大豆C4H基因克隆及生物信息学分析
摘要
大豆(Glycine max (L.) Merr.)是我国重要的粮食和油料作物,含有丰富的蛋白质和脂肪,综合利用价值高,而因为重迎茬问题引起了严重的连作障碍,导致大豆抗逆性降低,产量大幅度下降。近年来,研究表明次生代谢产物的化感效应也是引起连作障碍的重要原因之一,同时次生代谢已成为植物分子生物学研究的热点之一。苯丙烷代谢途径是植物最重要的次生代谢途径之一。肉桂酸-4-羟基化酶(C4H,EC 1.14.13.11),又称反式肉桂酸-4-单氧化酶,催化肉桂酸羟化作用产生4-香豆酸盐,是苯丙烷途径中继L-苯丙氨酸解氨酶(PAL)之后的第二个关键酶。为了从分子水平上揭示大豆C4H的结构特点,并为提高其表达活性提供理论依据,本研究以大豆绥农14为材料,利用RT-PCR技术进行大豆肉桂酸-4-羟基化酶基因mRNA(C4H mRNA);利用PCR技术进行大豆肉桂酸-4-羟基化酶基因组基因(C4H DNA)的分离克隆并结合RACE技术进行C4H基因3’末端基因的克隆。在此基础上,利用生物信息学手段对C4H mRNA推导出的蛋白质序列进行主要结构分析和功能预测。通过上述研究得到以下结果:
利用RT-PCR方法成功地从大豆叶片中克隆了肉桂酸-4-羟基化酶基因mRNA(C4H mRNA)(登录号为FJ770468)。获得的C4H mRNA基因长度为1766bp,编码区1521bp,与已公布的C4H mRNA序列(登录号为X92437)的编码区相似性达98.77%,两者只有1个碱基的差异(位于1237位的碱基),并与其它植物的该基因序列具有同源性,应用NCBI上的Blast X工具将C4H cDNA的测序结果推导出的蛋白质序列与已知的C4H蛋白质序列(登录号为Q42797)比较,相似性达99.80%,两者只有1个氨基酸的差异(位于396位的氨基酸),编码一个含有506个氨基酸残基的多肽。
运用PCR扩增技术首次克隆了大豆肉桂酸-4-羟基化酶基因组基因C4H DNA(登录号为HM036117)。测序结果表明,C4H DNA含有1个外显子,无内含子,外显子序列与获得的C4H mRNA序列完全一致。
结合RACE技术克隆了大豆C4H基因3’末端序列,并将登录号为FJ770468的大豆C4H基因mRNA升级到FJ770468 updata,长度为1799bp。
用ExPasy软件包和TOPPRED在线分析C4H的理化特性,结果表明C4H的理论等电点PI=5.30,Mw=39092;C4H是易溶、亲水性较强的蛋白,同时有两个明显的疏水峰,有2个跨膜肽段;并运用DNAMAN6.0软件构建了C4H的系统进化树。通过HNN分析二级结构预测结果显示,C4H的α螺旋(Alpha helix)(Hh):251,49.60%,β折叠(Extended strand)(Ee):52,10.28%,无规卷曲(Random coil)(Cc):203,40.12%;Geno3d模建预测,C4H蛋白中β折叠区有57个折叠,折叠区间有24个α螺旋,此外还有14个卷曲结构。氨基酸序列和结构分析显示C4H蛋白包含了一个保守区,即P450 domain。利用SignalP分析得到信号肽存在几率、信号肽锚定几率均为0.498,切割位点位于28和29位氨基酸之间。
The soybean (Glycine max (L.) Merr.) is an significant foodstuff and oil crop in our country, it contains full protein and fat, the valve of multiple utilization is high. The antireversion force and the yield of soybean decrease greatly because of the severe continuous cropping. For the past few years, we learned about the allelopathy of secondary metabolite is one important reason of continuous cropping obstacle from study and research. And the study of secondary metabolism have become one hot spot of molecular bioresearch in plants. Phenylpropanoid metabolic pathway is one of the most important secondary metabolism pathway in plants.trans-cinnamic acid-4-hydroxylase (C4H, EC 1.14.13.11), another name is trans-cinnamic acid-4- mono-oxygenase, it catalyzes trans-cinnamic acid into 4-coumaric acid salt by hydroxylation. It is the second key enzyme in phenylpropanoid metabolic pathway after L- phenylalnine ammonialyase (PAL). In order to reveal the construction features of soybean C4H in the molecular level and provide a theoretical basis of increasing the activity of its