植物磺肽素（PSK-α）化学磺化与酶法磺化初探及其生物功能应用
|
摘要
植物磺肽素α(Phytosulfokine-α,PSK-α)是近些年新发现的一种多肽类植物内源激素,由一个五肽和两个磺酸基团组成,它广泛存在于大多数植物中,具有促进细胞分裂和分化,促进植物遗传转化,调节花粉萌发以及提高植物抗性等多种重要的生理功能。而目前PSK-α的合成主要有化学合成和生物合成两种方法,其中,五肽的磺化是合成的关键因素。本研究的主要目的是利用化学和生物(酶法)的方法将磺酸基团加入到已有的五肽上,同时将获得的PSK-α应用于目前国内研究相对较少的植物抗衰老和植物快繁两个领域,证明了其生理功能。本研究主要开展了下面几个方面的工作:
1.利用化学方法,将磺酸基团加入到五肽上,对产物进行了分离纯化以及质谱分析,最终获得了目的产物PSK-α;并引入了金属Na+对磺化过程的稳定性进性了比较。
2.通过查阅大量文献资料发现在某些生物体中存在着一种芳基磺基转移酶,利用酶的化学反应轻松将所需要的磺酸基团转移到酪氨酸的苯环上,从而实现PSK-α的生物学活性。这样,我们查阅到了对人体有益的一种乳酸菌—植物乳杆菌(L.plantarum),从它的基因组中克隆出芳基磺基转移酶的基因片段,但是此基因片段存在着大量的非限制性片段干扰。
3.用获得的PSK-α对莴苣离体叶片进行了抗衰老实验,研究了PSK-α对莴苣离体叶片衰老过程中抗氧化酶系统(H2O2含量,SOD酶,CAT酶,Apx酶活性)以及衰老指标(MDA,叶绿素,蛋白质含量)的影响。发现一定浓度的PSK-α能够明显提高莴苣离体叶片的抗衰老能力。
4.用获得的PSK-α对小麦成熟胚和非洲菊离体叶片进行了组培实验,研究了PSK-α对小麦成熟胚和非洲菊离体叶片愈伤组织生长情况的影响,测定了其出愈率和鲜重等指标。发现一定浓度的PSK-α能够提高其出愈率和鲜重。
Phytosulfokine-αis a recent discovery of a new peptide hormones in plants, it contains a five-peptide and the two sulfur groups, it is in most plants widely and with the promotion of cell division and differentiation, promotion of plant genetic transformation, regulation of pollen germination and improve plant resistance and other important physiological function. Synthetic PSK-αis currently the main chemical synthesis and biosynthesis of two ways, in which five synthetic peptides key factor in sulfonated. The main purpose of this study is the use of chemical and biological(enzymatic) methods to sulfur groups to the five peptides. At the same time ,use the received PSK-αto study Anti-aging in plants and plant propagation two fields which few study in China. This study demonstrated the biological function of its. This study conducted the following aspects of work:
1. Use chemical methods to sulfur group to five peptides, the product is purified and mass spectrometry analysis, and finally obtained the product of PSK-α.At the same time, we study the introduction of the metal Na+, compare sulfonation process stability.
2. Through access to large number of documents found in some organisms there is a sulfortransferase. We use the enzymatic method to transfer the sulfur group to the benzene ring of tyrosine, in order to achieve the biological activity of PSK-α.We find a lactic acid bacteria (L.plantarum) beneficial to human. We clone sulfotransferase gene fragments from its genome cloned. However, this gene fragment have a lot of non-restriction fragments.
3. We use the PSK-αto study the detached leaves of lettuce on the anti-aging experiment. It contains the antioxidant enzyme system( the content of H2O2, the activity of SOD, CAT, APx) and the indicators of aging(the content of MDA, Chlorophyll, Protein). We find that a certain concentration of PSK-αcan significantly enhance the lettuce leaves in vitro anti-aging ability.
4. We use the PSK-αto study wheat mature embryos and gerbera leaves on the callus experiments . Study the vitro growth of callus of wheat mature embryos and gerbera leaves in different concentration of PSK-α.We measure the rate of callus and fresh weight, find that a certain concentration of PSK-αcan increase the rate of callus and fresh weight.
