油菜叶片蛋白质组对机械损伤应答的初步分析

doi:10.3773/j.issn.1005-264x.2008.01.026

植物生态学报 ›› 2008, Vol. 32 ›› Issue (1): 220-225.DOI: 10.3773/j.issn.1005-264x.2008.01.026

油菜叶片蛋白质组对机械损伤应答的初步分析

乐寅婷¹^,², 李梅³, 陈倩¹^,², 魏伟¹^,^*()

1 中国科学院植物研究所植被与环境变化国家重点实验室,北京 100093
2 中国科学院研究生院,北京 100049
3 青海省卫生职业技术学院,西宁 810000

收稿日期:2006-06-28 接受日期:2006-09-25 出版日期:2008-06-28 发布日期:2008-01-30
通讯作者: 魏伟
作者简介:* E-mail: weiwei@ibcas.ac.cn
第一联系人：
本文的部分实验工作在中国科学院北京基因组研究所华大研发中心完成并承蒙中国科学院植物研究所沈世华研究员提出宝贵修改意见,特此致谢
基金资助:
自然科学基金面上项目(30370228);中国科学院植物研究所蛋白质组前沿项目和中国科学院知识创新方向性项目(KSCX2-SW124)

ANALYSIS OF PROTEIN RESPONSE TO MECHANICAL WOUNDING IN LEAVES OF BRASSICA NAPUS CV. WESTAR

LE Yin-Ting¹^,², LI Mei³, CHEN Qian¹^,², WEI Wei¹^,^*()

¹Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
²Graduate University of Chinese Academy of Sciences, Beijing 100049, China
³Qinghai Professional and Technological College of Hygienics, Xining 810000, China

Received:2006-06-28 Accepted:2006-09-25 Online:2008-06-28 Published:2008-01-30
Contact: WEI Wei

摘要/Abstract

摘要：

植物在对抗昆虫的长期进化过程中形成了自我防御机制,能够产生特异的抗性蛋白来应对昆虫的取食。该文用机械损伤模拟害虫取食,研究和对比了油菜(Brassica napus cv. Westar)在机械损伤前后可溶性总蛋白的含量变化并试图通过蛋白质组学技术来检测可能发生变化的蛋白质。蛋白质定量检测发现,同一植株同一叶片损伤前后可溶性总蛋白含量差异显著,损伤后蛋白表达量增高。蛋白质双向凝胶电泳及其差异显示分析损伤前后的蛋白质组,表明有8个蛋白质点发生明显的上调或下调。选择其中2个差异蛋白点经过MALDI-TOF质谱鉴定,它们分别是Rubisco小亚基前体、果糖-1,6-二磷酸醛缩酶和粪卟啉-3-氧化酶,这些蛋白质可能在油菜叶片应答机械损伤过程中对维持植物的生理功能起到重要作用。

关键词: 蛋白质组学, 总蛋白, 双向电泳, 肽质指纹分析, 植物-昆虫互作关系

Abstract:

Aims Plants have evolved self-defense mechanisms against insects and can produce insecticidal protein and other compounds. The development of proteomics has enabled study of the molecular mechanism of this defense. Oilseed rape (Brassica napus cv. Westar) is an important oil crop in China and suffers from damage caused by insect pests and disturbances such as hail. Our objective is to investigate changes of total soluble protein and induced individual protein in B. napus using mechanical damage to mimic insect feeding.
Methods We took two leaf samples from the same leaf of each plant 4 h apart and treated the first sample as mechanical damage. Total soluble protein (TP) of samples was measured by Bio-rad Protein Assay and separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Up-regulated proteins and new proteins were discriminated on the gel after staining. Two of eight different protein spots were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and identified by Mascot in database of Matrix Science.
Important findings TP concentration in leaves increased when measured after wounding. The two proteins discriminated on the 2-D gel were the small subunit (SSU) precursor of ribulose-1,5-bisphosphate carboxylase (rubisco) and a mixture containing fructose-bisphosphate aldolase and coproporphyrinogen Ⅲ oxidase (COP). The sequence of rubisco SSU was first reported in B. napus, and the other two enzymes were reported in Arabidopsis thaliana, a relative of B. napus. All of these proteins have been shown to be involved in plant responses to stress. Thus we assume that these three proteins are important in maintaining plant physiological functions during wound-response in leaves of B. napus. This finding could be useful in understanding the relationship between plants and phytophagous insects at the proteomic level.

Key words: proteomics, total soluble protein, two-dimensional (2-D) polyacrylamide gel electrophoresis, MALDI-TOF-MS, plant-insect interaction

乐寅婷, 李梅, 陈倩, 魏伟. 油菜叶片蛋白质组对机械损伤应答的初步分析. 植物生态学报, 2008, 32(1): 220-225. DOI: 10.3773/j.issn.1005-264x.2008.01.026

LE Yin-Ting, LI Mei, CHEN Qian, WEI Wei. ANALYSIS OF PROTEIN RESPONSE TO MECHANICAL WOUNDING IN LEAVES OF BRASSICA NAPUS CV. WESTAR. Chinese Journal of Plant Ecology, 2008, 32(1): 220-225. DOI: 10.3773/j.issn.1005-264x.2008.01.026

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参考文献 16

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代号 Code	序列号 Accession No.	分子量(道尔顿) Mass (Da)	序列覆盖 Sequence coverage (%)	蛋白名称 Protein name
6	CAA30290	20 529	56	RuBP羧化酶小亚基前体Rubisco ssu precursor (Brassica napus)
3	混合物 Mixture NP-568049	43 132	30	果糖-1,6-二磷酸醛缩酶 Fructose-bisphosphate aldolase (Arabidopsis thaliana)
	CAD12661	44 111	32	粪卟啉-3-氧化酶 Coproporphyrinogen Ⅲ oxidase (A. thaliana)

代号 Code	序列号 Accession No.	分子量(道尔顿) Mass (Da)	序列覆盖 Sequence coverage (%)	蛋白名称 Protein name
6	CAA30290	20 529	56	RuBP羧化酶小亚基前体Rubisco ssu precursor (Brassica napus)
3	混合物 Mixture NP-568049	43 132	30	果糖-1,6-二磷酸醛缩酶 Fructose-bisphosphate aldolase (Arabidopsis thaliana)
	CAD12661	44 111	32	粪卟啉-3-氧化酶 Coproporphyrinogen Ⅲ oxidase (A. thaliana)

油菜叶片蛋白质组对机械损伤应答的初步分析

ANALYSIS OF PROTEIN RESPONSE TO MECHANICAL WOUNDING IN LEAVES OF BRASSICA NAPUS CV. WESTAR

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