水稻多聚半乳糖醛酸酶抑制蛋白基因（Ospgip1）克隆与表达
|
摘要
植物多聚半乳糖醛酸酶抑制蛋白(PGIP)是一种能够特异性识别和结合真菌内切多聚半乳糖醛酸酶(PG)并抑制其活性的蛋白,其富含亮氨酸重复(LRR),在植物抗真菌过程中发挥重要作用。
本研究以水稻品种Lemont、Jasmine85和YSBR1为材料,克隆水稻PGIP基因(Ospgip1),并进行原核表达,同时明确OsPGIP1对水稻纹枯病菌PG的抑制活性。另外,通过荧光定量PCR,分析Ospgip1在水稻不同抗、感纹枯病品种中的时空表达,以及黄化处理和病菌侵染对Ospgip1的诱导表达,从而探明PGIP表达量与品种抗病性的关系。具体结果如下:
1、通过PCR技术,克隆获得Ospgip1基因。该基因全长为1 184 bp,具有930 bp的完整开放阅读框(ORF),终止密码子为TAA,无内含子。生物信息学分析表明,OsPGIP1由309个氨基酸组成,含9个LRR重复,1个疏水性N端信号肽;二级结构LRR区含有α-螺旋和β-折叠各9个,分别位于LRR首尾,此外,不规则盘绕(卷曲)占有较高比例(53%);三级结构倾向于产生纤维状构象。
2、RT-PCR研究表明,Ospgip1在水稻抗、感纹枯病品种中均能表达,但在不同生育期、不同部位是否表达则有差异。根据Real Time PCR测定,水稻抗病品种YSBR1和Jasmine85中Ospgip1的表达量要明显高于感病品种Lemont,表明Ospgip1表达与品种抗病性有一定关系。
3、Real Time PCR研究显示,稻苗黄化处理后,无论是抗病品种还是感病品种的Ospgip1表达均显著提高。但是,纹枯病菌侵染使得抗病品种Ospgip1表达量大大增加,而对感病品种影响不大。
Polygalacturonase inhibiting proteins (PGIPs) are anti-fungal proteins with leucine-rich repeat (LRR), which could inhibit the catalytic activity of fungal endo-PG.
In this study, gene Ospgip1 encoding OsPGIP1 in rice plants was cloned by PCR. The sequence and structure of OsPGIP1 was infered by biological information analysis and its inhibition to the activity of Rhictonia solani PG was surveyed. Furthermore, Ospgip1 expression in E. coli, its time and spatial expression in rice plants and its expression induced by the yellowing treatment and the pathogen inoculation were conducted. The detailed results are as follows:
1. Ospgip1 was a sequence of 1 184 bp, which contained a complete ORF of 930bp in length and encoded a protein of 309 amino acids with hydrophobic N-terminal signal peptide and 9 LRR, called OsPGIP1. The secondary structure of OsPGIP1’s LRR had 9α-helixes and 9β-sheets that sat at the front or end of LRR, and more irregular coils (53%). At last, the protein formed one fibriform structure.
2. According to RT-PCR, OsPGIP1 could be constritutively expressed in rice varieties resistant and susceptible to R. solani, but there was some difference in the expression in different growth stages or organs. Real Time PCR indicated that OsPGIP1 was expressed in rice resistant varieties (YSBR1 and Jasmine85) significantly higher than in the susceptible one (Lemont), which showed that there was a postive relationship of OsPGIP1 expression with the resistance of rice varieties.
3. OsPGIP1 expression induced by the yellowing treatment and the pathogen inoculation was studied by Real Time PCR. The results showed that OsPGIP1 expressed in rice seedlings was recreased after the yellowing treatment. But, in the infection of R. solani, OsPGIP1 exprossion in the resistant varieties was significantly stronger than that in the control, and no change occurred in the exprossion in the susceptible one.
本研究以水稻品种Lemont、Jasmine85和YSBR1为材料,克隆水稻PGIP基因(Ospgip1),并进行原核表达,同时明确OsPGIP1对水稻纹枯病菌PG的抑制活性。另外,通过荧光定量PCR,分析Ospgip1在水稻不同抗、感纹枯病品种中的时空表达,以及黄化处理和病菌侵染对Ospgip1的诱导表达,从而探明PGIP表达量与品种抗病性的关系。具体结果如下:
1、通过PCR技术,克隆获得Ospgip1基因。该基因全长为1 184 bp,具有930 bp的完整开放阅读框(ORF),终止密码子为TAA,无内含子。生物信息学分析表明,OsPGIP1由309个氨基酸组成,含9个LRR重复,1个疏水性N端信号肽;二级结构LRR区含有α-螺旋和β-折叠各9个,分别位于LRR首尾,此外,不规则盘绕(卷曲)占有较高比例(53%);三级结构倾向于产生纤维状构象。
2、RT-PCR研究表明,Ospgip1在水稻抗、感纹枯病品种中均能表达,但在不同生育期、不同部位是否表达则有差异。根据Real Time PCR测定,水稻抗病品种YSBR1和Jasmine85中Ospgip1的表达量要明显高于感病品种Lemont,表明Ospgip1表达与品种抗病性有一定关系。
3、Real Time PCR研究显示,稻苗黄化处理后,无论是抗病品种还是感病品种的Ospgip1表达均显著提高。但是,纹枯病菌侵染使得抗病品种Ospgip1表达量大大增加,而对感病品种影响不大。
Polygalacturonase inhibiting proteins (PGIPs) are anti-fungal proteins with leucine-rich repeat (LRR), which could inhibit the catalytic activity of fungal endo-PG.
In this study, gene Ospgip1 encoding OsPGIP1 in rice plants was cloned by PCR. The sequence and structure of OsPGIP1 was infered by biological information analysis and its inhibition to the activity of Rhictonia solani PG was surveyed. Furthermore, Ospgip1 expression in E. coli, its time and spatial expression in rice plants and its expression induced by the yellowing treatment and the pathogen inoculation were conducted. The detailed results are as follows:
1. Ospgip1 was a sequence of 1 184 bp, which contained a complete ORF of 930bp in length and encoded a protein of 309 amino acids with hydrophobic N-terminal signal peptide and 9 LRR, called OsPGIP1. The secondary structure of OsPGIP1’s LRR had 9α-helixes and 9β-sheets that sat at the front or end of LRR, and more irregular coils (53%). At last, the protein formed one fibriform structure.
2. According to RT-PCR, OsPGIP1 could be constritutively expressed in rice varieties resistant and susceptible to R. solani, but there was some difference in the expression in different growth stages or organs. Real Time PCR indicated that OsPGIP1 was expressed in rice resistant varieties (YSBR1 and Jasmine85) significantly higher than in the susceptible one (Lemont), which showed that there was a postive relationship of OsPGIP1 expression with the resistance of rice varieties.
3. OsPGIP1 expression induced by the yellowing treatment and the pathogen inoculation was studied by Real Time PCR. The results showed that OsPGIP1 expressed in rice seedlings was recreased after the yellowing treatment. But, in the infection of R. solani, OsPGIP1 exprossion in the resistant varieties was significantly stronger than that in the control, and no change occurred in the exprossion in the susceptible one.
