Clon ing of PAL Gene from Sophora japon ica, Construction of Anti2Sense Geneof SjPAL and Its Genetic Tran sforma tion in A rabidopsis

XU Feng; ZHU Jun; ZHANG Feng-xia; WANG Yan; CHENG Shui-yuan; CHENG Shu-han

Volume 21 Issue 5

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Clon ing of PAL Gene from Sophora japon ica, Construction of Anti2Sense Geneof SjPAL and Its Genetic Tran sforma tion in A rabidopsis

1.
College of Horticulture and Gardening, Yangtze University, J ingzhou　434025, Hubei, China
2.
College of Horticulture Science and Engineering, Shandong Agricultural University, Taipan　271018, Shandong, China
3.
College of Life Science and Engineering, Huanggang Normal University, Huanggang　438000, Hubei, China

Received Date: 2007-12-23

Abstract

A PAL gene fragment with 866 bp length ( named as SjPAL ) was cloned from Sophora japonica by RTP-CR and using a pair of degenerated p rimers, which were designed basing on the sequence of other p lant PAL genesconserved region. The deduced SjPAL polypep tide showed high identities ( > 87% ) to other p lant PAL amino acidsvia sequence analysis, and contained the similar active sites in PAL p roteins of O ryza sativa and A rabidopsis.Phylogenetic tree analysis indicated that the SjPAL had a closer relationship with PAL p roteins from leguminous p lantspecies. RTP-CR analysis revealed that the relative abundance of SjPAL mRNA in root and stem was about threetimes as in leaf. Utilizing anti-sense RNA technology, SjPAL gene was inserted directionally into pB I121, a p lantexp ression vector, to construct an anti-sense fusion gene and a p lant exp ression vector pB I121-PAL. Genetictransformation to A rabidopsis was mediated by EHA105. Transgenic A rabidopsis lines were performed using PCR,Northern blot, PAL activity of per unit material, and total polyphenolic and flavonoid concentration analysis. Theresults showed that the exp ression level of PAL gene, PAL activities of per unitmaterial, and total polyphenolic andflavonoid concentration of transformed lines were all significantly lower than the wild control. The p resent studiesp rovided an experimental basis for further genetic transformation in S. japonica of SjPAL gene anti-sense exp ressionvector to imp rove its rooting ability in regeneration system via regulating its phenolic compound content.
- Sophora japonica,
- rooting ability,
- PAL gene,
- anti-sense exp ression vector,
- genetic transformation,
- totalpolyphenolic,
- flavonoid

