盐肤木苯丙氨酸解氨酶基因的克隆及其功能性质研究
|
摘要
盐肤木(Rhus chinensis)是漆树科(Anacardiaceae),盐肤木属(Rhus)落叶小乔木,它作为中国经济价值极高的树种,可供制药、榨油、观赏和做工业染料的原料等。目前对盐肤木的研究主要在其成分及药用价值、形态特征、栽培方法和技术等方面,而有关其体内防御酶的研究很少。
苯丙氨酸解氨酶(phenylalanine ammonia-lyase, PAL)是苯丙烷代谢途径中的关键酶和限速酶,植物的初级代谢和苯丙烷类代谢就是被它所连接,PAL是与植物的抗逆性密切相关的酶,是一种防御酶。
本论文以盐肤木为研究材料,对其PAL基因进行分子克隆,构建重组表达载体,经原核表达系统得到其重组蛋白,进一步对其性质和功能进行研究。主要研究结果如下：
1.本研究提取得到的盐肤木总RNA,28SrRNA和18SrRNA的电泳条带清晰,没有拖尾现象,完整性很好,能用于后续分子克隆等实验。
2.首次得到盐肤木PAL基因(RcPAL)的ORF序列。RcPAL基因全长cDNA的长度为2491bp,其中2124bp的ORF编码707个氨基酸,5’端有一段197bp的非编码区(5’UTR),3’端有一个170bp的非编码区(3'UTR)。RcPAL全长cDNA序列是漆树科中首次被报道的。
3.构建成功重组表达载体pET-28a-PAL,长度约为7500bp。在大肠杆菌中,此载体被诱导后得到约77kDa的重组蛋白。经50%咪唑洗脱液得到纯度及酶活性最高的RcPAL酶液。
4. RcPAL的最适pH为9.0,最适温度为45℃,Ea为8.03kcal/mol。其动力学曲线呈双曲线型,底物L-苯丙氨酸浓度增大,RcPAL的酶活性也增强,并从底物浓度为20mM开始酶活性趋于平稳。RcPAL的Km为7.9mM, Kcat为52.31s-1。
5.对3个重要位点进行定点突变,成功得到3个突变体,Phe126→His126(TTC→CAC)、Ser194→Trp194(TTC→CAC)、Asp374→Ala374(TTC→CAC),突变体M1、M2、M3的PAL酶活性为对照样品的25%、2%、20%,证明这3个位点是酶活性位点。将Phe126突变为His126后,突变体M1的PAL相对酶活性仅为对照的25%,而突变体M1的TAL相对酶活性却为对照的22倍,证明Phe126是此酶的底物特异性位点。
Rhus chinensis belonging to Anacardiaceae, Rhus, a small deciduous tree, is a major economic tree species in China. This tree can be used to make pharmaceuticals, extract oil and serve as the raw materials of industrial dye. At present the researches of the Rhus chinensis were about its composition, medicinal value, morphological characteristics, cultivation method and technique, but little attention has been paid to its defense enzyme.
PAL is the key enzyme that connects the plant primary metabolism and benzene propane metabolism, catalyzes the first step reaction of benzene propane metabolic, also is the key enzyme of phenylpropanoid pathway. It is directly related to plant resistance, and is a defense enzyme.
This paper used Rhus chinensis as research material to clone its PAL gene, build a recombinant expression vector, obtain its recombinant protein by the prokaryotic expression, and further research its function and quality. The main results were as follows:
1. The study got the total RNA of Rhus chinensis,28S rRNA and18S rRNA had clear stripe, and showed no degradation phenomenon, suggesting it can be used in the subsequent molecular cloning.
2. The study acquired the ORF sequence of RcPAL for the first time. The full-length cDNA of RcPAL was2491bp, including2124bp open reading box, which encoded707amino acids. Its5'end noncoding region was197bp (5'UTR) in length, while the3'end noncoding region was170bp (3'UTR). The cDNA sequence of Re PAL was the first report in Anacardiaceae.
3. The study built the recombinant expression vector pET-28a-PAL, with a length was7500bp and encoding a77kDa protein. We got the pure and high activity RcPAL enzyme by50%imidazole elution fluid.
4. RcPAL's optimum pH was9.0, the optimum temperature was45℃, and Ea was8.03kcal/mol. Its dynamics curve was hyperbolic. Its activity increased with increasing concentration of the substrate L-phenylalanine. Its Km was7.9mM, and Kcat52.31s-1.