引文
[1]辛翠花,李颖,刘庆昌等.马铃薯抗晚疫病转基因材料的获得及活性氧清除酶系与抗病过程关系分析[J].中国农业科学,2008,41(12):4023-4029.
[2]唐翠清.稻瘟病的防治技术[J].农村实用信息技术,2009,6:41.
[3]王文娟,张飞云.植物抗病分子机制研究进展[J].生物技术通报,2007,1:19-23.
[4]王长春,蔡新忠,徐幼平.番茄与叶霉菌互作的分子机理[J].植物病理学报,2006,36(5):385-391.
[5]张月娟,朱秀秀,陈新等.番茄细菌性斑点病菌无毒基因研究进展[J].植物保护学报,2008,34(4):12-18.
[6]张萍萍,吴丽芳.植物抗病基因工程的研究进展[J].激光生物学报,2004,13(1):57-61.
[7]谷守芹,范永山.植物抗病基因工程研究综述[J].保定师范专科学校学报,2004,17(4):32-35,45.
[8]张红梅,张艳华,王伟.植物抗病基因工程研究进展[J].黑龙江八一农垦大学学报,2004,16(1):23-26.
[9]李淮,王莱,武国凡等.植物抗病性的分子机制和信号传导[J].西北师范大学学报,2006,42(2):113-117.
[10] Johal G S, Briggs S P. Reductase activity encoded by the hm1, disease resistance gene in maize [J]. Science, 1992, 258 (5084): 985-987.
[11] Talbot N J, Ebbole D J, Hamer J E. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus magnaporthe grisea [J]. Plant Cell, 1993, 5: 1575-1590.
[12] Flor H H. Current status of the gene-for-gene concept [J]. Annu. Rev. Phytopathol., 1971, 9: 275-296.
[13] Keen N T. Gene-for-gene complementarity in plant-pathogen interactions [J]. Annu. Rev. Genet., 1990, 24: 447-463.
[14]张艳贞,魏松红,杜娟等.植物抗病分子机制及抗病基因工程研究进展[J].沈阳农业大学学报,2000,31(4):365-369.
[15] Tang X, Frederick R D, Zhou J, et al. Initiation of plant disease resistance by physical interaction of AvrPto and Pto Kinase [J]. Science, 1996, 274: 2060-2063.
[16] Jia Y, McAdams S A, Bryan G T, et al. Direct interaction of resistance gene and avirulence gene products confers rice blast resistance [J]. EMBO J., 2000, 19 (15): 4004-4014.
[17] Biezen E A V D, Jones J D G. Plant disease-resistance proteins and the gene-for-gene concept [J]. Trends Biochem. Sci., 1998, 23 (12): 454-456.
[18] Mackey D, Holt B F, Wiig A, et al. RIN4 interacts with Pseudomonas syringae typeⅢeffector molecules and is required for RPM1-mediated resistance in Arabidopsis [J]. Cell, 2002, 108 (6): 743-754.
[19] Dangl J L, Jones J D G. Plant pathogens and integrated defence responses to infection [J]. Nature, 2001, 411: 826-833.
[20] Mackey D, Belkhadir Y, Alonso J M, et al. Arabidopsis RIN4 is a target of the typeⅢvirulence effector AvrRpt2 and modulates RPS2-mediated resistance [J]. Cell, 2003, 112 (3): 379-389.
[21] Axtell M J, Staskawicz B J. Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4 [J]. Cell, 2003, 112 (3): 369-377.
[22] Shao F, Golstein C, Ade J, et al. Cleavage of Arabidopsis PBS1 by a bacterial typeⅢeffector [J]. Science, 2003, 301 (5637): 1230-1233.
[23]罗敏,朱友林,余潮等.植物抗病基因的克隆及其结构与功能[J].遗传,2000,22(6):429-433.
[24] Lamb C, Dixon R A. The oxidative burst in plant disease resistance [J]. Annu. Rev. Plant Physiol. Plant Mol. Biol., 1997, 48: 251-275.
[25] Wojtaszek P. Oxidative burst: an early plant response to pathogen infection [J]. Biochem., 1997, 322: 681-692.
[26] Delaney T P, Uknes S, Vernooij B, et al. A central role of salicylic acid in plant disease resistance [J]. Science, 1994, 18: 1247-1250.
[27] Pieterse C M J, Van Wees S C M, Van Pelt J A, et al. A novel signaling pathway controlling induced systemic resistance in Arabidopsis [J]. Plant Cell, 1998, 10: 1571-1580.
[28] Nakashita H, Yasuda M, Nitta T, et al. Brassinosteroid functions in a broad range of diseaseresistance in tobacco and rice [J]. Plant, 2003, 33 (5): 887-898.
[29]孙大业.植物细胞信号转导研究进展[J].植物生理学通讯,1996,32(2):81-91.
[30]谭向红.21世纪初基因工程现状与发展趋势[J].四川农业大学学报,2002,20(2):162-171.
[31]张德水,陈受宜.植物抗病性的分子生物学研究进展[J].植物病理学报,1997,27(2):97-103.
[32]张祥喜,罗林广.植物抗病基因研究进展[J].分子植物育种,2003,1(4):531-537.
[33]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998,6:10-20.
[34]赵世民,祖国诚,刘根齐等.通过农杆菌介导法将兔防御素NP-1基因导入毛白杨(Populus tomentosa)[J].遗传学报,1999,26(6):711-714.
[35]徐惠君,Stcinbiss H H, Sobn A等.用基因枪将欧洲小麦梭条花叶病毒外壳蛋白基因(WSSMV-CP)导入小麦[J].云南大学学报,1999,21:26-27.
[36]俞志华,祝水金,夏武英等.从植物抗病基因的克隆看其结构、功能和进化[J].浙江大学学报,2002,28(1):107-113.
[37]宋凤鸣,郑重.植物抗真菌和细菌病害基因工程的策略及其进展[J].植物生理学通讯,1996,32(l):64-70.
[38]刘进元,余荔华.植物抗病基因工程的研究进展[J].生物工程进展,1994,14(2):31-34.
[39] Sridevi G, Sabapathi N, Meena P, et al. Transgenic indica rice variety pusa basmati 1 constitutively expressing a rice chitinase gene exhibits enhanced resistance to Rhizoctonia solani [J]. J. Plant Biochem. Biotechnol., 2003, 12 (2): 93-101.
[40] Wally O, Jayaraj J, Punja Z. Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase,β-1, 3-glucanase and peroxidise [J]. Eur. J. Plant Pathol., 2009, 123 (3): 331-342.
[41]顾丽红,张树珍,杨本鹏等.几丁质酶和β-1, 3-葡聚糖酶基因导入[J].甘蔗分子植物育种,2008,6(2):277-280.
[42]陈艳,江明锋,叶煜辉等.溶菌酶的研究进展[J].生物学杂志,2009,26(2):64-66.
