摘要
雪莲PEBP基因是本实验室分离出的新抗寒相关基因，共有510个核苷酸，网上进行BLAST对比，与拟南芥(Arabidopsis thaliana)中的磷脂酰乙醇胺结合蛋白基因类似，相似性81％。本实验以实验室构建的Teasy-PEBP质粒为模板，扩增PEBP基因，克隆到表达载体pET30(+)和pPIC9k并导入大肠杆菌BL21(DE3)和毕赤酵母(Pichia pastoris)GS115细胞内表达，纯化表达产物，优化表达条件，主要结果如下：
根据pET30(+)多克隆位点设计PEBP基因特异性引物，引入EcoRⅠ和NcoⅠ酶切位点，以Teasy-PEBP质粒为模板，扩增PEBP基因，连接到原核表达载体pET30(+)上，转化感受态表达菌株BL21(DE3)，在低温下，低浓度IPTG诱导融合蛋白表达，纯化表达产物，Western blot鉴定目的蛋白，SDS-PAGE分析：其相对分子量约为28KD，与预期相符，表达量约占菌体蛋白的26．8％，并且通过亲和层析纯化了重组融合蛋白，Western blot鉴定为阳性。
根据pPIC9k多克隆位点设计PEBP基因引物，分别加入EcoRⅠ和NotⅠ酶切位点，以Teasy-PEBP质粒为模板，扩增PEBP基因，克隆到表达载体pPIC9k，得到重组质粒并命名为pPIC9k-PEBP，将pPIC9k-PEBP用SalⅠ进行酶切，线性化后的pPIC9k-PEBP以电转化法导入到酵母GS115细胞中，利用MM和MD平板筛选Mut~s(methanol utilization slow)表型，PCR鉴定重组酵母菌株，乙醇诱导表达。SDS-PAGE分析：其相对分子量约为20KD，蛋白表达量可占总蛋白的90％，上清液抗冻检测表明，PEBP能降低液体的冰点，具有一定的抗冻作用。
优化重组质粒pPIC9k-PEBP在毕赤酵母(Pichia pastoris)GS115中的表达条件，初步得到最佳的重组蛋白表达条件为：诱导剂甲醇浓度为0．5％，诱导培养基的PH为6．0，诱导温度为32℃，诱导时间为216h。
实验成功构建了表达载体pET-PEBP和pPIC9k-PEBP，获得了高效表达产物，并为进一步研究雪莲PEBP的抗冻功能奠定基础。
PEBP gene is new Antifreeze gene that was separated by our laboratory in Saussurea involucrate Kar.er kir. The length of PEBP is 510bp,Homology search with PEBP were performed against GenBank using BLAST, the identity with PEBP of Arabidopsis thaliana were 81%.ln this research, Using Teasy-PEBP plasmid as a template ,the PEBP gene was amplified by PCR .PEBP gene was inserted into pET30a(+) andpPIC9K, then expressed in E. coli BL21 and pichia pastoris GS115, respectively. The expression products were purified by affinity chromatography, and the optimal expression condition was obtained by preliminary research. The main results were as follows:
A pair of specific primers PEBP sense and PEBP anti-sense were designed based on the multi clone site (MCS) of pET30a(+), introduced restriction site EcoRⅠand NcoⅠrespectively. The PEBP gene was amplified from Teasy-PEBP plasmid by PCR and inserted into the pET30a(+) vector. The recombinant vector was transferred into Ecoli BL21(DE3)and induced the expression of protein by low concentration of IPTG and low temperature overnight. Fusion protein was purified by Ni-NTA affinity chromatography. SDS-PAGE and Western blot analysis showed that the fusion protein relative molecular weight was 28KD that was consistent with the theoretical value, and its product was 26.8% in total protein.