expression. Test materials is SN14 in this study, the soybean trans-cinnamic acid-4-hydroxylase mRNA(C4H mRNA)and genome DNA (C4H DNAA) was cloned by RT-PCR and PCR, respectively. In addition, 3’termination sequence of Soybean C4H was cloned by RACE technique. The main structure and function of protein which is translated by C4H mRNA was predicted by bioinformatics analysis. The results were as follows:
The trans-cinnamic acid-4- hydroxylase mRNA (C4H mRNA)(Accession Number: FJ770468) was cloned successfully from soybean leaves by RT-PCR. The acquired sequence is 1766bp in length and the sequence in coding region of C4H is 1521bp. Compare with the coding region of C4H mRNA announced(Accession Number: X92437), the resemblance is 98.77%, just one basic group is different(situate 1237position ), the homology of this gene order exists in other plants, the protein translated by the sequencing result compares with the foregone protein (Accession Number: Q42797), the resemblance is 99.80%, just one amino acids is different(situate 396 position ), a polypeptide was encoded by 506 amino acid residues.
The soybean trans-cinnamic acid-4- hydroxylase genome DNA (C4H DNA) (Accession Number: HM036117). The sequencing result shows that C4H DNA contain one extron and no intron, the sequence of extron and C4H mRNA acquired are the same, completely.
3’termination sequence of Soybean C4H was cloned by RACE technique, and updata information of FJ770468, the new sequence is 1799bp in length.
The physical chemistry characteristics of C4H were analyzed on-line by ExPasy and TOPPRED, it shows that theory value of C4H PI and Mw is 5.30 and 39092; respectively; C4H is a soluble, hydrophilic protein, while there are two significant hydrophobic peaks and two transmembrane peptide by TOPPRED, the phyletic evolution tree was constructed by DNAMAN6.0. The secondary structure of C4H various amino acid residues were predicted by HNN, respectively, Alpha helix: 251 is 49.60%, Extended strand: 52 is 10.28%, Random coil: 203 is 40.12%; 3D structure was predicted by Geno3d,it shows that 57 extended strands contained 24 alpha helices inβ-folding area and 14 random coils. According to the amino acid sequence and structural analysis, it shows that C4H protein contains one conserved domain, viz. P450 domain. The existing probability and the anchoring probability of signal peptide were both 0.498 which were analyzed by SignalP, the cleavage site was between 28 and 29 position amino acids.
