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

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。

Aims Plants have evolved serf-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 posts 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 demotion/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 phytophagons insects at the proteomic level.