[43] Fabienne M, Marie-No?lle B, Elisabeth C. Strategies to Improve Plant Resistance to BacterialDiseases Through Genetic Engineering [J]. Trends Biotechnol., 1998, 16 (5): 203-210.
[44]姜丹.高产细菌素菌株WJK84-1的诱变筛选及其对樱桃冠瘿瘤病原菌抑菌机理的研究[D].辽宁师范大学硕士学位论文,2003.
[45]单丽波,贾旭.核糖体失活蛋白及其在植物抗真菌病基因工程中的应用[J].生物工程进展,2000,20(6):74-78.
[46]付鸣佳,吴祖建,林奇英.美洲商陆抗病毒蛋白研究进展[J].生物技术通讯,2002,13(1):66-71.
[47]张宏,胡春香,张德禄等.植物防御素研究进展[J].西北师范大学学报,2006,42(5):112-117.
[48] Tian L, Yang X D, Liu J Y. Tissue and Induction Expression Profiles of Rice Phospholipid Hydroperoxide Glutathione Peroxidase at Protein Level [J]. Prog. Biochem. Biophys., 2009, 36 (1): 77-82.
[49] Wegener C, Bartling S, Olsen O, et al. Pectate lyase in transgenic potatoes confers pre-activation of defence against Erwinia carotovora [J]. Physiol. Mol. Plant Pathol., 1996, 49 (6): 359-376.
[50]林燕燕.HarpinEccs蛋白诱导植物防卫反应的研究[D].四川师范大学硕士学位论文,2008.
[51]汤浩茹.核桃的体细胞胚胎发生与转基因研究[D].四川农业大学博士学位论文,2000.
[52]胡京昂.小西葫芦黄花叶病毒外壳蛋白基因介导的对西瓜病毒抗性的研究[D].河南农业大学硕士学位论文,2005.
[53] Klein T M, Kornstein L, Sanford J C, et al. Genetic transformation of maize cells by particle bombardment [J]. Plant Physiol., 1989, 91: 440-444.
[54]吴丽芳,李红,冯慧云等.用低能氩离子束介导将水稻几丁质酶基因导入水稻[J].科学通报,2000,45(21):2316-2321.
[55] Antonelli N M, Stadler J. Genomic DNA can be used with cationic methods for highly efficient transformation of maize protoplasts [J]. Theor. Appl. Genet., 1990, 80: 395-401.
[56] Williamson B, Johnston D J, Ramanathan V, et al. A polygalacturonase inhibitor from immature raspberry fruits: a possible new approach to grey mould control [J]. Acta Hort. 1993,352: 601-606.
[57] Favaron F, Ovidio R D, Porceddu E, et al. Purification and molecular characterization of a soybean polygalacturonase inhibiting protein [J]. Planta, 1994, 195 (1): 80-87.
[58] Kajava A V. Structural diversity of leucine-rich repeat proteins [J]. J. Mol. Biol., 1998, 277: 519-527.
[59]杨崇林,陈章良.高等植物的LRR蛋白:结构与功能[J].生物工程进展,1997,17(6):20,43-47.
[60] Yao C, Conway W S, Ren R, et al. Gene encoding polygalacturonase inhibitor in apple fruit is developmentally regulated and activated by wounding and fungal infection [J]. Plant Mol. Biol., 1999, 39 (6): 1231-1241.
[61] Mattei B, Bernalda M S, Federici L, et al. Secondary structure and post- translational modifications of the leucine-rich repeat protein PGIP (polygalacturonase inhibiting protein) from Phaseolus vulgaris [J].Biochemistry, 2001, 40 (2): 569-576.
[62] Stotz H U, Contos J J, Powell A L T, et al. Structure and expression of an inhibitor of fungal polygalacturonases from tomato [J]. Plant Mol. Biol., 1994, 25: 607-617.
[63]万琳,周立.小麦多聚半乳糖醛酸酶抑制蛋白的部分结构[J].中国生物化学与分子生物学报,2002,18(2):197-201.
[64] Matteo A D, Federici L, Mattei B, et al. The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense [J]. Nat. Prod. Sci., 2003, 100 (17): 10124-10128.
[65]周立,阮期平,侯留记等.烟草内切多聚半乳糖醛酸酶抑制蛋白分离纯化研究[J].中国烟草科学,1998,(4):21-25.
[66] Hadfield K A, Bennett A B. Polygalacturonase: Many genes in search of a function [J]. Plant Physiol., 1998, 117: 337-343.
[67] Cervone F, Hahn M G, Lorenzo G D, et al. Host-pathogen interactions: XXXIII. A plant protein converts a fungal pathogenesis factor onto an elicitor of plant defense responses [J]. Plant Physiol., 1989, 90: 542-548.
[68] Leckie F, Mattei B, Capodicasa C, et al. The specificity of polygalacturonase inhibiting protein(PGIP): a single amino acid substitution in the solvent-exposedβ-strand/β-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability [J]. EMBO J., 1999, 18 (9): 2352-2363.
[69] Henrik U, Stotz, John G, et al. Identification of target amino acids that affect inter- actions of fungal polygalacturonases and their plant inhibitor [J]. Physiol. Mol. Plant Patholo., 2000, 56: 117-130.
[70]万琳.小麦PGIP部分结构分析及与双子叶植物PGIP的比较研究[D].四川大学硕士学位论文,2001.
[71] Janni M, Giovanni M D, Roberti S, et al. Characterization of expressed pgip genes in rice and wheat reveals similar extent of sequence variation to dicot PGIPs and identifies an active PGIP lacking an entire LRR repeat [J]. Theor. Appl. Genet., 2006, 113: 1233-1245.
[72] Johnston D J, Ramanathan V, Williamson B, et al. A protein from immature raspberry fruits which inhibits endo-polygalacturonases from Botrytis cinerea and other micro-organisms [J]. Exp. Bot. 1993, 44 (5): 971-976.
[73]阮期平,周立,郑远旗.PGIP在植物抗病方面的研究进展[J].植物学通报,2000,17(1):60-63.
[74] Lorenzo D G, Ovidio D R, Cervone F. The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi [J]. Annu. Rev. Phytopathol., 2001, 39: 313-335.
[75] Li R G, Rimmer R, Yu M, et al. Two Brassica napus polygalacturonase inhibitory protein genes are expressed at different levels in response to biotic and abiotic stresses [J]. Planta, 2003, 217: 299-308.
[76] Gotoh Y, Nalumpang S, Isshiki A, et al. A cDNA Encoding polygalacturonase inhibiting protein induced in citrus leaves by polygalacturonase of Altemaria citri [J]. J. Gen. Plant Pathol., 2002, 68: 57-61.
[77] Jang S, Lee B, Kim C, et sl. The OsFOR1 gene encodes a polygalacturonase inhibiting protein (PGIP) that regulates floral organ number in rice [J]. Plant Mol. Bio., 2003,53: 357-369.
[78] Ramanathan V, Simpson C G, Thow G, et al. cDNA cloning and expression of polygalacturonase inhibiting protein (PGIP) from raspberry (Rubus idaeus) [J]. J. Exp. Biol.,1997, 48 (311): 1185-1193.
