砀山酥梨果实PAL酶学特性的研究及PAL基因克隆
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摘要
砀山酥梨(Pyrus bretschneideri cv.)原产安徽省砀山县,是我国梨的主栽品种和主要出口品种之一。但是由于近年来管理不善、品种退化等原因,使得石细胞含量明显增加,肉质变粗、多渣,口感变差,严重降低了砀山酥梨的品质及其经济价值。因此,通过生物学等手段降低石细胞含量,提高砀山酥梨品质,以增强砀山酥梨市场竞争力成为亟待解决的问题。本实验以砀山酥梨果实为材料,研究砀山酥梨果实发育过程中石细胞含量、木质素含量及木质素合成关键酶苯丙氨酸解氨酶(PAL)活性变化,找到梨石细胞发育与PAL的关系。并对PAL的酶学特性进行研究,以期了解梨PAL的性质,同时克隆PAL基因,为从生理生化及分子生物学水平调控砀山酥梨石细胞发育提供理论基础,主要研究结果如下:
1、通过对五种梨果实切片观察发现砀山酥梨、贡梨和丰水梨石细胞团较大,分布较密集;砀山酥梨石细胞团最大,分布最密集;库尔勒香梨石细胞团较小,分布较疏散。苹果梨,石细胞团最小,分布最疏散。
2、在砀山酥梨果实整个发育过程中,石细胞含量于花后51天出现高峰,高峰值为17.83%;木质素含量于花后47天和花后63出现两个高峰,峰值分别为6.47%和7.00%;PAL活性最大值出现在花后27天,最大值为85.53∪/g·h。三者高峰期的出现在时间上表现出一定的时序性,PAL活性的高峰期最先出现,然后木质素含量出现高峰期,石细胞含量的高峰期最后出现。它们三者的变化趋势基本相同,总体上均呈先升高后下降的趋势,且三者之间均呈显著正相关。
3、砀山酥梨果实PAL的最适pH为9.2;在pH9.0左右时稳定性较高;用pH4.0和pH10.0的缓冲液处理均导致酶分子结构发生变化,活性降低;PAL最适反应温度为60℃左右,较耐热;当反应时间在180min内PAL的活性与反应时间呈线性关系;PAL对L-苯丙氨酸有较高的专一性,对L-酪氨酸也表现出一定的活性;最适底物浓度为0.0004～0.0012mol/L;砀山酥梨果实PAL的Km值有两个,Km_1为0.7500mmol/L,Km_2为1.0690 mmol/L。
4、克隆得到砀山酥梨果实PAL基因cDNA片段长度为585bp,推断编码195个氨基酸序列。该序列包含与其它植物PAL相同的活性催化位点,经序列分析和同源性比对,该氨基酸序列与其它植物PAL氨基酸序列的同源性在90%以上。系统进化树分析表明,砀山酥梨PAL氨基酸序列与蔷薇科的甜樱桃PAL氨基酸序列聚类关系最近。
Pyrus bretschneideri cv.originates in Dangshan, Anhui Province, which is one of the pear products for main cultivating and exporting. However, due to poor management, species degradation and many other factors in recent years, the stone cell content is significantly increased in the pear. What’s more, the flesh becomes thicker, more residue and taste variation than before, which severely decline the quality of Pyrus bretschneideri cv. and economic value. At the present time, reducing the stone cell content by utilizing biotechnology to improve the quality and enhance the market competitiveness of Pyrus bretschneideri cv. become an urgent question. In this experiment, we use Pyrus bretschneideri cv. fruit as research sample. We try to analyze the fluctuation of stone cell content, lignin content and the PAL activity, a critical enzyme to produce lignin, in the process of fruit development; and dissect the relationship between fruit stone cell development and PAL activity. In addition, in order to understand the nature of PAL, the characterization of the PAL was studied; and PAL gene was cloned at the same time. All of these provided a theoretical foundation for regulating the stone cell development from the level of physiological, biochemical and molecular biology. The main research results are as follows:
1. Slices observation of five types of pear fruit about the shone cell group and instensive situation of structure, the order is: Pyrus bretschneideri cv., Fung pear, Korla fragrant pear, Gong pear and apple pear. Among these pears, Pyrus bretschneideri cv. showed the largest stone cell group and the most densely distributed, Gong pear and Fung pear were secondary, Korla fragrant pear show the smaller stone cell group and distribute comparably scattered, and Apple pear show the smallest stone cell group and the most scattered distributed.