5. The study got three mutants, Phe126→His126(TTC→CAC) Ser194→Trp194(TCG→TGG). Asp374→Ala374(GAT→GCT). The activity of mutants M1, M2, M3is25%,2%,20%of CK, which proved that the three sites in catalytic reaction played an important role. Phe126→His126, mutant M1's PAL activity was only25%of CK, but its TAL activity was22times as much as that of CK. So Phe126was important substrate specificity site.
苯丙氨酸解氨酶(phenylalanine ammonia-lyase, PAL)是苯丙烷代谢途径中的关键酶和限速酶,植物的初级代谢和苯丙烷类代谢就是被它所连接,PAL是与植物的抗逆性密切相关的酶,是一种防御酶。
本论文以盐肤木为研究材料,对其PAL基因进行分子克隆,构建重组表达载体,经原核表达系统得到其重组蛋白,进一步对其性质和功能进行研究。主要研究结果如下：
1.本研究提取得到的盐肤木总RNA,28SrRNA和18SrRNA的电泳条带清晰,没有拖尾现象,完整性很好,能用于后续分子克隆等实验。
2.首次得到盐肤木PAL基因(RcPAL)的ORF序列。RcPAL基因全长cDNA的长度为2491bp,其中2124bp的ORF编码707个氨基酸,5’端有一段197bp的非编码区(5’UTR),3’端有一个170bp的非编码区(3'UTR)。RcPAL全长cDNA序列是漆树科中首次被报道的。
3.构建成功重组表达载体pET-28a-PAL,长度约为7500bp。在大肠杆菌中,此载体被诱导后得到约77kDa的重组蛋白。经50%咪唑洗脱液得到纯度及酶活性最高的RcPAL酶液。
4. RcPAL的最适pH为9.0,最适温度为45℃,Ea为8.03kcal/mol。其动力学曲线呈双曲线型,底物L-苯丙氨酸浓度增大,RcPAL的酶活性也增强,并从底物浓度为20mM开始酶活性趋于平稳。RcPAL的Km为7.9mM, Kcat为52.31s-1。
5.对3个重要位点进行定点突变,成功得到3个突变体,Phe126→His126(TTC→CAC)、Ser194→Trp194(TTC→CAC)、Asp374→Ala374(TTC→CAC),突变体M1、M2、M3的PAL酶活性为对照样品的25%、2%、20%,证明这3个位点是酶活性位点。将Phe126突变为His126后,突变体M1的PAL相对酶活性仅为对照的25%,而突变体M1的TAL相对酶活性却为对照的22倍,证明Phe126是此酶的底物特异性位点。
Rhus chinensis belonging to Anacardiaceae, Rhus, a small deciduous tree, is a major economic tree species in China. This tree can be used to make pharmaceuticals, extract oil and serve as the raw materials of industrial dye. At present the researches of the Rhus chinensis were about its composition, medicinal value, morphological characteristics, cultivation method and technique, but little attention has been paid to its defense enzyme.
PAL is the key enzyme that connects the plant primary metabolism and benzene propane metabolism, catalyzes the first step reaction of benzene propane metabolic, also is the key enzyme of phenylpropanoid pathway. It is directly related to plant resistance, and is a defense enzyme.
This paper used Rhus chinensis as research material to clone its PAL gene, build a recombinant expression vector, obtain its recombinant protein by the prokaryotic expression, and further research its function and quality. The main results were as follows:
1. The study got the total RNA of Rhus chinensis,28S rRNA and18S rRNA had clear stripe, and showed no degradation phenomenon, suggesting it can be used in the subsequent molecular cloning.
2. The study acquired the ORF sequence of RcPAL for the first time. The full-length cDNA of RcPAL was2491bp, including2124bp open reading box, which encoded707amino acids. Its5'end noncoding region was197bp (5'UTR) in length, while the3'end noncoding region was170bp (3'UTR). The cDNA sequence of Re PAL was the first report in Anacardiaceae.
3. The study built the recombinant expression vector pET-28a-PAL, with a length was7500bp and encoding a77kDa protein. We got the pure and high activity RcPAL enzyme by50%imidazole elution fluid.