[79] Chimwamurombe P M, Botha A M, Wingfield M J, et al. Molecular relatedness of the polygalacturonase inhibiting protein genes in Eucalyptus species [J]. Theor. Appl. Genet., 2001, 102: 645-650.
[80] Toubart P, Desiderio A, Salvi G, et al. Cloning and characterization of the gene encoding the endopolygalacturonase inhibiting protein (PGIP) of Phadeolus vulgaris L. [J]. Plant J., 1992, 2 (3): 367-373.
[81] Daolong D, Wang B S, Tang Y X, et al. Cloning and sequence analysis of a gene encoding polygalacturonase inhibiting protein from cotton [J]. Prog. Nat. Sci., 2003, 13 (2): 119-124.
[82]张军科,张开春,李嘉瑞等.马哈利樱桃PGIP cDNA克隆及序列分析[J].西北植物学报,2001,21(6):1123-1127.
[83] Simpson C G, MacRae E, Gardner R C. Cloning of a polygalacturonase inhibiting protein from Kiwifruit (Actinidia deliciosa) [J]. Plant Physiol., 1995, 108: 1748.
[84] Pressey R. Polygalacturonase inhibitors in bean pods [J]. Phytochemistry, 1996, 42 (5): 1267-1270.
[85] Desiderio A, Aracri B, Leckie F, et al. Polygalacturonase inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris [J]. Mol. Plant Microbe. Interact., 1997, 10 (7): 852-860.
[86] Gazendam I, Oelofse D, Berger D K. High-level expression of apple PGIP1 is not sufficient to protect transgenic potato against Verticillium dahliae [J]. Physio. Mol. Plant Pathol., 2004, 65: 145-155.
[87] Bennett A, Labavitch J M, Powell A, et al. Plant inhibitors of fungal polygalacturonases and their use to control fungal disease [J]. US Patent., 1996, 5: 569-830.
[88] Albersheim P, Anderson A J. Proteins from plant cell walls inhibit polygalacturo- nases secreted by plant pathogens [J]. Proc. Nat. Acad. Sci. USA, 1971, 68 (8): 1815-1819.
[89] Khanuja S P S, Shasany A K, Darokar M P, et al. Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils [J]. Plant Mol. Biol. Rep., 1999, 17: 1-7.
[90]巴克.分子生物学实验室工作手册[M].北京:科学出版社,2005:210.
[91] Scarafoni A, Duranti M. An approach to the critical assessment of the experimental conditions in practical molecular biology: isolation of plant DNA [J]. Biochem. Mol. Biol. Edu., 2001, 29: 21-23.
[92] Chung C T, Niemela S L, Miller R H. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution [J]. Proc. Natl. Acad. Sci. USA, 1989, 86 (8): 2172-2175.
[93]王金宏,刘娟妮,纪庆等.抗CD3 ScFv/抗PgP ScFv双功能抗体的高密度发酵与纯化[J].药物生物技术,2008,15(4):249-252.
[94]萨姆布鲁克,弗里奇,曼尼阿蒂斯.分子克隆实验指南[M].北京:科学出版社,1992:19-22.
[95] Cheng J, Randall A Z, Sweredoski M J, et al. SCRATCH: a protein structure and structural feature prediction server [J]. Nucleic Acids Rese., 2005, 33 (7):72-76.
[96] Primo M E, Sica M P, Risso V A, et al. Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2 [J]. Biochim. et Biophy. Acta, 2006, 1764: 174-181.
[97] Fong J H, Keating A E, Singh M. Predicting specificity in bZIP coiled-coil protein interactions [J]. Genome Biol., 2004, 5 (2): 2-10.
[98] Lambert C, Leonard N, Bolle X D, et al. ESyPred3D: Prediction of proteins 3D structures [J]. Bioinformatics, 2002, 18 (9): 1250-1256.
[99] Benkert P, Schwede T, Tosatto S C E. QMEANclust: estimation of protein model quality by combining a composite scoring function with structural density information [J]. BMC Struct. Biol., 2009, 9: 35-42.
[100] Gasteiger E, Hoogland C, Gattiker A, et al. The Proteomics Protocol Handbook [M]. Totow: Humana Press, 2005: 571-607.
[101] Harrison R G. Expression of soluble heterologous proteins via fusion with NusA protein [J]. Novation, 2000, 11: 4-7.
[102] Cserzo M, Eisenhaber F, Eisenhaber B, et al. On filtering false positive transmembraneprotein predictions [J]. Protein Eng., 2002, 15 (9): 745-752.
[103] Nakao M C, Nakai K. Improvement of PSORT II protein sorting prediction for mammalian Proteins [J]. Genome Inf., 2002, 13: 441-442.
[104] Bendtsen J D, Nielsen H, Heijne G V, et al. Improved prediction of signal peptides-SignalP 3.0 [J]. J. Mol. Biol., 2004, 340 (4): 783-795.
[105] Emanuelsson O, Brunak S, Heijne G V, et al. Locating proteins in the cell using TargetP, SignalP and related tools [J]. Nat. Protocols, 2007, 2: 953-971.
[106] Blom N, Gammeltoft S, Brunak S. Sequence and structure-based prediction of Eukaryotic protein phosphorylation sites [J]. J. Mol. Biol., 1999, 294: 1351-1362.
[107] Novagen. pET System Manual [OB/OL]. Merck-China: Novagen, 2002.
[108]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000:77-101.
[109]张梅,郭丽清,关夏玉等.AiiA融合蛋白包涵体变性和复性的研究[J].福建师范大学学报,2008,24(4):76-79.
[110] Clark E D B. Refolding of recombinant proteins [J]. Curr. Opinion Biotechnol., 1998, 9: 157-163.
[111]周立,刘勇,李建吾等.小麦多聚半乳糖醛酸酶抑制蛋白对几种病原真菌抑制作用的研究[J].植物病理学报,1998,28(2):107-112.
[112] Sella L, Castiglioni, Roberti Serena, et al. An endo-polygalacturonase (PG) of Fusarium moniliforme escaping inhibition by plant polygalacturonase-inhibiting proteins (PGIPs) provides new insights into the PG-PGIP interaction [J]. FEMS Microbiol. Letters, 2004, 240: 117-124.
[113] Taylor R J, Secor G A. An improved diffusion assay for quantifying the polygalacturonase content of Erwinia culture filtrates [J]. Phytopathol.,1988, 78: 1101-1103.
[114]王天龙,仇宏伟,陈海华等.3, 5-二硝基水杨酸法测定果胶酶活力的条件研究[J].食品与机械,2008,24(3):96-104.
[115]周立,李建吾,郑远旗等.还原糖法测定多聚半乳糖醛酸酶抑制蛋白活性的研究[J].四川大学学报,1996,33(3):311-315.
[116]王晓利.苹果多聚半乳糖醛酸酶抑制蛋白的分离纯化与抑菌作用研究[D].西北农林科技大学硕士论文,2008.