Expression primers PEBP sense and PEBP anti-sense were designed based on the MCS)of pPIC9K, introduced restriction site EcoRⅠand NotⅠrespectively. The PEBP gene was amplified from Teasy-PEBP plasmid by PCR and inserted into the Pichia pastoris expression vector. The recombinant plasmid named as pPIC9K-PEBP was linearized by SalⅠ,and men transformed into Pichia pastoris GS115. Mut~s (Methanol utilization slow) transformants were screened by MD and MM plates, confirmed by PCR. Supernatant of the culture was analyzed by SDS-PAGE, a size of specific strip was found, indicating that the PEBP gene was expressed and its relative molecular weight is 20KD, and target protein was 90% in total protein. The PEBP has been definiteanti-freeze character by the biological activity detection of the supernatant.
The optimal expression parameters of GS115 were 0.5% ( V/V ) CH_3OH concentration, PH 6.0 culture medium. 30℃culture temperature, 216 hours inducing time.
The prokaryotic and Eukaryotic expression system of PEBP is successfully constructed. It lays the foundation for the further study on the Antifreeze characters of the PEBP
引文
[1] Gutfreund H. Kinetics for the Life Sciences:Receptors, Transmitters and Catalysts,Cambridge University Press, 1995, 278-306
[2] Levitt J. Responses of plants to environmental stress. Chilling, Freezing, and High Temperature Stresses, 2Ed. Academic Press, New York, 1980
[3] Pearce RS and Ashworth EN. Cell shape and localization of ice in leaves of overwintering wheat during frost stress in the field. Planta, 1992,188:324-331
[4] Bixby JA and Brown GM. Ribosomal changes during induction of cold hardiness in black locust seedlings. Plant Physiol, 1975,56:617-621
[5] Laroche A and Hopkins WG.. Polysomes from winter rye seedling grown at low temperature.Plant Physiol, 1987, 85:648-654
[6] Lindow SE. Use of genetically altered bacteria to achieve plant frost control. In:NakasJP and Hagedorn C (eds) Biotechnology of Plant-Microbe Interactions. New York:McGraw-Hill, 1990,85-110
[7] Griffith M and Antikainen M. Extracellular ice formation in freezing-tolerant plants. Adv Low-temp Biol, 1996,3:107-139
[8] Mazur P. Cryobiology. The freezing of biological systems. Science, 1970, 168:939-947
[9] Steponkus PL. Preface. Advances In low temp boil, 1992, 1:ⅸ-ⅹⅰ
[10] Steponkus PL, Uemura M and Webb MS. A contrast of the cryostability of the plasma membrane of winter rye and spring oat. Two species that widely differ in their freezing tolerance and plasma membrane lipidcomposition. In PL Steponkus, ed, Advances in Low-temperature Biology, VoL2. JAI Press, London, 1993, 211-312
[11] Williams WP. Cold-induced lipid phase transitions. Phil Trans Royal Soc Lond Series B Bial Sci, 1990,326:555-570
[12] Pearce RS and Willison JHM. A freeze-etch study of the effect of extracellular freezing on the cellular membranes of wheat. Pianta, 1985,163:304-316
[13] Lyons JM, Raison JK(1970).Oxidative activity of mitochondria isolated from plant tissus sensitive and resistant to chilling injury.Plant Physiol 45(4):386-389
[14] 沈漫，王明麻，黄敏仁．植物抗寒机理研究进展．植物学通报，1997，14(2)：1-8