References

[1]	张晓英,王华芳,朱　祯,等. 国槐离体再生及抗虫基因sck的转导[J]. 林业科学, 2006, 42 (9) : 34 - 38
[2]	陈箐筠,干　信. 槐米中斛皮素的提取及对中枢神经系统作用的研究进展[J]. 化学与生物工程, 2007, 24 (3) : 11 - 13
[3]	Qi Y, Sun A, Liu R, et al. Isolation and purification of flavonoidand isoflavonoid compounds from the pericarp of Sophora japonica L.by adsorp tion chromatography on 12% cross-linked agarose gelmedia[J]. J ChromatogrA, 2007, 1140 (1) : 219 - 224
[4]	孟庆杰,王光全,孟庆军,等. 蝴蝶槐的组织培养和快速繁殖技术[J]. 林业科技, 2006, 31 (3) : 63 - 64
[5]	Zhang X, Wang H, YinW, et al. Agrobacterium tum efaciens-mediated GUS gene transfer to Sophora japonica L. [J] Forestry Studies inChina, 2006, 8 (4) : 6 - 9
[6]	孙书伟. 影响香花槐生根的几个因素[J]. 甘肃农业, 2006(1) : 206
[7]	Davis T D, Haissig B E: A historical evaluation of adventitious rooting research to 1993 [M] / /Davis TD, HaissigB E. Biology ofAdventitious Root Formation. Basic Life Science, vol 62, New York:Plenum Press, 1994: 274 - 335
[8]	Euch C E, Jay-Allemand C, PastugliaM, et al. Exp ression of antisense chalcone synthase RNA in transgenic hybrid walnut microcuttings. Effect on flavonoid content and rooting ability [J]. PlantMolBiol, 1998, 38 (3) : 467 - 479
[9]	Trobec M, Ltampar F, Veberiˇc R, et al. Fluctuations of differentendogenous phenolic compounds and cinnamic acid in the first daysof the rooting p rocess of cherry rootstock‘GiSelA 5’leafy cuttings[J]. J Plant Physiol, 2005, 162 (5) : 589 - 597
[10]	van Doorsselaere J, BaucherM, Chognot E, et al. A novel ligninin Pop lar treeswith a reduced caffeic acid /5-hydroxyferulic acid Omethyltransferase activity [J]. Plant J, 1995, 8 (1) : 855 - 864
[11]	van der Krol A R, Lenting P E, Veenstra J, et al. An antisensechalcone synthase gene in transgenic p lants inhibits flower p igmentation [J]. Nature, 1988, 333 (6179) : 866 - 869
[12]	Elomaa P, Honkanen J, Pusk R, et al. Agrobacterium-mediatedtransfer of antisense chalcone synthase cDNA to Gerbera hybrida inhibits flower p igmentation [J]. Bio /Technology, 1993, 11 ( 2 ) :508 - 511
[13]	Bachem CW B, Speckmann G J, van derLinde P C G, et al. Antisense exp ression of polyphenol oxidase genes inhibits enzymaticbrowning in potato tubers [J]. Bio /Technology, 1994, 12 ( 2 ) :1101 - 1105
[14]	Blee K, Choi JW, OpConnellA P, et al. Antisense and sense exp ression of cDNA coding for CYP73A15, a class II cinnamate 4hydroxylase, leads to a delayed and reduced p roduction of lignin intobacco [J]. Phytochemistry, 2001, 57 (7) : 1159 - 1166
[15]	程水源,陈昆松,刘卫红,等. 植物苯丙氨酸解氨酶( PAL)基因的表达调控与研究展望[J]. 果树学报, 2003, 20 ( 5 ) :351 - 357
[16]	Hahlbrock K, Scheel D. Physiology and molecular biology of phenylp ropanoid metabolism [J]. Annu Rev Plant Physiol PlantMolBiol, 1989, 40 (6) : 347 - 369
[17]	Holton T A, Cornish E. Genetics and biochemistry of anthocyaninbiosynthesis[J]. Plant Cell, 1995, 7 (9) : 1071 - 1083
[18]	Ritter H, Schulz G E. Structural basis for the entrance into thephenylp ropanoid metabolism catalyzed by phenylalanine ammonialyase [J]. Plant Cell, 2004, 16 (2) : 3426 - 3436
[19]	Butland S L, ChowM L, EllisB E. A diverse family of phenylalanine ammonia-lyase genes exp ressed in p ine trees and cell cultures[J]. PlantMol Biol, 1998, 37 (1) : 15 - 24
[20]	程水源,杜何为,许　锋,等. 银杏苯丙氨酸解氨酶基因的克隆和序列分析[J]. 林业科学研究, 2005, 18 (5) : 573 - 577
[21]	程水源,陈昆松,杜何为,等. 银杏RNA的提取[J]. 果树学报,2005, 22 (4) : 428 - 429
[22]	Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A LaboratoryManual [M]. Cold Sp ring Harbor Laboratory Press, ColdSp ring Harbor, 1989
[23]	Clough S J, BentA F. Floral dip: a simp lifiedmethod forAgrobacterium-mediated transformation of A rabidopsis thaliana [J]. PlantJ, 1998, 16 (6) : 735 - 743
[24]	Koukol J, Conn E E. The metabolism of aromatic compounds inhigher p lants. Purification and p roperties of the L-phenylalanine deaminase of Herdeum vulagare [J]. J Biol Chem, 1961, 236 (10) :2692 - 2698
[25]	Singleton V L, Rossi J r J A. Colorimetry of total phenolics withPhosphomolybdic-phosphotungstic acid reagents [J]. Am J EnolViticult, 1965, 16 (3) : 144 - 158
[26]	程水源,王　燕,李俊凯,等. 银杏叶黄酮含量变化及分布规律的研究[J]. 园艺学报, 2001, 28 (4) : 353 - 355
[27]	Wanner L A, Li G, Ware D, et al. The phenylalanine ammonialyase gene family in A rabidopsis thaliana [J]. Plant Mol Biol,1995, 27 (2) : 327 - 338
[28]	Zhu Q, Dabi T, Beeche A, et al. Cloning and p roperties of a ricegene encoding phenylalanine ammonia-lyase [J]. PlantMol Biol,1995, 29 (3) : 535 - 550
[29]	Kervinen T, Peltonen S, Utriainen M, et al. Cloning and characterization of cDNA clones encoding phenylalanine ammonia-lyase inbarely [J]. Plant Sci, 1997, 123 (1) : 143 - 150
[30]	Stefanie L, Monica L, Brian E. A diverse family of phenylalanineAmmonia-lyase genes exp ressed in p ine trees and cell cultures[J].PlantMol Biol, 1998, 37 (1) : 15 - 24
[31]	Osakabe K, Kawai S, Katayama Y. Characterization of the structure and determination ofmRNA levels of the Phenylalanine ammonia-lyase gene family from Populus kitakam iensis [J]. Plant MolBiol, 1995, 28 (6) : 1133 - 1141
[32]	Kervinen T, Peltonen S, Teeri T H, et al. Differential exp ressionof phenylalanine ammonia-lyase genes in barley induced by fungalinfection or elicitors [J]. New Phytol, 1998, 139 (3) : 293 - 300
[33]	Cochrane F C, Davin L B, LewisN G. The A rabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the fourPAL isoforms [J]. Phytochemistry, 2004, 65 (11) : 1557 - 1564
[34]	Izant J G, Weintraub H. Inhibition of thymidine kinase gene exp ression by antisense RNA: A molecular app roach to genetic analysis [J]. Cell, 1984, 36 (4) : 1007 - 1015