[117]万佳,况浩池,谢必武等.一个水稻钙调素结合蛋白基因OsCaMBP的克隆及表达分析[J].中国水稻科学,2008,22(3):243-248.
[118]王希成.生物化学[M].北京:清华大学出版社,2005:25.
[119] Kumar M, Sanford K J, Cuevas W A, et al. Designer protein-based performance materials [J]. Biomacromol., 2006, 7: 2543-2551.
[120] Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease [J]. Annu. Rev. Biochem., 1994, 63 (1): 345-382.
[121]陈丹英,赵允,赵大忠等.铁线蕨中间纤维的研究及某些植物类角蛋白的比较分析[J].植物学报,1998,40(9):790-795.
[122]黄京飞.蛋白质序列和结构的保守性与其功能的关系[J].动物学研究,1999,20(1):21-25.
[123] Bishop J G. Directed mutagenesis confirms the functional importance of positively selected sites in polygalacturonase inhibitor protein [J]. Mol. Biol. Evol., 2005, 22 (7): 1531-1534.
[124] Ferrari S, Vairo D, Ausubel F M, et al. Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection [J]. Plant Cell, 2003, 15:93-106.
[125]孙柏欣,刘长远,陈彦等.基因表达系统研究进展[J].现代农业科技,2008,2:205-209.
[126] Kiefhaber T, Rudolph R, Kohler H H, et al. Protein aggregation in vitro and in vivo: A quantitative model of the kinetic competition between folding and aggregation [J]. Nat. Biotechnol., 1991, 9: 825-829.
[127] Forciniti D. Protein refolding using aqueous two-phase systems [J]. J. Chromatogr., 1994, 668 (1): 95-100.
[128] Maeda Y, Ueda T, Imoto T. Effective renaturation of denatured and reduced immunoglobu- lin G in vitro without assistance of chaperone [J]. Protein Eng., 1996, 9 (1): 95-100.
[129] Xie Y, Wetlaufer D B. Control of aggregation in protein refolding: The temperature-leap tactic [J]. Protein Sci., 1996, 5: 517-523.
[130] Georgiou G, Valax P. Expression of correctly folded proteins in Escherichia coil [J]. Curr.Opin. Biotechnol., 1996, 7: 190-197.
[131]徐灵龙,石星明,王云峰等.优化的复合自动诱导培养基(CAI-4)对外源蛋白在大肠杆菌中表达的影响[J].微生物学报,2009,49(1):128-134.
[132] Lilie H, Schwarz E, Rudolph R. Advances in refolding of proteins produced in E. coli [J]. Curr. Opin. Biotechnol., 1998, 9: 497-501.
[133] Dabrowski M J, Chen J P, Shi H, et al. Strategies for protein-based nanofabrication: Ni2+-NTA as a chemical mask to control biologically imposed symmetry [J]. Chem. Biol., 1998, 5 (10): 689-697.
[134]张蓓,沈立松.实时荧光定量PCR的研究进展及其应用[J].国外医学临床生物化学与检验学分册,2003,24(6):327-329.
[135]左示敏,王子斌,陈夕军等.水稻纹枯病改良新抗源YSBR1的抗性评价[J].作物学报,2009,35(4):608-614.
[136]阮期平,周立,刘勇.小麦在与水杨酸诱导的应答过程中PGIP的积累[J].应用与环境生物学报,2000,6(4):313-316.
[137]杨晓宇,樊新萍,曹秋芬等.PGIP在作物抗病育种中的研究进展[J].山西农业科学,2008,36(7):86-89.
[138] Oelofse D, Dubery I A, Meryer R, et al. Apple polygalacturonase inhibiting protein 1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and theantracnose pathogen of lupins [J]. Phytochemistry, 2006, 67 (3): 255-263.
[2]唐翠清.稻瘟病的防治技术[J].农村实用信息技术,2009,6:41.
[3]王文娟,张飞云.植物抗病分子机制研究进展[J].生物技术通报,2007,1:19-23.
[4]王长春,蔡新忠,徐幼平.番茄与叶霉菌互作的分子机理[J].植物病理学报,2006,36(5):385-391.
[5]张月娟,朱秀秀,陈新等.番茄细菌性斑点病菌无毒基因研究进展[J].植物保护学报,2008,34(4):12-18.
[6]张萍萍,吴丽芳.植物抗病基因工程的研究进展[J].激光生物学报,2004,13(1):57-61.
[7]谷守芹,范永山.植物抗病基因工程研究综述[J].保定师范专科学校学报,2004,17(4):32-35,45.
[8]张红梅,张艳华,王伟.植物抗病基因工程研究进展[J].黑龙江八一农垦大学学报,2004,16(1):23-26.
[9]李淮,王莱,武国凡等.植物抗病性的分子机制和信号传导[J].西北师范大学学报,2006,42(2):113-117.
[10] Johal G S, Briggs S P. Reductase activity encoded by the hm1, disease resistance gene in maize [J]. Science, 1992, 258 (5084): 985-987.
[11] Talbot N J, Ebbole D J, Hamer J E. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus magnaporthe grisea [J]. Plant Cell, 1993, 5: 1575-1590.
[12] Flor H H. Current status of the gene-for-gene concept [J]. Annu. Rev. Phytopathol., 1971, 9: 275-296.
[13] Keen N T. Gene-for-gene complementarity in plant-pathogen interactions [J]. Annu. Rev. Genet., 1990, 24: 447-463.
[14]张艳贞,魏松红,杜娟等.植物抗病分子机制及抗病基因工程研究进展[J].沈阳农业大学学报,2000,31(4):365-369.
[15] Tang X, Frederick R D, Zhou J, et al. Initiation of plant disease resistance by physical interaction of AvrPto and Pto Kinase [J]. Science, 1996, 274: 2060-2063.
[16] Jia Y, McAdams S A, Bryan G T, et al. Direct interaction of resistance gene and avirulence gene products confers rice blast resistance [J]. EMBO J., 2000, 19 (15): 4004-4014.
[17] Biezen E A V D, Jones J D G. Plant disease-resistance proteins and the gene-for-gene concept [J]. Trends Biochem. Sci., 1998, 23 (12): 454-456.
[18] Mackey D, Holt B F, Wiig A, et al. RIN4 interacts with Pseudomonas syringae typeⅢeffector molecules and is required for RPM1-mediated resistance in Arabidopsis [J]. Cell, 2002, 108 (6): 743-754.
[19] Dangl J L, Jones J D G. Plant pathogens and integrated defence responses to infection [J]. Nature, 2001, 411: 826-833.
[20] Mackey D, Belkhadir Y, Alonso J M, et al. Arabidopsis RIN4 is a target of the typeⅢvirulence effector AvrRpt2 and modulates RPS2-mediated resistance [J]. Cell, 2003, 112 (3): 379-389.
[21] Axtell M J, Staskawicz B J. Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4 [J]. Cell, 2003, 112 (3): 369-377.
[22] Shao F, Golstein C, Ade J, et al. Cleavage of Arabidopsis PBS1 by a bacterial typeⅢeffector [J]. Science, 2003, 301 (5637): 1230-1233.
