鸭梨PAL克隆及其在果实发育和机械伤害过程中的表达

doi:10.3864/j.issn.0578-1752.2014.21.019

摘要/Abstract

摘要： 【目的】克隆鸭梨苯丙氨酸解氨酶基因（PAL）序列，并分析其在果实发育阶段和机械伤害时的表达模式，为研究鸭梨褐变机制提供理论依据。【方法】采用同源克隆方法，获得鸭梨PAL全长序列；利用在线生物信息学软件对其进行分析；采用MEGA 5.1软件构建PAL蛋白系统进化树；利用实时荧光定量PCR技术分析PAL在鸭梨果实发育阶段及受机械伤害过程中的表达。【结果】在鸭梨中获得了2个含完整CDS区的PAL，分别命名为PbPAL1和PbPAL2。PbPAL1 cDNA序列全长为2 232 bp，含2 160 bp完整开放阅读框、60 bp的5′非翻译区及12 bp的3′非翻译区，GenBank登录号为GU906268.1；PbPAL2 cDNA序列全长为2 387 bp，含2 163 bp完整开放阅读框、14 bp的5′非翻译区及210 bp的3′非翻译区，GenBank登录号为GU906269.1。系统进化分析表明PbPAL1和PbPAL2位于分子进化树的不同分支，PbPAL1与西洋梨（Pyrus communis）的PAL同源性较高，而PbPAL2与桃（Prunus persica）的PAL同源性较高。定量PCR分析表明，随着鸭梨果实发育成熟，果皮、果肉和果心的PbPAL1和PbPAL2表达量各异，但均呈下降趋势；且果实发育早期果心中的PbPAL1和PbPAL2表达量显著高于果皮和果肉。损伤处理可迅速诱导鸭梨的果肉褐变，果皮褐变出现较晚；同时，损伤刺激果皮和果肉组织中PbPAL1和PbPAL2表达上调。果皮中PbPAL1表达量在损伤处理后6 h达到高峰，而PbPAL2在损伤处理后48 h才显著上升；果肉中PbPAL1在损伤处理后1 h表达量开始显著上升，而PbPAL2在损伤处理后12 h才显著上升。【结论】鸭梨PbPAL1和PbPAL2属于2个不同的PAL类型，其表达随果实发育呈下降趋势，机械损伤过程中，两基因表达显著上调，表明其参与了损伤引起的组织褐变过程。

关键词: 鸭梨, 苯丙氨酸解氨酶, 褐变, 机械损伤, 果实发育

Abstract: 【Objective】 The objective of this study is to investigate the phenylalanine ammonia-lyase gene (PAL) sequence characters and expression pattern during fruit development and tissue browning in ‘Yali’ pear.【Method】Got full coding domain sequence of PAL by homology-based cloning, the bioinformatic characteristics of the cloned PAL were analyzed using online service, PAL protein phylogenetic tree was constructed by MEGA 5.1 software, analyzed the expression pattern of different development stages of fruit and tissue browning of ‘Yali’ pear by real-time quantitative RT-PCR. 【Result】 Two PAL genes containing full length CDS were obtained and termed PbPAL1 and PbPAL2, respectively. The full length cDNA of PbPAL1 was 2 232 bp, containing 2 160 bp open reading frame, 60 bp 5′ untranslated region and 12 bp 3′ untranslated region (GeneBank Access Number: GU906268.1). The full length cDNA of PbPAL2 was 2 387 bp, containing 2 163 bp open reading frame, 14 bp 5′ untranslated region and 210 bp 3′ untranslated region (GeneBank Access Number: GU906269.1). Phylogenetic analysis indicated that PbPAL1 and PbPAL2 were classified into two different phylogenetic tree branches. PbPAL1 was highly homologous to PAL of Pyrus communis, while PbPAL2 was highly homologous to PAL of Prunus persica. Although the expression levels of the PbPAL1 and PbPAL2 were different in the peel, flesh and core, all of the expression patterns showed a down trend along with fruit development, and were significantly higher in core than those in peel and flesh at early development stage. Wounding treatment could rapidly induce browning of flesh, while slowly induce the browning of peel. In both flesh and peel, the expression levels of PbPAL1 and PbPAL2 were markedly up-regulated by wounding treatment. In peel, the expression level of PbPAL1 appeared a peak at 6 h after wounding treatment, while PbPAL2 mRNA level significantly increased at 48 h after treatment. In flesh, the expression level of PbPAL1 obviously increased at 1 h after wounding treatment, and PbPAL2 began to rise at 12 h after wounding treatment.【Conclusion】PbPAL1 and PbPAL2 belong to different PAL gene types, and their expression showed a decreasing trend along with fruit development, and up-regulated by wounding, thus, they were involved in tissue browning induced by wounding in ‘Yali’ pear.

