真姬菇在高温胁迫下的差异表达蛋白分析

生物技术通报 ›› 2014, Vol. 0 ›› Issue (5): 142-147.

真姬菇在高温胁迫下的差异表达蛋白分析

刘琳¹ 贾培培¹ 卢伟东¹ 郭奇林² 郭立忠¹

(1.青岛农业大学生命科学学院山东省应用真菌重点实验室，青岛 266109；2.西北农林大学生命科学学院，杨凌 712100)

收稿日期:2013-12-23 出版日期:2014-05-23 发布日期:2014-05-24
作者简介:刘琳，女，博士，讲师，研究方向：微生物生物转化与分离；E-mail：liulin@qau.edu.cn；贾培培同为本文第一作者
基金资助:
国家自然科学基金项目(31172012/C1506)

Differential Protein Expression Analysis of Hypsizygus marmoreus Under High Temperature Stress

Liu Lin¹ Jia Peipei¹ Lu Weidong¹ Guo Qilin² Guo Lizhong¹

(1. Shandong Provincial Key Laboratory of Applied Mycology，College of Life Science，Qingdao Agricultural University，Qingdao 266109；2. College of Life Science，Northwest Agriculture & Forestry University，Yangling 712100)

Received:2013-12-23 Published:2014-05-23 Online:2014-05-24

摘要/Abstract

摘要： 采用双向电泳分析Tris-饱和酚法获得的真姬菇菌丝体全蛋白，利用PDQuest8.0软件分析双向电泳图谱，比较最适温度(25℃)和高温胁迫条件下(42℃)真姬菇菌丝体蛋白的表达差异，筛选出与高温胁迫相关的蛋白，并对获得的差异蛋白进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定。结果显示，共鉴定7个蛋白点中，Spa2同源结构域的表达量下调，DEHA2G15532p、肽酶M76家族、HSP70、SNF2家族的DNA修复蛋白、细胞色素p450及AM域家族和锌指蛋白的混合物的表达量上调。这些蛋白参与了信号转导、蛋白处理、DNA修复等多种抗逆反应生理过程。

关键词: 真姬菇, 双向电泳(2-DE), 高温胁迫, 差异蛋白, 基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)

Abstract: Mycelial total proteins in Hypsizygus marmoreus from Tris-saturated phenol isolation method was analyzed by two-dimensional electrophoresis(2-DE), then the 2-DE maps obtained were analyzed with software PDQuest8.0 to compare the protein expression difference between the optimum temperature(25℃)and high temperature stress conditions(42℃), and screen the high temperature stress-related proteins. The differential expression proteins were examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS), and seven protein spots were identified. They are the Spa2 homology domain, DEHA2G15532p, peptidase M76 family, HSP70、SNF2 family of DNA repair proteins, cytochrome P450 and mixture of SAM domain family and zinc protein. The point 2 is up-regulated and the other six points were down-regulated. These high temperature stress-related proteins were involved in signal transduction, protein process, DNA repair and other general stress tolerance.

Key words: Hypsizygus, Narmoreus Two-dimensional electrophoresis(2-DE), High temperature, Stress Differential, Protein expression, Natrix-assisted laser desorption/ionization time of flight, Nass, Spectrometry(MALDI-TOF-MS)

刘琳, 贾培培, 卢伟东, 郭奇林, 郭立忠. 真姬菇在高温胁迫下的差异表达蛋白分析[J]. 生物技术通报, 2014, 0(5): 142-147.

Liu Lin, Jia Peipei, Lu Weidong, Guo Qilin, Guo Lizhong. Differential Protein Expression Analysis of Hypsizygus marmoreus Under High Temperature Stress[J]. Biotechnology Bulletin, 2014, 0(5): 142-147.