1.利用化学方法,将磺酸基团加入到五肽上,对产物进行了分离纯化以及质谱分析,最终获得了目的产物PSK-α;并引入了金属Na+对磺化过程的稳定性进性了比较。
2.通过查阅大量文献资料发现在某些生物体中存在着一种芳基磺基转移酶,利用酶的化学反应轻松将所需要的磺酸基团转移到酪氨酸的苯环上,从而实现PSK-α的生物学活性。这样,我们查阅到了对人体有益的一种乳酸菌—植物乳杆菌(L.plantarum),从它的基因组中克隆出芳基磺基转移酶的基因片段,但是此基因片段存在着大量的非限制性片段干扰。
3.用获得的PSK-α对莴苣离体叶片进行了抗衰老实验,研究了PSK-α对莴苣离体叶片衰老过程中抗氧化酶系统(H2O2含量,SOD酶,CAT酶,Apx酶活性)以及衰老指标(MDA,叶绿素,蛋白质含量)的影响。发现一定浓度的PSK-α能够明显提高莴苣离体叶片的抗衰老能力。
4.用获得的PSK-α对小麦成熟胚和非洲菊离体叶片进行了组培实验,研究了PSK-α对小麦成熟胚和非洲菊离体叶片愈伤组织生长情况的影响,测定了其出愈率和鲜重等指标。发现一定浓度的PSK-α能够提高其出愈率和鲜重。
Phytosulfokine-αis a recent discovery of a new peptide hormones in plants, it contains a five-peptide and the two sulfur groups, it is in most plants widely and with the promotion of cell division and differentiation, promotion of plant genetic transformation, regulation of pollen germination and improve plant resistance and other important physiological function. Synthetic PSK-αis currently the main chemical synthesis and biosynthesis of two ways, in which five synthetic peptides key factor in sulfonated. The main purpose of this study is the use of chemical and biological(enzymatic) methods to sulfur groups to the five peptides. At the same time ,use the received PSK-αto study Anti-aging in plants and plant propagation two fields which few study in China. This study demonstrated the biological function of its. This study conducted the following aspects of work:
1. Use chemical methods to sulfur group to five peptides, the product is purified and mass spectrometry analysis, and finally obtained the product of PSK-α.At the same time, we study the introduction of the metal Na+, compare sulfonation process stability.
2. Through access to large number of documents found in some organisms there is a sulfortransferase. We use the enzymatic method to transfer the sulfur group to the benzene ring of tyrosine, in order to achieve the biological activity of PSK-α.We find a lactic acid bacteria (L.plantarum) beneficial to human. We clone sulfotransferase gene fragments from its genome cloned. However, this gene fragment have a lot of non-restriction fragments.
3. We use the PSK-αto study the detached leaves of lettuce on the anti-aging experiment. It contains the antioxidant enzyme system( the content of H2O2, the activity of SOD, CAT, APx) and the indicators of aging(the content of MDA, Chlorophyll, Protein). We find that a certain concentration of PSK-αcan significantly enhance the lettuce leaves in vitro anti-aging ability.
4. We use the PSK-αto study wheat mature embryos and gerbera leaves on the callus experiments . Study the vitro growth of callus of wheat mature embryos and gerbera leaves in different concentration of PSK-α.We measure the rate of callus and fresh weight, find that a certain concentration of PSK-αcan increase the rate of callus and fresh weight.
引文
[1] Pearce G,Strydom D,Johnson S et al.A polypeptide from tomato leaves induces wound-inducible proteinase-inhibitor proteins.Science,1991,253:895-898
[2]沈世华,朱至清。新型植物生长调节物质—激素性多肽的研究进展。植物学通报,1999,16:648-652
[3] Matsubayashi Y,Sakagami Y.Phytosulfokine,sulfated peptides that induce the proliferation of singlle mesophyll cells of Asparagus officinalisL.Proc Natl Acad Sci USA,1996,93:7623-7627
[4] MatsubayashiY,SakagamiY,TakagiL.Phytosulfokine,asulfatedpentapeptide tapeptide,stimulates the proliferation of rice cells by means of specifichigh-andlow-affinitybindingsites.ProcNatlAcadSciUSA,1997,94:13357-13362
[5] MatsubayashiY,Hannai H,Hara O,et al.Active fragments and analogs of the plant growthfactor,phytosulfokine:structure-activity-relationships[J].Biochem.Biophy.Res.Com.,1996,225:209-214
[6] MatsubayashiY,TakahataY,MoritaA,et al.Preparation and characterization of fully active biotinylated analogs of phytosulfokine-α[J].Biochem.Biophy.Res.Com.,1999,63:1847-1849
[7] Bahyrycz A,Matsubayashi Y,Ogawa M,et al.Plant peptide hormone phytosulfokine-α:synthesis of new analogues and their biological evalution[J].Pnas,1997,94:13357-13362
[8] Bahyrycz A,Matsubayashi Y,Ogawa M,et al.Further analogues of plant peptide hormone phytosulfokine-αand their biological evalution[J]..Pept.Sci.,2004,10:462-469
[9] MatsubayashiY,TakahataY,MoritaA et al.An LRR receptor kinase involved in perception of a peptide plant hormore,Phytosulfokine.Scinece,2002,296:1470-1472