[23]罗敏,朱友林,余潮等.植物抗病基因的克隆及其结构与功能[J].遗传,2000,22(6):429-433.
[24] Lamb C, Dixon R A. The oxidative burst in plant disease resistance [J]. Annu. Rev. Plant Physiol. Plant Mol. Biol., 1997, 48: 251-275.
[25] Wojtaszek P. Oxidative burst: an early plant response to pathogen infection [J]. Biochem., 1997, 322: 681-692.
[26] Delaney T P, Uknes S, Vernooij B, et al. A central role of salicylic acid in plant disease resistance [J]. Science, 1994, 18: 1247-1250.
[27] Pieterse C M J, Van Wees S C M, Van Pelt J A, et al. A novel signaling pathway controlling induced systemic resistance in Arabidopsis [J]. Plant Cell, 1998, 10: 1571-1580.
[28] Nakashita H, Yasuda M, Nitta T, et al. Brassinosteroid functions in a broad range of diseaseresistance in tobacco and rice [J]. Plant, 2003, 33 (5): 887-898.
[29]孙大业.植物细胞信号转导研究进展[J].植物生理学通讯,1996,32(2):81-91.
[30]谭向红.21世纪初基因工程现状与发展趋势[J].四川农业大学学报,2002,20(2):162-171.
[31]张德水,陈受宜.植物抗病性的分子生物学研究进展[J].植物病理学报,1997,27(2):97-103.
[32]张祥喜,罗林广.植物抗病基因研究进展[J].分子植物育种,2003,1(4):531-537.
[33]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998,6:10-20.
[34]赵世民,祖国诚,刘根齐等.通过农杆菌介导法将兔防御素NP-1基因导入毛白杨(Populus tomentosa)[J].遗传学报,1999,26(6):711-714.
[35]徐惠君,Stcinbiss H H, Sobn A等.用基因枪将欧洲小麦梭条花叶病毒外壳蛋白基因(WSSMV-CP)导入小麦[J].云南大学学报,1999,21:26-27.
[36]俞志华,祝水金,夏武英等.从植物抗病基因的克隆看其结构、功能和进化[J].浙江大学学报,2002,28(1):107-113.
[37]宋凤鸣,郑重.植物抗真菌和细菌病害基因工程的策略及其进展[J].植物生理学通讯,1996,32(l):64-70.
[38]刘进元,余荔华.植物抗病基因工程的研究进展[J].生物工程进展,1994,14(2):31-34.
[39] Sridevi G, Sabapathi N, Meena P, et al. Transgenic indica rice variety pusa basmati 1 constitutively expressing a rice chitinase gene exhibits enhanced resistance to Rhizoctonia solani [J]. J. Plant Biochem. Biotechnol., 2003, 12 (2): 93-101.
[40] Wally O, Jayaraj J, Punja Z. Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase,β-1, 3-glucanase and peroxidise [J]. Eur. J. Plant Pathol., 2009, 123 (3): 331-342.
[41]顾丽红,张树珍,杨本鹏等.几丁质酶和β-1, 3-葡聚糖酶基因导入[J].甘蔗分子植物育种,2008,6(2):277-280.
[42]陈艳,江明锋,叶煜辉等.溶菌酶的研究进展[J].生物学杂志,2009,26(2):64-66.
[43] Fabienne M, Marie-No?lle B, Elisabeth C. Strategies to Improve Plant Resistance to BacterialDiseases Through Genetic Engineering [J]. Trends Biotechnol., 1998, 16 (5): 203-210.
[44]姜丹.高产细菌素菌株WJK84-1的诱变筛选及其对樱桃冠瘿瘤病原菌抑菌机理的研究[D].辽宁师范大学硕士学位论文,2003.
[45]单丽波,贾旭.核糖体失活蛋白及其在植物抗真菌病基因工程中的应用[J].生物工程进展,2000,20(6):74-78.
[46]付鸣佳,吴祖建,林奇英.美洲商陆抗病毒蛋白研究进展[J].生物技术通讯,2002,13(1):66-71.
[47]张宏,胡春香,张德禄等.植物防御素研究进展[J].西北师范大学学报,2006,42(5):112-117.
[48] Tian L, Yang X D, Liu J Y. Tissue and Induction Expression Profiles of Rice Phospholipid Hydroperoxide Glutathione Peroxidase at Protein Level [J]. Prog. Biochem. Biophys., 2009, 36 (1): 77-82.
[49] Wegener C, Bartling S, Olsen O, et al. Pectate lyase in transgenic potatoes confers pre-activation of defence against Erwinia carotovora [J]. Physiol. Mol. Plant Pathol., 1996, 49 (6): 359-376.
[50]林燕燕.HarpinEccs蛋白诱导植物防卫反应的研究[D].四川师范大学硕士学位论文,2008.
[51]汤浩茹.核桃的体细胞胚胎发生与转基因研究[D].四川农业大学博士学位论文,2000.
[52]胡京昂.小西葫芦黄花叶病毒外壳蛋白基因介导的对西瓜病毒抗性的研究[D].河南农业大学硕士学位论文,2005.
[53] Klein T M, Kornstein L, Sanford J C, et al. Genetic transformation of maize cells by particle bombardment [J]. Plant Physiol., 1989, 91: 440-444.
[54]吴丽芳,李红,冯慧云等.用低能氩离子束介导将水稻几丁质酶基因导入水稻[J].科学通报,2000,45(21):2316-2321.
[55] Antonelli N M, Stadler J. Genomic DNA can be used with cationic methods for highly efficient transformation of maize protoplasts [J]. Theor. Appl. Genet., 1990, 80: 395-401.
[56] Williamson B, Johnston D J, Ramanathan V, et al. A polygalacturonase inhibitor from immature raspberry fruits: a possible new approach to grey mould control [J]. Acta Hort. 1993,352: 601-606.
[57] Favaron F, Ovidio R D, Porceddu E, et al. Purification and molecular characterization of a soybean polygalacturonase inhibiting protein [J]. Planta, 1994, 195 (1): 80-87.
[58] Kajava A V. Structural diversity of leucine-rich repeat proteins [J]. J. Mol. Biol., 1998, 277: 519-527.
[59]杨崇林,陈章良.高等植物的LRR蛋白:结构与功能[J].生物工程进展,1997,17(6):20,43-47.
[60] Yao C, Conway W S, Ren R, et al. Gene encoding polygalacturonase inhibitor in apple fruit is developmentally regulated and activated by wounding and fungal infection [J]. Plant Mol. Biol., 1999, 39 (6): 1231-1241.
[61] Mattei B, Bernalda M S, Federici L, et al. Secondary structure and post- translational modifications of the leucine-rich repeat protein PGIP (polygalacturonase inhibiting protein) from Phaseolus vulgaris [J].Biochemistry, 2001, 40 (2): 569-576.
[62] Stotz H U, Contos J J, Powell A L T, et al. Structure and expression of an inhibitor of fungal polygalacturonases from tomato [J]. Plant Mol. Biol., 1994, 25: 607-617.