利用RT-PCR方法成功地从大豆叶片中克隆了肉桂酸-4-羟基化酶基因mRNA(C4H mRNA)(登录号为FJ770468)。获得的C4H mRNA基因长度为1766bp,编码区1521bp,与已公布的C4H mRNA序列(登录号为X92437)的编码区相似性达98.77%,两者只有1个碱基的差异(位于1237位的碱基),并与其它植物的该基因序列具有同源性,应用NCBI上的Blast X工具将C4H cDNA的测序结果推导出的蛋白质序列与已知的C4H蛋白质序列(登录号为Q42797)比较,相似性达99.80%,两者只有1个氨基酸的差异(位于396位的氨基酸),编码一个含有506个氨基酸残基的多肽。
运用PCR扩增技术首次克隆了大豆肉桂酸-4-羟基化酶基因组基因C4H DNA(登录号为HM036117)。测序结果表明,C4H DNA含有1个外显子,无内含子,外显子序列与获得的C4H mRNA序列完全一致。
结合RACE技术克隆了大豆C4H基因3’末端序列,并将登录号为FJ770468的大豆C4H基因mRNA升级到FJ770468 updata,长度为1799bp。
用ExPasy软件包和TOPPRED在线分析C4H的理化特性,结果表明C4H的理论等电点PI=5.30,Mw=39092;C4H是易溶、亲水性较强的蛋白,同时有两个明显的疏水峰,有2个跨膜肽段;并运用DNAMAN6.0软件构建了C4H的系统进化树。通过HNN分析二级结构预测结果显示,C4H的α螺旋(Alpha helix)(Hh):251,49.60%,β折叠(Extended strand)(Ee):52,10.28%,无规卷曲(Random coil)(Cc):203,40.12%;Geno3d模建预测,C4H蛋白中β折叠区有57个折叠,折叠区间有24个α螺旋,此外还有14个卷曲结构。氨基酸序列和结构分析显示C4H蛋白包含了一个保守区,即P450 domain。利用SignalP分析得到信号肽存在几率、信号肽锚定几率均为0.498,切割位点位于28和29位氨基酸之间。
The soybean (Glycine max (L.) Merr.) is an significant foodstuff and oil crop in our country, it contains full protein and fat, the valve of multiple utilization is high. The antireversion force and the yield of soybean decrease greatly because of the severe continuous cropping. For the past few years, we learned about the allelopathy of secondary metabolite is one important reason of continuous cropping obstacle from study and research. And the study of secondary metabolism have become one hot spot of molecular bioresearch in plants. Phenylpropanoid metabolic pathway is one of the most important secondary metabolism pathway in plants.trans-cinnamic acid-4-hydroxylase (C4H, EC 1.14.13.11), another name is trans-cinnamic acid-4- mono-oxygenase, it catalyzes trans-cinnamic acid into 4-coumaric acid salt by hydroxylation. It is the second key enzyme in phenylpropanoid metabolic pathway after L- phenylalnine ammonialyase (PAL). In order to reveal the construction features of soybean C4H in the molecular level and provide a theoretical basis of increasing the activity of its expression. Test materials is SN14 in this study, the soybean trans-cinnamic acid-4-hydroxylase mRNA(C4H mRNA)and genome DNA (C4H DNAA) was cloned by RT-PCR and PCR, respectively. In addition, 3’termination sequence of Soybean C4H was cloned by RACE technique. The main structure and function of protein which is translated by C4H mRNA was predicted by bioinformatics analysis. The results were as follows:
The trans-cinnamic acid-4- hydroxylase mRNA (C4H mRNA)(Accession Number: FJ770468) was cloned successfully from soybean leaves by RT-PCR. The acquired sequence is 1766bp in length and the sequence in coding region of C4H is 1521bp. Compare with the coding region of C4H mRNA announced(Accession Number: X92437), the resemblance is 98.77%, just one basic group is different(situate 1237position ), the homology of this gene order exists in other plants, the protein translated by the sequencing result compares with the foregone protein (Accession Number: Q42797), the resemblance is 99.80%, just one amino acids is different(situate 396 position ), a polypeptide was encoded by 506 amino acid residues.
The soybean trans-cinnamic acid-4- hydroxylase genome DNA (C4H DNA) (Accession Number: HM036117). The sequencing result shows that C4H DNA contain one extron and no intron, the sequence of extron and C4H mRNA acquired are the same, completely.
3’termination sequence of Soybean C4H was cloned by RACE technique, and updata information of FJ770468, the new sequence is 1799bp in length.