4. RcPAL's optimum pH was9.0, the optimum temperature was45℃, and Ea was8.03kcal/mol. Its dynamics curve was hyperbolic. Its activity increased with increasing concentration of the substrate L-phenylalanine. Its Km was7.9mM, and Kcat52.31s-1.
5. The study got three mutants, Phe126→His126(TTC→CAC) Ser194→Trp194(TCG→TGG). Asp374→Ala374(GAT→GCT). The activity of mutants M1, M2, M3is25%,2%,20%of CK, which proved that the three sites in catalytic reaction played an important role. Phe126→His126, mutant M1's PAL activity was only25%of CK, but its TAL activity was22times as much as that of CK. So Phe126was important substrate specificity site.
引文
[1]方建新.多用途野生盐肤木林.林业实用技术[J],2005,1 1：44.
[2]中国科学院植物研究所主编,中国高等植物图鉴(第二册)[M],科学出版社,1983,632.
[3]林锦仪,连培华.角倍蚜虫夏寄主盐肤木物候期的观察研究.福建林学院学报[J],1996,16：139-142.
[4]郑勉,闪天禄.中国植物志(第45卷第1分册)[M].北京：科学出版社,1980,100-101.
[5]郑光武,陈芳清,郭彦荣.樟村坪磷矿废弃地先锋物种的光合生理特性.福建林业科学[J],2009,36：79-52.
[6]许又凯,刘宏茂.中国云南热带野生蔬菜[M].科学出版社,2002,175.
[7]江苏新医学院.中药大辞典[M].上海人民出版社,1997,1817-1819.
[8]李加林,吴素珍,卑占宇.盐肤木总黄酮提取工艺研究.时珍国医国药[J],2009,20：1116-1117.
[9]Cui CB, Zhao QC, Cai B, et al. Two new and four known polyphenolics obtained as new cell cycle inhibitors from Rubus aleaefolius Poir[J], J Asian Nat Prod Res,2002,4: 243.
[10]Cui CB, Yan SY, Cai B, et al. Carbazole alkaloids as new cell cycle inhibitor andapoptosis inducers from Clusena dunniana Levl[J], J Asian Nat Prod Res,2002, 4:233.
[11]朱俊彦,熊龙花HPLC法测定盐肤木中没食子酸的含量.江西中医学院学报[J],2006,18：27.
[12]朱俊义,赵凤娟,杨忠顺等.盐肤木茎的木材解剖学研究.植物研究[J],2005,25：155-158.
[13]刘春强.盐肤木基础研究及利用.中国林业[J],2008,12：53.
[14]张燕平,郑兴峰,杨力真等.角倍及盐肤小叶片形态构造解剖分析.南京林业大学学报(自然科学版)[J],2001,11：6-10.
[15]黄家德,丘风波.盐肤木种子繁殖试验.广西植物[J],1994,1:85-89.
[16]王琼,宋桂龙.盐肤木种子硬实与萌发特性研究.种子[J],2008,27：59-61.
[17]梅再美,游金平.盐肤木枝枯病的发生发展规律研究.贵州林业科技[J],1996,24：1-6.
[18]唐觉,蔡邦华.贵州泥潭五倍子的研究.昆虫学报[J],1957,7：131-140.
[19]程惠珍,林余霖,陈君.陕西省山阳县五倍子的资源调查.中国中药杂志[J],1991,16：8-9.
[20]李志国,杨文云,夏定久.中国五倍子研究现状.林业科学研究[J],2003,16：760-767.
[21]唐觉.五倍子及其繁殖增产的途径.昆虫学报[J],1976,19：252-296,
[22]王建平.野生盐肤木林改造技术研究.林业科技通讯[J],1992,12：10-13.
[23]邱明生,赵志模,徐学勤等,角倍倍园主要生态因子对五倍子产量的影响.生态农业研究[J],1997,5：19-23.
[24]张广学,乔格侠,钟铁森,张万玉.中国动物志[M],昆虫纲,第十四卷,同翅目,犷蚜科,瘦绵蚜科.北京：科学出版社,1999,256-273.
[25]张光学,钟铁森.中国经济昆虫志[M].第25册,同翅目,蚜虫类.北京：科学出版社,1983,78-79.
[26]Qiao,G-X and Zhang, G-X 2000a. A revision of Cieoica Hart.1894 from China(Homoptera:Aphididae:Pemphigidae) Acta Ento-tool.Sin.,44:79-87.