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Clon ing of PAL Gene from Sophora japon ica, Construction of Anti2Sense Geneof SjPAL and Its Genetic Tran sforma tion in A rabidopsis

1. College of Horticulture and Gardening, Yangtze University, J ingzhou　434025, Hubei, China

2. College of Horticulture Science and Engineering, Shandong Agricultural University, Taipan　271018, Shandong, China

3. College of Life Science and Engineering, Huanggang Normal University, Huanggang　438000, Hubei, China

Received Date: 2007-12-23

Abstract: A PAL gene fragment with 866 bp length ( named as SjPAL ) was cloned from Sophora japonica by RTP-CR and using a pair of degenerated p rimers, which were designed basing on the sequence of other p lant PAL genesconserved region. The deduced SjPAL polypep tide showed high identities ( > 87% ) to other p lant PAL amino acidsvia sequence analysis, and contained the similar active sites in PAL p roteins of O ryza sativa and A rabidopsis.Phylogenetic tree analysis indicated that the SjPAL had a closer relationship with PAL p roteins from leguminous p lantspecies. RTP-CR analysis revealed that the relative abundance of SjPAL mRNA in root and stem was about threetimes as in leaf. Utilizing anti-sense RNA technology, SjPAL gene was inserted directionally into pB I121, a p lantexp ression vector, to construct an anti-sense fusion gene and a p lant exp ression vector pB I121-PAL. Genetictransformation to A rabidopsis was mediated by EHA105. Transgenic A rabidopsis lines were performed using PCR,Northern blot, PAL activity of per unit material, and total polyphenolic and flavonoid concentration analysis. Theresults showed that the exp ression level of PAL gene, PAL activities of per unitmaterial, and total polyphenolic andflavonoid concentration of transformed lines were all significantly lower than the wild control. The p resent studiesp rovided an experimental basis for further genetic transformation in S. japonica of SjPAL gene anti-sense exp ressionvector to imp rove its rooting ability in regeneration system via regulating its phenolic compound content.

