蒺藜苜蓿MtLEA5B的克隆和功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2018-0095

摘要/Abstract

摘要： 胚胎发育晚期丰富蛋白（Late embryogenesis abundant protein,LEA protein）是一类参与植物抗逆性应答的亲水蛋白。旨在揭示异源表达LEA蛋白在非生物胁迫条件下对宿主的保护作用。从蒺藜苜蓿（Medicago truncatula）幼苗中克隆到一个编码LEA_5蛋白的基因,命名为MtLEA5B,通过半定量RT-PCR分析MtLEA5B的表达模式。利用SDS-PAGE蛋白电泳检测重组蛋白的耐热性和可溶性。构建原核表达载体,转化大肠杆菌过表达,检测大肠杆菌的生长存活情况。MtLEA5B的表达模式受到低温（4℃）、脱水、150 mmol/L NaCl胁迫和ABA处理的调控。SDS-PAGE蛋白电泳出现一条相比理论预测值较大,且沸水条件下可溶性未降低的条带。在大肠杆菌中过表达MtLEA5B蛋白能明显提升宿主菌在高温（55℃）和冷冻（-20℃）条件下的生存率。MtLEA5B是诱导性表达基因;MtLEA5B蛋白具有较高的亲水性和耐热性。在大肠杆菌中过表达可以明显提升其对温度胁迫的耐受性。

关键词: 蒺藜苜蓿, 胚胎发育晚期丰富蛋白, MtLEA

Abstract: Late embryogenesis abundant protein（LEA protein）is hydrophilic one that is involved in the responses to stress in plants. This work is aimed to unravel the protective effect of heterologous expressed LEA protein on host under abiotic stress. A novel gene encoding LEA_5 protein was cloned from Medicago truncatula seedlings,and designated as MtLES5B,and its expression patterns were analyzed by semi-quantitative RT-PCR. Then the heat resistance and solubility of recombinant protein were detected by SDS-PAGE protein electrophoresis. Further,the prokaryotic expression vector was constructed and transferred to Escherichia coli so as to induce MtLEA5B over expression in E. coli. The expression patterns of MtLEA5B gene was regulated by dehydration,low temperature,high salt stress,and ABA induction. There was a band by SDS-PAGE protein electrophoresis,which was larger than the theoretical prediction value and did not decrease in solubility under boiling water condition. The overexpression of MtLEA5B protein in E. coli obviously increased the survival rate of hosts at heat（55℃）and freeze（-20℃）. Moreover,MtLEA5B was an inducible expressed gene,MtLEA5B protein had high hydrophily and heat resistance,overexpression in E. coli may significantly enhance its tolerance to temperature stress.

Key words: Medicago truncatula, late embryogenesis abundant protein, MtLEA

张业猛, 沈迎芳, 王英芳, 姚品雅, 王海庆. 蒺藜苜蓿MtLEA5B的克隆和功能分析[J]. 生物技术通报, 2018, 34(7): 101-107.

ZHANG Ye-meng, SHEN Ying-fang, WANG Ying-fang, YAO Pin-ya, WANG Hai-qing. Cloning and Functional Analysis of the MtLEA5B Gene from Medicago truncatula[J]. Biotechnology Bulletin, 2018, 34(7): 101-107.

