叶用莴苣热激蛋白90（LsHsp90）基因的克隆及其在热激下的表达

doi:10.3864/j.issn.0578-1752.2013.16.023

摘要/Abstract

摘要： 【目的】克隆叶用莴苣热激蛋白Hsp90基因并探讨其响应热胁迫的相关机制，为揭示叶用莴苣抗热分子机制奠定理论基础。【方法】采用同源克隆和RACE 技术相结合，从叶用莴苣叶片总RNA 中克隆LsHsp90 cDNA 序列。通过实时荧光定量 PCR (qRT-PCR) 分析LsHsp90在叶用莴苣耐热品种Z36和热敏品种S106受37℃和42℃热激后叶片的表达差异。【结果】该基因cDNA序列全长2 330 bp，开放阅读框2 097 bp，编码698个氨基酸，推导的蛋白质分子量约79.8 kD。蛋白质结构预测及同源比对分析表明，LsHsp90基因编码蛋白含ATPase位点和Hsp90保守结构域，并与拟南芥（AY081302.1）、紫茎泽兰（EU269070.1）等多种物种的热激蛋白90高度同源；进化树分析表明，LsHsp90与紫茎泽兰Hsp90基因（EU269070.1）聚为一类。qRT-PCR分析表明，37℃热胁迫下，该基因在耐热品种和热敏品种的叶片中均上调表达；42℃高温胁迫下，热敏品种S106 中下调表达，而耐热品种Z36上调表达。【结论】成功从叶用莴苣叶片中分离克隆到LsHsp90，该基因具有已知物种Hsp90基因的特征，该基因在热胁迫下有不同的表达特征，预示该基因其可能在抗高温胁迫中起重要作用。

关键词: 叶用莴苣 , 热激蛋白90 , 基因克隆 , 高温胁迫 , 基因表达

Abstract: 【Objective】 LsHsp90 gene was cloned and used to analyze the expression profiles under heat shock stresses, in order to provide a foundation for investigating heat tolerant mechanisms of molecular regulation in stress tolerance of Lactuca sativa L.【Method】 With homology cloning approaches coupling with RACE techniques, the full-length cDNA of LsHsp90 was cloned from total RNA of the leaves of L. sativa L. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression patterns of this gene in leaves after heat shock unter 37℃ and 42℃.【Result】The full cDNA sequence of LsHsp90 is 2 330 bp，containing a 2 097 bp open reading flame (ORF) and encoding 698 amino acid residues with a predicted molecular mass of 79.8 kD．The LsHsp90 protein contains a predicted ATPase site and a Hsp90 conservative structure domain, which is highly conserved in plants and especially similar to Hsp90 from Ageratina adenophora (EU269070.1) and Arabidopsis thaliana (AY081302.1). Phylogenetic analysis showed that LsHsp90 and Hsp90 from Ageratina adenophora (EU269070.1) were clustered into one group. Real-time PCR analysis showed that, the expression of LsHsp90 in leaves of two varieties was upregulated under 37℃. LsHsp90 was induced down regulation in heat-sensitive variety S106 under 42℃, while the expression of LsHsp90 in heat-tolerant variety Z36 was upregulated.【Conclusion】LsHsp90 gene was firstly isolated and characterized from L. sativa L. This gene has genetic characteristics similar with all species and the induced expression profiling of LsHsp90 showed different patterns under heat stress. The results indicate that the LsHsp90 gene may play an important role in response to heat shock stress.

Key words: Lactuca sativa L. , Hsp90 , gene clone , heat stress , gene expression

任月, 韩莹琰, 李婷, 郝敬虹, 范双喜. 叶用莴苣热激蛋白90（LsHsp90）基因的克隆及其在热激下的表达[J]. 中国农业科学, 2013, 46(16): 3514-3522.