Key words: ‘Yali&rsquo, pear, phenylalanine ammonia-lyase, browning, wounding, fruit development

闫洪波，程玉豆，何近刚，葛文雅，杨坤，关军锋. 鸭梨PAL克隆及其在果实发育和机械伤害过程中的表达[J]. 中国农业科学, 2014, 47(21): 4341-4348.

YAN Hong-bo, CHENG Yu-dou, HE Jin-gang, GE Wen-ya, YANG Kun, GUAN Jun-feng. Cloning of PAL Gene in ‘Yali’ Pear and Its Expression During Fruit Development and Wounding[J]. Scientia Agricultura Sinica, 2014, 47(21): 4341-4348.

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参考文献

[1] 吴耕西, 周宏伟, 汪建民. 鸭梨酶促褐变的生化机制及底物鉴定. 园艺学报, 1992, 19(3):198-202.

Wu G X, Zhou H W, Wang J M. Biochemical mechanism and substances determination of enzymic browning of ‘Yali’ pear (Pyrus Bretschneideri Rehd.). Acta Horticulture Sinica, 1992, 19(3): 198-202. (in Chinese)

[2] Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W. Lignin biosynthesis and structure. Plant Physiology, 2010, 153(3): 895-905.

[3] Achnine D R. Natural products and plant disease resistance. Nature, 2001, 411, 843-847.

[4] Holgre R, Georg E. Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia- lyase. The Plant Cell, 2004, 16(12): 3426-3436.

[5] Achnine L, Blancaflor E B, Rasmussen S, Dixon R A. Colocalization of L-phenylalanine ammonialyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. The Plant Cell, 2004, 16(11): 3098-3109.

[6] 程水源, 陈昆松, 刘卫红, 杜何为. 植物苯丙氨酸解氨酶基因的表达调控与研究展望. 果树学报, 2003, 20(5): 351-357.

Cheng S S, Chen K S, Liu W H, Du H W. Regulation and expression of the PAL in plant and its outlook. Journal of Fruit Science, 2003, 20(5): 351-357. (in Chinese)

[7] 鞠志国, 刘成连, 原永兵, 戴洪义, 阎升平, 徐军. 莱阳茌梨酚类物质合成的调节及其对果实品质的影响. 中国农业科学, 1993, 26(4): 44-48.

Ju Z G, Liu C L, Yuan Y B, Dai H Y, Yan S P, Xu J. Regulation of phenolics synthesis and its effects on fruit quality of Laiyang Chili (pear). Scientia Agricultura Sinica, 1993, 26(4): 44-48. (in Chinese)

[8] Huang J L, Gu M, Lai Z, Fan B, Shi K, Zhou Y H, Yu J Q, Chen Z. Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress. Plant Physiology, 2010, 153(4): 1526-1538.

[9] Reichert A I, He X Z, Dixon R A. Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): Characterization of the four tobacco PAL genes and active heterotetrameric enzymes. Biochemical Journal, 2009, 424(2): 233-242.

[10] Chang A, Lim M H, Lee S W, Robb E J, Nazar R N. Tomato phenylalanine ammonia-lyase gene family, highly redundant but strongly underutilized. The Journal of Biological Chemistry, 2008, 283(48): 33591-33601.

[11] Liang X W, Dron M, Cramer C L, Dixon R A, Lamb C J. Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues. The Journal of Biological Chemistry, 1989, 264(24): 14486-14492.

[12] 荣瑞芬, 郭堃, 厉重先, 李丽云, 李祖明. 紫外照射诱导采后番茄苯丙氨酸解氨酶的分离纯化. 北方园艺, 2007, 12: 1-4.

Rong R F, Guo K, Li C X, Li L Y, Li Z M. Purification and properties of PAL from harvested tomato induced by UV-C irradiation. Northern Horticulture, 2007, 12: 1-4. (in Chinese)

[13] 马俊彦, 杨汝德, 敖利刚. 植物苯丙氨酸解氨酶的生物学研究进展. 现代食品科技, 2007, 23(7): 71-74.

Ma J Y, Yang R D, Ao L G. Progress in biological research of phenylalanine ammonialyase. Modern Food Science and Technology, 2007, 23(7): 71-74. (in Chinese)

[14] Kumar A, Ellis B E. The phenylalanine ammonia-lyase gene family in raspberry. Structure, expression, and evolution. Plant Physiology, 2001, 127(1): 230-239.

[15] 李正国, 高雪, 樊晶, 杨迎伍, 李道高, Kanellis A K. 奉节脐橙果实苯丙氨酸解氨酶活性及其基因表达与果皮褐变的关系. 植物生理与分子生物学学报, 2006, 32(3): 381-386.