[10] Matsubayashi Y,Sakagami Y.120-and160-Kda receptors of endogenous mitogenicpeptede,phytosulfokine-α,in rice plasma membrances.[J] Biol Chem,2000,275:15520-15525
[11] Matsubayashi Y, MoritaA,Matsunaga E et al.Physiological relationships between anxin,cytokinin,and apeptide growth factor,phytosulfokine-α,instimulation of asparagus cell proliferation.Planta,1999,207:559-565
[12] Matsubayashi Y,Goto T.Sakagami Y.Chemical nurising:phytosulfokine improve genetic transformation efficiency bi promoting the proliferation of suiviving cells on selective media[J].Plant Cell Rep.,2004,23:155-158
[13] Chen D,Xu Z,et al.Effect of phytosulfokine-αon Agrbacterium-mediated transfomation in rice[J].Rice Sci.,2005,12:255-260
[14] Sasaki K,IshiseT,Shimomura K,et al.Effects of phytosulfokine-αon growth and tropane aklaloid production in transformen roots of Atropabelladonna[J].Plant Growth Reg.,2002,26:87-89
[15] Kimb J,Gibson D M,Shuler M L.Effect of the plant peptide regulator,phytosulfokine-α,on the growth and Taxol production from Taxus sp.suspension cultures[J].Biotech.Bioengi.,2006,95:8-14
[16] Yang H,Matsubayashi Y,Nzkamura K,et al.Oryzasativa PSK gene encodes a precursor of phytosulfokine-α,a sulfated pepetide growth factor found in plants[J].Pnas,1999,96:13 13560-13565
[17] Yang H,MatsubayashiY,HannaiH,et al.Molecular cloning and characterization of Os PS K,a gene encoding a p recursor for phytosulfokine-α,required for rice cell proliferation[J].Plant Mol.B iol.,2000,635:635-647
[18] Yang H,Matsubayashi Y,Nzkamura K,et al. Diversit y of A rabidopsis genes encoding precursors for phytosulfokine-aαpepetide growth factor[J].Pl ant Physiol.,2001,127:842-851
[19] Rosenbergrd,Shworaknw,L iu J,et al.Heparan sulfate proteoglycans of the cardiovascular system.Specific structures emergebut how is synthesis regulated[J].J.Cli n.I nvest.,1997,99:2 062-2 070
[20] Hanai H,MatsunoT,YamamotoM,et al.A secreted peptide growthfactor,phytosulfo kine,acting as a stimulatory factor of carrot somatic embryo formation[J].Pl ant Cell Physiol.,2000,41:27-32
[21] Matsubayashi Y,Sakagami Y.Characterization of specific binding sites for a mitogenic sulfated peptide,phytosulfokine-α,in the plasma membrane fraction derived from Oryza sativa L[J].Eur.J.B iochem.,1999,262:666-671
[22] YamakawaS,et al.Promotive effects of the peptidyl plant growth factor, p hytosulfokine-αon the growth and chlorophyll content of A rabidopsis seedlings under high night-time temperature conditions[J]. Biosci.Biotech.Biochem.,1999,63:2 240-2 243.
[23] Eun C,Ko S,MatsubayashiY,et al.Phytosulfokine-αrequires auxin to stimulate carrot non-embryogenic cell proliferation[J].Plant Physiol. Biochem,2003,41:447-452
[24] ChenY F,et al.Peptide growth factor phytosulfokine-αcontributes to the pollen population effect[J].Plant a,2000,211:752-755
[25] Lorbiecke R,et al.Comparative analysis of PSK peptide growth factor precursor ho mologs[J].Plant Sci.,2002,163:321-332
[26] SturataR,Street H E.Studies on t he growth in cult ure of plant cells.The initiation of division in suspensions of stationary-phasecells of Acer pseudopllatanus L.[J].J.Exp.B ot.,2005,20:556-571
[27] Bodanszky M.Natarajan S,HahneW,Gardner JD. Choleeystokinin(Pancreozymin).3.synthesis and properties of ananalogue of the C-tenninal heptapeptide with serine sulfate rlacing tyrosine sulfate.Joumal of MedicinalChemistry,1977,20:1047-1050
[28] Meldal M.Preparation of novel O-sulfated amino aeid building bloeks withi mproved acid stability for Fmoc-based solid-phase peptide synthesis.Journal of Chemistry Soeiety,PerkinTransactionsl,2002,682-686
[29] CarpinoLA.New Methods of Introducing the Carbo-t-butoxy Proteetive Group.Preparation of t-Butyl Cyanoformate.Journal of the AinerieanChemieal Soeiety,1960,82:2725-2727
[30] CarpinoLA,Han GY The 9-Fluorenylmethoxycarbonyl Amino-Proteeting Group.The Journal of organic Chemistry,1972,37:3404-3409
[31] CarpinoLA,Imazumi H,El-Faham A,Ferrer F J,Zhang C,Lee Y Foxman B M,Henklein P,Hanay C,Mugge C,Wenschuh H,Klose J,Beyennann M,Bienert M.The Uronium/Guanidinium Peptide CouPling Reagents:Finally theTrue UroniumSalts.Angewandte Chemie International Edition,2002,41:441-445
[32] CarpinoL A,Imazurni H,El-faham,A.Uronium and irnmonium salts for peptide couPling.US Patent,6825347,2002-05-21