The physical chemistry characteristics of C4H were analyzed on-line by ExPasy and TOPPRED, it shows that theory value of C4H PI and Mw is 5.30 and 39092; respectively; C4H is a soluble, hydrophilic protein, while there are two significant hydrophobic peaks and two transmembrane peptide by TOPPRED, the phyletic evolution tree was constructed by DNAMAN6.0. The secondary structure of C4H various amino acid residues were predicted by HNN, respectively, Alpha helix: 251 is 49.60%, Extended strand: 52 is 10.28%, Random coil: 203 is 40.12%; 3D structure was predicted by Geno3d,it shows that 57 extended strands contained 24 alpha helices inβ-folding area and 14 random coils. According to the amino acid sequence and structural analysis, it shows that C4H protein contains one conserved domain, viz. P450 domain. The existing probability and the anchoring probability of signal peptide were both 0.498 which were analyzed by SignalP, the cleavage site was between 28 and 29 position amino acids.
引文
陈安和,李加纳,柴友荣等. 2007.羽衣甘蓝中一个突变型肉桂酸-4-羟化酶基因的克隆及分析[J].园艺学报, 34 ( 4): 915～922
陈安和. 2006.甘蓝型油菜及其亲本物种C4H基因家族克隆及比较基因组学研究[J].博士学位论文
戴勋. 2002.植物次生代谢[J].昭通师范高等专科学校学报, 24(5):35～38
巩相景,吕福堂. 2006.化感作用及其在农业生产中的应用[J].生物技术通报, 2006年增刊:116～119
韩驰. 2000.茶叶防癌作用研究[J].中国肿瘤, 9 (1) :18～20
何水林. 2002.植保素代谢与植物防御反应[M].广州:广东科技出版社: 1～25
李波,梁颖,柴友荣. 2006.植物肉桂酰辅酶A还原酶(CCR)基因的研究进展[J].分子植物育种, 2006, 4(3S): 55-65
李莉,赵越,马君兰. 2006.苯丙氨酸代谢途径关键酶:PAL, C4H, 4CL研究新进展[J].生物信息学:187～189
李玉花,刘靖华,徐启江等. 2006.现代分子生物学模块实验指南[M] .北京:高等教育出版社: 295～311
林元震. 2006.甜杨葡萄糖-6-磷酸脱氢酶基因克隆及结构分析与功能鉴定(D).北京林业大学博士学位论文:56～57
刘志胜,李里特,辰巳英三. 2000.大豆异黄酮及其生理功能研究进展[J].食品工业科技, 21(1):78
J曼. 1983.次生代谢作用[M].曹日强译.北京.科学出版社
欧阳光察,薛应龙. 1988.植物苯丙烷类代谢的生理意义及其调控[J].植物生理学通讯. (3): 9～16
石碧,狄莹. 1999.植物多酚[M],北京:科学出版社, 171～180 ,285～290
孙啸.陆祖宏. 2005.生物信息学基础[M].北京:清华大学出版社
唐建军,项田夫,张禄源,等. 1995.植物次生代谢、离体培养条件下次生代谢产物积累及其调控研究进展[J].中国野生植物资源, 17(4):1～6
阎秀峰. 2001.植物次生代谢生态学[J].植物生态学报, 25 (5) : 639～640
张成岗,贺福初. 2002.生物信息学方法与实践[M].北京:科学出版社
张奕. 2000.连作大豆化感作用及化感物质种类的鉴定[D].东北师范大学学位论文:1～3
赵淑娟,刘涤,胡之壁. 2003.植物次生代谢基因工程[J].中国生物工程杂志, 23(7): 52～56
中国科学院植物研究所. 1979.中国高等植物图鉴[M].北京:科学出版社
钟扬,王莉,张亮. 2003.生物信息学[M].北京:高等教育出版社
Arimura GI. 2000. Herbivory-induced volatiles elicit defense gene in lima bean leaves[J].Nature. 406 :512～515
Baldi P, Chauvin Y, Hunkapiller T, etal. 1994. Hidden Markov models of biological primary sequence information[J]. Proc. Nat. Acad. Sci. (USA),91(3):1059～1063
Barber M. S., and Mitchell H. J. 1997. Regulation of phenylpropanoid metabolism in relation to lignin biosynthesis in plants [J]. Int. Rev. Cytol. 172: 243-293.