2. In the whole process of Pyrus bretschneideri cv. fruit development, stone cell content peaked at the stage of DPA51, the peak value is 17.83%; lignin content appeared two peak at the stage of DPA47 and DPA63, the peak value are 6.47% and 7.00% respectively; PAL activity peaked at the stage of DPA27, the value is 85.53U/g·h. The emergences of these three peaks showed some sequential relationship. PAL activity was first to reach the peak, then lignin content, the final one was stone cell content. Their trends were basically same, generally was first increased then declined. Besides, all of them showed significantly positive correlation.
3. The most suitable pH for Pyrus bretschneideri cv. fruit PAL activity was 9.2. The PAL activity was comparably stable at around pH9.0. The PAL activity depressed remarkably both at pH4.0 and pH10.0 buffer due to the conformational destruction. The most suitable reaction temperature was about 60℃.The PAL activity and reaction time present linear relationship when the reaction time within 180min. In addition, PAL has a high specificity on L-phenylalanine, and also shows some activity on L-Tyrosine. The most suitable substrate concentration of PAL is 0.0004~0.0012 mol/L. Dangshan pear fruit PAL has two Km values, Km1 is 0.7500mmol/L and Km2 is 1.0690mmol/L.
4. The PAL gene was cloned from the fruit of Pyrus bretschneideri cv.by RT-PCR. The sequence of the cDNA fragment was 582 base pairs and encoded a 195 amino acid protein. And it was found to have 90% homology with those of other plants PAL genes via multiple alignments. The catalytic active sites in PAL protein of other plants were also found in the deduced PAL protein. Phylogenetic tree analysis revealed that the PAL amino acid of the Pyrus bretschneideri cv. had closer relationship with the PAL amino acid of the Pyrunus avium. than other plants.
1、通过对五种梨果实切片观察发现砀山酥梨、贡梨和丰水梨石细胞团较大,分布较密集;砀山酥梨石细胞团最大,分布最密集;库尔勒香梨石细胞团较小,分布较疏散。苹果梨,石细胞团最小,分布最疏散。
2、在砀山酥梨果实整个发育过程中,石细胞含量于花后51天出现高峰,高峰值为17.83%;木质素含量于花后47天和花后63出现两个高峰,峰值分别为6.47%和7.00%;PAL活性最大值出现在花后27天,最大值为85.53∪/g·h。三者高峰期的出现在时间上表现出一定的时序性,PAL活性的高峰期最先出现,然后木质素含量出现高峰期,石细胞含量的高峰期最后出现。它们三者的变化趋势基本相同,总体上均呈先升高后下降的趋势,且三者之间均呈显著正相关。
3、砀山酥梨果实PAL的最适pH为9.2;在pH9.0左右时稳定性较高;用pH4.0和pH10.0的缓冲液处理均导致酶分子结构发生变化,活性降低;PAL最适反应温度为60℃左右,较耐热;当反应时间在180min内PAL的活性与反应时间呈线性关系;PAL对L-苯丙氨酸有较高的专一性,对L-酪氨酸也表现出一定的活性;最适底物浓度为0.0004～0.0012mol/L;砀山酥梨果实PAL的Km值有两个,Km_1为0.7500mmol/L,Km_2为1.0690 mmol/L。
4、克隆得到砀山酥梨果实PAL基因cDNA片段长度为585bp,推断编码195个氨基酸序列。该序列包含与其它植物PAL相同的活性催化位点,经序列分析和同源性比对,该氨基酸序列与其它植物PAL氨基酸序列的同源性在90%以上。系统进化树分析表明,砀山酥梨PAL氨基酸序列与蔷薇科的甜樱桃PAL氨基酸序列聚类关系最近。
Pyrus bretschneideri cv.originates in Dangshan, Anhui Province, which is one of the pear products for main cultivating and exporting. However, due to poor management, species degradation and many other factors in recent years, the stone cell content is significantly increased in the pear. What’s more, the flesh becomes thicker, more residue and taste variation than before, which severely decline the quality of Pyrus bretschneideri cv. and economic value. At the present time, reducing the stone cell content by utilizing biotechnology to improve the quality and enhance the market competitiveness of Pyrus bretschneideri cv. become an urgent question. In this experiment, we use Pyrus bretschneideri cv. fruit as research sample. We try to analyze the fluctuation of stone cell content, lignin content and the PAL activity, a critical enzyme to produce lignin, in the process of fruit development; and dissect the relationship between fruit stone cell development and PAL activity. In addition, in order to understand the nature of PAL, the characterization of the PAL was studied; and PAL gene was cloned at the same time. All of these provided a theoretical foundation for regulating the stone cell development from the level of physiological, biochemical and molecular biology. The main research results are as follows:
1. Slices observation of five types of pear fruit about the shone cell group and instensive situation of structure, the order is: Pyrus bretschneideri cv., Fung pear, Korla fragrant pear, Gong pear and apple pear. Among these pears, Pyrus bretschneideri cv. showed the largest stone cell group and the most densely distributed, Gong pear and Fung pear were secondary, Korla fragrant pear show the smaller stone cell group and distribute comparably scattered, and Apple pear show the smallest stone cell group and the most scattered distributed.
