油菜黑芥子酶基因的大肠杆菌工程菌构建和表达研究
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摘要
黑芥子酶(EC3.2.1.147)存在于十字花科植物中。在一定的条件下,黑芥子酶水解硫代葡萄糖苷,生成葡萄糖、硫酸盐、异硫代氰酸盐、乙腈、硫氰酸盐等物质。其中,酶解产物异硫代氰酸盐具有很好的抗肿瘤生物活性。但异硫代氰酸盐在水相中不稳定,易降解失去生物活性。硫代葡萄糖苷的稳定性较好,且黑芥子酶被固定化后,也有很好的稳定性。不过从植物中提取黑芥子酶,产量低,不能满足产品生产的需要。因此,我们利用转基因技术构建合适的工程菌,以期得到大量的、纯度高、有生物活性的黑芥子酶。
本实验中,我们尝试将油菜中MYR1基因克隆到原核表达载体中进行异源表达。为了高效表达黑芥子酶,我们采用了pGEX-4T-1表达系统。
实验首先从油菜幼苗中提取总RNA,采用RT-PCR法将MYR1编码的成熟肽链基因序列扩增出来。经过克隆测序,与NCBI序列库对比同源性,得知该基因与NO.60214有99%的同源性,氨基酸仅有一个不同。再利用SalⅠ位点与pGEX-4T-1连接,构建成重组子pGEX-4T-1-MYR1。经过PCR鉴定、SalⅠ酶切及EcoRⅠ酶切鉴定以及测序,筛选出重组大肠杆菌。将之在在OD600为0.8、37℃、IPTG浓度为0.08mmol/L的条件下诱导表达2.5h,表达产物经SDS-PAGE初步检测,与空质粒pGEX-4T-1表达产物相比较,其在90KDa处有表达量很高的一条带,但没有获得可溶性的GST融合蛋白,推测是GST融合蛋白大量表达聚集形成了包涵体。通过改变培养条件,在20℃、OD_(600)为0.8左右、IPTG浓度0.02mM下诱导2.5h,液氮破碎后经过浓缩,再用少量的pH6的磷酸缓冲液溶解,我们获得少量可溶的、有活性的GST融合蛋白,以SINIGRIN做底物,测得融合蛋白的比活力为3.1×10~(-3)U/mg。
Myrosinase(EC3.2.1.147),which exists in Cruciferae family,can hydrolyze glucosinolates into glucose、sulfat、isothiocyanates、nitrile and thiocyanate.Thereinto,The enzymatic products of isothiocynate have several biological activities.But the isothiocynate is unstale in water,it easily degrade and lose activities.Glucosinolates have a good stabilization,and also myrosinase have a good stabilization by immobilized.But the economic efficiency of withdrawing myrosinase from the plant is low,cannot satisfy the production needed.Therefore,we use transgene technology to construct the appropriate project bacteria,to obtain high purity and activity myrosinase.
In this experiment,we attempt to hetorologous expression throug the MYR1 gene cloning to prokaryotic expression vector.In order to highly effective expresses myrosinase,we use the pGEX-4T-1 express system.
At first,we withdraw total RNA from the rape seedling,uses the RT-PCR to expand the MYR1 encoded mature peptide gene sequence increases.By sequencing after the clone,contrasting with the NCBI sequence,we know this gene there has 99%homology with NO.60214, the amino acid only one is difference.Again,we use the SalⅠspot to connect with the pGEX-4T-1,constructing recombinant pGEX-4T-1-MYR1.Confirmating by PCR,SalⅠenzyme cutter and EcoRⅠenzyme cutter、sequencing,We screen the correct recombinant.Expression product at OD_(600)0.8、37℃、0.08mmol/LIPTG、and 2.5h,and it expressed highly at 90KDa contrasting with the product of pGEX-4T-1 when run in the SDS-PAGE.But it had not obtained the soluble GST fusion protein,by supposing the GST fusion protein massive expressions gathering and forming inclusion body.After changing conditions,we induced 2.5h at 20℃,OD_(600)0.8,IPTG 0.02mM,after lysised cell by fluid nitrogen and concentrated,then used a little of phosphoric buffer(pH=6).Finally,we obtain a little of dissolve activity GST fusion protein,as SINIGRIN is substrate,The specific activity of the fusion protein is 3.1×10~(-3)U/mg.
