溶杆菌属Lysobacter yanansis sp. nov. 胞内外蛋白双向电泳条件优化及图谱建立

生物技术通报 ›› 2013, Vol. 0 ›› Issue (4): 140-146.

溶杆菌属Lysobacter yanansis sp. nov. 胞内外蛋白双向电泳条件优化及图谱建立

王斐斐武坤毅郭玲崔浪军任靖

陕西师范大学生命科学学院陕西师范大学药用植物资源与天然药物化学教育部重点实验室，西安 710062

收稿日期:2012-10-16 修回日期:2013-04-22 出版日期:2013-04-22 发布日期:2013-04-22
作者简介:王斐斐，女，硕士，研究方向：微生物蛋白质组学；E-mail ：wangfeifei488@163.com
基金资助:
国家自然科学基金项目（31101481），中央高校基本科研业务费专项资金（GK200902032），2012 年度陕西师范大学实验技术研究立项项目（SYJS201213），陕西师范大学青年项目（201001005），陕西师范大学2011 年校级大学生创新实验计划项目（cx11072）

Optimization and Construction of the Intracellular and Extracellular Proteomic Map of Lysobacter yanansis sp. nov.

Wang Feifei, Wu Kunyi, Guo Ling, Cui Langjun, Ren Jing

Key Laboratory of Medicinal Plant Resources and Natural Pharmaceutical Chemistry

Received:2012-10-16 Revised:2013-04-22 Published:2013-04-22 Online:2013-04-22

摘要/Abstract

摘要： 通过对溶杆菌属细菌Lysobacter yanansis sp. nov. 胞外蛋白上样量和聚焦程序两个方面进行双向凝胶电泳条件的优化，分析了其对溶杆菌Lysobacter yanansis sp. nov. 胞外分泌蛋白的双向凝胶电泳图谱的影响。结果表明，150 μg 的上样量和聚焦程序Ⅱ 获得的双向电泳图谱蛋白点数较多且清晰。同时，建立了溶杆菌属细菌Lysobacter yanansis sp. nov. 胞外和胞内蛋白双向电泳图谱，经过硝酸银染色和PDQuest 图像分析检测到了100 种胞外蛋白和204 种胞内蛋白。

关键词: 溶杆菌属, 双向电泳, 生物防治

Abstract: In this study, we separated the extracellular protein excreted by Lysobacter yanansis sp. nov. through the two-dimensional （2-D）electrophoresis, and analyzed the effect of isoelectric focusing programs and loading volumes on 2-D proteomic maps. The results showed that a clearly 2-DE map was obtained when the 150 μg loading volumes and isoelectric focusing programs Ⅱ were used. Furthermore, 2-D proteomic maps of extract intracellular and extracellular protein from Lysobacter yanansis sp. nov. were constructed. The separated protein spots in gels were analyzed by PDQuest. 100 spots of extracellular proteins and about 204 spots of intracellular proteins had been separated by 2-DE.

Key words: Lysobacter, Two-dimensional electrophoresis（2-DE）, Biodefence

王斐斐武坤毅郭玲崔浪军任靖. 溶杆菌属Lysobacter yanansis sp. nov. 胞内外蛋白双向电泳条件优化及图谱建立[J]. 生物技术通报, 2013, 0(4): 140-146.

Wang Feifei, Wu Kunyi, Guo Ling, Cui Langjun, Ren Jing . Optimization and Construction of the Intracellular and Extracellular Proteomic Map of Lysobacter yanansis sp. nov.[J]. Biotechnology Bulletin, 2013, 0(4): 140-146.

