Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (8): 1486-1494.doi: 10.3864/j.issn.0578-1752.2017.08.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Function Analysis of Heat-Shock-Protein LsHsp70-2711 Gene Under High Temperature Stress in Leaf Lettuce (Lactuca sativa L.)

LI YaBo, LI Ting, HAN YingYan, FAN ShuangXi   

  1. College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206
  • Received:2016-09-13 Online:2017-04-16 Published:2017-04-16

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Abstract: 【Objective】Through cloning of genes related to Hsp70, and using VIGS to analyze Hsp70 expression and morphological change of leaf lettuce under heat stress to lay a foundation for analysis of the response mechanism and molecular mechanism of heat shock protein under heat stress.【Method】The full-length cDNA gene of LsHsp70-2711 gene was obtained by homologous cloning and RACE technology. By real-time fluorescence quantitative PCR (qRT-PCR), the expression of the gene from P-S11and G-S59 was analyzed. According to the VIGS technology, the pTRV-LsHsp702711 vector was constructed and transformed into Agrobacterium GV1301. The leaves of lettuce were infected by the injection method. After three weeks, the positive plants were identified by PCR. The gene expression and morphology were compared between the control group and positive group, and the expression characteristics of LsHsp70-2711 were analyzed again after heat stress and drought treatment. 【Result】The sequence analysis indicated that the full-length cDNA was 2226 bp, the open reading frame was 2154 BP, encoding 718 amino acids, and its homology compared with that of Arabidopsis (NP_187864.1), Tomato (NP_001266213.1), Saussurea Medusa (AAB99745.1), and other species. qRT-PCR results showed that the expression of the gene in the two cultivars was up-regulated, and the expression level of heat resistant cultivars was significantly higher than the heat sensitive cultivar, and the gene expression of P-S11 was inhibited by the increase of stress time, and the expression level of G-S59 was higher in the heat resistant variety. In heat resistant variety, the maximum expression of LsHsp70-2711 appeared at 42℃, 60 min, and the maximum expression of heat sensitive variety reached at 37℃, 60 min. Prediction of subcellular localization showed that LsHsp70-2711 was mainly in the cytoplasm. The constructed vector was used to invade the leaves of lettuce, and the positive plants were obtained after identification. LsHsp70-2711 expression decreased after it was silenced by VIGS, compared with the control, the stem length of the positive plants was significantly increased. After heat stress and drought treatment, the expression of LsHsp70-2711 was significantly lower than the control, and the influence of high temperature treatment on LsHsp70-2711 was greater than drought stress.【Conclusion】 LsHsp70-2711 gene belongs to Hsp70 gene family, and is related to the heat resistance in lettuce. The research results of the study will provide theoretical supports for the analysis of function of high temperature in bolting of lettuce.