[27]漆云庆,邱建生,杨光勇.角倍蚜种群动态的研究[J].贵州林业科技,1990,4,14-24.
[28]邱明生,赵志模,徐学勤等.角倍蚜与其寄主间营养关系的研究[J].西南农业大学学报,1996,6,613-617.
[29]Takagi G. life-cycle of Schlechtendalia chinensis (Bell) and its gall-formation by artificial inoculation with the stem-mother[J]. Zool. Mag.,1934,46,473-481.
[30]李勇民,田泽君.国外五倍子研究进展[[J].四川林业科技,1993,3,35-39.
[31]Takada H. Does the Sexual Female of Schlechtendalia chinensis (BELL)(Homoptera:Pemphigidae) "Viviparously" Produce the Fundatrix[J]. Appl. Ent.Zool.,1991,1,117-121.
[32]王定卿,王贤斌,明安觉等.角倍蚜人工培育技术[J].湖北林业科技,2007,143, 70-71.
[33]Guo GX, Liu Y, Yang JJ, et al. Identification, activity and function determination of several salivary enzymes secreted by Macrosiphum avenae. Acta Entomologica Sinica,2006,49:768-774.
[34]Kessler A, Baldwin IT. Defensive function of herbivore-induced plan t volatile emissions in nature. Science,2001,291:2141-214.
[35]Kessler A, Baldwin IT. Plant responses to insect herbiovory:The emerging molecular analysis. Annual Review of Plant Biology,2002,53:299-328.
[36]Kranthi S, Kranthi KR, Wanjari RR. Influence of semilooper damage on cotton host-plant resistance to Helicoverpa armigera (Hub.).Plant Science, 2003,164:157-163.
[37]Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Science,1997,2:379-383.
[38]Li JB, Ren ZHM. Genetic diversity among Schlechtendalia chinensis individuals revealed by Cytb sequences. Journal of Fudan University (Natural Science), 2009,48:59-65.
[39]Li JB, Ren ZHM, Ma ENB. Comparative genetic diversity of Schlechtendalia Chinensis and their hosts Rhus Chinensis population. Journal of Shanxi University (Nature Science Edition).2009,32:298-303.
[40]Li XG, Liu HX, Huang J. Molecular mechanisms of insect pests induced plant defense. Chinese Journal of Applied Ecology,2008,19:893-900.
[41]江昌俊,余有本.苯丙氨酸解氨酶的研究进展[J].安徽农业大学学报,2001,28：425-430.
[42]Ritter H, Schulz G E. Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase [J]. The Plant Cell, 2004,16:3426-3436.
[43]Elkind Y, Edwards R, Mavandad M, et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyasc gene [J]. Proc. Nadl. Acad. Sci, 1990,81:9057-9061.
[44]程水源,陈昆松,刘卫红等.植物苯丙氨酸解氨酶基因的表达调控与研究展望[J].果树学报,2003,20：351-357.
[45]贺立红,张进标,宾金华.苯丙氨酸解氨酶的研究进展[J].食品科技,2006,7.
[46]江苏新医学院.中药大辞典[M].上海人民出版社,1997,1817-1819.
[47]Wanner, L.A., Li, G., Ware, D., Somssich, I.E., Davis, K.R. The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana. Plant Mol. Biol.1995,27, 327-338.
[48]Song, J., Wang, Z. Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmPAL1) from Salvia miltiorrhiza. Mol. Biol. Rep.2009,36,939-952.
[49]姜红林,梁颖.甘蓝型油菜苯丙氨酸解氨酶的分离纯化与性质研究[J].植物生理科学,2006,22：282-286.
[50]Mario Monzdesa,etal. Rapid activation of PhenylProPanoid metabolism in elieitor-treated hybrid Poplar Suspension-cultured cells[J].Plant Physiology,1992, 98:728-737.
[51]高旭辉.论茶树抗病性的生化基础[J].茶叶通讯,1998：27-29.
[52]Bolwell G P,Cramer C L,Lamb C J, et al. L-Phenylalanine ammonia-lyase from Phaseolus vulgaris:partial degradation of enzyme subunits in vitro and in vivo[J].Biochimica et Biophysica Acta,1986,881:210-217.
[53]孟延发,辛嘉英.植物苯丙氨酸解氨酶的研究,鹰嘴豆PAL的纯化及其基本性质[J].兰州大学学报(自然科学版),1990,26：109-113.