REN Yue, HAN Ying-Yan, LI Ting, HAO Jing-Hong, FAN Shuang-Xi. Molecular Cloning and Expression Analysis of Heat-Shock-Protein90 (LsHsp90) Gene from Leaf Lettuce (Lactuca sativa L.) Under Heat Shock[J]. Scientia Agricultura Sinica, 2013, 46(16): 3514-3522.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.16.023

https://www.chinaagrisci.com/CN/Y2013/V46/I16/3514

参考文献

[1]Boston R S, Viitanen P V, Vierling E. Molecular chaperones and protein folding in plants. Plant Molecular Biology, 1996, 32(1/2): 191-222.

[2]Miernyk J A. Protein folding in the plant cell. Plant Physiology, 1999, 121(3): 695-703.

[3]Mukhopadhyay I, Nazir A, Saxena D K, Chowdhuri D K. Heat shock response: hsp70 in environmental monitoring. Journal of Biochemical and Molecular Toxicology, 2003, 17(5): 249-254.

[4]Timperio A M, Egidi M G, Zolla L. Proteomics applied on plant abiotic stresses: role of heat shock proteins (HSP). Journal of Proteomics, 2008, 71(4): 391-411.

[5]Sun W, Van M M, Verbruggen N. Small heat shock proteins and stress tolerance in plants. Biochimica et Biophysica Acta, 2002, 1577(1): 1-9.

[6]Carper S W, Duffy J J, Gerner E W. Heat shock protein in thermotolerance and other cellular processes. Cancer Research, 1987, 47(20): 5249-5255.

[7]Emelyanov V V. Phylogenetic relationships of organellar Hsp90 homologs reveal fundamental differences to organellar Hsp70 and Hsp60 evolution. Gene, 2002, 299(1-2): 125-133.

[8]Pearl L H, Prodromou C. Structure and mechanism of the Hsp90 molecular chaperone machinery. Annual Review of Biochemistry, 2006, 75: 271-294.

[9]Terasawa K, Minami M, Minami Y. Constantly up-dated knowledge of Hsp90. The Journal of Biochemistry, 2005, 137(4): 443-447.

[10]Wegele H, Wandinger S K, Schmid A B, Reinstein J, Buchner J. Substrate transfer from the chaper-one Hsp70 to Hsp90. Journal of Molecular Biology, 2006, 356(3): 802-811.

[11]Milioni D, Hatzopoulos P. Genomic organization of hsp90 gene family in Arabidopsis. Plant Molecular Biology, 1997, 35: 955-961.

[12]Krishna P, Gloor G. The Hsp90 family of proteins in Arabidopsis thaliana. Cell Stress Chaperones, 2001, 6: 238-246.

[13]Chen B, Zhong D B, Monteiro A. Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms. BMC Genomics, 2006, 7: 156.

[14]Yonehara M, Minami Y, Kawata Y, Nagai J, Yahara I. Heat-induced chaperone activity of HSP90. Journal of Biological Chemistry, 1996, 271(5): 2641-2645.

[15]Neuer A, Spandorfer S D, Giraldo P, Dieterle S, Rosenwaks Z, Witkin S S. The role of heat shock proteins in reproduction. European Society of Human Reproduction and Embryology, 2000, 6(2): 149-159．

[16]宫伟娜.低温胁迫过程中入侵植物紫茎泽兰热激蛋白基因的作用[D]. 北京: 中国农业科学院, 2009.

Gong W N. The effect of the heat shock protein genes of invasive alien weed Ageratina adenophora (compositae) under low temperature stress[D]. Beijing: Chinese Academy of Agricultural Sciences, 2009. (in Chinese)

[17]高丽红, 尚庆茂, 马海艳. 两种不同耐热性菜豆品种在高温胁迫下叶绿素a 荧光参数的差异. 中国农学通报, 2004, 20(1): 173-176.

Gao L H, Shang Q M, Ma H Y. The difference of chlorophyll a fluorescence parameter in two common beans varieties with heat tolerance under high temperature stress. Chinese Agricultural Science Bulletin, 2004, 20(1): 173-176. (in Chinese)

[18]吴斌. 萝卜耐热性鉴定与热激蛋白基因克隆[D]. 南京农业大学, 2009.

