The Cloning and Functional Expression of a Kunitz Trypsin Inhibitor Gene from Populus alba var.pyramidalis

LIANG Wen-xing; FAN Zai-feng; LI Yong; SONG Li-min; LI Huai-fang; TIAN Guo-zhong

Volume 19 Issue 4

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The Cloning and Functional Expression of a Kunitz Trypsin Inhibitor Gene from Populus alba var.pyramidalis

1.
Plant Pathology Department, China Agricultural University, Beijing 100094, China
2.
Research Institute of Forest Ecology, Environment and Protection, CAF, Key Laboratory of Forestry Protection, State Forestry Administration, Beijing 100091, China

Received Date: 2005-06-07

Abstract

Wounding of poplar trees leads to the accumulation of several mRNA species that encode proteins with putative defensive function.A Kuntiz trypsin inhibitor gene was isolated from Populus alba var.pyramidalis by RT-PCR.The gene was expressed in E.coli as recombinant protein and its product was shown to have inhibitor activity to trypsin in vitro.No evident affinity was found between the recombinant protein and bovine trypsin.Western blotting analysis showed that the recombinant protein could strongly recognize the antibody specifically for PtTI2 from trembling aspen.
- trypsin inhibitor(TI)gene,
- Populus alba var.pyramidalis,
- wound inducement

References

[1]	Ryan C A. Systemic response to wounding[A]. In: Denno R, McClure M. Variable Plants and Herbivores in Natural and ManagedSystems[M]. Academic Press, New York, 1983: 43 ～ 60
[2]	Ryan C A. Proteinase inhibitor gene families: Strategies for transformation to imp rove p lant defenses against herbivores [J]. BioEssays, 1989, 10: 20 ～ 24
[3]	Ryan C A. Protease inhibitors in p lants: genes for imp roving defenses against insects and pathogens [J]. Annu Rev Phytopathol,1990, 28: 425 ～ 449
[4]	Lipke H, Fraenkel G S, Liener I. Effect of soybean inhibitors ongrowth of Tribolium confusum [J]. Agric Food Chem, 1954, 2: 410～ 414
[5]	Peng L H, Black L L. Increased p roteinase inhibitor activity in response to infection of resistant tomato p lants by Phytophthora infestans[J]. Phytopathol, 1976, 66: 958 ～ 963
[6]	Geoffroy P, LegrandM, FritigB. Isolation and characterization of ap roteinaceous inhibitor of microbial p roteinases induced during thehypersensitive reaction of tobacco to tobacco mosaic virus [J]. MolPlant2Microbe In, 1990, 3: 327 ～ 333
[7]	Pautot V, Holzer FM, Walling L L. Differential exp ression of tomato p roteinase inhibitor Ⅰand Ⅱ genes during bacterial pathogeninvasion and wounding [J]. Mol Plant-Microbe Interact, 1991, 4:284 ～ 292
[8]	Roby D, Toppan A, Esquerre2Tugaye M T. Cell surface in p lantmicroorganism interactions. Ⅷ. Increased p roteinase inhibitor activity inmelon p lants in response to infection by Colletotrichum lagenarium or to treatment with an elicitor fraction from this fungus [J].PhysioMol Plant Pathol, 1987, 30: 453 ～ 460