本实验中,我们尝试将油菜中MYR1基因克隆到原核表达载体中进行异源表达。为了高效表达黑芥子酶,我们采用了pGEX-4T-1表达系统。
实验首先从油菜幼苗中提取总RNA,采用RT-PCR法将MYR1编码的成熟肽链基因序列扩增出来。经过克隆测序,与NCBI序列库对比同源性,得知该基因与NO.60214有99%的同源性,氨基酸仅有一个不同。再利用SalⅠ位点与pGEX-4T-1连接,构建成重组子pGEX-4T-1-MYR1。经过PCR鉴定、SalⅠ酶切及EcoRⅠ酶切鉴定以及测序,筛选出重组大肠杆菌。将之在在OD600为0.8、37℃、IPTG浓度为0.08mmol/L的条件下诱导表达2.5h,表达产物经SDS-PAGE初步检测,与空质粒pGEX-4T-1表达产物相比较,其在90KDa处有表达量很高的一条带,但没有获得可溶性的GST融合蛋白,推测是GST融合蛋白大量表达聚集形成了包涵体。通过改变培养条件,在20℃、OD_(600)为0.8左右、IPTG浓度0.02mM下诱导2.5h,液氮破碎后经过浓缩,再用少量的pH6的磷酸缓冲液溶解,我们获得少量可溶的、有活性的GST融合蛋白,以SINIGRIN做底物,测得融合蛋白的比活力为3.1×10~(-3)U/mg。
Myrosinase(EC3.2.1.147),which exists in Cruciferae family,can hydrolyze glucosinolates into glucose、sulfat、isothiocyanates、nitrile and thiocyanate.Thereinto,The enzymatic products of isothiocynate have several biological activities.But the isothiocynate is unstale in water,it easily degrade and lose activities.Glucosinolates have a good stabilization,and also myrosinase have a good stabilization by immobilized.But the economic efficiency of withdrawing myrosinase from the plant is low,cannot satisfy the production needed.Therefore,we use transgene technology to construct the appropriate project bacteria,to obtain high purity and activity myrosinase.
In this experiment,we attempt to hetorologous expression throug the MYR1 gene cloning to prokaryotic expression vector.In order to highly effective expresses myrosinase,we use the pGEX-4T-1 express system.
At first,we withdraw total RNA from the rape seedling,uses the RT-PCR to expand the MYR1 encoded mature peptide gene sequence increases.By sequencing after the clone,contrasting with the NCBI sequence,we know this gene there has 99%homology with NO.60214, the amino acid only one is difference.Again,we use the SalⅠspot to connect with the pGEX-4T-1,constructing recombinant pGEX-4T-1-MYR1.Confirmating by PCR,SalⅠenzyme cutter and EcoRⅠenzyme cutter、sequencing,We screen the correct recombinant.Expression product at OD_(600)0.8、37℃、0.08mmol/LIPTG、and 2.5h,and it expressed highly at 90KDa contrasting with the product of pGEX-4T-1 when run in the SDS-PAGE.But it had not obtained the soluble GST fusion protein,by supposing the GST fusion protein massive expressions gathering and forming inclusion body.After changing conditions,we induced 2.5h at 20℃,OD_(600)0.8,IPTG 0.02mM,after lysised cell by fluid nitrogen and concentrated,then used a little of phosphoric buffer(pH=6).Finally,we obtain a little of dissolve activity GST fusion protein,as SINIGRIN is substrate,The specific activity of the fusion protein is 3.1×10~(-3)U/mg.
引文
[1] SUSANNA ERIDSSON,BO EK,JIPING XUE,LARS RASK,JOHAN MEIJER. Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba[J].Physiologiaalntarum, 2001, 111: 353-364.
[2] LUDIKHUYZE L,OOMS V, WEEMAES C, et al. Kinetic study of the irreversible thermal and pressure inactivation of myrosinase from broccoli(Brassica oleracca L.Cv.Italica) [J]. Journal of Agricultural and Food Chemistry, 1999, 47: 1541-1548.
[3] SUSANNA ER1DSSON,ERIK ANDREASSON,BARBARA EKBOM.Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins[J].Plant physiol, 2002, 129: 1592-1599.
[4] ROSA E A S, HEANEY R K, FENWICK G R, et al. Glucosinolates in crop plants[J]. Horticultural Reviews, 1997, 19:99-215.
[5] FALK A,EK B,RASK L.Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae)[J].Plant Mol Biol,1995,27(5):863-874.
[6] DANIEL KLIEBENSTEN1N,DEANA PEDERSEN,BRIDGET BARKER,THOMAS MITCHELL-OLDS.Comparative analysis of quantitative trait loci controlling glucosinolates myrosinase and insect resistance in arabidopsis thaliana[J].Genetics, 2002,161:325-332
[7] BONES A,ROSSITER J T.The myrosinase-glucosinolate system,its organization and biochemistry[J].Physiol Plant,1996,97:194-208.
[8] KOZLOWSKA H J, NOWAK H, NOWAK J. Characterisation of myrosinase in polish of rapeseed[J]. Journal of the Science of Food and Agriculture, 1983, 34(11): 1171-1178.
[9] MCGREGOR D I, MULLIN W J. Review of analysis of glucosinolates[J]. Journal of the Association of Official Analvtical Chemists, 1983, 66: 825-849.