Battle M., etal. 2000. Global carbon sinks and their variability inferred from atmospheric O2 and d13C[J]. Science. 287:2467.
Bell-Lelong D. A., Cusumano J. C., Meyer K., and Chapple C. 1997.
Cinnamate-4-hydroxylase expression in Arabidopsis (Regulation in response to development and the environment)[J]. Plant Physiol.113: 729-738.
Bruin J , Dicke M & Sabelis MW. 1992. Plants are better protected against spirder-mites after exposure to volatiles from infested conspecifics[J]. Experentia. 48 :525～529
Chappell J. 1995. Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants[J]. Annu Rev Plant Physiol Plant Mol Biol, 46 :521～547
Chapple C. 1998. Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases[J]. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 311-343
Crock J, Wildung M, Croteau R. 1997. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint ( Menthax piperita L. ) that produces the aphid alarm pheromone ( E)-β-farne- sene[J]. Proc Natl Acad Sci USA, 94 (24) : 12833～12838
De Moraes CM , Mescher MC , Tumlinson J H. 2001. Caterpillar-induced plant nocturnal plant volatiles repel conspecific females. Nat ure. 410 :577～580
Degtyarenko KN, Archakov AI. 1993. Molecular evolution of P450 superfamily and P450-containing monooxygenase systems[J]. FEBS Lett. 332(1-2):1-8
Dicke M. 1999. Evolution of induced indirect defence of plants[J]. In : Tollrian R , Harvell CD. The Ecology and Evolution of Inducible Defenses. Princeton : Princeton University Press. 62～88
Dixon RA, Paiva N L. 1995. Stress-induced phenylpropanoid metabolism[J]. Plant Cell. 7 :1085～1097
Facchini PJ. 2001. Alkaloid biosynthesis in plants: biochemistry, cell biolo-gy, molecular regulation, and metabolic engineering applications[J]. A nnu Rev Plant Physiol Plant Mol Biol. 52 : 29～66
Fahrendorf T, Dixon R A. 1993. Stress responses in alfalfa(Medicago sativa L.)XVIII.Molecular cloning and expression of the elicitor-inducible cinnamic acid 4-hydroxylase cytochrome P450[J].Arch Biophys, (305): 509- 515.
Graham-Lorence S, Amarneh B, White RE, Peterson JA, Simpson ER.1995. A three-dimensional model of aromatase cytochrome P450[J]. Protein Sci. 4(6):1065-1080
Guengerich FP. 1991. Reactions and significance of cytochrome P-450 enzymes[J]. J. Biol. Chem. 266(16):10019-10022
Hartmut K Lichtenthaler. 1999. The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants[J]. A nnu Rev Plant Physiol Plant Mol Biol , 50 :47～65
Hatfield R., Vermerris W. 2001. Lignin Formation in Plants. The Dilemma of linkage Specificity[J]. Plant Physiol, 126:1351～1357
Hu XF (胡秀芳), Shen SG(沈生荣), Piao ZR(朴宰日), Yang XQ(杨贤强). 1999. Review on antioxidative mechanism of tea ployphenols[J]. J Tea Sci (茶叶科学), 19 :41～48
Ikeda I, Masto Y, Sasaki E, Nakayama M, Nagao H, Takeo T, Yayabe F, Sugano M. 1992.