Key words: Lactuca sativa L., Hsp70, clone, temperature stress, VIGS

[1]    Mulabagal V, Ngouajio M, Nair A. In vitro evaluation of red and green lettuce (Lactuca sativa) for functional food properties. Food Chemistry, 2010, 118: 300-306.
[2]    许俊强, 汤青林, 宋明, 王志敏. 蔬菜抽薹的遗传规律及机理研究. 长江蔬菜, 2011(8): 7-10.
XU J Q, TANG Q L, SONG M, WANG Z M. Research on inheritance and mechanism of bolting in vegetables. Changjiang Vegetables, 2011(8): 7-10. (in Chinese)
[3]    王荣青, 万红建, 李志邈, 叶青静, 阮美颖, 周国治, 姚祝平, 杨悦俭. 番茄Hsp70基因鉴定及系统发育关系分析. 核农学报, 2014, 28(3): 378-385.
WANG R Q, WAN H J, LI Z M, YE Q J, Ruan M Y, Zhou G Z, Yao Z P, Yang Y J. Identification and phylogenetic relationships of Hsp70 genes in tomato. Nuclear Agricultural Sciences, 2014, 28(3): 378-385. (in Chinese)
[4]    齐妍, 徐兆师, 李盼松, 陈明, 李连城, 马有志. 植物热激蛋白70的分子作用机理及其利用研究进展. 植物遗传资源学报, 2013, 14(3): 507-511.
QI Y, XU Z S, LI P S, CHEN M, LI L C, MA Y Z. Research progress on Molecular Mechanism and applicatio of HSP70 in plants. Plant Genetic Resources, 2013, 14(3): 507-511. (in Chinese)
[5]    BOSTON R S, VIITANEN P V, VIERLING E. Molecular chaperones and protein folding in plant. Plant Molecular Biology, 1996, 32(1/2): 191-222.
[6]    POLIER S, DRAGOVIC Z, HARTL F U. Strucrural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding. Cell, 2008, 133: 1068-1079.
[7]    SCHUERMANN J P, JIANG J, Cuellar J. Structure of the Hsp70: Hsc70 nucleotide exchange machine. Molecular Cell, 2008, 31: 232-243.
[8]    LEE G J, POKALA N, VIERLING E. Structure and in vitro molecular chaperone activity of cytosolic small heat shock proteins from pea. Biological Chemistry, 1995, 270(18): 10432-10438.
[9]    SANMIYA K, SUZUKI K, EGAWA Y, SHONO M. Mitochondrial small heat-shock protein enhances thermotolerance in tobacco plants . FEBS Letters, 2004, 55(1/3): 265-268.
[10]   AHN Y J, CLAUSSEN K, ZIMMERMAN J L. Genotypic differences in the heat-shock response and thermotolerance in four potato cultivars . Plant Science, 2004, 166(4): 901-911.
[11]   肖艳萍, 宫伟娜, 万方浩, 李正跃, 谢丙炎. 紫茎泽兰热激蛋白70基因的克隆与序列分析. 中国农业科技导报, 2010, 12(1): 111-117.
XIAO Y P, GONG W N, WAN F G, LI Z Y, XIE B Y. Cloning and sequence analysis of heat shock protein 70 gene from Ageratina adenophora. Agricultural Science and Technology, 2010, 12(1): 111-117. (in Chinese)
[12]   安艳秋, 蔺瑞明, 芦冯晶, 王风涛, 徐世昌, 许玉凤. 小麦热激蛋白基因TaHSP70克隆及其在植物防卫和抗逆反应中的表达分析. 分子植物育种, 2011, 9(4): 402-409.
AN Y Q, LIN R M, FENG J, WANG F T, XU S C, XU Y F. Cloning of heat shock protein gene TaHSP70 from wheat and its expression in plant defense and anti reverse reaction. Molecular Plant Breeding, 2011, 9(4): 402-409. (in Chinese)
[13]   SUN Y, MACRAE T H. The Small heat shock proteins and their role in human disease. Febs Journal, 2005, 272(11): 2613-2627.
[14]   TANAKA K I, NAMBA T, ARAI Y, FUJIMOTO M, ADACHI H, SOBUE G, TAKEUCHI K, NAKAI A, MIZUSHIMA T. Genetic evidence for a protective role for heat shock factor and heat shock protein 70 against colitis. Biological Chemistry, 2007, 282: 23240-23252.
[15]   FRACY E, SERPENTINI A, Fiévet B, LEBEL J M. Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primaryculture. Comparative Biochemistry and Physiology Part B, 2007, 146: 540-550.
[16]   赵雁, 毕玉芬, 邵辰光. 牧草和饲料作物蛋白质变化与耐热性的关系.云南农业大学学报(自然科学版), 2012, 27(3): 440-446.
ZHAO Y, BI Y F, SHAO C G. The relationship between protein changes and heat tolerance of forage and feed crops. Yunnan Agricultural University (Natural Science Edition), 2012, 27(3): 440-446. (in Chinese)
[17]   HARTL F, HAYER-HARTL M. Molecular chaperones in the cytosol from nascent chain to folded protein. Science. 2002, 295: 1852-1858.
[18]   JOLLY C, MORIMOTO R. Stress and the cell nucleus: Dynamics of gene expression and structural reorganization. Gene Expression, 1999, 7: 261-270.
[19]   MYCKO, CWIKLINSKA M P, WALCZAK H , LIBERT A, RAINE C, SELMAJ C S, KrzySZTOF W. A heat shock protein gene (Hsp70) is critically involved in the generation of the immune response to myelin antigen. Eurpean Journal of Immunology, 2008, 38: 1999-2013.
[20]   SUNG D Y, GUY C. Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis evidence for pleiortopic consequences. Plant Physiology, 2003, 132: 979-987.
[21]   SCAFARO A P, HAYNES P A, ATWELL B J. Physiological and molecular changes in Oryza meridionalis Ng., A heat-tolerant species of wild rice. Journal of Experimental Botany, 2010, 61(1): 191-202.
[22]   Qi Y C, Wang H J, Zou Y. Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice. FEBS Journal, 2011, 585: 231-239.
[23]   李慧聪, 郭秀林, 王冬梅. 玉米热激蛋白70基因对温度胁迫的响应. 河北农业大学学报, 2010, 33(6): 12-15.
LI H C, GUO X L, WANG D M. Responses of HSP70 gene expression to temperature stresses in maize. Journal of Agricultural University of Hebei,2010, 33(6): 12-15. (in Chinese)
[24]   胡秀丽, 李艳辉, 杨海荣. HSP70可提高干旱高温复合胁迫诱导的玉米叶片抗氧化防护能力. 作物学报, 2010, 36(4): 636-644.
HU X L, LI Y H, YANG H R. Heat shock protein 70 may improve the ability of antioxidant defense induced by the combination of drought and heat in maize leaves. Acta Agronomica Sinica, 2010, 36(4): 636-644. (in Chinese)
[25]   PRATT W B, TOFT D O. Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Experimental Biology and Medicine, 2003, 228: 111-133.
[26]   YOUNG J C, BARRAL J M, ULRICH HARTL F. More than folding: localized functions of cytosolicchaperones. Trends in Biochemical Sciences, 2003, 28: 541-547.
[27]   SUNG D, VIERLING E, GUY C L. Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiology, 2001, 126(2): 789-800.
[28]   卢承琼, 曾其伟, 向仲怀, 何宁佳. 桑树小分子热激蛋白基因的鉴定及在逆境胁迫下的表达. 蚕业科学, 2014, 40(6): 965-973.
LU C Q, ZENG Q W, XIANG Z H, HE N J. Identification of Mulberry small heat shock protein genes and their expression under abiotic stresses. Science of Sericulture,2014, 40(6): 965-973. (in Chinese)
[29]   宋洪兵, 宋小明, 周晶晶, 刘环环, 李英, 侯喜林. 不结球白菜BcHSP70-1基因的克隆与进化及其表达分析. 西北植物学报, 2013, 33(8): 1508-1515.
SONG H B, SONG X M, ZHOU J J, LIU H H, LI Y, HOU X L. Phylogenetic and expression analysis of BcHSP70-1 gene from brassica campestris ssp.chinensis. Acta Botanica Boreali-Occidentalia Sinica, 2013, 33(8): 1508-1515. (in Chinese)
[30]   SRIKANTHBABU V, GANESHKUMAR, KRISHNAPRASAD B T. Identification of pea genotypes with enhanced thermotolerance using temperature induction response technique (TIR). Plant Physiology, 2002, 159: 535-545.
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