[33] Challa H,Woski S A.Solution Phase Synthesis of Potential DNA-Binding Moleeules based on the PNA Baekbone.Tetrahedron Letters,1999,40:419-422
[34] Coste J,Frerot E,Jouin P,Castro B.Oxybenzotriazole free peptide coupling reagents for N-methylated amino acids.Tetralledron Letters,1991,32:1967-1970
[35] Coste J,Frerot E,Patrick J.Coupling N-methylated amino acids using PyBroP and PyCloP halogenoPhoSPhonium salts:mechanism and fields of applieation.The Joumal of Organic Chemistry)1994,59:2437-2446
[36] Dhaon M K,Olsen R K,Ramasamy K.Esterification of N-protected alPha-amino acids with alcohol/earbodiimide/4-(dimethylamino)pyridine.Racemization of asPartic and glutamic acid derivatives.The Joumal of Organic Chemistry,1982, 47:1962-1965
[37] Durieux P,Fernandez-Cameado J,Tuehseherer G.Sythesis of biotinylated glycosulfopeptides by chemoseleetive ligation.Tetrahedron Letters,2001,42: 2297-2299
[38] Falkiewiez B.Comparison of the effieiency of variouse coupling systems in the aeylation of model seeondary amines with thymin-1-ylacetic acid.Nueleie Acids Symposium Series,1999,42:153-154
[39] Fields G B.Methods for Removing the Fmoc Group.In:Pennington M W,Dunn B Meds.,peptide Sythesis Protocols.Totowa:HumanaPress,1994,17-27
[40] Frerot E,Coste J,Poncet J,Jouin P,Castro B.N-methyl N-carboxyanhydride:an unexpeeted by-produc twhen eoupling boe-N-methyl amino acids.TetrahedronLetters,1992,33:2815-2816
[41] Gibson F S,Park M S,RapoPort H.Bis[[4-(2,2-dimethyl-1,3-dioxolyl)]methyl]- -carbodiimide(BDDC) and its application to residue- free esterifications,peptide eouplings,and dehydrations.The Journal of organic Chemistry-1994,59:7503-7507
[42] Gilbert E E.The reaetion of sulfur trioxide and of its adduets with organic compounds Chemical Reviews,1962,62:549- 589
[43] Han H,Wolfe M M,Brenner S,Janda K D.Liquld-Phasee combinatorial synthesis. Proceedings of the National Aeademy of Scienees of the United States of America 1995,92:6419-6423
[44] Han S Y and KimY A.Reeent development of peptide coupling reagents in organic synthesis.Tbtrahedron,2004,60:2447-2467
[45] Izdebski J,Kunee D.Evaluation of CarbodiimidesUsing a Competition Method. Joumal of peptide Seience,1997,3:141-144
[46] Jarowieki K and Kocienski P.Protecting groups.Joumal of Chemical Soeiety Perkin Transactionsl,2001,2109-2135
[47] Kice J L,Anderson J M.The mechanism of the acid hydrolysis of sodium arylsulfates.Journal of American Chemical Soeiety,1966,88:5242-5245
[48] Li P,Xu J C.New and highly effieient irnmonium type peptide coupling reagent: synthesis,meehanism and applieation.Tetrahedron,2000a,56:4437-4445
[49] Li P,Xu J C.Studies of novel and higly efficient peptide coupling reagents.Journal of the Graduate Sehool of the Chinese Aeademy of seiences,2004,21:125-134
[50] Ondetti M A,Pluizec J,Sabo E F,Sheehan J T,Williams N.Synthesis choleeystokinin-Panereozyminin.I.The C-terminal dodecapeptide.Joumal of American Chemistry Soeiety,1970,92:195-199
[51]张祎颖,洪汝科,张锦文,等。花药培养获得优质抗稻瘟病水稻[J].西南农业学报, 2008, 21(1): 75-79.
[52]柴卫淑,师佳,陈联秀,等.液体培养基在水稻花药培养中的应用研究[J].西南农业学报, 2004, 17(增刊): 78-80.
[53]李合生,孙群,赵世杰,章文华.植物生理生化实验原理及技术[M].北京:高等教育出版社,2000:167-169.
[54]张良,瞿伟菁.植物生理学实验指导(第三版)[M].北京:高等教育出版社,2003:123-124.
[55]陈金印,陈明.果实后熟哀老与植物激素的关系研究进展.江西农业大学学报2003,25(4):537-543
[56]董发刁,苗深.小麦根系过氧化氢积累与耐盐性的关系.武汉植物学研究,2002,20:293-298
[57]郭英,宋宪亮,王庆材,李玉静,孙学振.施钾对棉花苗期叶片内源激素与氧自由基代谢的影响.华北农学报,2006,21:59-62
[58]何长征,艾辛,熊先军,宋勇.硝酸银对黄瓜植株茎尖保护酶类活性的影响.湖南农业大学学报(自然科学版),2003,29(3):243-245
[59]何萍,金继运.氮钾营养对春玉米叶片衰老过程中激素变化与活性氧代谢的影响.植物营养与肥料学报,1999,5:289-296
[60]黄玉山,罗广华,关栗文.福诱导植物的自由基过氧化损伤.植物学报,1997,39:522-526
[61]谈建中.植物中的“多肤激素”.生物学通报,
[62]汤日圣,刘晓忠,陈以峰.4PU-30延缓杂交水稻叶片衰老的效果与作用.作用学报,1998,24:231-233
[63]阎秀峰,李晶.干旱胁迫对红松幼苗保护酶活性及脂质过氧化作用的影响.生态学报,1999,]9:850- 854
[64]杨淑慎,高俊风.活性氧、自由基与植物的衰老.西北植物学报,2001,21(2) :215-220
[65]杨晓红,陈晓阳,刘克锋.细胞分裂素对植物衰老的延缓作用.热带亚热带植物学报,2006,14(3):256-262
[66]张海明,王茅雁.干旱对玉米过氧化氢,MDA含量及SOD,CAT活性的影响.内蒙古农牧学院学报,1993,14:92-95
[67]张玉,李方,陈昆松,张上隆.生物自由基与植物组织衰老.园艺学进展2000:4,14-18
[68]赵春江,康书江,王纪华.植物内源激素对小麦叶片衰老的调控机理研究.华北农学报,2000,15:53-56
[69]周春华,刘红霞,韦军,活性氧与果实成熟衰老.上海交通大学学报,2002,20(l):77-84
[70]朱中华,段留生,冯雪梅.内源激素对小麦叶片衰老调控的系统分析.作物学报,1998,24:176-181
[71]于康宁.植物硫肽素_1_3_PSK的液相合成研究,华中农业大学研究生毕业论文,2009
[72]分子克隆实验指南,美,J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯著金冬雁,黎孟枫等译.