[9]	Hilder V A, Gatehouse A M R, Sheerman S E, et al. A novelmechanism of insect resistance engineered into tobacco [J]. Nature, 1987, 330: 160 ～ 163
[10]	Bradshaw H D J r, Hollick J B, Parsons T J, et al. Systemicallywound2responsive genes in pop lar trees encode p roteins similar tosweet potato sporamins and legume Kunitz tryp sin inhibitors [J].PlantMol Biol, 1989, 14: 51 ～ 59
[11]	Parsons T J, Bradshaw H D J r, Gordon M P. Systemic accumulation of specific mRNAs in respone to wounding in pop lar trees [J].Proc Natl Acad Sci, USA, 1989, 86: 7895 ～ 7899
[12]	Hollick J B, Gordan M P. A pop lar tree p roteinase inhibitor-likegene p romoter is responsive to wounding in transgenic tobacco [J].PlantMol Biol, 1993, 22: 561 ～ 572
[13]	HarutaM, Major I T, ChristopherM E, et al. A Kunitz tryp sin inhibitor gene family from trembling aspen (Populus trem uloidesMichx.) : cloning, functional exp ression, and induction by wounding and herbivory [J]. PlantMol Biol, 2001, 46: 347 ～ 359
[14]	Nishiguchi M. A genomic DNA clone encoding tryp sin inhibitorhomolog [D].
[15]	秦锡祥, 高瑞桐, 曹桂洪, 等. 抗光肩天牛杨树品种的筛选研究[J]. 林业科学研究, 1993, 6 (专刊) : 42 ～ 45
[16]	Larson P R, Isebrands J G. The p lastochron index as app lied to developmental studies of cottonwood [J]. Can J Forest Res, 1971,1: 1 ～ 11
[17]	MurrayM G, Thomp son W F. Rap id isolation of high molecularweight p lant DNA [J]. Nucl Acids Res, 1980, 8: 4321 ～ 4325
[18]	Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A LaboratoryManual [M]. Cold Sp ring Harbor Press, 1989
[19]	Towbin H. Electrophoretic transfer of p rotein from polyacrylamidegels to nitrocellulose sheets: p rocedure and some app lication [J].Proc Natl Acad Sci, USA, 1979, 76: 4350 ～ 4354
[20]	方敏,黄华樑. 包涵体蛋白质体外复性的研究进展[J]. 生物工程学报, 2001, 17: 608 ～ 612
[21]	Bradford M. A rap id and sensitive method for the quantitation ofmicrogram quantities of p rotein utilizing the p rincip les of p rotein-dyebinding [J]. Anal. Biochem, 1976, 71: 248 ～ 254
[22]	Kim S H, Hara S, Hase S, et al. Comparative study on amino acidsequences of Kunitz-type soybean tryp sin inhibitors, Tia, Tib, andTic1 [J]. J Biochem, 1985, 98: 435 ～ 448
[23]	Richardon M. Seed storage p roteins: the enzyme inhibitors [J].Meth Plant Biochem, 1991, 5: 259 ～ 305
[24]	赵天锡,陈章水. 中国杨树集约栽培[M]. 北京:中国科学技术出版社, 1994: 269 ～ 307
[25]	Yeh KW, Chen J C, Lin M I, et al. Functional activity of sporamin from sweet potato (Ipom oea batatas Lam.) : a tuber storagep rotein with tryp sin inhibitor activity [J]. PlantMol Biol, 1997,33: 565 ～ 570
[26]	ConfalonieriM, Allegro G, Balestrazzi A, et al. Regeneration ofPopulus nigra transgenic p lants exp ression a Kunitz p roteinase inhibitor (KTi3) gene [J]. Mol Breed, 1998, 4: 137～145