参考文献

[1] Dure l, Greevway SC, Galau GA. Developmental biochemistry of cotton seed embryogenesis and germination:changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis[J]. Biochemistry, 1981, 20(14):4162-4168.
[2] Gram J, Bartels D.The molecular basis of dehydration tolerance in plant[J]. Annual Review of plant biology, 1999, 50(1):571-599.
[3] Battista JR, Park MJ, Mclemore AE.Incativation of two homologues of proteins presumed to be involved in the desiccation tolerance of plant sensitizes Deinococcus radiodurans R1 to desiccation[J]. Cryobiology, 2001, 43(2):133-139.
[4] Campos FC, Cuevas-Velazquez C, et al.Group 1 LEA proteins, an ancestral plant protein group, are also present in other eukaryotes, and in the archeae and bacteria domains[J]. Molecular Genetic Genomics, 2013, 288:503-517.
[5] Wise MJ, Tunnacliffe A.POPP the question:what do LEA proteins do?[J]. Trends in Plant Science, 2004, 9(1):13-17.
[6] Browne J, Tunacliffe A, Burnell A.Anhydrobiosis:plant desiccation gene found in a nematode[J]. Nature, 2002, 416(6876):38.
[7] Bartels D, Salamini F.Desiccation tolerance in the resurrection plant Craterostigma Plantagineum. A contribution to the study of drought tolerance at the molecular level[J]. Plant Physiology, 2001, 127(4):1346-1353.
[8] 李剑, 赵常玉, 张富生, 等. LEA蛋白与植物抗逆性[J]. 植物生理学通讯, 2010, 46(11):1101-1108.
[9] zhao Y, wang Y, liu Q, et al. Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco[J]. Plant Cell Tissue and Organ Culture, 2017, 130:379-391.
[10] Wang L, Li X, Chen S, et al.Enhanced drought tolerance in transgenic Leymus chinensis TaLEA3[J]. Biotechnology Letter, 2009, 31(2):313-319.
[11] Baker J, Van Dennsteele C, Dure L.Sequence and characterization of 6 lea proteins and their genes from cotton[J]. Plant Mol Biol, 1989, 12(5):475-486.
[12] Bray EA.Molecular responses to water deficit[J]. Plant Physiology, 1993, 103(4):1035-1040.
[13] Tunnacliffe A, Wise MJ.The continuing conundrum of the LEA proteins[J]. Naturwissenschaften, 2007, 94:791-812.
[14] Battaglia M, Olvera-Carrillo Y, Garciarrybio A, et al, The enigmatic LEA proteins and other hydrophilins[J]. Plant Physiology, 2008, 148:6-24.
[15] Goyal K, Walton LJ, Tunnacliffe A.LEA proteins prevent protein aggregation due to water stress[J]. Biochemistry Journal, 2005, 388(1):151-157.
[16] Manfre AJ, Lanni LM, Marcotte WR.The Arabidopsis group 1 late embryogenesis abundant protein AtEM6 is required for normal seed development[J]. Plant Physiology, 2006, 140:140-149.
[17] Tang W, Page M.Overexpression of the Arabidopsis AtEm6 gene enhances salt tolerance in transgenic rice cell lines[J]. Plant Cell Tissue and Organ Culture, 2013, 114:339-350.
[18] Hand SC, Menzea MA, Tonerb M, et al.LEA proteins during water stress:not just for plants anymore[J]. Annual Review of Physiology, 73:115-134.
[19] Moons A, De Keyser A, Van Montagu M.A group 3 LEA cDNA of rice, responsive to abscisic acid, but not to jasmonic acid, shows variety-specific differences in salt stress response[J]. Gene, 1997, 1119(97):197-204.
[20] Liu Y, Wang L, Cai GH, et al.Response of tobacco to the Pseudomonas syringae pv. tomato DC3000 is mainly dependent on salicylic acid signaling pathway[J]. FEMS Microbiology Letter, 2013, 344(1):77-85
[21] Liu Y, Wang L, Xing X, et al.ZmLEA3, a multifunctional group 3 LEA protein from maize(Zea mays L. ), is involved in biotic and abiotic stresses[J]. Plant Cell Physiology, 2013, 54(6):944-959.
[22] Amara L, Capellades M, Ludevid MD.Enhance water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene[J]. Plant Physiology, 2013, 170(9):864-873.