[73]杨洁彬,郭兴华,等.乳酸菌—生物学基础和应用[M].北京:中国轻工业出版社, 1996.135-146
[74]冉艳红,杨春哲,黄雪松.乳酸菌在果蔬加工中的应用现状与前景[J].中国调味品,2001,(11):3-8
[75]凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工出版社,1999.55-78
[76]施安辉,周波.乳酸菌分类,生理特性及在食品酿造工业上的应用[J].中国调味品,2001,(11):3-8
[78]冉艳红,黄雪松,于淑娟.自然发酵泡菜汁中主要成分的变化[J].中国调味品.1997,(83):248-258
[79]郭本桓.益生菌.化学工业出版社,2004:381-428
[80]胡国庆等.植物乳杆菌在不同基质中转化生成共轭亚油酸的研究.郑州工程学院学报,2004,6:53-56
[81]陆胜民等.不同发酵方法对泡雷笋品质的影响.食品与发酵工业,2004,30 (30-32)
[82]张中义等.产共轭亚油酸乳酸菌的筛选及产物分析.中国农业大学学报,2004,9 (3):5-8
[83]李平兰等.产细菌素植物乳杆菌菌株的筛选及其细菌素生物学特征研究.食品与发酵工业,1999,25 (1):1-4
[84]王立梅等.发酵香肠的工艺研究院.吉林农业大学学报,1999,21(3):104-107
[85]高云等.植物乳杆菌发酵鲜整枣的工艺研究.食品科技,2002,23 (1):23-25
[86]孙军勇,李芸.麦汁的生物酸化技术[J].食品与生物技术学报,2006,1:70-73.
[87]龚庆芳,顾国贤.生物酸化技术在啤酒生产中的应用[J].酿酒科技,2005,11:61-64.
[88]丁燕,杜金华.乳酸菌及其代谢产物在啤酒工业中的应用[J].酿酒,2001,28 (4):102-103.
[89]庞钦.酒精浓醪发酵酒糟生产乳酸的研究[D].无锡:江南大学,2002.
[90]沈萍,范秀容.微生物学实验[M].北京:高等教育出版社,1996.
[91]张波,王厚军,卞杰.啤酒厌氧菌的检测[J].中国酿造,2004,4:32-35.
[92]陈晓平,刘华英,魏小川.自然发酵酸菜汁中乳酸菌的分离筛选与鉴定研究[J].食品科学,2006,2:91-94.
[93]叶淑红,何连芳.植物乳杆菌的筛选及其在泡菜中的应用[J].中国酿造,2004,7:19-21.
[94]杨王君.食用菌与人体健康[J].四川食品与发酵,1998 (1/2):45.
[95]张虹,张卓.食用菌药用研究综述[J].大庆高等专科学校学报,1998,18 (4):78-81.
[96]中国微生物资源中心.乳酸细菌及其它益生细菌的生物学特性和应用,微生态制剂中乳酸细菌及其它益生细菌的生物学特性学习班讲义.1997:10-11,20-21,56-57
[97]黄伟坤等.食品检验与分析.北京:中国轻工业出版社,1995.454-455
[98]广东省微生物研究所.天然调味料—酵母抽提物中试报告.1987
[99]于信令等.味精工业手册.北京:轻工业出版社,1995,453-459
[100]彭小平等.食品研究与开发,1998,(4):13-15
[2]沈世华,朱至清。新型植物生长调节物质—激素性多肽的研究进展。植物学通报,1999,16:648-652
[3] Matsubayashi Y,Sakagami Y.Phytosulfokine,sulfated peptides that induce the proliferation of singlle mesophyll cells of Asparagus officinalisL.Proc Natl Acad Sci USA,1996,93:7623-7627
[4] MatsubayashiY,SakagamiY,TakagiL.Phytosulfokine,asulfatedpentapeptide tapeptide,stimulates the proliferation of rice cells by means of specifichigh-andlow-affinitybindingsites.ProcNatlAcadSciUSA,1997,94:13357-13362
[5] MatsubayashiY,Hannai H,Hara O,et al.Active fragments and analogs of the plant growthfactor,phytosulfokine:structure-activity-relationships[J].Biochem.Biophy.Res.Com.,1996,225:209-214
[6] MatsubayashiY,TakahataY,MoritaA,et al.Preparation and characterization of fully active biotinylated analogs of phytosulfokine-α[J].Biochem.Biophy.Res.Com.,1999,63:1847-1849
[7] Bahyrycz A,Matsubayashi Y,Ogawa M,et al.Plant peptide hormone phytosulfokine-α:synthesis of new analogues and their biological evalution[J].Pnas,1997,94:13357-13362
[8] Bahyrycz A,Matsubayashi Y,Ogawa M,et al.Further analogues of plant peptide hormone phytosulfokine-αand their biological evalution[J]..Pept.Sci.,2004,10:462-469
[9] MatsubayashiY,TakahataY,MoritaA et al.An LRR receptor kinase involved in perception of a peptide plant hormore,Phytosulfokine.Scinece,2002,296:1470-1472
[10] Matsubayashi Y,Sakagami Y.120-and160-Kda receptors of endogenous mitogenicpeptede,phytosulfokine-α,in rice plasma membrances.[J] Biol Chem,2000,275:15520-15525
[11] Matsubayashi Y, MoritaA,Matsunaga E et al.Physiological relationships between anxin,cytokinin,and apeptide growth factor,phytosulfokine-α,instimulation of asparagus cell proliferation.Planta,1999,207:559-565
[12] Matsubayashi Y,Goto T.Sakagami Y.Chemical nurising:phytosulfokine improve genetic transformation efficiency bi promoting the proliferation of suiviving cells on selective media[J].Plant Cell Rep.,2004,23:155-158
[13] Chen D,Xu Z,et al.Effect of phytosulfokine-αon Agrbacterium-mediated transfomation in rice[J].Rice Sci.,2005,12:255-260