2. In the whole process of Pyrus bretschneideri cv. fruit development, stone cell content peaked at the stage of DPA51, the peak value is 17.83%; lignin content appeared two peak at the stage of DPA47 and DPA63, the peak value are 6.47% and 7.00% respectively; PAL activity peaked at the stage of DPA27, the value is 85.53U/g·h. The emergences of these three peaks showed some sequential relationship. PAL activity was first to reach the peak, then lignin content, the final one was stone cell content. Their trends were basically same, generally was first increased then declined. Besides, all of them showed significantly positive correlation.
3. The most suitable pH for Pyrus bretschneideri cv. fruit PAL activity was 9.2. The PAL activity was comparably stable at around pH9.0. The PAL activity depressed remarkably both at pH4.0 and pH10.0 buffer due to the conformational destruction. The most suitable reaction temperature was about 60℃.The PAL activity and reaction time present linear relationship when the reaction time within 180min. In addition, PAL has a high specificity on L-phenylalanine, and also shows some activity on L-Tyrosine. The most suitable substrate concentration of PAL is 0.0004~0.0012 mol/L. Dangshan pear fruit PAL has two Km values, Km1 is 0.7500mmol/L and Km2 is 1.0690mmol/L.
4. The PAL gene was cloned from the fruit of Pyrus bretschneideri cv.by RT-PCR. The sequence of the cDNA fragment was 582 base pairs and encoded a 195 amino acid protein. And it was found to have 90% homology with those of other plants PAL genes via multiple alignments. The catalytic active sites in PAL protein of other plants were also found in the deduced PAL protein. Phylogenetic tree analysis revealed that the PAL amino acid of the Pyrus bretschneideri cv. had closer relationship with the PAL amino acid of the Pyrunus avium. than other plants.
引文
[1]张力,于润卿.梨优良品种及其丰产优质栽培技术[M].北京:中国林业出版社, 2002, 192-199
[2]张兴旺.我国梨产销现状、存在的问题和解决措施[J].柑橘与亚热带果树信息, 2005, 21(2): 8-10
[3]李绍华.世界果树生产状况及提高我国果品市场竞争力对策[J].中国农业大学学报, 2003, 1(8): 7-13
[4]陶书田,张绍玲,乔永进,等.梨果实发育过程中石细胞团及几种相关酶活性变化的研究[J].果树学报, 2004, 6(21): 512-520
[5]刘小阳,李玲,高贵珍,等.光照对砀山酥梨果实发育过程中POD、PAL、PPO酶活性的影响研究[J].激光生物学报, 2008, 17(3): 95-298
[6]李玲,蔡永萍,刘小阳.梨果实的石细胞[J].植物生理学通讯, 2004, 40(5): 629-632
[7]中国农业百科全书编辑部.中国农业百科全书(果树卷)[M].北京:农业出版社, 1993
[8]张振铭,胡化广.不同品种梨果实石细胞含量的比较研究[J].安徽农学通报, 2007, 13(16): 28-29
[9]何凤仁.酥梨、鸭梨果实石细胞群研究[J].江苏农学院学报, 1988, 9(1): 35-36
[10]陶世蓉.梨果实结构与耐贮性及品质关系的研究[J].西北植物学报, 2000, 20(4): 544-548.
[11]山东省莱阳农学院编著.梨[M].科学出版社, 1978.
