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蛋白质组与翻译后修饰蛋白质组共同研究甜菜耐盐胁迫

2017-09-26
中科新生命
2862

单体附加系M14甜菜 —— 具有耐盐性,能够在500mM的盐浓度下耐受7天:利用2倍体栽培甜菜(Beta vulgaris. VV,2n=18)与4倍体野生白花甜菜(B.corolliflora Zoss. CCCC,2n=4X=36)进行种间杂交获得异源3倍体甜菜,栽培甜菜与异源3倍体甜菜连续回交获得了1套栽培甜菜附加了白花甜菜染色体的单体附加系。其染色体组组成为栽培甜菜染色体组附加了一条白花甜菜第九号染色体。

Quantitative proteomics and phosphoproteomics of sugar beet monosomic addition line M14 in response to salt stress

Journal of Proteomics 143 (2016) 286–297 【IF=3.914】 

本文作者同时利用蛋白质组学(label-free)+翻译后修饰组学(label-free磷酸化蛋白质组)通过定量分析甜菜M14短期响应盐胁迫的磷酸化蛋白质组学和总蛋白组的改变,解析甜菜M14短期响应盐胁迫的机制,为培育耐盐性的作物提供分子基础。

▲▲ 图:文章技术路线

结果讨论:

1. 之前的研究报道显示SOD和POD酶活性是植物对盐响应和耐受的关键指示剂,作者检测了200mM和400mM NaCl处理播种三周后甜菜M14幼苗不同时间段的酶活,发现200mM处理的甜菜SOD和POD酶活性在30分钟达到最高值,说明此时的甜菜对盐胁迫的响应最高,而400mM处理的甜菜SOD和POD酶活性在60分钟达到最高值,因此,作者采取了200mM盐浓度处理M14 30分钟以及400mM处理60分钟的叶片。


Fig. 1. Time course of the increases of SOD and POD activities compared to respective controls in sugar beet M14 leaves after NaCl treatments. (A) SOD in 200mMand 400mMNaCl stress.(B) POD in 200 mM and 400 mM NaCl stress. Reported values are the mean of three independent experiments from different samples with standard errors.


2. 对200mM NaC处理30分钟以及400mM处理60分钟的M14叶片进行蛋白质和磷酸化蛋白质组学进行定量分析筛选出114个差异蛋白以及189个差异磷酸化蛋白,并对这些差异蛋白分别进行GO功能注释。


Fig. 2. Functional classification of the identified differentially accumulated proteins and phosphoproteins based on the uniprot database, related publications and Blast2Go. (A) 114 differentially accumulated proteins; (B) 189 differential phosphoproteins.

3. 以磷蛋白磷酸酶2C家族蛋白(PP2C) (gi|224144954)以及ABA响应原件结合蛋白(AREB) (gi|479277783)为例,显示M14响应盐胁迫的蛋白磷酸化位点二级质谱图。


Fig. 3. Phosphopeptide MS/MS spectra map. (A) MS/MS spectrum of VNpTLLNLPR from PP2C. (B) MS/MS spectrum of DFGpSMNMDELLK from AREB.

4. qPCR对一些差异蛋白以及差异磷酸化蛋白进行数据验证



Fig. 4. Real-time PCR analysis of the genes encoding the differential phosphoproteins and proteins in signal transduction pathways. The x-axis is NaCl concentration. The y-axis is the relative expression of each gene encoding the phosphoproteins and proteins.

5. 根据差异蛋白以及差异修饰蛋白的KEGG通路以及相关的文献报道,作者推导出甜菜耐受不同盐浓度的分子机理。

Fig. 5. Schematic presentation of systematic salt tolerance mechanisms in M14 leaves. The identified proteins were put into subcellular locations and KEGG pathways. The red and green highlighted proteins indicate increased and decreased phosphoproteins, respectively, and those underlined red and green highlighted proteins with increased and decreased levels, respectively. (A) Salt response mechanisms under 200 mM NaCl treatment; (B) Salt response mechanisms under 400 mM NaCl treatment. Please refer to Supplemental Table 7 for protein IDs.

本文是非常经典的利用蛋白质组学和修饰蛋白组学解析耐盐机理的文章,从最初合适材料的获得,利用蛋白质组学和修饰蛋白质组学筛选差异蛋白,然后从蛋白表达以及蛋白翻译后修饰这俩个不同层面对差异蛋白以及差异蛋白进行GO功能注释,接下来再进行qPCR数据验证,最后结合KEGG通路注释以及已有的文献报道推导出甜菜M14的耐盐机理。文章结构简单,思路清晰,是非常值得借鉴的思路。

注意!!!

目前蛋白质组学以及修饰蛋白质组学文章对于数据验证要求更加严格,需要在蛋白层面进行验证,建议采用WB(有相应抗体)或者PRM(无相应抗体)进行数据验证。