白菜型冬油菜叶片质外体蛋白提取及响应低温胁迫的蛋白表达分析

doi:10.7668/hbnxb.2016.04.008

华北农学报 ›› 2016, Vol. 31 ›› Issue (4): 44-50. doi: 10.7668/hbnxb.2016.04.008

所属专题： 油料作物；白菜；

白菜型冬油菜叶片质外体蛋白提取及响应低温胁迫的蛋白表达分析

袁金海², 刘自刚^1,2, 孙万仓^1,2, 曾秀存^1,3, 马骊², 方园², 刘海卿², 郭仁迪², 王治江², 陈奇², 王凯音², 刘林波²

1. 甘肃省油菜工程技术研究中心, 甘肃兰州 730070;
2. 甘肃农业大学农学院, 甘肃省作物遗传改良与种质创新重点实验室, 甘肃省干旱生境作物学重点实验室, 甘肃兰州 730070;
3. 河西学院农业与生物技术学院, 甘肃张掖 734000

收稿日期:2016-03-12 出版日期:2016-08-28
通讯作者: 刘自刚(1975-),男,甘肃天水人,副教授,博士,主要从事油菜遗传育种研究。
作者简介:袁金海(1988-),男,甘肃靖远人,在读硕士,主要从事油菜遗传育种研究。
基金资助:
国家“863”高技术研究发展计划项目（2011AA10A104）；国家自然科学基金项目（31460356）；国家“973”计划项目（2015CB150206）；国家农业科技成果转化项目（2014G10000317）；国家自然基金项目（31560397）；甘肃省自然基金项目（145RJZG050）

Extraction of Winter Turnip Rape Leaf Apoplast and Analysis of Protein Expression in Response to Low Temperature Stress

YUAN Jinhai², LIU Zigang^1,2, SUN Wancang^1,2, ZENG Xiucun^1,3, MA Li², FANG Yuan², LIU Haiqing², GUO Rendi², WANG Zhijiang², CHEN Qi², WANG Kaiyin², LIU Linbo²

1. Gansu Engineering and Technology Research Center of Rapeseed, Lanzhou 730070, China;
2. Agronomy College, Gansu Agricultural University, Gansu Key Laboratory of Crop Improvement & Germplasm Enhancement, Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070, China;
3. College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, China

Received:2016-03-12 Published:2016-08-28

摘要/Abstract

摘要： 为了研究质外体蛋白在低温胁迫下白菜型冬油菜抗寒的作用机理，以陇油7号五叶期叶片为试材，采用2种不同提取液（提取液A：0.1 mol/L pH值=7.6 Tris-HCl、0.2 mol/L KCl、1 mmol/L PMSF，提取液B：0.05 mol/L pH值=7.6 Tris-HCl、0.01 mol/L EDTA-Na₂、0.02 mol/L Vc、1 mmol/L PMSF）进行质外体蛋白提取效果比较。结果表明：提取液A的蛋白提取率达到了（0.073±0.004 6） mg/g，比提取液B提高了42.88%；经六磷酸葡萄糖脱氢酶（G₆PDH）活性检测，提取液A提取的蛋白质污染率较提取液B低0.91%；双向电泳第一向等电聚焦IEF中，提取液A提取的蛋白质不仅除盐时间较提取液B短4.5 h，且获得的凝胶图谱清晰；通过丰度计算，得到陇油7号低温胁迫（4℃，48 h）后具有显著差异的蛋白点339个，其中表达量变化在2倍以上的蛋白点为46个，包括上调表达蛋白点18个，下调表达蛋白点21个，特异性表达蛋白点7个，推测这些差异蛋白点可能与低温胁迫有关。对特异表达的7个蛋白点进行质谱分析鉴定，得到与低温胁迫相关的蛋白，为白菜型冬油菜超强抗寒品种陇油7号抗寒相关分子机理的研究奠定了基础理论。

关键词: 质外体, 陇油7号, 蛋白, 双向电泳

Abstract: In order to explore the mechanism of super cold resistant varieties of Winter turnip rape,select super cold resistant varieties of Winter turnip rape Longyou No.7 leaves as materials,using two different extraction liquids(Extraction A:0.1 mol/L Tris-HCl (pH=7.6),0.2 mol/L KCl,1 mmol/L PMSF,Extraction B:0.05 mol/L Tris-HCl (pH=7.6),0.01 mol/L EDTA-Na₂,0.02 mol/L Vc,1 mmol/L PMSF) to compare the extracted effect.The experiment showed that the extraction A liquid protein yield reached (0.073±0.004 6) mg/g,and the extraction ratio increased by 42.88% compared to liquid B.Through the Glucose-6-phosphate dehydrogenize (G₆PDH) activity detection and the extraction A was lower than B 0.91 percent;not only the extraction A extract protein was shorter than B 4.5 hours in the first oelectric focusing electrophoresis of two-dimensional electrophoresis,but also was get clear gel map;through the calculation of abundance,there were total 339 differentially expressed proteins discovered in Longyou No.7 under low temperature stress(4℃,48 h)later,changes in expression levels of more than two times the protein spots of 46,which 18 up-regulated protein expression spots,21 down-regulated protein expression spots,7 specific protein expression spots,these differential protein spots were suggested to be related to low temperature stress.This laid the foundation for the research of cold resistant varieties in Winter turnip rape Longyou No.7 proteomics aspect.

Key words: Apoplast, Longyou No.7, Protein, Two-dimensional electrophoresis

中图分类号:

Q945.78
Q946

袁金海, 刘自刚, 孙万仓, 曾秀存, 马骊, 方园, 刘海卿, 郭仁迪, 王治江, 陈奇, 王凯音, 刘林波. 白菜型冬油菜叶片质外体蛋白提取及响应低温胁迫的蛋白表达分析[J]. 华北农学报, 2016, 31(4): 44-50. doi: 10.7668/hbnxb.2016.04.008.

YUAN Jinhai, LIU Zigang, SUN Wancang, ZENG Xiucun, MA Li, FANG Yuan, LIU Haiqing, GUO Rendi, WANG Zhijiang, CHEN Qi, WANG Kaiyin, LIU Linbo. Extraction of Winter Turnip Rape Leaf Apoplast and Analysis of Protein Expression in Response to Low Temperature Stress[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(4): 44-50. doi: 10.7668/hbnxb.2016.04.008.

http://www.hbnxb.net/CN/Y2016/V31/I4/44

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白菜型冬油菜叶片质外体蛋白提取及响应低温胁迫的蛋白表达分析

Extraction of Winter Turnip Rape Leaf Apoplast and Analysis of Protein Expression in Response to Low Temperature Stress

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白菜型冬油菜叶片质外体蛋白提取及响应低温胁迫的蛋白表达分析

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