[12]颐模,林凤起,张冰冰.梨果肉结构的解剖研究[J].中国果树, 1989(4): 32-34
[13]刘小阳,李玲,宗梅,等.梨果实石细胞含量及其对梨品质的影响[J].安徽农业大学学报, 2004, 31(1): 106-196
[14]牟其芸,李文香,张华云,等.梨果实中石细胞含量测定及与果实品质相关性的研究[J].落叶果树,1996, 1: 7-9
[15]刘小阳,高贵珍.梨石细胞研究进展[J].安徽农业科学, 2007, 35(33): 10572-10574
[16]刘小阳,付金木,高贵珍.梨石细胞与石果病[J].安徽农业科学, 2008, 36 (2): 632-933
[17]阿拉木萨,李宝江.梨果实石细胞团的发育、分布及其对果实品质的影响[J].北方果树, 1994, 4: 4-6
[18]张华云,王善光,牟其芸,等.套袋对莱阳梨果皮结构和PPO、POD活性的影响[J].园艺学报, 1996, 23(1): 23-26
[19]赵广杰.木材细胞壁的构造及其主要成分的堆积过程[J].北京林业大学学报, 1999, 21(1): 74-78
[20]马俊颜,杨汝德,敖利刚.植物苯丙氨酸解氨酶生物学研究进展[J].现代食品科技, 2007, 23(7): 71-75
[21]李莉,赵越,马君兰.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, 2006, 5(4): 187-189
[22]贺立红,张进标,宾金华.苯丙氨酸解氨酶的研究进展[J].现代食品科技, 2006, 23(7): 71-75
[23]程水源,陈昆松,刘卫红,等.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, 5(20): 351-357
[24]张宽朝,金青,蔡永萍,等.苯丙氨酸解氨酶与其在重要次生代谢产物调控中的作用研究进展[J].中国农学通报, 2008, 24(12): 59-62
[25] Holger Ritter, Georg E. Schulz. Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase[J]. The Plant Cell, 2004, 16: 3426-3436
[26] Hanxiao Jiang, Karl V.Wood, John A.Morgan. Metabolic Engineering of the Phenylpropanoid Pathway in Saccharomyces cerevisiae[J]. Applied and Environmental Microbiology, 2005, 71(6): 2962-2969
[27] Lahoucine Achnine, Elison B.Blancaflor, Susanne Rasmussen, et al. Colocalization of L-Phenylalanine Ammonia-lyase and Cinnamate-4-Hydroxylase for Metabolic Channeling in Phenylpropanoid Biosynthesis[J]. The Plant Cell, 2004, 16: 3098-3109
[28]章霄云,郭安平,贺立卡,等.木质素生物合成及其基因调控的研究进展[J].分子植物育种, 2006, 4(3): 431-437
[29] P. Whiting, D. A. Goring. Chemical characterization of tissue fractions from the middlel -amella and secondary wall of black spruce tracheids[J]. Wood Science Technology, 1982, 16: 261-267
[30]付伟,廖祥儒,王俊峰,等.植物体内的木质素[J].生物学通报, 2004, 39(2): 12-14
[31]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, 4(28): 425-430
[32] Aldwin M. Anterola, Jae-Heung Jeon, Laurence B. Davin, et al. Transcriptional control of monolignol biosynthesis in Pinus taeda factors affecting monolignolratios and carbon allocation in phenylpropanoid metabolism[J]. Journal of Biology and Chemistry, 2002, 277( 21): 18272-18280
[33] Vincent J. H. Sewalt, Weiting Ni, Jack W. Blount, et al. Reduced lignin content and altered lignin composition in transgenic tobacco downregulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase[J]. Plant Physiol., 1997, 115: 41-50
[34] Jane Koukol, P. Miljanich, Eric E. Conn. The metabolism of aromatic compounds in higher plants. IV.Purification and properties of the-phenylalanine deaminase of Herdeum vulagare[J]. Journal of Biology and Chemistry, 1962, 237(10): 3223-3228
[35]薛应龙,欧阳光察,澳绍根,等.植物苯丙氨酸解氨酶的研究IV.水稻幼苗中PAL活性的动态变化[J].植物生理学报, 1983, 3(9): 301-305.