Tea catechins decrease micellar solubility and intestinal sbsorption of cholestezol in rats[J]. Biochem Biophys Acta , 1127 (2) :141～147
Kessler A , Baldwin IT. 2001. Defensive function of herbivore-induced plant volatile emission in nature[J]. Science. 291 :2141～2144
Koopman E., Logemann E., and Hahlbrock K. (1999) Regulation and functional expression of cinnamate 4-hydroxylase from parsley [J]. Plant Physiol. 119: 49-55
Kosuge T Gilehrist D.G. 1980. Aromatic amino acid biosynthesis and its regulateon[J]. Biochem. Plant. 507～331
Kutchan ToniM. 2001. Ecological A rsenal and Developmental Dispatcher The Paradigm of Secondary Metabolism[J]. Plant Physiol, 125(1): 58～601
Mizutani M, Ward E, DiMaio J , etal. 1993. Molecular cloning and sequencing of a cDNA encoding mung bean cytochrome P450 possessing cinnamate-4-hydroxylase acticity[J]. Biocbean Biopbys Res Commum, 190: 875- 880
Mizutani M.,Ward E., Di Maio J., etal. 1993. Molecular cloning and sequencing of a cDNA encoding mung bean cytochrome P450(P450C4H)possessing cinnamate-4-hydroxylase activity [J]. Biochem. Biophys. Res. Commun. 190:875-880
Nebert DW, Gonzalez FJ. 1987. P450 genes: structure, evolution, and regulation[J]. Annu. Rev. Biochem. 56:945-993
Nedelkina S., Jupe S. C. Blee K. A., Schalk M., Werck Reichhart D., and Bolwell G. P. 1999.
Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast [J]. Plant Mol. Biol. 39: 1079-1090
Nelson DR, Kamataki T, Waxman DJ, et al. 1993. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature[J]. DNA Cell Biol. 12(1):1-51
Russell D. W, Conn E. E. 1967. The cinnamic acid 4-hydroxylase of pea seedlings [J]. Arch. Biochem. Biophys. 122:256-258
Teutsch H. G, Hasenfratz M. P., Lesot A., etal. 1993. Isolation and sequence of a cDNA encoding the Jerusalem artichke cinnamate 4-hydroxylase,a major plant cytochrome P450 involved in the general phenylpropanoid pathway [J]. Proc. Natl. Acad. Sci. USA 90:119-126
Teutsch H. G., Hasenfratz M. P., Lesot A., Stoltz C., Gamier J. M., Jeltsch J. M., Durst F. and Werck Reichhart D. 1993. Isolation and sequence of a cDNA encoding the Jerusalem artichokecinnamate-4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway[J]. Proc. Natl. Acad. Sci. USA 90: 4102-4107
Turlings TC , Tumlinson J H. 1992. Systemic release of chemical signals by herbivore injured corn[J]. Proc Natl Acad Sci USA. 89 : 8399～8402
Werck-Reichhart D, Feyereisen R. 2000. Cytochromes P450: a success story[J]. Genome Biol. 1(6):REVIEWS3003
陈安和. 2006.甘蓝型油菜及其亲本物种C4H基因家族克隆及比较基因组学研究[J].博士学位论文
戴勋. 2002.植物次生代谢[J].昭通师范高等专科学校学报, 24(5):35～38
巩相景,吕福堂. 2006.化感作用及其在农业生产中的应用[J].生物技术通报, 2006年增刊:116～119
韩驰. 2000.茶叶防癌作用研究[J].中国肿瘤, 9 (1) :18～20
何水林. 2002.植保素代谢与植物防御反应[M].广州:广东科技出版社: 1～25
李波,梁颖,柴友荣. 2006.植物肉桂酰辅酶A还原酶(CCR)基因的研究进展[J].分子植物育种, 2006, 4(3S): 55-65
李莉,赵越,马君兰. 2006.苯丙氨酸代谢途径关键酶:PAL, C4H, 4CL研究新进展[J].生物信息学:187～189
李玉花,刘靖华,徐启江等. 2006.现代分子生物学模块实验指南[M] .北京:高等教育出版社: 295～311
林元震. 2006.甜杨葡萄糖-6-磷酸脱氢酶基因克隆及结构分析与功能鉴定(D).北京林业大学博士学位论文:56～57