[10] RASK L,ANDREASSON E,EKBOM B,ERIDSSON S,PONTOPPIDAN B,MEIJER J.Myrosinase:Gene family evolution and herbivore defense in Brassicaceae[J].Plant Mol Biol,2000,42:93-113.
[11] STEVEN F V, MARK A B, Glucosinolate hydrolysis products from various plant sources: pH effects, isolation and purification[J]. Industrial Crops and Products, 2005, 21: 193-202.
[12] LENMAN M,FALK A,et al.Differential expression of myrosinase gene families[J].Plant Mol Biol,1993,103:703-711.
[13] XUE J,LENMAN M,FALK A,RASK L.The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family[J].Plant Mol Biol, 1992,18:387-398.
[14] THANGSTAD O P,WINGE P,HUSEBYE H,BONES A.The myrosinase(thioglucoside glucohydrolase)gene family in Brassicaceae[J].Plant Mol Biol, 1993,23:511-524.
[15] FALK A,XUE J,LENMAN M,RASK L.Sequence of a cDNA clone enconding the enzyme myrosinase and expression of myrosinase in different tissues of Brassica napus[J].Plant Sci,1992,83:181-186.
[16] FALK A,EK B,RASK L.Chaaracterization of a new myrosinase in Brassica napus[J].Plant Mol Biol, 1995,27:863-874.
[17]. JAWNNY E W, ELSAYED S T, RASHAD M M, et al.Myrosinase from roots o-f Raphanus sativus[J].Phytochemistry, 1995,39(6): 1301-1303.
[18] BJORKMAN R,LONNERDAL B.Studies on myrosinases Ⅲ Enzymatic properties of myrosinases from Sinapis alba and Brassica napus seeds[J].Biochim. Biophys. Acta, 1973,327:121-131.
[19] BO PONTOPPIDAN,BARBARA EKBOM,SUSANNA ETIKSSON,JOHAN MEIJER.Purification and characterization of myrosinase from the cabbage aphid(Brevicor-yne bras sicae),a brassica herbivore[J].Eur.J.Biochem,2001,268:1041-1048.
[20] LENMAN M,FALK A,et al.Differential expression of myrosinase gene families[J].Plant Mol Biol,1993,103:703-711.
[21] SHIKITA M, FAHEY J W, GOLDEN T R, et al. An unusual case of uncompetitive activation by ascorbic acid:purificarion and kinetic properties of a myrosinase from Raphanus sativus seedlings[J]. Biochemical Journal of London, 1999, 341(3): 725-732.
[22] LENMAN M,FALK A,XUE J,RASK L.Characterization of Brassica napus myrosinase pseudogene:myrosinases are members of the BGAfamily of β-glycosidases[J].Plant Mol Biol, 1993,21:463-474.
[23] BUCHWALDT L,LARSEN L M,et al.Fast polymer liquid chromatography isolation and characterization of plant myrosinase, β-thioglucoside glucohydrolase,isoenzymes[J].Chromatogr, 1986,363:71 -80.
[24] BONES A M.Distribution of β-thioglucosidsae activity in intact plants,cell and tissue cultures and tissue cultures and regenerant plants of Brassica napus[J].Exp Bot,1990,41:737-744.
[25]RASK L,EKBOM B,ERIKSSON S,et al.Myrosinase:Gene family evolution and herbivore defense in Brassicaceae[J].Plant Mol Biol,20003,42:93-113.
[26]DURHAM P L,POULTON J E.Biochemistry of myrosinase and glucosinolates[J].Z.Naturforsch,1990,45:173-178.
[27]SERKADIS M,GETAHUN,FUNGLUNG CHUNG.Conversion of Glucosinolates to Isothiocyanates in Humans after Ingestion of Cooked Watercress[J].Cancer Epidemiology Biomarkers & Prevention,1999,8:447-451.
[28]JOHNSON I T.Brassica vegetables and human health:glucosinolates in the food chain[J].Acta Horticulturae,2000,539:39-46.
[29]DEKKER M,VERKERK R,JONGEN W M F.Predictive modelling of health aspects in the food production chain:A case study on glucosinolates in cabbage[J].Trends in Food Science & Technology,2000,11:174-181.
[30]SIXUE CHEN,BARBARA ANN HALKIER.Functional expression and characterization of the myrosinase MYR1from Brassica napus in Saccharomyces cerevisiae[J].Protein Expression and Purification,1999,17:414-420.
[31]SCOFIELD A M,ROSSITER J T,WITHAM P,et al.Inhibition of thioglucosida-secatalysed glucosinolate hydrolysis by catanospermine and related alkaloids[J].Pytochemistry,1990,29(1):107-109.
[32]刘月萍.黑芥子酶的提取分离、性质及固定化研究[D].杭州:浙江工商大学食品与生物学院,2006:21-47.
[33]XIAN LI,MOSBAH M KUSHAD.Purification and characterization of myrosinase from horseradish(Armoracia rusticana)roots[J].Plant Physiology and Biochemistry,2005,43:503-511.