[36] Rajagopal Subramaniam, Susanne Reinold, Elizabeth K. Molitor, et a1. Structure, inheritance, and expression of hybrid poplar(Populus trichocarpa×Popu-lusdeltoids)phenylalanine ammonia-lyase genes[J]. Plant Physio1ogy, 1993, 102: 71-83
[37]胡美娇,刘秀娟,黄圣明.热处理后芒果、香蕉果皮PAL活性变化与炭疽病发生的关系[J].热带作物学报, 2000, 4(21): 63-68
[38]江柯,卢涛,赵德立,等.红酵母苯丙氨酸解氨酶的分离纯化及性质研究[J].四川大学学报(自然科学版), 2004, 4(41): 865-868
[39]冯守千,陈学森,张春雨,等.砂梨品种"满天红"及其芽变品系"奥冠"花青苷合成与相关酶活性研究[J].中国农业科学, 2008, 41(10): 3184-3190
[40]曹建康,姜微波.采后ASM诱导处理对鸭梨果实黑霉病的控制[J].园艺学报, 2005, 32(5): 783-78
[41]田晓艳,刘延吉.脱落酸对南果梨色素及部分合成关键酶的影响[J].北方园艺, 2008, 12: 155-156
[42] Amrita Kumar, Brian E. Ellis. The Phenylalanine Ammonia-Lyase Gene Family in Raspberry. Structure, Expression, and Evolution[J]. Plant Physiology, 2001, 127: 230–239
[43]徐晓梅,杨署光.苯丙氨酸解氨酶研究进展[J].安徽农业科学, 2009, 37(31): 15115-15119
[44] Cramer CL,Edwards K,Dron M. et al. Phenylalanine ammonia-lyase gene organization and structure[J]. Plant Molecular Biology, 1989, 12: 367-383
[45] Lois R, Dietrich A, Hahlbrock K, Schulz W.. A phenylalanine ammonia-lyasegene from parsley: structure. regulation, and identification of elicitor and light responsive cis-acting element[J]. European Molecular Biology Organization Journal, 1989, 8(6): 1641-1648
[46] YeoY. S, Lee S. W, Kim Y. H et al. Resteiction mapping of phenylalanine annonia-lyade gene family on tomato (Lycopersicon esculentum) [J]. Rural Development Administration Journal of Agricultural Science, Biotechnology, 1994, 36(2): 187~192
[47] Joos H. J, Hahlbroxk K. Phenylalanine ammonia-lyase in potato (Solanum tuberosum L.):genomic complexity, structural comparison of two selected genes and modes of expression[J]. European Journal of Biochemistry, 1992, 204(2): 621-629.
[48] Fukazawa-Akada T, Kung S. D, Watson J. C. Phenylalanine ammonia-lyase gene , structure, expression, and evolution in Nicitiana[J]. Plant Molecular Biology, 1996, 30: 711~722.
[49] Whetten R.W, Sederoff R. R. Phenylalanine ammonia-lyase from loblolly pine[J]. Plant Physiology, 1992, 98: 380~386.
[50] Gowri G, Paiva N. L, Dixon R. A. Stress responses in alfalfa (Medicago sativa L.) Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants[J]. Plant Molecular Biology, 1991, 17(3): 415~429.
[51] Ei-ichi Minami, Yoshihiro Ozeki, Makoto Matsuoka, et al . Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants[J]. European Journal of Bio-chemistry, 1989, 185: 19~25
[52] Liang X, Dron M, Schmid J, et al. Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environ mental cues[J]. The Journal of Biological Chemistry, 1989, 264(24): 14486~14492
[53]陈新,万德光,严寿云,等.银杏苯丙氨酸解氨酶(PAL)基因的克隆[J].成都中医药大学学报,2004,27(1):32-34
[54]王燕,许锋,杜何为.夹竹桃苯丙氨酸解氨酶的基因克隆与序列分析[J].华北农学报, 2007, 4(22): 19-24
[55]陈雅平,陈云凤,陈启助,大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析[J].热带亚热带植物学报, 2007, 5(15): 421-427
[56]许锋,陈柳吉,蔡荣,等.海桐花苯丙氨酸解氨酶的基因克隆与序列分析[J].西北农业学报, 2008, 2(17): 218-224
[57]薛永常,李金花,卢孟柱,等. 107杨次生木质部PAL基因的RT-PCR扩增及其鉴定[J].林业科学, 2004, 40(4): 193-197
[58]王燕,张凤霞,朱俊,等.核桃苯丙氨酸结案酶的基因克隆与序列分析[J].湖北农业科学, 2008, 47(6): 622-626
[59] Feng Xu, Rong Cai, Shuiyuan Cheng, et al. Molecular cloning,characterization and expression of phenylalanine ammonia-lyase gene from Ginkgo biloba[J]. African Journal of Biotechnology, 2008, 6(7): 721-729
[60] L. Pellegrini, O. Rohfritsch, B. Fritig and M., et al. Phenylalanine Ammonia-Lyase in Tobacco (Molecular Cloning and Gene Expression during the Hypersensitive Reaction to Tobacco Mosaic Virus and the Response to a Fungal Elicitor)[J]. Plant Physiology, 1994, 106: 877-886
[61]徐义流.砀山酥梨[M].中国农业出版社,2009.