刘志胜,李里特,辰巳英三. 2000.大豆异黄酮及其生理功能研究进展[J].食品工业科技, 21(1):78
J曼. 1983.次生代谢作用[M].曹日强译.北京.科学出版社
欧阳光察,薛应龙. 1988.植物苯丙烷类代谢的生理意义及其调控[J].植物生理学通讯. (3): 9～16
石碧,狄莹. 1999.植物多酚[M],北京:科学出版社, 171～180 ,285～290
孙啸.陆祖宏. 2005.生物信息学基础[M].北京:清华大学出版社
唐建军,项田夫,张禄源,等. 1995.植物次生代谢、离体培养条件下次生代谢产物积累及其调控研究进展[J].中国野生植物资源, 17(4):1～6
阎秀峰. 2001.植物次生代谢生态学[J].植物生态学报, 25 (5) : 639～640
张成岗,贺福初. 2002.生物信息学方法与实践[M].北京:科学出版社
张奕. 2000.连作大豆化感作用及化感物质种类的鉴定[D].东北师范大学学位论文:1～3
赵淑娟,刘涤,胡之壁. 2003.植物次生代谢基因工程[J].中国生物工程杂志, 23(7): 52～56
中国科学院植物研究所. 1979.中国高等植物图鉴[M].北京:科学出版社
钟扬,王莉,张亮. 2003.生物信息学[M].北京:高等教育出版社
Arimura GI. 2000. Herbivory-induced volatiles elicit defense gene in lima bean leaves[J].Nature. 406 :512～515
Baldi P, Chauvin Y, Hunkapiller T, etal. 1994. Hidden Markov models of biological primary sequence information[J]. Proc. Nat. Acad. Sci. (USA),91(3):1059～1063
Barber M. S., and Mitchell H. J. 1997. Regulation of phenylpropanoid metabolism in relation to lignin biosynthesis in plants [J]. Int. Rev. Cytol. 172: 243-293.
Battle M., etal. 2000. Global carbon sinks and their variability inferred from atmospheric O2 and d13C[J]. Science. 287:2467.
Bell-Lelong D. A., Cusumano J. C., Meyer K., and Chapple C. 1997.
Cinnamate-4-hydroxylase expression in Arabidopsis (Regulation in response to development and the environment)[J]. Plant Physiol.113: 729-738.
Bruin J , Dicke M & Sabelis MW. 1992. Plants are better protected against spirder-mites after exposure to volatiles from infested conspecifics[J]. Experentia. 48 :525～529
Chappell J. 1995. Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants[J]. Annu Rev Plant Physiol Plant Mol Biol, 46 :521～547
Chapple C. 1998. Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases[J]. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 311-343
Crock J, Wildung M, Croteau R. 1997. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint ( Menthax piperita L. ) that produces the aphid alarm pheromone ( E)-β-farne- sene[J]. Proc Natl Acad Sci USA, 94 (24) : 12833～12838
De Moraes CM , Mescher MC , Tumlinson J H. 2001. Caterpillar-induced plant nocturnal plant volatiles repel conspecific females. Nat ure. 410 :577～580
Degtyarenko KN, Archakov AI. 1993. Molecular evolution of P450 superfamily and P450-containing monooxygenase systems[J]. FEBS Lett. 332(1-2):1-8
Dicke M. 1999. Evolution of induced indirect defence of plants[J]. In : Tollrian R , Harvell CD. The Ecology and Evolution of Inducible Defenses. Princeton : Princeton University Press. 62～88
Dixon RA, Paiva N L. 1995. Stress-induced phenylpropanoid metabolism[J]. Plant Cell. 7 :1085～1097
Facchini PJ. 2001. Alkaloid biosynthesis in plants: biochemistry, cell biolo-gy, molecular regulation, and metabolic engineering applications[J]. A nnu Rev Plant Physiol Plant Mol Biol. 52 : 29～66
Fahrendorf T, Dixon R A. 1993. Stress responses in alfalfa(Medicago sativa L.)XVIII.Molecular cloning and expression of the elicitor-inducible cinnamic acid 4-hydroxylase cytochrome P450[J].Arch Biophys, (305): 509- 515.