参考文献

［1］ Matthew C. Biopiracy rules should not block biological control [J].
Nature, 2010, 467（7314）：369-369.
［2］ Luo Y, Zhang DD, Dong XW, et al. Antimicrobial peptaibols induce defense responses and systemic resistance in tobacco against tobacco mosaic virus [J]. FEMS Microbiol Lett, 2010, 313（2）：120-126.
［3］ Tofazzal Islam M. Mode of antagonism of a biocontrol bacterium Lysobacter sp. SB-K88 toward a damping-off pathogen aphanomyces cochlioides [J]. World J Microbiol Biotechnol, 2010, 26 ：629-637.
［4］ Postma J, Stevens LH, Wiegers GL, et al. Biological control of Pythium aphanidermatum in cucumber with a combined application of Lysobacter enzymogenes strain 3.1T8 and chitosan [J]. Biol Control, 2009, 48（3）：301-309.
［5］ Vasilyeva NV, Tsfasman IM, Suzina NE, et al. Secretion of bacteriolytic endopeptidase L5 of Lysobacter sp. XL1 into the medium by means of outer membrane vesicles [J]. FEBS J, 2008, 275（15）：3827-3835.
［6］ Kilic-Ekici O, Gary Y. Induced resistance as a mechanism of biological control by Lysobacter enzymogenes strain C3 [J].
Biological Control, 2003, 93（9）：1103-1110.
［7］胡苏莹. 一株具有抗菌活性远志根际细菌的鉴定, 及其代谢产物的研究［D］. 西安：陕西师范大学, 2012.
［8］ Ong SE, Pandey A. An evaluation of the use of two-dimensional gel electrophoresis in proteomics [J]. Biomolecular Engineering, 2001, 18（5）：195-205.
［9］ Simpson RJ. Purifying proteins for proteomics ［M］. America ：Cold Spring Harbor Laboratory Press, 2004 ：392-394.
［10］ O’Farrell PH. High resolution two-dimensional electrophoresis of proteins [J]. The Journal of Biological Chemistry, 1975, 250（10）: 4007-4021.
［11］ Molloy MP. Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients [J]. Analytical Biochemistry, 2000, 280（1）：1-10.
［12］ Rabilloud T. Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis [J]. Electrophoresis, 1998, 19（5）：758-760.
［13］ Galvani M, Rovatti L, Hamdan M, et al. Protein alkylation in the presence/absence of thiourea in proteome analysis ：A matrix assisted laser desorption/ionization-time off light-mass spectrometry investigation [J]. Electrophoresis, 2001, 22（10）：2066-2074.
［14］ Shaw MM, Riederer BM. Sample preparation for two-dimensional gel electrophoresis [J]. Proteomics, 2003, 3（8）：1408-1417.
［15］ Rabilloud T, Adessi C, Giraudel A, Lunardi J. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients [J]. Electrophoresis, 1997, 18（3-4）: 307-316.
［16］ Diwu Z, Haugland RP, Patton WF. Background-free, high sensitivity staining of proteins in one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex [J]. Electrophoresis, 2000, 21 ：2509-2521.
［17］ Lopez MF, Berggren K, Chernokalskaya E. A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two—dimensional gels and identification by peptide mass profiling [J]. Electrophoresis, 2000, 21（17）：3673-3683.
［18］ Candiano G, Bruschi M, Musante L, et al. Blue silver ：A very sensitive colloidal coomassie G-250 staining for proteome analysis [J]. Electrophoresis, 2004, 25（9）：1327-1333.
［19］丁承强, 马丹, 王绍华, 丁艳锋. 水稻蛋白质组双向电泳优化流程及方法[J]. 植物学报, 2011, 46（1）：67-73.
［20］ Boguth G, Harger A, Scheibe B. The current state of two— dimensional electrophoresis with immobilized pH gradients [J].
Electrophoresis, 2000, 21 ：1037-10553.
［21］ Parker R, Flowers TJ, Moore AL, et al. An accurate and reproducible method for proteome profiling of the effects of salt stress in the rice leaf lamina [J]. Journal of Experimental Botany, 2006, 57（5）：1109-1118.
［22］谈旭翡, 陈智. 蛋白质组学研究中双向电泳的样品制备[J].
医学分子生物学杂志, 2008, 5（5）：462-465.
［23］ Nandakumar MP, Cheung A, Marten MR. Proteomic analysis of extracellular proteins from Escherichia coli W3110 [J]. Journal of Proteome Research, 2006, 5（5）：1155-1161.
［24］ Yamaguchi Y, Pfeiffer SE. Highly basic myelin, and oligodendrocyte proteins analyzed by NEPHGE-two-dimensional gel electrophoresis ：Recognition of novel developmentally regulated proteins [J].
Journal of Neuroscience Research, 1999, 56（2）：199-205.
［25］ Ducret A, Desponts C, Desmarais S. A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini twodimensional gel electrophoresis [J]. Electrophoresis, 2000, 21 （11）：2196-2208.
［26］ Patton WF, Schulenberg B, Steinberg TH. Two-dimensional gel electrophoresis ；better than a poke in the ICAT? [J]. Protein Technologies and Commercial Enzymes, 2002, 13（4）：321-328.
［27］ Alban A, David SO, Bjorkesten L. A novel experimental design for comparative two-dimensional gel analysis ：Two-dimensional difference gel electrophoresis incorporating a pooled internal standard [J]. Proteomics, 2003, 3（1）：36-44.

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