[62] Shutian Tao, Shahrokh K. hanizadeh, Hua Zhang. 2009. Anatomy, ultrastructure and lignin distribution of stone cells in two Pyrus species[J]. Plant Science, 176: 413-419
[63]吴少华.梨果肉石细胞的研究[J].福建农业大学学报, 1996, 25(1): 29-32
[64]聂敬全,蔡永萍,张士鸿,等.砀山酥梨果实石细胞与薄壁细胞发育关系的解剖学研究[J].园艺学报, 2009, 36(8): 1209-1214
[65]聂继云,李静,杨振锋,等.冷冻法测定梨的石细胞含量[J].果树学报, 2006, 23(1): 133-135
[66]贾彩红,王宏芝,杜克久,等.抑制4CL基因表达的转基因毛白杨中木质素含量与茎秆颜色的关系[J].农业生物技术学报, 2004,12(6): 621-624
[67]樊洪泓,李廷春,林毅,等.石斛总RNA提取方法的研究[J].激光生物学报, 2007, 16(1): 105-108
[68]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].中国农学通报, 2006, 22(7): 282-286.
[69]江力,袁怀波,张世杰,等.山药苯丙氨酸解氨酶特性的研究[J].食品科学, 2006, 27(10): 36-40.
[70]王镜岩,朱圣庚,徐长法.生物化学[M].高等教育出版社, 2002.
[71]欧阳光察,应初衍,沃绍根,等.植物苯丙氨酸解氨酶的研究.Ⅵ.水稻、小麦PAL的纯化及基本特性[J].植物生理学报, 1985, l1 (2): 204-214
[2]张兴旺.我国梨产销现状、存在的问题和解决措施[J].柑橘与亚热带果树信息, 2005, 21(2): 8-10
[3]李绍华.世界果树生产状况及提高我国果品市场竞争力对策[J].中国农业大学学报, 2003, 1(8): 7-13
[4]陶书田,张绍玲,乔永进,等.梨果实发育过程中石细胞团及几种相关酶活性变化的研究[J].果树学报, 2004, 6(21): 512-520
[5]刘小阳,李玲,高贵珍,等.光照对砀山酥梨果实发育过程中POD、PAL、PPO酶活性的影响研究[J].激光生物学报, 2008, 17(3): 95-298
[6]李玲,蔡永萍,刘小阳.梨果实的石细胞[J].植物生理学通讯, 2004, 40(5): 629-632
[7]中国农业百科全书编辑部.中国农业百科全书(果树卷)[M].北京:农业出版社, 1993
[8]张振铭,胡化广.不同品种梨果实石细胞含量的比较研究[J].安徽农学通报, 2007, 13(16): 28-29
[9]何凤仁.酥梨、鸭梨果实石细胞群研究[J].江苏农学院学报, 1988, 9(1): 35-36
[10]陶世蓉.梨果实结构与耐贮性及品质关系的研究[J].西北植物学报, 2000, 20(4): 544-548.
[11]山东省莱阳农学院编著.梨[M].科学出版社, 1978.
[12]颐模,林凤起,张冰冰.梨果肉结构的解剖研究[J].中国果树, 1989(4): 32-34
[13]刘小阳,李玲,宗梅,等.梨果实石细胞含量及其对梨品质的影响[J].安徽农业大学学报, 2004, 31(1): 106-196
[14]牟其芸,李文香,张华云,等.梨果实中石细胞含量测定及与果实品质相关性的研究[J].落叶果树,1996, 1: 7-9
[15]刘小阳,高贵珍.梨石细胞研究进展[J].安徽农业科学, 2007, 35(33): 10572-10574
[16]刘小阳,付金木,高贵珍.梨石细胞与石果病[J].安徽农业科学, 2008, 36 (2): 632-933
[17]阿拉木萨,李宝江.梨果实石细胞团的发育、分布及其对果实品质的影响[J].北方果树, 1994, 4: 4-6
[18]张华云,王善光,牟其芸,等.套袋对莱阳梨果皮结构和PPO、POD活性的影响[J].园艺学报, 1996, 23(1): 23-26
[19]赵广杰.木材细胞壁的构造及其主要成分的堆积过程[J].北京林业大学学报, 1999, 21(1): 74-78
[20]马俊颜,杨汝德,敖利刚.植物苯丙氨酸解氨酶生物学研究进展[J].现代食品科技, 2007, 23(7): 71-75
[21]李莉,赵越,马君兰.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, 2006, 5(4): 187-189
[22]贺立红,张进标,宾金华.苯丙氨酸解氨酶的研究进展[J].现代食品科技, 2006, 23(7): 71-75
[23]程水源,陈昆松,刘卫红,等.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, 5(20): 351-357
[24]张宽朝,金青,蔡永萍,等.苯丙氨酸解氨酶与其在重要次生代谢产物调控中的作用研究进展[J].中国农学通报, 2008, 24(12): 59-62
[25] Holger Ritter, Georg E. Schulz. Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase[J]. The Plant Cell, 2004, 16: 3426-3436
[26] Hanxiao Jiang, Karl V.Wood, John A.Morgan. Metabolic Engineering of the Phenylpropanoid Pathway in Saccharomyces cerevisiae[J]. Applied and Environmental Microbiology, 2005, 71(6): 2962-2969
[27] Lahoucine Achnine, Elison B.Blancaflor, Susanne Rasmussen, et al. Colocalization of L-Phenylalanine Ammonia-lyase and Cinnamate-4-Hydroxylase for Metabolic Channeling in Phenylpropanoid Biosynthesis[J]. The Plant Cell, 2004, 16: 3098-3109
[28]章霄云,郭安平,贺立卡,等.木质素生物合成及其基因调控的研究进展[J].分子植物育种, 2006, 4(3): 431-437