[34]张树政.酶制剂工业(上册)[J].北京:科学出版社,1998,36-49.
[35]胡人卫,胡述辉等,1990:气谱内标法测定油菜硫代葡萄糖甙总量的研究,西南农业学报3:96-99
[36]袁丽风,王志刚,郭伟强.硫代葡萄糖苷的HPLC-MS分离鉴定研究[J].浙江科技学院学报,2003,15:87-89.
[37] Renato Iori, Patrick Rollin, Harald Streicher, Joachim Thiem, Sandro Palmieri, 1996: The myrosinase-gluxosinolate interaction mechanism studied using some synthetic competitive inhibitors. FEBS letters. 385: 87-90
[38] FRAUKE V.HARTEL,ANDERS BRAND.Characterization of a Brassica napus myrosinase expressed and secreted by Pichia Pastoris[J].Protein Expression and Purification,2002,24:221-226.
[39] LENMAN M,RODIN J,JOSEFSSON L G,RASK L.Immunological characterization of rapeseed myrosinase[J].Eur.J.Biochem, 1990,194:747-753.
[40] TAIPALEENSUU J,FALK A,RASK L.A wound and methyl jasmonate-inducible transcript coding for a myrosinase-associated protein with similarities to an early nodulin[J].Plant Physiol, 1996,110:483-491.
[41] TAIPALEENSUU J,ANDREASSON E,ERIKSSON S.RASK L.Regulation of the wound induced myrosinase-associated protein transcript in Brassica napus pIants[J].Eur.J.Biochem, 1997,247:963-971.
[42] TAIPALEENSUU J,ERIKSSON S, RASK L.The myrosinase-binding protein from Brassica napus seeds possesses lectin activity and has a highly simimar vegetatively expressed wound inducible counterpart[J].Eur.J.Biochem, 1997,250:680-688.
[43] GESHI N,BRANDT A.Two jasmonate-inducible myrosinase-binding proteins from Brassica napus L seedlings with homology to jacalin[J].Planta,1998,204:295-304.
[44] GESHI N,ANDREASSON E,MEIJER J,RASK L,BRANDT A.Co-localization of myrosinase-binding proteins in grains of myrosin cells in cotyledom of Brassica napus seedlings[J]. 1998,36:583-590.
[45] ERIKSSON S,EK B,XUE J,RASK L,MEIJER J.Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis aIba[J].Physiol plant,2001,111:353-364.
[46] RASK L,ANDREASSON E,EKBOM B,ERIKSSON S,PONTOPPIDAN B,MEIJER J.Myrosinase:Gene family evolution and herbivore defense in Brassicaceae[J].Plant Mol.Biol,42: 93-113
[47] BONES A M.Distribution of β-thioglucosidase activity in intact plants,cell and tissue cultures and regenerant plants of Brassica naous 1[J].J Exp.Bot,41:737-744.
[48]BURMEISTER W P,et al.The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase[J].Structure,1997,5:663-675.
[49]THANGSTAD O.P,WINGE P,HUSEBYE H,BONES A.The thioglucoside glucohydrolase(myrosinase)gene family in Brassicaceae[J].Plant Mol boil,1993,23:511-524
[50]XUE J,LENMAN M,FALK A,RASK L.The glucosinolate-degrading enzyme myros-inase in Brassicaceae is encoded by a gene family[J].Plant Mol boil,1992,18:387-398.
[51]周宇苟,魏东芝,王二力.融合表达载体pGEX及其应用[J].生命科学,1998,3:122-124.
[52]SANG JUNG AHN,JUNG SOO SEO,MOO-SANG KIN,SOO JIN JEON,et al.Cloning,site-directed mutagenesis and expression of cathepsin L-like cysteine protease from U -ronema marinum(Ciliophora:Scuticociliatida)[J].Molecular and Biochemical Parasitolo -gy,2007,156:191-198.
[53]CHEN WU,YUAN WANG,MINJI ZOU,YAOJUN SHAN,GUANGYIN YAO,PING WEI,GUANGYU CHEN,JIAXI WANG,DONGGANG XU.Prokaryotic expression,purificatio -n,and production of polyclonal antibody against human polypeptide N-acetylgalactosa minyltransferase 14[J].Molecular and Biochemical Parasitology,2007,24:
[54]苏力坦·阿巴白克力,白勇,朱玉贤.GDA2基因的克隆、原核表达与融合蛋白的纯化[J].农业生物技术学报,2003,11:6-10.
[55]陈哲宇,柴延丰,何成,路长林,吴祥莆.垂体腺苷酸环化酶激活肽基因的合成表达与活性研究[J].生物化学与生物物理进展,2001,192-197.
[56]王见冬,袁其朋,钱忠明.萝卜硫素研究进展[J].食品与发酵工业,2002,29(2):76-80.