[63]万琳,周立.小麦多聚半乳糖醛酸酶抑制蛋白的部分结构[J].中国生物化学与分子生物学报,2002,18(2):197-201.
[64] Matteo A D, Federici L, Mattei B, et al. The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense [J]. Nat. Prod. Sci., 2003, 100 (17): 10124-10128.
[65]周立,阮期平,侯留记等.烟草内切多聚半乳糖醛酸酶抑制蛋白分离纯化研究[J].中国烟草科学,1998,(4):21-25.
[66] Hadfield K A, Bennett A B. Polygalacturonase: Many genes in search of a function [J]. Plant Physiol., 1998, 117: 337-343.
[67] Cervone F, Hahn M G, Lorenzo G D, et al. Host-pathogen interactions: XXXIII. A plant protein converts a fungal pathogenesis factor onto an elicitor of plant defense responses [J]. Plant Physiol., 1989, 90: 542-548.
[68] Leckie F, Mattei B, Capodicasa C, et al. The specificity of polygalacturonase inhibiting protein(PGIP): a single amino acid substitution in the solvent-exposedβ-strand/β-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability [J]. EMBO J., 1999, 18 (9): 2352-2363.
[69] Henrik U, Stotz, John G, et al. Identification of target amino acids that affect inter- actions of fungal polygalacturonases and their plant inhibitor [J]. Physiol. Mol. Plant Patholo., 2000, 56: 117-130.
[70]万琳.小麦PGIP部分结构分析及与双子叶植物PGIP的比较研究[D].四川大学硕士学位论文,2001.
[71] Janni M, Giovanni M D, Roberti S, et al. Characterization of expressed pgip genes in rice and wheat reveals similar extent of sequence variation to dicot PGIPs and identifies an active PGIP lacking an entire LRR repeat [J]. Theor. Appl. Genet., 2006, 113: 1233-1245.
[72] Johnston D J, Ramanathan V, Williamson B, et al. A protein from immature raspberry fruits which inhibits endo-polygalacturonases from Botrytis cinerea and other micro-organisms [J]. Exp. Bot. 1993, 44 (5): 971-976.
[73]阮期平,周立,郑远旗.PGIP在植物抗病方面的研究进展[J].植物学通报,2000,17(1):60-63.
[74] Lorenzo D G, Ovidio D R, Cervone F. The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi [J]. Annu. Rev. Phytopathol., 2001, 39: 313-335.
[75] Li R G, Rimmer R, Yu M, et al. Two Brassica napus polygalacturonase inhibitory protein genes are expressed at different levels in response to biotic and abiotic stresses [J]. Planta, 2003, 217: 299-308.
[76] Gotoh Y, Nalumpang S, Isshiki A, et al. A cDNA Encoding polygalacturonase inhibiting protein induced in citrus leaves by polygalacturonase of Altemaria citri [J]. J. Gen. Plant Pathol., 2002, 68: 57-61.
[77] Jang S, Lee B, Kim C, et sl. The OsFOR1 gene encodes a polygalacturonase inhibiting protein (PGIP) that regulates floral organ number in rice [J]. Plant Mol. Bio., 2003,53: 357-369.
[78] Ramanathan V, Simpson C G, Thow G, et al. cDNA cloning and expression of polygalacturonase inhibiting protein (PGIP) from raspberry (Rubus idaeus) [J]. J. Exp. Biol.,1997, 48 (311): 1185-1193.
[79] Chimwamurombe P M, Botha A M, Wingfield M J, et al. Molecular relatedness of the polygalacturonase inhibiting protein genes in Eucalyptus species [J]. Theor. Appl. Genet., 2001, 102: 645-650.
[80] Toubart P, Desiderio A, Salvi G, et al. Cloning and characterization of the gene encoding the endopolygalacturonase inhibiting protein (PGIP) of Phadeolus vulgaris L. [J]. Plant J., 1992, 2 (3): 367-373.
[81] Daolong D, Wang B S, Tang Y X, et al. Cloning and sequence analysis of a gene encoding polygalacturonase inhibiting protein from cotton [J]. Prog. Nat. Sci., 2003, 13 (2): 119-124.
[82]张军科,张开春,李嘉瑞等.马哈利樱桃PGIP cDNA克隆及序列分析[J].西北植物学报,2001,21(6):1123-1127.
[83] Simpson C G, MacRae E, Gardner R C. Cloning of a polygalacturonase inhibiting protein from Kiwifruit (Actinidia deliciosa) [J]. Plant Physiol., 1995, 108: 1748.
[84] Pressey R. Polygalacturonase inhibitors in bean pods [J]. Phytochemistry, 1996, 42 (5): 1267-1270.
[85] Desiderio A, Aracri B, Leckie F, et al. Polygalacturonase inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris [J]. Mol. Plant Microbe. Interact., 1997, 10 (7): 852-860.
[86] Gazendam I, Oelofse D, Berger D K. High-level expression of apple PGIP1 is not sufficient to protect transgenic potato against Verticillium dahliae [J]. Physio. Mol. Plant Pathol., 2004, 65: 145-155.
[87] Bennett A, Labavitch J M, Powell A, et al. Plant inhibitors of fungal polygalacturonases and their use to control fungal disease [J]. US Patent., 1996, 5: 569-830.
[88] Albersheim P, Anderson A J. Proteins from plant cell walls inhibit polygalacturo- nases secreted by plant pathogens [J]. Proc. Nat. Acad. Sci. USA, 1971, 68 (8): 1815-1819.
[89] Khanuja S P S, Shasany A K, Darokar M P, et al. Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils [J]. Plant Mol. Biol. Rep., 1999, 17: 1-7.
[90]巴克.分子生物学实验室工作手册[M].北京:科学出版社,2005:210.
[91] Scarafoni A, Duranti M. An approach to the critical assessment of the experimental conditions in practical molecular biology: isolation of plant DNA [J]. Biochem. Mol. Biol. Edu., 2001, 29: 21-23.
[92] Chung C T, Niemela S L, Miller R H. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution [J]. Proc. Natl. Acad. Sci. USA, 1989, 86 (8): 2172-2175.
[93]王金宏,刘娟妮,纪庆等.抗CD3 ScFv/抗PgP ScFv双功能抗体的高密度发酵与纯化[J].药物生物技术,2008,15(4):249-252.
[94]萨姆布鲁克,弗里奇,曼尼阿蒂斯.分子克隆实验指南[M].北京:科学出版社,1992:19-22.
[95] Cheng J, Randall A Z, Sweredoski M J, et al. SCRATCH: a protein structure and structural feature prediction server [J]. Nucleic Acids Rese., 2005, 33 (7):72-76.
[96] Primo M E, Sica M P, Risso V A, et al. Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2 [J]. Biochim. et Biophy. Acta, 2006, 1764: 174-181.
[97] Fong J H, Keating A E, Singh M. Predicting specificity in bZIP coiled-coil protein interactions [J]. Genome Biol., 2004, 5 (2): 2-10.
