国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化

许锋; 朱俊; 张风霞; 王燕; 程水源; 程述汉

国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化

1.
长江大学园艺园林学院,湖北荆州　434025
2.
山东农业大学园艺科学与工程学院,山东泰安　271018
3.
黄冈师范学院生命科学与工程学院,湖北黄冈　438000
基金项目:
教育部新世纪优秀人才支持计划(NCET-04-0746 ) , 湖北省教育厅重大项目( Z200627002 ) , 湖北省自然科学基金(2002AB094)和湖北省青年杰出人才基金(2003AB 014)

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

摘要: 根据其它植物PAL基因的保守区域,设计一对兼并引物,采用RT-PCR方法从国槐中克隆了一个长866 bp的PAL基因片段,命名为SjPAL。序列分析发现SjPAL 多肽与其它植物的PAL 氨基酸序列高度同源(87%以上) ,且包含与水稻和拟南芥的PAL类似的活性位点。系统进化树分析表明SjPAL 与豆科植物的PAL 亲缘关系较近。RTP-CR结果显示, SjPAL 在根和茎的表达量约为叶中的3倍。利用反义RNA技术将SjPAL 基因克隆至植物表达载体pB I121,构建了SjPAL反义RNA植物表达载体pB I121-PAL ,通过根癌农杆菌EHA105将其导入拟南芥基因组,对获得的抗性植株进行PCR鉴定、Northern杂交分析、PAL 活性分析以及总多酚含量和类黄酮含量分析。结果表明,该反义RNA已整合到拟南芥基因组中,转基因拟南芥的PAL 基因表达量、单位材料PAL 活性、总多酚含量和类黄酮含量均显著低于野生型对照。本研究为下一步利用该基因反义表达载体转化国槐,通过调节酚类物质含量提高其在再生体系中的生根能力提供了试验依据。
- 国槐
- / 生根能力
- / PAL 基因
- / 反义表达载体
- / 遗传转化
- / 总多酚
- / 类黄酮
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

[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

[1]	汪阳东 , 李春秀 , 齐力旺 , 张守攻 . 中间锦鸡儿fad2基因片段克隆、反义表达载体构建及遗传转化初步研究. 林业科学研究, 2007, 20(1): 6-9.
[2]	刘明靖 , 李纪元 , 范正琪 , 田敏 , 范妙华 . 根癌农杆菌介导的反义VeFAD2基因对少根根霉的遗传转化及其影响因子. 林业科学研究, 2010, 23(4): 592-596.
[3]	余义勋 , 包满珠 , 孙振元 . 香石竹ACC氧化酶重复基因植物表达载体的构建及转基因植株的获得. 林业科学研究, 2004, 17(1): 1-5.
[4]	刘会超 , 刘孟刚 , 郭丽娟 , 贾文庆 . 农杆菌介导的SeNHX1基因转化月季愈伤组织的研究. 林业科学研究, 2010, 23(4): 617-621.
[5]	沈植国 , 程建明 , 武方方 , 丁鑫 . 不同花色类型蜡梅花被片靶向类黄酮代谢组学分析. 林业科学研究, 2023, 36(4): 82-89. doi: 10.12403/j.1001-1498.20220537
[6]	张川红 , 沈应柏 , 尹伟伦 . 盐胁迫对国槐和核桃幼苗光合作用的影响. 林业科学研究, 2002, 15(1): 34-40.
[7]	刘文汝 , 冀胜鑫 , 甄志先 . 感染国槐带化病植株内源激素及转录组分析. 林业科学研究, 2022, 35(1): 141-149. doi: 10.13275/j.cnki.lykxyj.2022.01.016
[8]	赵策 , 邱尔发 , 马俊丽 , 黄帅帅 . 行道树国槐不同形态重金属富集效能研究. 林业科学研究, 2019, 32(3): 142-151. doi: 10.13275/j.cnki.lykxyj.2019.03.019
[9]	汪跃 , 金开璇 . 国槐带化病中发现类菌原体(MLO). 林业科学研究, 1992, 5(1): 109-110.
[10]	张冰玉 , 苏晓华 , 李义良 , 张永安 , 曲良建 , 王玉珠 , 田颖川 . 转双价抗蛀干害虫基因杨树的获得及其抗虫性鉴定. 林业科学研究, 2005, 18(3): 364-368.
[11]	林荣呈 , 陈龙清 , 包满珠 , 孙振元 . 提高根癌农杆菌介导的香石竹遗传转化效率的研究. 林业科学研究, 2003, 16(2): 123-128.
[12]	王民炎 , 俞文仙 , 赵耘霄 , 陈益存 , 高暝 , 吴立文 , 吴善群 , 汪阳东 . 农杆菌介导的山苍子遗传转化体系的构建. 林业科学研究, 2022, 35(5): 71-80. doi: 10.13275/j.cnki.lykxyj.2022.005.008
[13]	张俊卫 , 包满珠 , 孙振元 . 草坪草的遗传转化研究进展. 林业科学研究, 2003, 16(1): 87-94.
[14]	李辛雷 , 王佳童 , 孙振元 , 王洁 , 殷恒福 , 范正琪 , 李纪元 . 金花茶花朵和叶片UPLC-QTOF-MS分析. 林业科学研究, 2018, 31(6): 83-88. doi: 10.13275/j.cnki.lykxyj.2018.06.012
[15]	耿涌杭 , 许新桥 , 倪建伟 , 苏上 . 不同采收时期对香椿饲用品质的影响. 林业科学研究, 2019, 32(2): 145-151. doi: 10.13275/j.cnki.lykxyj.2019.02.021
[16]	范正琪 , 卢孟柱 , 李纪元 , 田敏 , 李辛雷 , 吴开云 , 陈东亮 , 范妙华 . 麻疯树pepc基因正义、反义植物表达载体构建与功能初步分析. 林业科学研究, 2011, 24(2): 165-170.
[17]	谢丽华 , 蒋晶 , 刘明英 , 乔桂荣 , 邱文敏 , 杨惠琴 , 卓仁英 . ptc-miR213的人工microRNA植物表达载体的构建及遗传转化. 林业科学研究, 2013, 26(1): 29-33.
[18]	吴晓娟 , 鲁俊倩 , 常英英 , 钟姗辰 , 苏晓华 , 张冰玉 . AtMET1基因在84K杨中的遗传转化及诱导表达分析. 林业科学研究, 2020, 33(3): 63-69. doi: 10.13275/j.cnki.lykxyj.2020.03.008
[19]	叶小真 , 杨婕 , 冯丽贞 , 江仲鹏 , 陆芝 , 刘雨菁 , 李丽红 , 陈全助 . 桉树PAL基因克隆及焦枯病菌诱导下的表达分析. 林业科学研究, 2019, 32(6): 99-105. doi: 10.13275/j.cnki.lykxyj.2019.06.013
[20]	魏艳秀 , 刘攀峰 , 杜庆鑫 , 杜红岩 . 不同种质杜仲叶中多酚和黄酮含量差异性分析. 林业科学研究, 2016, 29(4): 529-535.

点击查看大图

计量

文章访问数: 3112
HTML全文浏览量: 148
PDF下载量: 1156
被引次数: 0

姓名
邮箱
手机号码
标题
留言内容
验证码

留言板

国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化

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

计量

国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化

English Abstract

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

全文HTML

目录

留言板

国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化

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

计量

出版历程

国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化

English Abstract

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

全文HTML

目录