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Reference (34)

[1]	张晓英,王华芳,朱　祯,等. 国槐离体再生及抗虫基因sck的转导[J]. 林业科学, 2006, 42 (9) : 34 - 38
[2]	陈箐筠,干　信. 槐米中斛皮素的提取及对中枢神经系统作用的研究进展[J]. 化学与生物工程, 2007, 24 (3) : 11 - 13
[3]	Qi Y, Sun A, Liu R, et al. Isolation and purification of flavonoidand isoflavonoid compounds from the pericarp of Sophora japonica L.by adsorp tion chromatography on 12% cross-linked agarose gelmedia[J]. J ChromatogrA, 2007, 1140 (1) : 219 - 224
[4]	孟庆杰,王光全,孟庆军,等. 蝴蝶槐的组织培养和快速繁殖技术[J]. 林业科技, 2006, 31 (3) : 63 - 64
[5]	Zhang X, Wang H, YinW, et al. Agrobacterium tum efaciens-mediated GUS gene transfer to Sophora japonica L. [J] Forestry Studies inChina, 2006, 8 (4) : 6 - 9
[6]	孙书伟. 影响香花槐生根的几个因素[J]. 甘肃农业, 2006(1) : 206
[7]	Davis T D, Haissig B E: A historical evaluation of adventitious rooting research to 1993 [M] / /Davis TD, HaissigB E. Biology ofAdventitious Root Formation. Basic Life Science, vol 62, New York:Plenum Press, 1994: 274 - 335
[8]	Euch C E, Jay-Allemand C, PastugliaM, et al. Exp ression of antisense chalcone synthase RNA in transgenic hybrid walnut microcuttings. Effect on flavonoid content and rooting ability [J]. PlantMolBiol, 1998, 38 (3) : 467 - 479
[9]	Trobec M, Ltampar F, Veberiˇc R, et al. Fluctuations of differentendogenous phenolic compounds and cinnamic acid in the first daysof the rooting p rocess of cherry rootstock‘GiSelA 5’leafy cuttings[J]. J Plant Physiol, 2005, 162 (5) : 589 - 597
[10]	van Doorsselaere J, BaucherM, Chognot E, et al. A novel ligninin Pop lar treeswith a reduced caffeic acid /5-hydroxyferulic acid Omethyltransferase activity [J]. Plant J, 1995, 8 (1) : 855 - 864
[11]	van der Krol A R, Lenting P E, Veenstra J, et al. An antisensechalcone synthase gene in transgenic p lants inhibits flower p igmentation [J]. Nature, 1988, 333 (6179) : 866 - 869
[12]	Elomaa P, Honkanen J, Pusk R, et al. Agrobacterium-mediatedtransfer of antisense chalcone synthase cDNA to Gerbera hybrida inhibits flower p igmentation [J]. Bio /Technology, 1993, 11 ( 2 ) :508 - 511
[13]	Bachem CW B, Speckmann G J, van derLinde P C G, et al. Antisense exp ression of polyphenol oxidase genes inhibits enzymaticbrowning in potato tubers [J]. Bio /Technology, 1994, 12 ( 2 ) :1101 - 1105
[14]	Blee K, Choi JW, OpConnellA P, et al. Antisense and sense exp ression of cDNA coding for CYP73A15, a class II cinnamate 4hydroxylase, leads to a delayed and reduced p roduction of lignin intobacco [J]. Phytochemistry, 2001, 57 (7) : 1159 - 1166
[15]	程水源,陈昆松,刘卫红,等. 植物苯丙氨酸解氨酶( PAL)基因的表达调控与研究展望[J]. 果树学报, 2003, 20 ( 5 ) :351 - 357
[16]	Hahlbrock K, Scheel D. Physiology and molecular biology of phenylp ropanoid metabolism [J]. Annu Rev Plant Physiol PlantMolBiol, 1989, 40 (6) : 347 - 369
[17]	Holton T A, Cornish E. Genetics and biochemistry of anthocyaninbiosynthesis[J]. Plant Cell, 1995, 7 (9) : 1071 - 1083
[18]	Ritter H, Schulz G E. Structural basis for the entrance into thephenylp ropanoid metabolism catalyzed by phenylalanine ammonialyase [J]. Plant Cell, 2004, 16 (2) : 3426 - 3436