[15] 简令成．植物冻害和抗寒性的细胞生物学研究．植物生理生化进展．1996
[16] Thomashow MF. Role of cold-responsive genes in plant freezing tolerance. Plant Physiol, 1998, 118: 1-7
[17] Weiser C J.Cold resistance and injury in woodu plant.Science 1970, 169:1269-1278
[18] Guy C.Moleeular responses of plants to cold shock and cold acclimation. [J].Mol. Microbio. 1999
[19] Thomashow, MF.So What'sNew in the Field of Plant Cold Acclimation?Lots!Plant Physiol., 2001, 125:89-93
[20] 王艇，苏应娟，刘良文．植物低温诱导蛋白和低温诱导基因的表达调控．武汉植物学研究，1997，15(1)：80-90
[21] Kodama H, Horiguchi G, Nishiuchi T, et al. Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves. Plant Physiol, 1995,107(4): 1177-1185
[22] Hughes MA, Dunn MA.The molecular biology of plant acclimation to low tenperature.JExp Bot, 1996, 7:377-403
[23] Yamaguchi-Shinozaki K, Shinozaki K.A Novel cis-Acting Element in an Arabidopis Gene Is Involved in Responsiveness to Drought,Low Temperature,or Hihg-Salt Stress.Plant Cell, 1995, 6:251-264
[24] Worrall D, Elias L, Ashford D, et al. Acarrot leucinerich repeat proein that inhibits ice recrystallization[J]. Science, 1998, 282(2): 115—117
[25] 尹明安，崔鸿文，樊代明，等．抗冻蛋白基因的克隆及测序[J]．园艺学报，2001，28(2)：173—174
[26] Huang T, DuiTlan J G. Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA——bind—ing activity from winter bittersweet nightshade, Solanumdulcarnara[J]. Plant Mol Bid, 2002, 48(4): 339—350
[27] Sidelmttomm C, Worrall D. Heat stable antifreeze protein from grass[J]. Nature, 2000, 406: 256—263
[28] Yen S, Griffith M. Chitinase genes responsive to mid en—coden antifreeze proeins in winter cereals[J]. Plant Physiol, 2000, 124:1251—1264
[29] 范云，刘兵，王宏斌，等．胡萝卜抗冻蛋白基因的克隆及其在E．coli中的表达[J]．中山大学学报(自然科学版)，2002，41(3)：52—55
[30] LinYZ, ZhangZY, et al. One rapid an defficient method for total RNA from shoots regenerated invitro of Populous suaveolens[J]. Forestry Studies in China, 2003(6): 18—21
[31] Pihakaski—Maunsbach K, Tamminen L, Pietinen M, et al. Antifreeze proteins are secreted by winter rye cellsin suspension culture[J]. Physiol Plant, 2003, 118: 393—398
[32] Griffith M,Ala P, Yang D S C, et al. Antifreeze proteins produced endogenously in winter rye leaves[J]. Plant Physiol, 1992, 100: 593—596
[33] Hon W C, Giffith M, Mlyrm-z A, et al. Antifreeze proteins in winter rye are similar to pathogenesis—related proteins[J]. Plant Physiol, 1995, 109(3): 879—889
[34] Duman J G. Purification and characterization of a thermal hysteresis protein from a plant, the bittersweet nightshade(Sol—num dulcamara)[J]. BiochimBiophysActa, 1994, 1206(1): 129—135
[35] 江勇，魏令波，费云标，等．分离和鉴定沙冬青抗冻蛋白质[J]．植物学报，1999，41(9)：967—971
[36] Michael W, Robert W, Ron B, et al. Purification, immunolocalization cryoprotective, and antifreeze activityofPCA60: A dehydrin from peach(Prunuspersica)[J]. Physiologia Plantarum, 1999, 105: 600—608
[37] Sidebottomm C, Worrall D. Heat stable antifreeze pro—tein fmmgrass[J]. NanIre, 2000, 406:256—263
[38] 祭美菊，安黎哲，陈拓，等．天山寒区冰缘植物珠芽蓼叶片抗冻蛋白的发现[J]．冰川冻土，2001，23(4)：210—216
[39] Nelly M T, Brian L P, Viswanathan V K, et al. Dynsmics of antifreeze glycoptoteins in the presence of ice[J]. Biophys, 2002, 82(1): 464—473
