应用2D-DIGE技术分析柑橘衰退病毒诱导的甜橙差异表达蛋白  

Identification of differentially expressed proteins in sweet oranges induced by Citrus Tristeza Virus using 2D-DIGE

作　　者：杨方云[1,2] 李中安[2] 周常勇[2] 周彦[2] 

机构地区：[1]西南大学园艺园林学院 [2]中国农业科学院柑桔研究所,重庆400700

出　　处：《果树学报》2013年第1期16-21,I0002,共7页Journal of Fruit Science

基　　金：国家自然科学基金(30771485);长江学者和创新团队发展计划项目(PCSIRT;IRT0976);公益性行业(农业)科研专项(201203076)

摘　　要：【目的】为了研究柑橘衰退病毒(Citrus tristeza virus,CTV)诱导甜橙差异表达蛋白的分离和鉴定,【方法】以接种了CTV的甜橙植株为实验组,健康植株为对照组,利用双向荧光差异凝胶电泳(2D-DIGE)技术分析CTV诱导的甜橙差异表达蛋白。【结果】以t检验P<0.05和相对表达量≥1.5的水平作为判别标准,获得了91个CTV诱导的差异表达蛋白点,在实验组中含量高于对照组的蛋白56个,含量低于对照组的蛋白35个。通过MALDI-TOF质谱分析成功鉴定从中选择的37个蛋白点,其中19个蛋白功能明确,16个为预测或假定具有某种蛋白功能,包括与抗氧化相关的硫氧还蛋白、铁氧还蛋白、超氧化物歧化酶和抗坏血酸过氧化酶,与代谢相关的磷酸甘油酸酯水解酶、木葡聚糖内糖基转移酶、变味蛋白,分子伴侣热激蛋白,折叠酶肽酰脯氨酰顺反式异构酶,以及与光合代谢相关的蛋白等。【结论】CTV的侵染影响到甜橙各种复杂的生物学过程。【Objective】To determine the probability of 2D-DIGE technology to identify the differentially expressed proteins in sweet oranges induced by Citrus tristeza virus,CTV.【Method】 The same weight leaf samples of each plant were mixed from CTV-inoculated sweet orange plants and healthy plants,respectively.Total proteins were extracted from above mixed samples with three replications.Each protein sample was labeled with three different CyDyes Cy2,Cy3 and Cy5,and Cy2-labeled sample was used as an internal standard pooled from all the samples.Labeled protein samples were separated with 2-D DIGE and differential protein spots were picked out.MALDI-TOF-MS and bioinformatics were adopted to identify and interpret the significance of differentially expressed proteins.【Result】Total 91 Differential protein spots were detected with statistical variance of two groups（relative average volume ratio ≥1.5;t-test,P0.05）.Among these proteins,56 protein volumes of CTV group were higher than that of healthy group,and 35 proteins lower.PMFs of 37 proteins were obtained by MAIDI-TOF-MS analysis.Through searching NCBI,19 protein functions were clear,16 proteins predicted or putative,and 2 proteins unknown.The identified proteins were involved mainly in metabolism（including 2-Phospho-D-glycerate hydrolase,NADP-isocitrate dehydrogenase,Xyloglucan endotransglycosylase,Adenylate kinase）,antioxidant activity（including Thioredoxin H-type 5,Ferredoxin-NADP reductase,L-ascorbate peroxidase T,Iron superoxide dismutase）,photosynthesis（including Ribulose 1,5-bisphosphate carboxylase/oxygenase large or small subunit,Oxygen-evolving enhancer protein 1 and 2,Chlorophyll a/b-binding protein precursor） and molecular chaperone（such as Heat shock protein）.【Conclusion】 2D-DIGE can be used to identify the differentially expressed proteins in sweet oranges induced by different pathologic CTV strains in order to clarify the mechanism of CTV pathogenesis.

关 键 词：柑橘 衰退病毒 蛋白 双向荧光差异凝胶电泳 

分 类 号：S666[农业科学—果树学]