[54]王燕,刘卫红,杜何为等.银杏叶中苯丙氨酸解氨酶基本特性的研究[J].湖北农业科学,2004,：72-74.
[55]李艳华,王庆国.芦笋过氧化物酶和苯丙氨酸解氨酶特性及抑制条件的研究[J].食品与发酵工业,2007,33：55-59.
[2]中国科学院植物研究所主编,中国高等植物图鉴(第二册)[M],科学出版社,1983,632.
[3]林锦仪,连培华.角倍蚜虫夏寄主盐肤木物候期的观察研究.福建林学院学报[J],1996,16：139-142.
[4]郑勉,闪天禄.中国植物志(第45卷第1分册)[M].北京：科学出版社,1980,100-101.
[5]郑光武,陈芳清,郭彦荣.樟村坪磷矿废弃地先锋物种的光合生理特性.福建林业科学[J],2009,36：79-52.
[6]许又凯,刘宏茂.中国云南热带野生蔬菜[M].科学出版社,2002,175.
[7]江苏新医学院.中药大辞典[M].上海人民出版社,1997,1817-1819.
[8]李加林,吴素珍,卑占宇.盐肤木总黄酮提取工艺研究.时珍国医国药[J],2009,20：1116-1117.
[9]Cui CB, Zhao QC, Cai B, et al. Two new and four known polyphenolics obtained as new cell cycle inhibitors from Rubus aleaefolius Poir[J], J Asian Nat Prod Res,2002,4: 243.
[10]Cui CB, Yan SY, Cai B, et al. Carbazole alkaloids as new cell cycle inhibitor andapoptosis inducers from Clusena dunniana Levl[J], J Asian Nat Prod Res,2002, 4:233.
[11]朱俊彦,熊龙花HPLC法测定盐肤木中没食子酸的含量.江西中医学院学报[J],2006,18：27.
[12]朱俊义,赵凤娟,杨忠顺等.盐肤木茎的木材解剖学研究.植物研究[J],2005,25：155-158.
[13]刘春强.盐肤木基础研究及利用.中国林业[J],2008,12：53.
[14]张燕平,郑兴峰,杨力真等.角倍及盐肤小叶片形态构造解剖分析.南京林业大学学报(自然科学版)[J],2001,11：6-10.
[15]黄家德,丘风波.盐肤木种子繁殖试验.广西植物[J],1994,1:85-89.
[16]王琼,宋桂龙.盐肤木种子硬实与萌发特性研究.种子[J],2008,27：59-61.
[17]梅再美,游金平.盐肤木枝枯病的发生发展规律研究.贵州林业科技[J],1996,24：1-6.
[18]唐觉,蔡邦华.贵州泥潭五倍子的研究.昆虫学报[J],1957,7：131-140.
[19]程惠珍,林余霖,陈君.陕西省山阳县五倍子的资源调查.中国中药杂志[J],1991,16：8-9.
[20]李志国,杨文云,夏定久.中国五倍子研究现状.林业科学研究[J],2003,16：760-767.
[21]唐觉.五倍子及其繁殖增产的途径.昆虫学报[J],1976,19：252-296,
[22]王建平.野生盐肤木林改造技术研究.林业科技通讯[J],1992,12：10-13.
[23]邱明生,赵志模,徐学勤等,角倍倍园主要生态因子对五倍子产量的影响.生态农业研究[J],1997,5：19-23.
[24]张广学,乔格侠,钟铁森,张万玉.中国动物志[M],昆虫纲,第十四卷,同翅目,犷蚜科,瘦绵蚜科.北京：科学出版社,1999,256-273.
[25]张光学,钟铁森.中国经济昆虫志[M].第25册,同翅目,蚜虫类.北京：科学出版社,1983,78-79.
[26]Qiao,G-X and Zhang, G-X 2000a. A revision of Cieoica Hart.1894 from China(Homoptera:Aphididae:Pemphigidae) Acta Ento-tool.Sin.,44:79-87.
[27]漆云庆,邱建生,杨光勇.角倍蚜种群动态的研究[J].贵州林业科技,1990,4,14-24.
[28]邱明生,赵志模,徐学勤等.角倍蚜与其寄主间营养关系的研究[J].西南农业大学学报,1996,6,613-617.