[57]李志邈,曹家树.蔬菜的抗癌特性[J].北方园艺,2001,4:4-6.
[58]ZHANG Y,KENSLER T W,CHO C G,et al.Anticarcinogenic activities of sulforaphane and structurally related synthetic norbomyl isothiocyanates[J].Proceedings of the National Academy of Sciences of the USA,1994,91(8):3147-3150.
[59]DINKOVA-KOSTOVA A T,HOLTZCLAW W D,COLER R N,et al.Direct evidence that sulthydryl groups of Keap 1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants[J].Proceedings of the National Academy of Sciences of the USA,2002,99(18):11908-11913.
[60]陈谦,张小智.食道癌的化学预防[J].肿瘤防治研究,2003,30(2):165-169.
[61]SRINIBAS D,AMRISH K T,HARJIT K.Cancer modulation by glucosinolates:a review[J].Current Science,2000,79(12):1665-1671.
[62]Koenraad F,Bart P,Margreet B,et al.Study of the Role of Antimicrobial Glucosinolate-Derived Isothiocyanates in Resistance of Arabidopsis to Microbial Pathogens[J].Plant Physiol,2001,125:1688-1699.
[63]Xiangqun Gao,Albena T.Dinkova-Kostova,Paul Talalay.Powerful and prolonged pro tection of human retinal pigment epithelial cells,keratinocytes,and mouse leukemia c ells against oxidative damage:The indirect antioxidant effects of sulforaphane[J].PNAS,2001,98(26):15221-15226.
[64]GRAHAM P B,YONGPING B,GARY W.Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronsyl transferase(UGT1A1)and glutathione transferase(GSTA1)in cultured cell[J].Carcinogenesis,2002,23(8):1399-1404.
[65]ZHU M,ZHANG Y,COOPER S,et al.Phase Ⅱ enzyme inducer,sulforaphane,inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism[J].Molecular Carcinogenesis,2004,41(3):179-186.
[66]GRAHAM P B,YONGPING B,GARY W.Sulforaphane and its glutathione conjug-ate but not sulforaphane nitrile induce UDP-glucuronsyl transferase(UGT1A1)and glutathione transferase(GSTA1)in cultured cell[J].Carcinogenesis,2002,23(8):1399-1404.
[67]苏光耀.西兰花种子中硫苷酶解产物萝卜硫素的提取分离与结构鉴定[D].杭州:浙江工商大学食品与生物学院,2006:7
[68]郑晓飞,et al.RNA实验技术手册[M].北京:科学出版社,2004:21-23.
[69]萨姆布鲁克 J,拉塞尔DW.分子克隆实验指南(第三版)黄培堂等译[M].北京:科学出版社,2002:582-585.
[70]郑小飞.RNA实验技术手册[M].北京:科学出版社,2004:24-26.
[71]J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子克隆实验指南[M].北京:科学出版社,1999:304.
[72]F.奥斯伯,R.布伦特,R.E 金斯顿,D.D.穆尔,J.G塞德曼,J.A.史密斯,K.斯特拉尔.精编分子生物学实验指南[M].北京:科学出版社,1998:41
[73]J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子克隆实验指南[M].北京:科学出版社,1999:55-56。
[74]谢磊,孙建波,张世清,2004:大肠杆菌表达系统及其研究进展,华南热带农业大学学报,(6):21-24
[75]A.S.Kolaskarl,Urmila Kulkarni-Kale,1999:Prediction of Three-Dimensional Structure and Mapping of Conformational Epitopes of Envelope Glycoprotein of Japanese Encephalitis[J].Virus and Virology,261:31-42
[76]Amersham,GST Gene Fusion Protion System Handbook
[77]J E.科林根,et al.精编蛋白质科学实验指南[M].北京:科学出版社,2007:177-179.
[78]张树政,et al.酶制剂工业[M].北京:科学出版社,1998:218-223.
[79]吴乃虎.基因工程原理(下册)[M]北京:科学出版社,2001:138-148.
[2] LUDIKHUYZE L,OOMS V, WEEMAES C, et al. Kinetic study of the irreversible thermal and pressure inactivation of myrosinase from broccoli(Brassica oleracca L.Cv.Italica) [J]. Journal of Agricultural and Food Chemistry, 1999, 47: 1541-1548.
[3] SUSANNA ER1DSSON,ERIK ANDREASSON,BARBARA EKBOM.Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins[J].Plant physiol, 2002, 129: 1592-1599.
[4] ROSA E A S, HEANEY R K, FENWICK G R, et al. Glucosinolates in crop plants[J]. Horticultural Reviews, 1997, 19:99-215.
[5] FALK A,EK B,RASK L.Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae)[J].Plant Mol Biol,1995,27(5):863-874.