Wu B. Identification of heat resistances and cloning of HSP gene in radish (Raphanus sativus L.) [D]. Nanjing: Nanjing Agricultural University, 2009. (in Chinese)

[19]陈以博, 侯喜林, 陈晓峰. 不结球白菜幼苗耐热性机制初步研究. 南京农业大学学报, 2010, 33(1): 27-31.

Chen Y B, Hou X L, Chen X F. Studies on heat tolerance mechanism of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Journal of Nanjing Agricultural University, 2010, 33(1): 27-31. (in Chinese)

[20]周群初, 马艳青. 蔬菜耐热性研究现状及展望. 长江蔬菜, 2000(3): 1-4.

Zhou Y C, Ma Y Q. Progress and present research of heat resistance in vegetables. Journal of Changjiang Vegetables, 2000(3): 1-4. (in Chinese)

[21]胡俏强, 陈龙正, 张永吉, 徐海, 宋波, 苏小俊, 袁希汉. 普通白菜苗期耐热性鉴定方法研究. 中国蔬菜, 2011(2): 56-61.

Hu Q Q, Chen L Z, Zhang Y J, Xu B, Song B, Su X J, Yuan X H. Studies on heat tolerance identification method for non-heading Chinese cabbage [Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee]. Chinese Vegetables, 2011(2): 56-61. (in Chinese)

[22]吴韩英, 寿森炎, 朱祝军, 杨信廷. 高温胁迫对甜椒光合作用和叶绿素荧光的影响. 园艺学报, 2001, 28(6): 517-521.

Wu H Y, Shou S Y, Zhu Z J, Yang X T. Effects of high temperature stress on photosynthesis and chlorophyll fluorescence in sweet pepper (Capsicumf ructescens L.). Acta Horticulturae Sinica, 2001, 28(6): 517-521. (in Chinese)

[23]Prodromou C, Roe S M, O'Brien R, Ladbury J E, Piper P W, Pearl L H. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell, 1997, 90(1): 65-75.

[24]Gupta R S. Phylogenetic analysis of the 90 kD heat shock family of protein sequences and an examination of the relationship among animals, plants, and fungi species. Molecular Biology and Evolution, 1995, 12(6): 1063-1073.

[25]Minami Y, Kimura Y, Kawasaki H, Suzuki K, YaharaI. The carboxy-teminal region of mammalian Hsp90 is required for its dimerization and function in vivo. Molecular and Cellular Biology, 1994, 14(2): 1459-1464.

[26]Zhao R M, Houry W A. Hsp90: a chaperone for protein folding and gene regulation. Biochemistry and Cell Biology, 2005, 83(6): 703-710.

[27]Song H M, Fan P X, Shi W L, Zhao R M, Li Y X. Expression of five AtHsp90 genes in Saccharomyces cerevisiae reveals functional differences of AtHsp90s under abiotic stresses. Journal of Plant Physiology, 2010, 167(14):1172-1178.

[28]Krebs R A, Holbrook S H. Reduced enzyme activity following Hsp70 overexpression in Drosophila melanogaster. Biochemical Genetics, 2001, 39(1/2): 73-82.

[29]Weng J, 周恒. 耐热与热敏感小麦品种对热冲击的不同反应. 麦类作物学报, 1993(2): 28-31.

Weng J, Zhou H. The different reactions of the heat-tolerant and heat-sensitive wheat varieties thermal shock. Journal of Triticeae Crops, 1993(2): 28-31. (in Chinese)

[30]周向红, 李信书, 王萍, 阎斌伦, 滕亚娟, 易乐飞. 条斑紫菜HSP90基因的克隆与表达分析. 水产学报, 2010, 34(12): 1844-1852.

Zhou X H, Li X S, Wang P, Yan B L, Teng Y J, Yi L F. Molecular cloning and expression analysis of HSP90 gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta). Journal of Fisheries of China, 2010, 34(12): 1844-1852. (in Chinese)