[19]	Butland S L, ChowM L, EllisB E. A diverse family of phenylalanine ammonia-lyase genes exp ressed in p ine trees and cell cultures[J]. PlantMol Biol, 1998, 37 (1) : 15 - 24
[20]	程水源,杜何为,许　锋,等. 银杏苯丙氨酸解氨酶基因的克隆和序列分析[J]. 林业科学研究, 2005, 18 (5) : 573 - 577
[21]	程水源,陈昆松,杜何为,等. 银杏RNA的提取[J]. 果树学报,2005, 22 (4) : 428 - 429
[22]	Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A LaboratoryManual [M]. Cold Sp ring Harbor Laboratory Press, ColdSp ring Harbor, 1989
[23]	Clough S J, BentA F. Floral dip: a simp lifiedmethod forAgrobacterium-mediated transformation of A rabidopsis thaliana [J]. PlantJ, 1998, 16 (6) : 735 - 743
[24]	Koukol J, Conn E E. The metabolism of aromatic compounds inhigher p lants. Purification and p roperties of the L-phenylalanine deaminase of Herdeum vulagare [J]. J Biol Chem, 1961, 236 (10) :2692 - 2698
[25]	Singleton V L, Rossi J r J A. Colorimetry of total phenolics withPhosphomolybdic-phosphotungstic acid reagents [J]. Am J EnolViticult, 1965, 16 (3) : 144 - 158
[26]	程水源,王　燕,李俊凯,等. 银杏叶黄酮含量变化及分布规律的研究[J]. 园艺学报, 2001, 28 (4) : 353 - 355
[27]	Wanner L A, Li G, Ware D, et al. The phenylalanine ammonialyase gene family in A rabidopsis thaliana [J]. Plant Mol Biol,1995, 27 (2) : 327 - 338
[28]	Zhu Q, Dabi T, Beeche A, et al. Cloning and p roperties of a ricegene encoding phenylalanine ammonia-lyase [J]. PlantMol Biol,1995, 29 (3) : 535 - 550
[29]	Kervinen T, Peltonen S, Utriainen M, et al. Cloning and characterization of cDNA clones encoding phenylalanine ammonia-lyase inbarely [J]. Plant Sci, 1997, 123 (1) : 143 - 150
[30]	Stefanie L, Monica L, Brian E. A diverse family of phenylalanineAmmonia-lyase genes exp ressed in p ine trees and cell cultures[J].PlantMol Biol, 1998, 37 (1) : 15 - 24
[31]	Osakabe K, Kawai S, Katayama Y. Characterization of the structure and determination ofmRNA levels of the Phenylalanine ammonia-lyase gene family from Populus kitakam iensis [J]. Plant MolBiol, 1995, 28 (6) : 1133 - 1141
[32]	Kervinen T, Peltonen S, Teeri T H, et al. Differential exp ressionof phenylalanine ammonia-lyase genes in barley induced by fungalinfection or elicitors [J]. New Phytol, 1998, 139 (3) : 293 - 300
[33]	Cochrane F C, Davin L B, LewisN G. The A rabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the fourPAL isoforms [J]. Phytochemistry, 2004, 65 (11) : 1557 - 1564
[34]	Izant J G, Weintraub H. Inhibition of thymidine kinase gene exp ression by antisense RNA: A molecular app roach to genetic analysis [J]. Cell, 1984, 36 (4) : 1007 - 1015

Clon ing of PAL Gene from Sophora japon ica, Construction of Anti2Sense Geneof SjPAL and Its Genetic Tran sforma tion in A rabidopsis

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通讯作者: 陈斌, bchen63@163.com

Proportional views

Clon ing of PAL Gene from Sophora japon ica, Construction of Anti2Sense Geneof SjPAL and Its Genetic Tran sforma tion in A rabidopsis

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