[6] DANIEL KLIEBENSTEN1N,DEANA PEDERSEN,BRIDGET BARKER,THOMAS MITCHELL-OLDS.Comparative analysis of quantitative trait loci controlling glucosinolates myrosinase and insect resistance in arabidopsis thaliana[J].Genetics, 2002,161:325-332
[7] BONES A,ROSSITER J T.The myrosinase-glucosinolate system,its organization and biochemistry[J].Physiol Plant,1996,97:194-208.
[8] KOZLOWSKA H J, NOWAK H, NOWAK J. Characterisation of myrosinase in polish of rapeseed[J]. Journal of the Science of Food and Agriculture, 1983, 34(11): 1171-1178.
[9] MCGREGOR D I, MULLIN W J. Review of analysis of glucosinolates[J]. Journal of the Association of Official Analvtical Chemists, 1983, 66: 825-849.
[10] RASK L,ANDREASSON E,EKBOM B,ERIDSSON S,PONTOPPIDAN B,MEIJER J.Myrosinase:Gene family evolution and herbivore defense in Brassicaceae[J].Plant Mol Biol,2000,42:93-113.
[11] STEVEN F V, MARK A B, Glucosinolate hydrolysis products from various plant sources: pH effects, isolation and purification[J]. Industrial Crops and Products, 2005, 21: 193-202.
[12] LENMAN M,FALK A,et al.Differential expression of myrosinase gene families[J].Plant Mol Biol,1993,103:703-711.
[13] XUE J,LENMAN M,FALK A,RASK L.The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family[J].Plant Mol Biol, 1992,18:387-398.
[14] THANGSTAD O P,WINGE P,HUSEBYE H,BONES A.The myrosinase(thioglucoside glucohydrolase)gene family in Brassicaceae[J].Plant Mol Biol, 1993,23:511-524.
[15] FALK A,XUE J,LENMAN M,RASK L.Sequence of a cDNA clone enconding the enzyme myrosinase and expression of myrosinase in different tissues of Brassica napus[J].Plant Sci,1992,83:181-186.
[16] FALK A,EK B,RASK L.Chaaracterization of a new myrosinase in Brassica napus[J].Plant Mol Biol, 1995,27:863-874.
[17]. JAWNNY E W, ELSAYED S T, RASHAD M M, et al.Myrosinase from roots o-f Raphanus sativus[J].Phytochemistry, 1995,39(6): 1301-1303.
[18] BJORKMAN R,LONNERDAL B.Studies on myrosinases Ⅲ Enzymatic properties of myrosinases from Sinapis alba and Brassica napus seeds[J].Biochim. Biophys. Acta, 1973,327:121-131.
[19] BO PONTOPPIDAN,BARBARA EKBOM,SUSANNA ETIKSSON,JOHAN MEIJER.Purification and characterization of myrosinase from the cabbage aphid(Brevicor-yne bras sicae),a brassica herbivore[J].Eur.J.Biochem,2001,268:1041-1048.
[20] LENMAN M,FALK A,et al.Differential expression of myrosinase gene families[J].Plant Mol Biol,1993,103:703-711.
[21] SHIKITA M, FAHEY J W, GOLDEN T R, et al. An unusual case of uncompetitive activation by ascorbic acid:purificarion and kinetic properties of a myrosinase from Raphanus sativus seedlings[J]. Biochemical Journal of London, 1999, 341(3): 725-732.
[22] LENMAN M,FALK A,XUE J,RASK L.Characterization of Brassica napus myrosinase pseudogene:myrosinases are members of the BGAfamily of β-glycosidases[J].Plant Mol Biol, 1993,21:463-474.
[23] BUCHWALDT L,LARSEN L M,et al.Fast polymer liquid chromatography isolation and characterization of plant myrosinase, β-thioglucoside glucohydrolase,isoenzymes[J].Chromatogr, 1986,363:71 -80.
[24] BONES A M.Distribution of β-thioglucosidsae activity in intact plants,cell and tissue cultures and tissue cultures and regenerant plants of Brassica napus[J].Exp Bot,1990,41:737-744.
[25]RASK L,EKBOM B,ERIKSSON S,et al.Myrosinase:Gene family evolution and herbivore defense in Brassicaceae[J].Plant Mol Biol,20003,42:93-113.
[26]DURHAM P L,POULTON J E.Biochemistry of myrosinase and glucosinolates[J].Z.Naturforsch,1990,45:173-178.
[27]SERKADIS M,GETAHUN,FUNGLUNG CHUNG.Conversion of Glucosinolates to Isothiocyanates in Humans after Ingestion of Cooked Watercress[J].Cancer Epidemiology Biomarkers & Prevention,1999,8:447-451.
[28]JOHNSON I T.Brassica vegetables and human health:glucosinolates in the food chain[J].Acta Horticulturae,2000,539:39-46.