[14] Sasaki K,IshiseT,Shimomura K,et al.Effects of phytosulfokine-αon growth and tropane aklaloid production in transformen roots of Atropabelladonna[J].Plant Growth Reg.,2002,26:87-89
[15] Kimb J,Gibson D M,Shuler M L.Effect of the plant peptide regulator,phytosulfokine-α,on the growth and Taxol production from Taxus sp.suspension cultures[J].Biotech.Bioengi.,2006,95:8-14
[16] Yang H,Matsubayashi Y,Nzkamura K,et al.Oryzasativa PSK gene encodes a precursor of phytosulfokine-α,a sulfated pepetide growth factor found in plants[J].Pnas,1999,96:13 13560-13565
[17] Yang H,MatsubayashiY,HannaiH,et al.Molecular cloning and characterization of Os PS K,a gene encoding a p recursor for phytosulfokine-α,required for rice cell proliferation[J].Plant Mol.B iol.,2000,635:635-647
[18] Yang H,Matsubayashi Y,Nzkamura K,et al. Diversit y of A rabidopsis genes encoding precursors for phytosulfokine-aαpepetide growth factor[J].Pl ant Physiol.,2001,127:842-851
[19] Rosenbergrd,Shworaknw,L iu J,et al.Heparan sulfate proteoglycans of the cardiovascular system.Specific structures emergebut how is synthesis regulated[J].J.Cli n.I nvest.,1997,99:2 062-2 070
[20] Hanai H,MatsunoT,YamamotoM,et al.A secreted peptide growthfactor,phytosulfo kine,acting as a stimulatory factor of carrot somatic embryo formation[J].Pl ant Cell Physiol.,2000,41:27-32
[21] Matsubayashi Y,Sakagami Y.Characterization of specific binding sites for a mitogenic sulfated peptide,phytosulfokine-α,in the plasma membrane fraction derived from Oryza sativa L[J].Eur.J.B iochem.,1999,262:666-671
[22] YamakawaS,et al.Promotive effects of the peptidyl plant growth factor, p hytosulfokine-αon the growth and chlorophyll content of A rabidopsis seedlings under high night-time temperature conditions[J]. Biosci.Biotech.Biochem.,1999,63:2 240-2 243.
[23] Eun C,Ko S,MatsubayashiY,et al.Phytosulfokine-αrequires auxin to stimulate carrot non-embryogenic cell proliferation[J].Plant Physiol. Biochem,2003,41:447-452
[24] ChenY F,et al.Peptide growth factor phytosulfokine-αcontributes to the pollen population effect[J].Plant a,2000,211:752-755
[25] Lorbiecke R,et al.Comparative analysis of PSK peptide growth factor precursor ho mologs[J].Plant Sci.,2002,163:321-332
[26] SturataR,Street H E.Studies on t he growth in cult ure of plant cells.The initiation of division in suspensions of stationary-phasecells of Acer pseudopllatanus L.[J].J.Exp.B ot.,2005,20:556-571
[27] Bodanszky M.Natarajan S,HahneW,Gardner JD. Choleeystokinin(Pancreozymin).3.synthesis and properties of ananalogue of the C-tenninal heptapeptide with serine sulfate rlacing tyrosine sulfate.Joumal of MedicinalChemistry,1977,20:1047-1050
[28] Meldal M.Preparation of novel O-sulfated amino aeid building bloeks withi mproved acid stability for Fmoc-based solid-phase peptide synthesis.Journal of Chemistry Soeiety,PerkinTransactionsl,2002,682-686
[29] CarpinoLA.New Methods of Introducing the Carbo-t-butoxy Proteetive Group.Preparation of t-Butyl Cyanoformate.Journal of the AinerieanChemieal Soeiety,1960,82:2725-2727
[30] CarpinoLA,Han GY The 9-Fluorenylmethoxycarbonyl Amino-Proteeting Group.The Journal of organic Chemistry,1972,37:3404-3409
[31] CarpinoLA,Imazumi H,El-Faham A,Ferrer F J,Zhang C,Lee Y Foxman B M,Henklein P,Hanay C,Mugge C,Wenschuh H,Klose J,Beyennann M,Bienert M.The Uronium/Guanidinium Peptide CouPling Reagents:Finally theTrue UroniumSalts.Angewandte Chemie International Edition,2002,41:441-445
