树皮组织蛋白点质谱分析

Table 1 Identification of representative proteins in apple branches with MALDI-TOF/TOF and database searching

Spot No.	Protein name	Accession No.	Theoretical MW (kDa)/pI	Species	Protein score
1	ATP synthase subunit beta	gi\|470126069	60.12/6.01	Fragaria vesca	431
2	Actin	gi\|355329944	40.37/5.67	Malus domestica	587
3	Oxygen-evolving enhancer protein	gi\|470129332	35.35/6.1	Fragaria vesca	265
4	Oxygen-evolving enhancer protein 1chloroplastic-like	gi\|470129332	35.35/6.1	Fragaria vesca	569
5	Uncharacterized protein At2g37660	gi\|470133785	35.44/5.86	Fragaria vesca	91
6	PRUPE_ppa007855mg	gi\|462397824	37.65/8.09	Prunus persica	62
7	PRUPE_ppa006513mg	gi\|462411618	42.18/5.82	Prunus persica	289
8	Acetyl-CoA carboxylase 1 isoform 3	gi\|508724180	24.65/6.17	Theobroma cacao	64
9	Cytosolic malate dehydrogenase	gi\|78216493	35.97/6.01	Malus domestica	334
10	PRUPE_ppa007772mg	gi\|462411755	39.12/5.88	Prunus persica	222

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苹果属植物树皮组织总蛋白提取及分离方法的建立

Extraction and Separation Analysis of Total Protein from the Bark of Apple Branches

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