[29]DEKKER M,VERKERK R,JONGEN W M F.Predictive modelling of health aspects in the food production chain:A case study on glucosinolates in cabbage[J].Trends in Food Science & Technology,2000,11:174-181.
[30]SIXUE CHEN,BARBARA ANN HALKIER.Functional expression and characterization of the myrosinase MYR1from Brassica napus in Saccharomyces cerevisiae[J].Protein Expression and Purification,1999,17:414-420.
[31]SCOFIELD A M,ROSSITER J T,WITHAM P,et al.Inhibition of thioglucosida-secatalysed glucosinolate hydrolysis by catanospermine and related alkaloids[J].Pytochemistry,1990,29(1):107-109.
[32]刘月萍.黑芥子酶的提取分离、性质及固定化研究[D].杭州:浙江工商大学食品与生物学院,2006:21-47.
[33]XIAN LI,MOSBAH M KUSHAD.Purification and characterization of myrosinase from horseradish(Armoracia rusticana)roots[J].Plant Physiology and Biochemistry,2005,43:503-511.
[34]张树政.酶制剂工业(上册)[J].北京:科学出版社,1998,36-49.
[35]胡人卫,胡述辉等,1990:气谱内标法测定油菜硫代葡萄糖甙总量的研究,西南农业学报3:96-99
[36]袁丽风,王志刚,郭伟强.硫代葡萄糖苷的HPLC-MS分离鉴定研究[J].浙江科技学院学报,2003,15:87-89.
[37] Renato Iori, Patrick Rollin, Harald Streicher, Joachim Thiem, Sandro Palmieri, 1996: The myrosinase-gluxosinolate interaction mechanism studied using some synthetic competitive inhibitors. FEBS letters. 385: 87-90
[38] FRAUKE V.HARTEL,ANDERS BRAND.Characterization of a Brassica napus myrosinase expressed and secreted by Pichia Pastoris[J].Protein Expression and Purification,2002,24:221-226.
[39] LENMAN M,RODIN J,JOSEFSSON L G,RASK L.Immunological characterization of rapeseed myrosinase[J].Eur.J.Biochem, 1990,194:747-753.
[40] TAIPALEENSUU J,FALK A,RASK L.A wound and methyl jasmonate-inducible transcript coding for a myrosinase-associated protein with similarities to an early nodulin[J].Plant Physiol, 1996,110:483-491.
[41] TAIPALEENSUU J,ANDREASSON E,ERIKSSON S.RASK L.Regulation of the wound induced myrosinase-associated protein transcript in Brassica napus pIants[J].Eur.J.Biochem, 1997,247:963-971.
[42] TAIPALEENSUU J,ERIKSSON S, RASK L.The myrosinase-binding protein from Brassica napus seeds possesses lectin activity and has a highly simimar vegetatively expressed wound inducible counterpart[J].Eur.J.Biochem, 1997,250:680-688.
[43] GESHI N,BRANDT A.Two jasmonate-inducible myrosinase-binding proteins from Brassica napus L seedlings with homology to jacalin[J].Planta,1998,204:295-304.
[44] GESHI N,ANDREASSON E,MEIJER J,RASK L,BRANDT A.Co-localization of myrosinase-binding proteins in grains of myrosin cells in cotyledom of Brassica napus seedlings[J]. 1998,36:583-590.
[45] ERIKSSON S,EK B,XUE J,RASK L,MEIJER J.Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis aIba[J].Physiol plant,2001,111:353-364.
[46] RASK L,ANDREASSON E,EKBOM B,ERIKSSON S,PONTOPPIDAN B,MEIJER J.Myrosinase:Gene family evolution and herbivore defense in Brassicaceae[J].Plant Mol.Biol,42: 93-113
[47] BONES A M.Distribution of β-thioglucosidase activity in intact plants,cell and tissue cultures and regenerant plants of Brassica naous 1[J].J Exp.Bot,41:737-744.
[48]BURMEISTER W P,et al.The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase[J].Structure,1997,5:663-675.
[49]THANGSTAD O.P,WINGE P,HUSEBYE H,BONES A.The thioglucoside glucohydrolase(myrosinase)gene family in Brassicaceae[J].Plant Mol boil,1993,23:511-524
[50]XUE J,LENMAN M,FALK A,RASK L.The glucosinolate-degrading enzyme myros-inase in Brassicaceae is encoded by a gene family[J].Plant Mol boil,1992,18:387-398.
[51]周宇苟,魏东芝,王二力.融合表达载体pGEX及其应用[J].生命科学,1998,3:122-124.
[52]SANG JUNG AHN,JUNG SOO SEO,MOO-SANG KIN,SOO JIN JEON,et al.Cloning,site-directed mutagenesis and expression of cathepsin L-like cysteine protease from U -ronema marinum(Ciliophora:Scuticociliatida)[J].Molecular and Biochemical Parasitolo -gy,2007,156:191-198.