[32] CarpinoL A,Imazurni H,El-faham,A.Uronium and irnmonium salts for peptide couPling.US Patent,6825347,2002-05-21
[33] Challa H,Woski S A.Solution Phase Synthesis of Potential DNA-Binding Moleeules based on the PNA Baekbone.Tetrahedron Letters,1999,40:419-422
[34] Coste J,Frerot E,Jouin P,Castro B.Oxybenzotriazole free peptide coupling reagents for N-methylated amino acids.Tetralledron Letters,1991,32:1967-1970
[35] Coste J,Frerot E,Patrick J.Coupling N-methylated amino acids using PyBroP and PyCloP halogenoPhoSPhonium salts:mechanism and fields of applieation.The Joumal of Organic Chemistry)1994,59:2437-2446
[36] Dhaon M K,Olsen R K,Ramasamy K.Esterification of N-protected alPha-amino acids with alcohol/earbodiimide/4-(dimethylamino)pyridine.Racemization of asPartic and glutamic acid derivatives.The Joumal of Organic Chemistry,1982, 47:1962-1965
[37] Durieux P,Fernandez-Cameado J,Tuehseherer G.Sythesis of biotinylated glycosulfopeptides by chemoseleetive ligation.Tetrahedron Letters,2001,42: 2297-2299
[38] Falkiewiez B.Comparison of the effieiency of variouse coupling systems in the aeylation of model seeondary amines with thymin-1-ylacetic acid.Nueleie Acids Symposium Series,1999,42:153-154
[39] Fields G B.Methods for Removing the Fmoc Group.In:Pennington M W,Dunn B Meds.,peptide Sythesis Protocols.Totowa:HumanaPress,1994,17-27
[40] Frerot E,Coste J,Poncet J,Jouin P,Castro B.N-methyl N-carboxyanhydride:an unexpeeted by-produc twhen eoupling boe-N-methyl amino acids.TetrahedronLetters,1992,33:2815-2816
[41] Gibson F S,Park M S,RapoPort H.Bis[[4-(2,2-dimethyl-1,3-dioxolyl)]methyl]- -carbodiimide(BDDC) and its application to residue- free esterifications,peptide eouplings,and dehydrations.The Journal of organic Chemistry-1994,59:7503-7507
[42] Gilbert E E.The reaetion of sulfur trioxide and of its adduets with organic compounds Chemical Reviews,1962,62:549- 589
[43] Han H,Wolfe M M,Brenner S,Janda K D.Liquld-Phasee combinatorial synthesis. Proceedings of the National Aeademy of Scienees of the United States of America 1995,92:6419-6423
[44] Han S Y and KimY A.Reeent development of peptide coupling reagents in organic synthesis.Tbtrahedron,2004,60:2447-2467
[45] Izdebski J,Kunee D.Evaluation of CarbodiimidesUsing a Competition Method. Joumal of peptide Seience,1997,3:141-144
[46] Jarowieki K and Kocienski P.Protecting groups.Joumal of Chemical Soeiety Perkin Transactionsl,2001,2109-2135
[47] Kice J L,Anderson J M.The mechanism of the acid hydrolysis of sodium arylsulfates.Journal of American Chemical Soeiety,1966,88:5242-5245
[48] Li P,Xu J C.New and highly effieient irnmonium type peptide coupling reagent: synthesis,meehanism and applieation.Tetrahedron,2000a,56:4437-4445
[49] Li P,Xu J C.Studies of novel and higly efficient peptide coupling reagents.Journal of the Graduate Sehool of the Chinese Aeademy of seiences,2004,21:125-134
[50] Ondetti M A,Pluizec J,Sabo E F,Sheehan J T,Williams N.Synthesis choleeystokinin-Panereozyminin.I.The C-terminal dodecapeptide.Joumal of American Chemistry Soeiety,1970,92:195-199
[51]张祎颖,洪汝科,张锦文,等。花药培养获得优质抗稻瘟病水稻[J].西南农业学报, 2008, 21(1): 75-79.
[52]柴卫淑,师佳,陈联秀,等.液体培养基在水稻花药培养中的应用研究[J].西南农业学报, 2004, 17(增刊): 78-80.
[53]李合生,孙群,赵世杰,章文华.植物生理生化实验原理及技术[M].北京:高等教育出版社,2000:167-169.
[54]张良,瞿伟菁.植物生理学实验指导(第三版)[M].北京:高等教育出版社,2003:123-124.