Li Z G, Gao X, Fan J, Yang Y W, Li D G, Kanellis A K. The Relationship between activity and gene expression of phenylalanine ammonia-lyase and peel pitting in ‘Fengjie’ navel orange fruits. Journal of Plant Physiology and Molecular Biology, 2006, 32(3): 381-386. (in Chinese)

[16] 张士鸿, 金青, 樊洪, 黄蓓, 蔡永萍, 林毅, 张金云, 徐义流. 砀山酥梨果实苯丙氨酸解氨酶基因cDNA克隆及序列分析. 激光生物学报, 2010, 19(1): 97-103.

Zhang S H, Jin Q, Fan H, Huang B, Cai Y P, Lin Y, Zhang J Y, Xu Y L. Cloning and sequencing of a phenylalanine ammonia-lyase cDNA from Pyrus bretschneideri cv. Acta Laser Biology Sinica, 2010, 19(1): 97-103. (in Chinese)

[17] Gasic K, Hernandez A, Korban S S. RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Molecular Biology Reporter, 2004, 22(4): 437-438.

[18] 林智华, 林程忠, 梁红, 周玲艳. 猕猴桃基因组DNA提取的研究. 安徽农业科学, 2009, 37(3): 996-998.

Lin Z H, Lin C Z, Liang H, Zhou L Y. Study on extraction of Actinidia genomic DNA. Journal of Anhui Agricultural Sciences, 2009, 37(3): 996-998. (in Chinese)

[19] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology Evolution, 2011, 28(10): 2731-2739.

[20] 闫洪波, 葛文雅, 杨坤, 程玉豆, 关军锋. 鸭梨肌动蛋白2基因克隆和表达分析. 河北农业大学学报, 2012, 35(1): 27-30.

Yan H B, Ge W Y, Yang K, Cheng Y D, Guan J F. Cloning and expression of actin2 gene in ‘Yali’ pear. Journal of Hebei Agricultural University, 2012, 35(1): 27-30. (in Chinese)

[21] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 2001, 25(4): 402-408.

[22] 张振铭, 施泽斌, 张绍铃, 乔勇进, 陶书田. 砀山酥梨不同发育时期套袋对石细胞发育的影响. 园艺学报, 2007, 43(3): 565-568.

Zhang Z M, Shi Z B, Zhang S L, Qiao Y J, Tao S T. Bagging at different developmental stages on sclereid formation in ‘Dangshan su’ pear fruit. Acta Horticulturae Sinica, 2007, 43(3): 565-568. (in Chinese)

[23] 王斌, 张楠, 闫冲冲, 金青, 林毅, 蔡永萍, 张金云. 套袋对砀山酥梨果实石细胞发育及木质素代谢的影响. 园艺学报, 2013, 40(3): 531-539.

Wang B, Zhang N, Yan C C, Jin Q, Lin Y, Cai Y P, Zhang Y J. Bagging for the development of stone cell and metabolism of lignin in Pyrus bretschneideri ‘Dangshan Suli’. Acta Horticulturae Sinica, 2013, 40(3): 531-539. (in Chinese)

[24] 俞雅琼, 董明, 王旭东, 张国庆, 李娜. 机械损伤对砀山酥梨采后生理生化变化的影响. 保鲜与加工, 2011, 11(3): 10-15.

Yu Y Q, Dong M, Wang X D, Zhang G Q, Li N. Effects of mechanical damage on postharvest physiology and biochemistry of ‘Dangshan’ pear. Storage and Process, 2011, 11(3): 10-15. (in Chinese)

[25] 魏敏, 周会玲, 陈小利, 辛付存. 低温贮藏对鲜切富士苹果褐变的影响. 西北林学院学报, 2011, 26(5): 131-134.

Wei M, Zhou H L, Chen X l, Xin F C. Effect s of storage temperature on browning of fresh cut ’Fuji’ apples. Journal of Northwest A & F University, 2011, 26(5): 131-134. (in Chinese)

[26] Nguyen T B T, Ketsa S, Van Doorn W G. Relationship between browning and the activities of polyphenol oxidase and phenylalanine ammonia lyase in banana peel during low temperature storage. Postharvest Biology and Technology, 2003, 30(2): 187-193.

[27] López-Galvez G, Saltveit M, Cantwell M. Wound-induced phenylalanine ammonia lyase activity, factors affecting its induction and correlation with the quality of minimally processed lettuces. Postharvest Biology and Technology, 1996, 9(2):223-233.

[28] Kato M, Hayakawa Y, Hyodo H, Ikoma Y, Yano M. Wound induced ethylene synthesis and expression and formation of 1-aminocyclopropane- 1-carboxylate (ACC) synthase, ACC oxidase, phenylalanine ammonia- lyase, and peroxidase in wounded mesocarp tissue of Cucurbita maxima. Plant Cell Physiology, 2000, 41(4): 440-447.

[29] Kao Y, Harding S A, Tsai C. Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking aspen. Plant Physiology, 2002, 130(2): 796-807.