[53]CHEN WU,YUAN WANG,MINJI ZOU,YAOJUN SHAN,GUANGYIN YAO,PING WEI,GUANGYU CHEN,JIAXI WANG,DONGGANG XU.Prokaryotic expression,purificatio -n,and production of polyclonal antibody against human polypeptide N-acetylgalactosa minyltransferase 14[J].Molecular and Biochemical Parasitology,2007,24:
[54]苏力坦·阿巴白克力,白勇,朱玉贤.GDA2基因的克隆、原核表达与融合蛋白的纯化[J].农业生物技术学报,2003,11:6-10.
[55]陈哲宇,柴延丰,何成,路长林,吴祥莆.垂体腺苷酸环化酶激活肽基因的合成表达与活性研究[J].生物化学与生物物理进展,2001,192-197.
[56]王见冬,袁其朋,钱忠明.萝卜硫素研究进展[J].食品与发酵工业,2002,29(2):76-80.
[57]李志邈,曹家树.蔬菜的抗癌特性[J].北方园艺,2001,4:4-6.
[58]ZHANG Y,KENSLER T W,CHO C G,et al.Anticarcinogenic activities of sulforaphane and structurally related synthetic norbomyl isothiocyanates[J].Proceedings of the National Academy of Sciences of the USA,1994,91(8):3147-3150.
[59]DINKOVA-KOSTOVA A T,HOLTZCLAW W D,COLER R N,et al.Direct evidence that sulthydryl groups of Keap 1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants[J].Proceedings of the National Academy of Sciences of the USA,2002,99(18):11908-11913.
[60]陈谦,张小智.食道癌的化学预防[J].肿瘤防治研究,2003,30(2):165-169.
[61]SRINIBAS D,AMRISH K T,HARJIT K.Cancer modulation by glucosinolates:a review[J].Current Science,2000,79(12):1665-1671.
[62]Koenraad F,Bart P,Margreet B,et al.Study of the Role of Antimicrobial Glucosinolate-Derived Isothiocyanates in Resistance of Arabidopsis to Microbial Pathogens[J].Plant Physiol,2001,125:1688-1699.
[63]Xiangqun Gao,Albena T.Dinkova-Kostova,Paul Talalay.Powerful and prolonged pro tection of human retinal pigment epithelial cells,keratinocytes,and mouse leukemia c ells against oxidative damage:The indirect antioxidant effects of sulforaphane[J].PNAS,2001,98(26):15221-15226.
[64]GRAHAM P B,YONGPING B,GARY W.Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronsyl transferase(UGT1A1)and glutathione transferase(GSTA1)in cultured cell[J].Carcinogenesis,2002,23(8):1399-1404.
[65]ZHU M,ZHANG Y,COOPER S,et al.Phase Ⅱ enzyme inducer,sulforaphane,inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism[J].Molecular Carcinogenesis,2004,41(3):179-186.
[66]GRAHAM P B,YONGPING B,GARY W.Sulforaphane and its glutathione conjug-ate but not sulforaphane nitrile induce UDP-glucuronsyl transferase(UGT1A1)and glutathione transferase(GSTA1)in cultured cell[J].Carcinogenesis,2002,23(8):1399-1404.
[67]苏光耀.西兰花种子中硫苷酶解产物萝卜硫素的提取分离与结构鉴定[D].杭州:浙江工商大学食品与生物学院,2006:7
[68]郑晓飞,et al.RNA实验技术手册[M].北京:科学出版社,2004:21-23.
[69]萨姆布鲁克 J,拉塞尔DW.分子克隆实验指南(第三版)黄培堂等译[M].北京:科学出版社,2002:582-585.
[70]郑小飞.RNA实验技术手册[M].北京:科学出版社,2004:24-26.
[71]J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子克隆实验指南[M].北京:科学出版社,1999:304.
[72]F.奥斯伯,R.布伦特,R.E 金斯顿,D.D.穆尔,J.G塞德曼,J.A.史密斯,K.斯特拉尔.精编分子生物学实验指南[M].北京:科学出版社,1998:41
[73]J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.分子克隆实验指南[M].北京:科学出版社,1999:55-56。
[74]谢磊,孙建波,张世清,2004:大肠杆菌表达系统及其研究进展,华南热带农业大学学报,(6):21-24
[75]A.S.Kolaskarl,Urmila Kulkarni-Kale,1999:Prediction of Three-Dimensional Structure and Mapping of Conformational Epitopes of Envelope Glycoprotein of Japanese Encephalitis[J].Virus and Virology,261:31-42
[76]Amersham,GST Gene Fusion Protion System Handbook
[77]J E.科林根,et al.精编蛋白质科学实验指南[M].北京:科学出版社,2007:177-179.
[78]张树政,et al.酶制剂工业[M].北京:科学出版社,1998:218-223.
[79]吴乃虎.基因工程原理(下册)[M]北京:科学出版社,2001:138-148.