[23] Larkin MA, Blackshields G, Brown NP, et al.Clustal W and Clustal X version 2. 0[J], Bioinformatics Applications Note, 2001, 23(21):2947-2948.
[24] Liu ZY, Li XL, Qi X, et al.Alfalfa science research by Chinese scholars science 1950:history and main topics[J]. Acta Prataculturas Sinica, 2015, 24(10):58-69.
[25] Battaglia M, Covarrubias AA. Late Embryogenesis Abundant(LEA)proteins in Legumes[J]. Front Plant Sci, 2013, 4:190.
[26] Kalemba EM, Bagniewska-Zadworna A, Ratajczak E.Multiple subcellular localizations of teins in the embryonic axes of common beech(Fagus sylvatica L. )seeds during maturation and dry storage[J]. Journal of Plant Growth Regulation, 2015, 34(1):137-149.
[27] Grahman LN, Chen L, Nazim S, et al.Interactions of intrinsically disordered Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes-synergistic effects of lipid composition and temperature on secondary structure[J]. Biochemistry and Cell biology, 2010, 88(5):791-807.
[28] Baker I, Steele C, Dure L.Sequence and characterization of 6 LEA proteins and their genes from cotton[J]. Plant Mol Biol, 1988, 11:277-291.
[29] Galau GA, Wang HY, Hughes, et al. Cotton Lea5 and Lea74 encode atypical late embryogenesis-abundant proteins[J]. Plant Physiology, 1993, 101:695-696.
[30] Liu R, Liu M, Liu J, et al.Heterologous expression of a Ammopiptanthus mongolocus late embryogensis abundant protein gene(Am-LEA)enhance Escherichia coli viability under cold and heat stress[J]. Plant Growth Regulation, 2010, 60(2):163-168.
[31] Wang WG, Li R, Liu B, et al.Alternatively spliced transcripts of group 3 late embryogenesis abundant protein from Pogonatherum paniceum confer different abiotic stress tolerance in Escherichia coli[J]. Journal of Plant Physiology, 2012, 169(15):1559-1564.
[32] Ochoa-Alfaro AE, Rodríguez-Kessler M, Pérez-Morales MB, et al.Functional characterization of an acidic SK3 dehydrin isolated from an Opuntia streptacantha cDNA library[J]. Planta, 2012, 235(3):565-578.
[33] Lakshmi TV, Varalaxmi Y, et al.Metabolic engineering of SK2-type of dehydrinⅠ(DHN1)gene isolated from Sorghum bicolor enhances tolerance to water-deficit and NaCl stresses in transgenic tobacco[J]. Plant Omics, 2015, 8(6):556.
[34] Shi J, Liu M, Chen Y, et al.Heterologous expression of the dehydrin-like protein gene AmCIP from Ammopiptanthus mongolicus enhance viability of Escherichia coli and tobacco under cold stress[J]. Plant Growth Regulation, 2015, 79(1):1-10.
[35] Rodriguez-Salazar J, Moreno S, Espín g. LEA proteins are involved in cyst desiccation resistance and other abiotic stresses in Azotobacter vinelandii[J]. Cell Stress and Chaperones, 2017, 22:397-408.
[36] Wu Y, Liu C, Kuang J, et al.Overexpression of SmLEA enhance salt and drought tolerance in Escherichia coli and Salvia miltiorrhiza[J]. Protoplasma, 2011:1-9.
[37] Zhang L, Oht A, Takagi M, et al.Expression of plant group 2 and group 3 lea gene in Saccharomyces cerevisiae revealed functional divergence among LEA proteins[J]. Journal of Biochemistry, 2000, 127(4):611-616.
[38] Lan T, Cai D, Zhang YZ.Expression of three different group soybean lea genes enhance stress tolerance in Escherichia coli[J]. Journal of Integrative Plant Biology, 2005, 42(5):613-621.
[39] Park SC, Im YH, Jeong JC, et al.Sweetpotato late embryogensis abundant 14(IbLEA14)gene influences lignification and increases osmotic-and salt stress-tolerance of transgenic calli[J]. Planta, 2011, 233(3):621-634.
[40] Salleh FM, Evans K, Goodall B, et al.A novel function for a redox-related LEA protein(SAG21/AtLEA5)in root development and biotic stress responses[J]. Plant, Cell＆Environment, 2012, 35(2):418-429.