[29] P. Whiting, D. A. Goring. Chemical characterization of tissue fractions from the middlel -amella and secondary wall of black spruce tracheids[J]. Wood Science Technology, 1982, 16: 261-267
[30]付伟,廖祥儒,王俊峰,等.植物体内的木质素[J].生物学通报, 2004, 39(2): 12-14
[31]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, 4(28): 425-430
[32] Aldwin M. Anterola, Jae-Heung Jeon, Laurence B. Davin, et al. Transcriptional control of monolignol biosynthesis in Pinus taeda factors affecting monolignolratios and carbon allocation in phenylpropanoid metabolism[J]. Journal of Biology and Chemistry, 2002, 277( 21): 18272-18280
[33] Vincent J. H. Sewalt, Weiting Ni, Jack W. Blount, et al. Reduced lignin content and altered lignin composition in transgenic tobacco downregulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase[J]. Plant Physiol., 1997, 115: 41-50
[34] Jane Koukol, P. Miljanich, Eric E. Conn. The metabolism of aromatic compounds in higher plants. IV.Purification and properties of the-phenylalanine deaminase of Herdeum vulagare[J]. Journal of Biology and Chemistry, 1962, 237(10): 3223-3228
[35]薛应龙,欧阳光察,澳绍根,等.植物苯丙氨酸解氨酶的研究IV.水稻幼苗中PAL活性的动态变化[J].植物生理学报, 1983, 3(9): 301-305.
[36] Rajagopal Subramaniam, Susanne Reinold, Elizabeth K. Molitor, et a1. Structure, inheritance, and expression of hybrid poplar(Populus trichocarpa×Popu-lusdeltoids)phenylalanine ammonia-lyase genes[J]. Plant Physio1ogy, 1993, 102: 71-83
[37]胡美娇,刘秀娟,黄圣明.热处理后芒果、香蕉果皮PAL活性变化与炭疽病发生的关系[J].热带作物学报, 2000, 4(21): 63-68
[38]江柯,卢涛,赵德立,等.红酵母苯丙氨酸解氨酶的分离纯化及性质研究[J].四川大学学报(自然科学版), 2004, 4(41): 865-868
[39]冯守千,陈学森,张春雨,等.砂梨品种"满天红"及其芽变品系"奥冠"花青苷合成与相关酶活性研究[J].中国农业科学, 2008, 41(10): 3184-3190
[40]曹建康,姜微波.采后ASM诱导处理对鸭梨果实黑霉病的控制[J].园艺学报, 2005, 32(5): 783-78
[41]田晓艳,刘延吉.脱落酸对南果梨色素及部分合成关键酶的影响[J].北方园艺, 2008, 12: 155-156
[42] Amrita Kumar, Brian E. Ellis. The Phenylalanine Ammonia-Lyase Gene Family in Raspberry. Structure, Expression, and Evolution[J]. Plant Physiology, 2001, 127: 230–239
[43]徐晓梅,杨署光.苯丙氨酸解氨酶研究进展[J].安徽农业科学, 2009, 37(31): 15115-15119
[44] Cramer CL,Edwards K,Dron M. et al. Phenylalanine ammonia-lyase gene organization and structure[J]. Plant Molecular Biology, 1989, 12: 367-383
[45] Lois R, Dietrich A, Hahlbrock K, Schulz W.. A phenylalanine ammonia-lyasegene from parsley: structure. regulation, and identification of elicitor and light responsive cis-acting element[J]. European Molecular Biology Organization Journal, 1989, 8(6): 1641-1648
[46] YeoY. S, Lee S. W, Kim Y. H et al. Resteiction mapping of phenylalanine annonia-lyade gene family on tomato (Lycopersicon esculentum) [J]. Rural Development Administration Journal of Agricultural Science, Biotechnology, 1994, 36(2): 187~192
[47] Joos H. J, Hahlbroxk K. Phenylalanine ammonia-lyase in potato (Solanum tuberosum L.):genomic complexity, structural comparison of two selected genes and modes of expression[J]. European Journal of Biochemistry, 1992, 204(2): 621-629.
