张琛，杨建军，王晨，马洁，杨涓，刘根红，郑蕊，郑国琦

摘要：【目的】探索適合宁夏枸杞果实细胞壁蛋白提取方法，为后续进行宁夏枸杞蛋白质组学研究提供技术支持。【方法】以宁夏枸杞果实为试验材料，采用六步洗脱法去除胞浆蛋白，然后通过定量和电泳比较氯化钙提取法、氯化锂提取法、氯化钙加氯化锂结合提取法和苯酚提取法对细胞壁蛋白的提取效果，通过双向电泳与液相色谱对所得细胞壁蛋白进行验证。【结果】六步洗脱法可较好地除去胞浆蛋白与其他干扰物质，4种方法对宁夏枸杞果实细胞壁蛋白的提取效果排序为：氯化钙提取法（272 μg/g）>苯酚提取法（261 μg/g）>氯化钙加氯化锂结合提取法（217 μg/g）>氯化锂提取法（176 μg/g）。裂解液I[7 mol/L尿素、2 mol/L硫脲、30 g/L 3-[3-（胆酰胺丙基）二甲氨基]丙磺酸内盐（CHAPS）、50 g/L 二硫苏糖醇（DTT）]溶解蛋白质效果更优，蛋白质定量浓度更高、电泳条带更多、更清晰。氯化钙提取所得蛋白质样品双向电泳结果较好，蛋白质点数较多、清晰度和分辨率较高;通过液相色谱可看出氯化钙提取的细胞壁蛋白肽段种类多，且大部分相对丰度较高，并含有低丰度蛋白质。所测蛋白质分类中有细胞壁定位的蛋白质。【结论】氯化钙单独提取蛋白质样品的方法是宁夏枸杞果实细胞壁蛋白提取最优方法，所得蛋白质样品已达到进行蛋白质组学研究的标准。

关键词： 宁夏枸杞;细胞壁蛋白;提取方法

中图分类号： S567.19                             文献标志码： A 文章编号：2095-1191（2021）09-2534-09

Optimization of extraction method of the cell wall protein of Lycium barbarum L.

ZHANG Chen1， YANG Jian-jun2， WANG Chen1， MA Jie1， YANG Juan1，

LIU Gen-hong1， ZHENG Rui1， ZHENG Guo-qi1*

（1College of Life Science， Ningxia University， Yinchuan  750021， China; 2Ningxia Rural Economic

Management Station， Yinchuan  750002， China）

Abstract：【Objective】To explore the suitable extraction scheme of cell wall protein from Lycium barbarum L. fruit，which provided technical support for the subsequent proteomic research of L. barbarum L. 【Method】Using L. barbarum L. fruit as experimental material and six-step elution method was used to remove cytoplasm proteins，then comparing cell wall protein extraction effect with calcium chloride extraction method，lithium chloride extraction method，calcium chloride plus lithium chloride extraction method and phenol extraction method by protein quantitation and electrophoresis. Cell wall proteins were examined by two dimensional electrophoresis（2-DE） and liquid chromatography（LC）. 【Result】Cytoplasmic protein and other interfering substances could be removed better using the six-step elution method. The extraction effect of the four methods on cell wall protein of L. barbarum L. fruit was ranked as follows： calcium chloride extraction method（272 μg/g）>phenol extraction method（261 μg/g）>calcium chloride plus lithium chloride extraction me-thod（217 μg/g）>lithium chloride extraction method（176 μg/g）. The dissolution effect of lysis buffer I[7 mol/L urea，2 mol/L thiourea，30 g/L 3-[3-（cholamidopropyl） dimethylamino]-1-propanesulfonate （CHAPS），50 g/L dithiothreitol（DTT）] was better，the quantitative concentration of protein was higher，and the electrophoretic bands were more and clearer. The results of two-dimensional electrophoresis of the protein samples extracted by calcium chloride were better，with more protein spots，higher clarity and resolution，and the liquid chromatography showed that there were many kinds of cell wall protein peptides extracted by calcium chloride method，and most of them had high relative abundance，and contained low abundance proteins. In the classification of the tested proteins，some proteins located on the cell wall. 【Conclusion】The method of extracting protein sample by calcium chloride alone is the best method to extract cell wall proteins from Ningxia L. barbarum L. fruit，and the obtained protein sample has reached the standard of proteomics research.

Key words： Lycium barbarum L.; cell wall proteins; extraction method

Foundation item： National Natural Science Foundation of China（81760682，31660363）;Ningxia Natural Science Foundation （2020AAC03097）;Ningxia Lycium barbarum Industry Development Center Project（2020）

0 引言

【研究意義】宁夏枸杞（Lycium barbarum L.）为茄科枸杞属植物，是我国重要的药用植物资源（国家药典委员会，2015）。其果实干燥后……