[29]Takagi G. life-cycle of Schlechtendalia chinensis (Bell) and its gall-formation by artificial inoculation with the stem-mother[J]. Zool. Mag.,1934,46,473-481.
[30]李勇民,田泽君.国外五倍子研究进展[[J].四川林业科技,1993,3,35-39.
[31]Takada H. Does the Sexual Female of Schlechtendalia chinensis (BELL)(Homoptera:Pemphigidae) "Viviparously" Produce the Fundatrix[J]. Appl. Ent.Zool.,1991,1,117-121.
[32]王定卿,王贤斌,明安觉等.角倍蚜人工培育技术[J].湖北林业科技,2007,143, 70-71.
[33]Guo GX, Liu Y, Yang JJ, et al. Identification, activity and function determination of several salivary enzymes secreted by Macrosiphum avenae. Acta Entomologica Sinica,2006,49:768-774.
[34]Kessler A, Baldwin IT. Defensive function of herbivore-induced plan t volatile emissions in nature. Science,2001,291:2141-214.
[35]Kessler A, Baldwin IT. Plant responses to insect herbiovory:The emerging molecular analysis. Annual Review of Plant Biology,2002,53:299-328.
[36]Kranthi S, Kranthi KR, Wanjari RR. Influence of semilooper damage on cotton host-plant resistance to Helicoverpa armigera (Hub.).Plant Science, 2003,164:157-163.
[37]Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Science,1997,2:379-383.
[38]Li JB, Ren ZHM. Genetic diversity among Schlechtendalia chinensis individuals revealed by Cytb sequences. Journal of Fudan University (Natural Science), 2009,48:59-65.
[39]Li JB, Ren ZHM, Ma ENB. Comparative genetic diversity of Schlechtendalia Chinensis and their hosts Rhus Chinensis population. Journal of Shanxi University (Nature Science Edition).2009,32:298-303.
[40]Li XG, Liu HX, Huang J. Molecular mechanisms of insect pests induced plant defense. Chinese Journal of Applied Ecology,2008,19:893-900.
[41]江昌俊,余有本.苯丙氨酸解氨酶的研究进展[J].安徽农业大学学报,2001,28：425-430.
[42]Ritter H, Schulz G E. Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase [J]. The Plant Cell, 2004,16:3426-3436.
[43]Elkind Y, Edwards R, Mavandad M, et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyasc gene [J]. Proc. Nadl. Acad. Sci, 1990,81:9057-9061.
[44]程水源,陈昆松,刘卫红等.植物苯丙氨酸解氨酶基因的表达调控与研究展望[J].果树学报,2003,20：351-357.
[45]贺立红,张进标,宾金华.苯丙氨酸解氨酶的研究进展[J].食品科技,2006,7.
[46]江苏新医学院.中药大辞典[M].上海人民出版社,1997,1817-1819.
[47]Wanner, L.A., Li, G., Ware, D., Somssich, I.E., Davis, K.R. The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana. Plant Mol. Biol.1995,27, 327-338.
[48]Song, J., Wang, Z. Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmPAL1) from Salvia miltiorrhiza. Mol. Biol. Rep.2009,36,939-952.
[49]姜红林,梁颖.甘蓝型油菜苯丙氨酸解氨酶的分离纯化与性质研究[J].植物生理科学,2006,22：282-286.
[50]Mario Monzdesa,etal. Rapid activation of PhenylProPanoid metabolism in elieitor-treated hybrid Poplar Suspension-cultured cells[J].Plant Physiology,1992, 98:728-737.
[51]高旭辉.论茶树抗病性的生化基础[J].茶叶通讯,1998：27-29.
[52]Bolwell G P,Cramer C L,Lamb C J, et al. L-Phenylalanine ammonia-lyase from Phaseolus vulgaris:partial degradation of enzyme subunits in vitro and in vivo[J].Biochimica et Biophysica Acta,1986,881:210-217.
[53]孟延发,辛嘉英.植物苯丙氨酸解氨酶的研究,鹰嘴豆PAL的纯化及其基本性质[J].兰州大学学报(自然科学版),1990,26：109-113.
[54]王燕,刘卫红,杜何为等.银杏叶中苯丙氨酸解氨酶基本特性的研究[J].湖北农业科学,2004,：72-74.
[55]李艳华,王庆国.芦笋过氧化物酶和苯丙氨酸解氨酶特性及抑制条件的研究[J].食品与发酵工业,2007,33：55-59.