[48] Fukazawa-Akada T, Kung S. D, Watson J. C. Phenylalanine ammonia-lyase gene , structure, expression, and evolution in Nicitiana[J]. Plant Molecular Biology, 1996, 30: 711~722.
[49] Whetten R.W, Sederoff R. R. Phenylalanine ammonia-lyase from loblolly pine[J]. Plant Physiology, 1992, 98: 380~386.
[50] Gowri G, Paiva N. L, Dixon R. A. Stress responses in alfalfa (Medicago sativa L.) Sequence analysis of phenylalanine ammonia-lyase (PAL) cDNA clones and appearance of PAL transcripts in elicitor-treated cell cultures and developing plants[J]. Plant Molecular Biology, 1991, 17(3): 415~429.
[51] Ei-ichi Minami, Yoshihiro Ozeki, Makoto Matsuoka, et al . Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants[J]. European Journal of Bio-chemistry, 1989, 185: 19~25
[52] Liang X, Dron M, Schmid J, et al. Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environ mental cues[J]. The Journal of Biological Chemistry, 1989, 264(24): 14486~14492
[53]陈新,万德光,严寿云,等.银杏苯丙氨酸解氨酶(PAL)基因的克隆[J].成都中医药大学学报,2004,27(1):32-34
[54]王燕,许锋,杜何为.夹竹桃苯丙氨酸解氨酶的基因克隆与序列分析[J].华北农学报, 2007, 4(22): 19-24
[55]陈雅平,陈云凤,陈启助,大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析[J].热带亚热带植物学报, 2007, 5(15): 421-427
[56]许锋,陈柳吉,蔡荣,等.海桐花苯丙氨酸解氨酶的基因克隆与序列分析[J].西北农业学报, 2008, 2(17): 218-224
[57]薛永常,李金花,卢孟柱,等. 107杨次生木质部PAL基因的RT-PCR扩增及其鉴定[J].林业科学, 2004, 40(4): 193-197
[58]王燕,张凤霞,朱俊,等.核桃苯丙氨酸结案酶的基因克隆与序列分析[J].湖北农业科学, 2008, 47(6): 622-626
[59] Feng Xu, Rong Cai, Shuiyuan Cheng, et al. Molecular cloning,characterization and expression of phenylalanine ammonia-lyase gene from Ginkgo biloba[J]. African Journal of Biotechnology, 2008, 6(7): 721-729
[60] L. Pellegrini, O. Rohfritsch, B. Fritig and M., et al. Phenylalanine Ammonia-Lyase in Tobacco (Molecular Cloning and Gene Expression during the Hypersensitive Reaction to Tobacco Mosaic Virus and the Response to a Fungal Elicitor)[J]. Plant Physiology, 1994, 106: 877-886
[61]徐义流.砀山酥梨[M].中国农业出版社,2009.
[62] Shutian Tao, Shahrokh K. hanizadeh, Hua Zhang. 2009. Anatomy, ultrastructure and lignin distribution of stone cells in two Pyrus species[J]. Plant Science, 176: 413-419
[63]吴少华.梨果肉石细胞的研究[J].福建农业大学学报, 1996, 25(1): 29-32
[64]聂敬全,蔡永萍,张士鸿,等.砀山酥梨果实石细胞与薄壁细胞发育关系的解剖学研究[J].园艺学报, 2009, 36(8): 1209-1214
[65]聂继云,李静,杨振锋,等.冷冻法测定梨的石细胞含量[J].果树学报, 2006, 23(1): 133-135
[66]贾彩红,王宏芝,杜克久,等.抑制4CL基因表达的转基因毛白杨中木质素含量与茎秆颜色的关系[J].农业生物技术学报, 2004,12(6): 621-624
[67]樊洪泓,李廷春,林毅,等.石斛总RNA提取方法的研究[J].激光生物学报, 2007, 16(1): 105-108
[68]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].中国农学通报, 2006, 22(7): 282-286.
[69]江力,袁怀波,张世杰,等.山药苯丙氨酸解氨酶特性的研究[J].食品科学, 2006, 27(10): 36-40.
[70]王镜岩,朱圣庚,徐长法.生物化学[M].高等教育出版社, 2002.
[71]欧阳光察,应初衍,沃绍根,等.植物苯丙氨酸解氨酶的研究.Ⅵ.水稻、小麦PAL的纯化及基本特性[J].植物生理学报, 1985, l1 (2): 204-214