参考文献

[1] 郑宇, 林兴生, 陈福如.真姬菇生物学特性研究初报[J].食用菌, 2001, 23(3)：12-13.
[2] Lam SK, Ng TB. Hypsin a novel thermostable ribosome-inactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus[J]. Biochemical and Biophysical Research Communications, 2001, 285：1071-1075.
[3] Kazutaka T, Yutaka A, Shoji O, et al. Isolation of a novel collagen-binding protein from the mushroom, Hypsizigus marmoreus, which inhibits the lewis lung carcinoma cell adhesion to type IV collagen[J]. The Journal of Biological Chemistry, 1995, 270(4)：1481-1484.
[4] Tetsuro I. Beneficial effects of edible and medicinal mushrooms on health care[J]. International Journal of Medicinal Mushrooms, 2001(3)：291-298.
[5] Ikemizu S, Kishimoto M, Konishi H. Angiotensin-converting enzyme inhibitors of bunashimeji and enokitake[P]. JP：08099895, 1996, gen Im.
[6] 李翠翠.真姬菇热激蛋白HmHSP70基因克隆及其原核表达分[D].青岛：青岛农业大学, 2009.
[7] 孙国庆, 郝涤非.苏北地区发展真姬菇生产可行性研究[J].食用菌, 2011(1)：5-7.
[8] Yao Y, Yang YW, Liu JY. An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE[J]. Electrophoresis, 2006, 27：4559-4569.
[9] Wang W, Scali M, Vignani R, et al. Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds[J]. Electrophoresis, 2003, 24：2369-2375.
[10] Grinyer J, McKay A, Nevalainen Herbert BR, Saraste M. Fungal proteomics：initial mapping of biological control strain Trichoderma harzianum[J]. Current Genet, 2004, 45：163-169.
[11] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochemistry, 1976, 72：248-254.
[12] 郭尧君.蛋白质电泳实验技术[M]. 第2版. 北京：科学出版社, 2005.
[13] Kumiko SK, Tadatoshi K, Noriko F, et al. Screening system for D-Asp-containing proteins using D-aspartyl endopeptidase and two-dimensional gel electrophoresis[J]. Amino Acids, 2009, 36：125-129.
[14] Sheu YJ, Santos B, Fortin N, et al. Spa2p interacts with cell polarity proteins and signaling components involved in yeast cell morphogenesis[J]. Molecular Cell Biology, 1998, 18：4053-4069.
[15] Van DF, Peter M. Spa2 functions as a scaffold-like protein to recruit the Mpk1p MAP kinase module to sites of polarized growth[J]. Current Biology, 2002, 12：1698-1703.
[16] Ryan JH, Bradford CB. The multifunctional GIT family of proteins[J]. Journal of Cell Science, 2006, 119：1469-1475.
[17] Arkowiz RA, Lowe N. A small conserved domain in the yeast spa2p is necessary and sufficient for its polarized localization[J]. Journal of Cell Biology, 1997, 138(1)：17-36.
[18] Schultz J, Bork P, Ponting CP, Hofmann K. SAM as a protein interaction domain involved in developmental regulation[J]. Protein Science, 1997, 6(1)：249-253.
[19] Baltimore D, Mayer BJ, Ren R, Clark KL. A putative modular domain present in diverse signaling proteins[J]. Cell, 1993, 73(4)：629-630.
[20] Gibson T, Musacchio A, Thompson J, et al. The PH domain：a common piece in the structural pathwork of signaling proteins[J]. Trends Biochemical Science, 1993, 18(9)：343-348.
[21] Wang DS, Shaw R, Winkelmann JC, Shaw G. Binding of PH domains of β-adrenergic receptor kinase and β-spectrin to WD40/β-transducin repeat containing regions of the β-subunit of trimeric G-proteins[J]. Biochemical and Biophysical Research Communications, 1994, 203(1)：29-35.
[22] Torrecilla I, Leganes F, Bonilla I. Use of recombinant aequorin to study calcium transients in response to heat and cold in cyanobacterial[J]. Plant Physiology, 2000, 123(1)：161-175.
[23] Page MJ, Enrico DC. Evolution of peptidase diversity[J]. Journal of Biological Chemistry, 2008, 283：30010-30014.
[24] 侯学文, 郭勇.泛肽与植物逆境响应[J].植物生理学通讯, 1998, 6：474-478.
[25] 姜周庚, 张延昭, 冯梦诗, 等.高温胁迫对盛铃期Bt棉棉铃中杀虫蛋白表达量及氮代谢的影响[J].中国棉花, 2012, 39(4)：13-15.
[26] Sorensen JG, Kristensen TN, Loeschcke V. The evolutionary and ecological role of heat shock proteins[J]. Ecology Letters, 2003, 6(11)：1025-1037.
[27] Krebs RA, Holbrook SH. Reduced enzyme activity following Hsp70 over expression I Drosophila melanogaster[J]. Biochemical Genetics, 2001, 39(1-2)：73-82.
[28] Waters ER, Lee GJ, Vierling E. Evolution, structure and function of the small heat shock proteins in plants[J]. Journal of Experimental Botany, 1996, 47(3)：325-338.
[29] 陆兆明, 徐祯, 王珂, 等.双孢蘑菇耐温差异蛋白质组学研究[J].厦门大学学报：自然科学版, 2009, 48(4)：590-593.
[30] 王海燕, 图力故尔, 陈强, 黄晨阳.真菌细胞色素P450研究进展[J].食用菌学报, 2010, 17(2)：97-102.
[31] 徐理, 朱龙付, 张献龙.棉花抗黄萎病机制研究进展[J].作物学报, 2012, 38(9)：1553-1560.