鸭梨PAL克隆及其在果实发育和机械伤害过程中的表达

【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。

Cloning of PAL Gene in ‘Yali’ Pear and Its Expression DuringFruit Development and Wounding

【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.