[98] Lambert C, Leonard N, Bolle X D, et al. ESyPred3D: Prediction of proteins 3D structures [J]. Bioinformatics, 2002, 18 (9): 1250-1256.
[99] Benkert P, Schwede T, Tosatto S C E. QMEANclust: estimation of protein model quality by combining a composite scoring function with structural density information [J]. BMC Struct. Biol., 2009, 9: 35-42.
[100] Gasteiger E, Hoogland C, Gattiker A, et al. The Proteomics Protocol Handbook [M]. Totow: Humana Press, 2005: 571-607.
[101] Harrison R G. Expression of soluble heterologous proteins via fusion with NusA protein [J]. Novation, 2000, 11: 4-7.
[102] Cserzo M, Eisenhaber F, Eisenhaber B, et al. On filtering false positive transmembraneprotein predictions [J]. Protein Eng., 2002, 15 (9): 745-752.
[103] Nakao M C, Nakai K. Improvement of PSORT II protein sorting prediction for mammalian Proteins [J]. Genome Inf., 2002, 13: 441-442.
[104] Bendtsen J D, Nielsen H, Heijne G V, et al. Improved prediction of signal peptides-SignalP 3.0 [J]. J. Mol. Biol., 2004, 340 (4): 783-795.
[105] Emanuelsson O, Brunak S, Heijne G V, et al. Locating proteins in the cell using TargetP, SignalP and related tools [J]. Nat. Protocols, 2007, 2: 953-971.
[106] Blom N, Gammeltoft S, Brunak S. Sequence and structure-based prediction of Eukaryotic protein phosphorylation sites [J]. J. Mol. Biol., 1999, 294: 1351-1362.
[107] Novagen. pET System Manual [OB/OL]. Merck-China: Novagen, 2002.
[108]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2000:77-101.
[109]张梅,郭丽清,关夏玉等.AiiA融合蛋白包涵体变性和复性的研究[J].福建师范大学学报,2008,24(4):76-79.
[110] Clark E D B. Refolding of recombinant proteins [J]. Curr. Opinion Biotechnol., 1998, 9: 157-163.
[111]周立,刘勇,李建吾等.小麦多聚半乳糖醛酸酶抑制蛋白对几种病原真菌抑制作用的研究[J].植物病理学报,1998,28(2):107-112.
[112] Sella L, Castiglioni, Roberti Serena, et al. An endo-polygalacturonase (PG) of Fusarium moniliforme escaping inhibition by plant polygalacturonase-inhibiting proteins (PGIPs) provides new insights into the PG-PGIP interaction [J]. FEMS Microbiol. Letters, 2004, 240: 117-124.
[113] Taylor R J, Secor G A. An improved diffusion assay for quantifying the polygalacturonase content of Erwinia culture filtrates [J]. Phytopathol.,1988, 78: 1101-1103.
[114]王天龙,仇宏伟,陈海华等.3, 5-二硝基水杨酸法测定果胶酶活力的条件研究[J].食品与机械,2008,24(3):96-104.
[115]周立,李建吾,郑远旗等.还原糖法测定多聚半乳糖醛酸酶抑制蛋白活性的研究[J].四川大学学报,1996,33(3):311-315.
[116]王晓利.苹果多聚半乳糖醛酸酶抑制蛋白的分离纯化与抑菌作用研究[D].西北农林科技大学硕士论文,2008.
[117]万佳,况浩池,谢必武等.一个水稻钙调素结合蛋白基因OsCaMBP的克隆及表达分析[J].中国水稻科学,2008,22(3):243-248.
[118]王希成.生物化学[M].北京:清华大学出版社,2005:25.
[119] Kumar M, Sanford K J, Cuevas W A, et al. Designer protein-based performance materials [J]. Biomacromol., 2006, 7: 2543-2551.
[120] Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease [J]. Annu. Rev. Biochem., 1994, 63 (1): 345-382.
[121]陈丹英,赵允,赵大忠等.铁线蕨中间纤维的研究及某些植物类角蛋白的比较分析[J].植物学报,1998,40(9):790-795.
[122]黄京飞.蛋白质序列和结构的保守性与其功能的关系[J].动物学研究,1999,20(1):21-25.
[123] Bishop J G. Directed mutagenesis confirms the functional importance of positively selected sites in polygalacturonase inhibitor protein [J]. Mol. Biol. Evol., 2005, 22 (7): 1531-1534.
[124] Ferrari S, Vairo D, Ausubel F M, et al. Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection [J]. Plant Cell, 2003, 15:93-106.
[125]孙柏欣,刘长远,陈彦等.基因表达系统研究进展[J].现代农业科技,2008,2:205-209.
[126] Kiefhaber T, Rudolph R, Kohler H H, et al. Protein aggregation in vitro and in vivo: A quantitative model of the kinetic competition between folding and aggregation [J]. Nat. Biotechnol., 1991, 9: 825-829.
[127] Forciniti D. Protein refolding using aqueous two-phase systems [J]. J. Chromatogr., 1994, 668 (1): 95-100.
[128] Maeda Y, Ueda T, Imoto T. Effective renaturation of denatured and reduced immunoglobu- lin G in vitro without assistance of chaperone [J]. Protein Eng., 1996, 9 (1): 95-100.
[129] Xie Y, Wetlaufer D B. Control of aggregation in protein refolding: The temperature-leap tactic [J]. Protein Sci., 1996, 5: 517-523.
[130] Georgiou G, Valax P. Expression of correctly folded proteins in Escherichia coil [J]. Curr.Opin. Biotechnol., 1996, 7: 190-197.
[131]徐灵龙,石星明,王云峰等.优化的复合自动诱导培养基(CAI-4)对外源蛋白在大肠杆菌中表达的影响[J].微生物学报,2009,49(1):128-134.
[132] Lilie H, Schwarz E, Rudolph R. Advances in refolding of proteins produced in E. coli [J]. Curr. Opin. Biotechnol., 1998, 9: 497-501.
[133] Dabrowski M J, Chen J P, Shi H, et al. Strategies for protein-based nanofabrication: Ni2+-NTA as a chemical mask to control biologically imposed symmetry [J]. Chem. Biol., 1998, 5 (10): 689-697.
[134]张蓓,沈立松.实时荧光定量PCR的研究进展及其应用[J].国外医学临床生物化学与检验学分册,2003,24(6):327-329.
[135]左示敏,王子斌,陈夕军等.水稻纹枯病改良新抗源YSBR1的抗性评价[J].作物学报,2009,35(4):608-614.
[136]阮期平,周立,刘勇.小麦在与水杨酸诱导的应答过程中PGIP的积累[J].应用与环境生物学报,2000,6(4):313-316.
[137]杨晓宇,樊新萍,曹秋芬等.PGIP在作物抗病育种中的研究进展[J].山西农业科学,2008,36(7):86-89.
[138] Oelofse D, Dubery I A, Meryer R, et al. Apple polygalacturonase inhibiting protein 1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and theantracnose pathogen of lupins [J]. Phytochemistry, 2006, 67 (3): 255-263.