[55]陈金印,陈明.果实后熟哀老与植物激素的关系研究进展.江西农业大学学报2003,25(4):537-543
[56]董发刁,苗深.小麦根系过氧化氢积累与耐盐性的关系.武汉植物学研究,2002,20:293-298
[57]郭英,宋宪亮,王庆材,李玉静,孙学振.施钾对棉花苗期叶片内源激素与氧自由基代谢的影响.华北农学报,2006,21:59-62
[58]何长征,艾辛,熊先军,宋勇.硝酸银对黄瓜植株茎尖保护酶类活性的影响.湖南农业大学学报(自然科学版),2003,29(3):243-245
[59]何萍,金继运.氮钾营养对春玉米叶片衰老过程中激素变化与活性氧代谢的影响.植物营养与肥料学报,1999,5:289-296
[60]黄玉山,罗广华,关栗文.福诱导植物的自由基过氧化损伤.植物学报,1997,39:522-526
[61]谈建中.植物中的“多肤激素”.生物学通报,
[62]汤日圣,刘晓忠,陈以峰.4PU-30延缓杂交水稻叶片衰老的效果与作用.作用学报,1998,24:231-233
[63]阎秀峰,李晶.干旱胁迫对红松幼苗保护酶活性及脂质过氧化作用的影响.生态学报,1999,]9:850- 854
[64]杨淑慎,高俊风.活性氧、自由基与植物的衰老.西北植物学报,2001,21(2) :215-220
[65]杨晓红,陈晓阳,刘克锋.细胞分裂素对植物衰老的延缓作用.热带亚热带植物学报,2006,14(3):256-262
[66]张海明,王茅雁.干旱对玉米过氧化氢,MDA含量及SOD,CAT活性的影响.内蒙古农牧学院学报,1993,14:92-95
[67]张玉,李方,陈昆松,张上隆.生物自由基与植物组织衰老.园艺学进展2000:4,14-18
[68]赵春江,康书江,王纪华.植物内源激素对小麦叶片衰老的调控机理研究.华北农学报,2000,15:53-56
[69]周春华,刘红霞,韦军,活性氧与果实成熟衰老.上海交通大学学报,2002,20(l):77-84
[70]朱中华,段留生,冯雪梅.内源激素对小麦叶片衰老调控的系统分析.作物学报,1998,24:176-181
[71]于康宁.植物硫肽素_1_3_PSK的液相合成研究,华中农业大学研究生毕业论文,2009
[72]分子克隆实验指南,美,J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯著金冬雁,黎孟枫等译.
[73]杨洁彬,郭兴华,等.乳酸菌—生物学基础和应用[M].北京:中国轻工业出版社, 1996.135-146
[74]冉艳红,杨春哲,黄雪松.乳酸菌在果蔬加工中的应用现状与前景[J].中国调味品,2001,(11):3-8
[75]凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工出版社,1999.55-78
[76]施安辉,周波.乳酸菌分类,生理特性及在食品酿造工业上的应用[J].中国调味品,2001,(11):3-8
[78]冉艳红,黄雪松,于淑娟.自然发酵泡菜汁中主要成分的变化[J].中国调味品.1997,(83):248-258
[79]郭本桓.益生菌.化学工业出版社,2004:381-428
[80]胡国庆等.植物乳杆菌在不同基质中转化生成共轭亚油酸的研究.郑州工程学院学报,2004,6:53-56
[81]陆胜民等.不同发酵方法对泡雷笋品质的影响.食品与发酵工业,2004,30 (30-32)
[82]张中义等.产共轭亚油酸乳酸菌的筛选及产物分析.中国农业大学学报,2004,9 (3):5-8
[83]李平兰等.产细菌素植物乳杆菌菌株的筛选及其细菌素生物学特征研究.食品与发酵工业,1999,25 (1):1-4
[84]王立梅等.发酵香肠的工艺研究院.吉林农业大学学报,1999,21(3):104-107
[85]高云等.植物乳杆菌发酵鲜整枣的工艺研究.食品科技,2002,23 (1):23-25
[86]孙军勇,李芸.麦汁的生物酸化技术[J].食品与生物技术学报,2006,1:70-73.
[87]龚庆芳,顾国贤.生物酸化技术在啤酒生产中的应用[J].酿酒科技,2005,11:61-64.
[88]丁燕,杜金华.乳酸菌及其代谢产物在啤酒工业中的应用[J].酿酒,2001,28 (4):102-103.
[89]庞钦.酒精浓醪发酵酒糟生产乳酸的研究[D].无锡:江南大学,2002.
[90]沈萍,范秀容.微生物学实验[M].北京:高等教育出版社,1996.
[91]张波,王厚军,卞杰.啤酒厌氧菌的检测[J].中国酿造,2004,4:32-35.
[92]陈晓平,刘华英,魏小川.自然发酵酸菜汁中乳酸菌的分离筛选与鉴定研究[J].食品科学,2006,2:91-94.
[93]叶淑红,何连芳.植物乳杆菌的筛选及其在泡菜中的应用[J].中国酿造,2004,7:19-21.
[94]杨王君.食用菌与人体健康[J].四川食品与发酵,1998 (1/2):45.
[95]张虹,张卓.食用菌药用研究综述[J].大庆高等专科学校学报,1998,18 (4):78-81.
[96]中国微生物资源中心.乳酸细菌及其它益生细菌的生物学特性和应用,微生态制剂中乳酸细菌及其它益生细菌的生物学特性学习班讲义.1997:10-11,20-21,56-57
[97]黄伟坤等.食品检验与分析.北京:中国轻工业出版社,1995.454-455
[98]广东省微生物研究所.天然调味料—酵母抽提物中试报告.1987
[99]于信令等.味精工业手册.北京:轻工业出版社,1995,453-459
[100]彭小平等.食品研究与开发,1998,(4):13-15