云锦杜鹃苯丙氨酸解氨酶基因的克隆及功能分析

Cloning and functional analysis of the phenylalaninammo-nialyase gene from Rhododendron fortunei

摘要:

苯丙氨酸解氨酶(phenylalaninammo-nialyase，PAL) 是植物香气化合物中苯甲酸甲酯合成途径的关键酶。为探究云锦杜鹃Rhododendron fortunei RhPAL基因的功能，利用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends，RACE) 技术克隆RhPAL基因全长cDNA序列，并应用高效液相色谱-质谱(liquid chromatography-mass spectrometry，LC-MS) 联用技术分析云锦杜鹃不同发育阶段的不同组织中RhPAL基因表达水平和代谢物含量变化关系。结果表明，从云锦杜鹃花cDNA中克隆得到RhPAL基因，含有完整的cDNA开放阅读框(open reading frame，ORF) 序列长2 145 bp，编码715个氨基酸，与其他物种的PAL蛋白质氨基酸序列具有较高的同源性；qRT-PCR分析表明，在不同花期的花瓣中，RhPAL表达量呈上升趋势，在衰败期表达量最高，雌蕊的表达量远远高于雄蕊，同时，新叶高于老叶，叶片的表达量高于花瓣和花蕊。通过酶联检疫法检测分析云锦杜鹃不同花期的花瓣中PAL酶活性，在花苞期酶活性最高，衰败期最低，总体呈下降趋势。通过LC-MS技术，检测分析云锦杜鹃中苯丙氨酸(phenylalanine，L-Phe) 和肉桂酸(cinnamic acid，Ca) 的含量，发现在云锦杜鹃不同组织中苯丙氨酸和肉桂酸的含量变化趋势与RhPAL基因表达水平相一致，且均与RhPAL基因表达量呈高度相关。推测RhPAL在云锦杜鹃花香物质合成中发挥关键作用，本研究结果可为研究杜鹃花香气物质分子调控机制提供理论依据。

关键词:杜鹃;高效液相色谱-质谱联用;苯丙氨酸解氨酶;基因克隆;生物信息学分析;肉桂酸

Abstract:

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.

Key words:Rhododendron;LC-MS;PAL;gene clone;bioinformatics analysis;cinnamic acid