[40] Bravo L A, Grifith M. Characterization of antifreeze activity in Antarctic plants[J]. Journal of Experimental Botany, 2005, 56(414): 1189—1196
[41] ZhouS, Sauv6R J, Mmbaga M T. Adaptation of Pachysandra terminalis Sie& Zucc to freezing tempera—tures by the accumulation of Mrna and cold——induced pmteins [J]. Hort Science, 2005, 40(2): 346—347
[42] Griffith M, Marentes E, Ala P, et al. The role of ice binding proteins in frost tolerance of winter rye[A]. LiPH eds. Advance in plant cold hardness[C]. Boca Ra. ton/Ann Arlmr/London/]1Okyo: C R C Press. 1993. 178—186
[43] Urrutia M E, DutTlan J G, Knight CA. Plant thermal—hysteresis proteins[J]. Biochem Biophy Acta, 1992,1121(1): 199—206
[44] An tikainen M, Griffith M, Zhang J, et al. Immunolo—calization of an tifreege proteins in-rater rye leaves, crown, and root by tissue printing[J]. Plant Physiol, 1996, 110: 845—857
[45] Meyer K, Keil M, Naldrett M J. A leucine—rich repeat protein of carrot that exhibits antifreeze activity[J]. FEBS Letters, 1999, 447:171—178
[46] 尹明安，崔鸿文，樊代明，等．胡萝卜抗冻蛋白基因可隆及植物表达载体构建[J]．西北农业科技大学学报(自然科学版)，2001，29(1)：6—10
[47] Fan Y, Liu B, Wang H B, et al. Cloning of an antifreeze protein gene from carrot and its influence on cold tolerance in transgenic tobacco plants[J]. Plant Cell Rep, 2002, 21: 296—301
[48] Cregg J M, Barringer K J, Hessler A Y, et al. Pichia pastoris as a host system for transformations[J]. Mol Cell Biol, 1985, 5(12): 3376-3385
[49] Hollenberg C P, Gellissen G. Production of recombinant proteins by methylotrophicyeasts[J]. Curt Opin Biotechnol, 1997, 8(5): 554-560
[50] 贾向志，袁汉英，马文煜．人溶菌酶基因的克隆及其在毕赤酵母中的表达[J]．第四军医大学学报，2001，22(22)：2068-2072
[51] 周祥山，范为民，张元兴．不同甲醇流加策略对重组毕赤酵母高密度发酵生产水蛭素的影响[J]．生物工程学报，2002，18(3)：348-351
[52] G Gellissen,M Piontek.U Dahlems et al.Recombinant hansenula polymorpha as a biocatalyst: coexpression of the spinach glycolate oxidase(GO) and the S cerevisiae catalase T(CTT1) gene[J].Appl Microbiol Biotechnol,1996, 46:46-54
[53] 费云标，高素琴，王维香，魏令波．植物冻害与抗冻研究对策探讨．科学新闻周刊，2001，13：42-43
[54] Steponkus PL and Webb MS. Freeze-induced dehydration and membrane destabilization in plants.In G Somero,B Osmond,eds, Water and Life:Comparative Analysis of WaterRelationships at the Organismic, Cellular and molecular Level. Springer-Verlag, Berlin, 1992, 338-362
[55] Li P(李萍)，Ai XL(艾秀莲)，Wang ZF(王志方)etal．Construction of Plant Expression Vector pXLPBP[J]. BIOTECHNOLOGY (生物技术), 2006, 16(2): 11-13
[56] Joseph Sambrook, David W.Russell.Molecular Clonging: a Laboratory Manual[M].Third Ed.New York: Cold Spring Harbor Laboratory Press, 2002: 1-50.
[57] 李美茹，刘鸿先，王以柔．Advances in Researches on Molecular Biology of Plant Cold Resistance [J].热带亚热带植物学报，2000，8(1)：70—80
[58] 林善枝，张志毅，林元震．植物抗冻蛋白分子改良[J]．植物生理与分子生物学报，2004，30(3)：251-260
[59] 郭建强，李运敏，岳丽丽等．Molecular Clonging and Expression of Extremely Thermostable and Acid-stable Amylase Gene in Pichia pastoris [J].生物工程学报，2006，22(2)：237-242
[60] Levitt J. Responses of Plant to Environmental Stresses: Chilling, Freezing, and High Temperature Stresses[M].2nd ed. New York: Academic Press, 1980.