Graham-Lorence S, Amarneh B, White RE, Peterson JA, Simpson ER.1995. A three-dimensional model of aromatase cytochrome P450[J]. Protein Sci. 4(6):1065-1080
Guengerich FP. 1991. Reactions and significance of cytochrome P-450 enzymes[J]. J. Biol. Chem. 266(16):10019-10022
Hartmut K Lichtenthaler. 1999. The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants[J]. A nnu Rev Plant Physiol Plant Mol Biol , 50 :47～65
Hatfield R., Vermerris W. 2001. Lignin Formation in Plants. The Dilemma of linkage Specificity[J]. Plant Physiol, 126:1351～1357
Hu XF (胡秀芳), Shen SG(沈生荣), Piao ZR(朴宰日), Yang XQ(杨贤强). 1999. Review on antioxidative mechanism of tea ployphenols[J]. J Tea Sci (茶叶科学), 19 :41～48
Ikeda I, Masto Y, Sasaki E, Nakayama M, Nagao H, Takeo T, Yayabe F, Sugano M. 1992.
Tea catechins decrease micellar solubility and intestinal sbsorption of cholestezol in rats[J]. Biochem Biophys Acta , 1127 (2) :141～147
Kessler A , Baldwin IT. 2001. Defensive function of herbivore-induced plant volatile emission in nature[J]. Science. 291 :2141～2144
Koopman E., Logemann E., and Hahlbrock K. (1999) Regulation and functional expression of cinnamate 4-hydroxylase from parsley [J]. Plant Physiol. 119: 49-55
Kosuge T Gilehrist D.G. 1980. Aromatic amino acid biosynthesis and its regulateon[J]. Biochem. Plant. 507～331
Kutchan ToniM. 2001. Ecological A rsenal and Developmental Dispatcher The Paradigm of Secondary Metabolism[J]. Plant Physiol, 125(1): 58～601
Mizutani M, Ward E, DiMaio J , etal. 1993. Molecular cloning and sequencing of a cDNA encoding mung bean cytochrome P450 possessing cinnamate-4-hydroxylase acticity[J]. Biocbean Biopbys Res Commum, 190: 875- 880
Mizutani M.,Ward E., Di Maio J., etal. 1993. Molecular cloning and sequencing of a cDNA encoding mung bean cytochrome P450(P450C4H)possessing cinnamate-4-hydroxylase activity [J]. Biochem. Biophys. Res. Commun. 190:875-880
Nebert DW, Gonzalez FJ. 1987. P450 genes: structure, evolution, and regulation[J]. Annu. Rev. Biochem. 56:945-993
Nedelkina S., Jupe S. C. Blee K. A., Schalk M., Werck Reichhart D., and Bolwell G. P. 1999.
Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast [J]. Plant Mol. Biol. 39: 1079-1090
Nelson DR, Kamataki T, Waxman DJ, et al. 1993. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature[J]. DNA Cell Biol. 12(1):1-51
Russell D. W, Conn E. E. 1967. The cinnamic acid 4-hydroxylase of pea seedlings [J]. Arch. Biochem. Biophys. 122:256-258
Teutsch H. G, Hasenfratz M. P., Lesot A., etal. 1993. Isolation and sequence of a cDNA encoding the Jerusalem artichke cinnamate 4-hydroxylase,a major plant cytochrome P450 involved in the general phenylpropanoid pathway [J]. Proc. Natl. Acad. Sci. USA 90:119-126
Teutsch H. G., Hasenfratz M. P., Lesot A., Stoltz C., Gamier J. M., Jeltsch J. M., Durst F. and Werck Reichhart D. 1993. Isolation and sequence of a cDNA encoding the Jerusalem artichokecinnamate-4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway[J]. Proc. Natl. Acad. Sci. USA 90: 4102-4107
Turlings TC , Tumlinson J H. 1992. Systemic release of chemical signals by herbivore injured corn[J]. Proc Natl Acad Sci USA. 89 : 8399～8402
Werck-Reichhart D, Feyereisen R. 2000. Cytochromes P450: a success story[J]. Genome Biol. 1(6):REVIEWS3003