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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The Cloning and Functional Expression of a Kunitz Trypsin Inhibitor Gene from Populus alba var.pyramidalis

1. Plant Pathology Department, China Agricultural University, Beijing 100094, China

2. Research Institute of Forest Ecology, Environment and Protection, CAF, Key Laboratory of Forestry Protection, State Forestry Administration, Beijing 100091, China

Received Date: 2005-06-07

Abstract: Wounding of poplar trees leads to the accumulation of several mRNA species that encode proteins with putative defensive function.A Kuntiz trypsin inhibitor gene was isolated from Populus alba var.pyramidalis by RT-PCR.The gene was expressed in E.coli as recombinant protein and its product was shown to have inhibitor activity to trypsin in vitro.No evident affinity was found between the recombinant protein and bovine trypsin.Western blotting analysis showed that the recombinant protein could strongly recognize the antibody specifically for PtTI2 from trembling aspen.

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Reference (26)

[1]	Ryan C A. Systemic response to wounding[A]. In: Denno R, McClure M. Variable Plants and Herbivores in Natural and ManagedSystems[M]. Academic Press, New York, 1983: 43 ～ 60
[2]	Ryan C A. Proteinase inhibitor gene families: Strategies for transformation to imp rove p lant defenses against herbivores [J]. BioEssays, 1989, 10: 20 ～ 24
[3]	Ryan C A. Protease inhibitors in p lants: genes for imp roving defenses against insects and pathogens [J]. Annu Rev Phytopathol,1990, 28: 425 ～ 449
[4]	Lipke H, Fraenkel G S, Liener I. Effect of soybean inhibitors ongrowth of Tribolium confusum [J]. Agric Food Chem, 1954, 2: 410～ 414
[5]	Peng L H, Black L L. Increased p roteinase inhibitor activity in response to infection of resistant tomato p lants by Phytophthora infestans[J]. Phytopathol, 1976, 66: 958 ～ 963
[6]	Geoffroy P, LegrandM, FritigB. Isolation and characterization of ap roteinaceous inhibitor of microbial p roteinases induced during thehypersensitive reaction of tobacco to tobacco mosaic virus [J]. MolPlant2Microbe In, 1990, 3: 327 ～ 333
[7]	Pautot V, Holzer FM, Walling L L. Differential exp ression of tomato p roteinase inhibitor Ⅰand Ⅱ genes during bacterial pathogeninvasion and wounding [J]. Mol Plant-Microbe Interact, 1991, 4:284 ～ 292
[8]	Roby D, Toppan A, Esquerre2Tugaye M T. Cell surface in p lantmicroorganism interactions. Ⅷ. Increased p roteinase inhibitor activity inmelon p lants in response to infection by Colletotrichum lagenarium or to treatment with an elicitor fraction from this fungus [J].PhysioMol Plant Pathol, 1987, 30: 453 ～ 460
[9]	Hilder V A, Gatehouse A M R, Sheerman S E, et al. A novelmechanism of insect resistance engineered into tobacco [J]. Nature, 1987, 330: 160 ～ 163
[10]	Bradshaw H D J r, Hollick J B, Parsons T J, et al. Systemicallywound2responsive genes in pop lar trees encode p roteins similar tosweet potato sporamins and legume Kunitz tryp sin inhibitors [J].PlantMol Biol, 1989, 14: 51 ～ 59
[11]	Parsons T J, Bradshaw H D J r, Gordon M P. Systemic accumulation of specific mRNAs in respone to wounding in pop lar trees [J].Proc Natl Acad Sci, USA, 1989, 86: 7895 ～ 7899
[12]	Hollick J B, Gordan M P. A pop lar tree p roteinase inhibitor-likegene p romoter is responsive to wounding in transgenic tobacco [J].PlantMol Biol, 1993, 22: 561 ～ 572
[13]	HarutaM, Major I T, ChristopherM E, et al. A Kunitz tryp sin inhibitor gene family from trembling aspen (Populus trem uloidesMichx.) : cloning, functional exp ression, and induction by wounding and herbivory [J]. PlantMol Biol, 2001, 46: 347 ～ 359
[14]	Nishiguchi M. A genomic DNA clone encoding tryp sin inhibitorhomolog [D].
[15]	秦锡祥, 高瑞桐, 曹桂洪, 等. 抗光肩天牛杨树品种的筛选研究[J]. 林业科学研究, 1993, 6 (专刊) : 42 ～ 45
[16]	Larson P R, Isebrands J G. The p lastochron index as app lied to developmental studies of cottonwood [J]. Can J Forest Res, 1971,1: 1 ～ 11
[17]	MurrayM G, Thomp son W F. Rap id isolation of high molecularweight p lant DNA [J]. Nucl Acids Res, 1980, 8: 4321 ～ 4325
[18]	Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A LaboratoryManual [M]. Cold Sp ring Harbor Press, 1989
[19]	Towbin H. Electrophoretic transfer of p rotein from polyacrylamidegels to nitrocellulose sheets: p rocedure and some app lication [J].Proc Natl Acad Sci, USA, 1979, 76: 4350 ～ 4354
[20]	方敏,黄华樑. 包涵体蛋白质体外复性的研究进展[J]. 生物工程学报, 2001, 17: 608 ～ 612
[21]	Bradford M. A rap id and sensitive method for the quantitation ofmicrogram quantities of p rotein utilizing the p rincip les of p rotein-dyebinding [J]. Anal. Biochem, 1976, 71: 248 ～ 254
[22]	Kim S H, Hara S, Hase S, et al. Comparative study on amino acidsequences of Kunitz-type soybean tryp sin inhibitors, Tia, Tib, andTic1 [J]. J Biochem, 1985, 98: 435 ～ 448
[23]	Richardon M. Seed storage p roteins: the enzyme inhibitors [J].Meth Plant Biochem, 1991, 5: 259 ～ 305
[24]	赵天锡,陈章水. 中国杨树集约栽培[M]. 北京:中国科学技术出版社, 1994: 269 ～ 307
[25]	Yeh KW, Chen J C, Lin M I, et al. Functional activity of sporamin from sweet potato (Ipom oea batatas Lam.) : a tuber storagep rotein with tryp sin inhibitor activity [J]. PlantMol Biol, 1997,33: 565 ～ 570
[26]	ConfalonieriM, Allegro G, Balestrazzi A, et al. Regeneration ofPopulus nigra transgenic p lants exp ression a Kunitz p roteinase inhibitor (KTi3) gene [J]. Mol Breed, 1998, 4: 137～145

The Cloning and Functional Expression of a Kunitz Trypsin Inhibitor Gene from Populus alba var.pyramidalis

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通讯作者: 陈斌, bchen63@163.com

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The Cloning and Functional Expression of a Kunitz Trypsin Inhibitor Gene from Populus alba var.pyramidalis

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