甘蔗苯丙氨酸解氨酶基因（PAL）的克隆和表达分析

doi:10.3864/j.issn.0578-1752.2013.14.002

中国农业科学 ›› 2013, Vol. 46 ›› Issue (14): 2856-2868.doi: 10.3864/j.issn.0578-1752.2013.14.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

甘蔗苯丙氨酸解氨酶基因（PAL）的克隆和表达分析

宋修鹏1, 黄杏2, 莫凤连1, 田丹丹3, 杨丽涛12, 李杨瑞12, 陈保善1

1.广西大学农学院/亚热带农业生物资源保护与利用国家重点实验室，南宁 530004
2.中国农业科学院甘蔗研究中心/农业部广西甘蔗生物技术与遗传改良重点实验室/广西农业科学院甘蔗研究所/广西甘蔗遗传改良重点实验室，南宁 530007
3.广西农业科学院生物技术研究所，南宁 530007

收稿日期:2013-03-05 出版日期:2013-07-15 发布日期:2013-05-15
通讯作者: 通信作者杨丽涛，E-mail：liyr@gxu.edu.cn
作者简介:宋修鹏，E-mail：xiupengsong@163.com
基金资助:
国家“863”计划课题（2013AA102604）、国家国际合作项目(2013DFA31600)、广西科学研究与技术开发计划项目（桂科产1123008-1，桂科攻1222009）、自治区主席科技资金项目（11166-02）

Cloning and Expression Analysis of Sugarcane Phenylalanin Ammonia-lyase (PAL) Gene

SONG Xiu-Peng-1, HUANG Xing-2, MO Feng-Lian-1, TIAN Dan-Dan-3, YANG Li-Tao-12, LI Yang-Rui-12, CHEN Bao-Shan-1

1.College of Agriculture, Guangxi University/State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Nanning 530004
2.Sugarcane Research Center, Chinese Academy of Agricultural Sciences/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture/Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences / Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007
3.Biotechnology Research Institute, Guangxi Academy of Agriculture Sciences, Nanning 530007

Received:2013-03-05 Online:2013-07-15 Published:2013-05-15

摘要/Abstract

摘要： 【目的】克隆甘蔗的苯丙氨酸解氨酶基因（PAL），分析其序列特征及其在不同组织和5种胁迫条件下的表达情况，为此基因在甘蔗抗逆育种中的应用提供理论支撑。【方法】利用串联飞行时间质谱仪对差异表达蛋白质进行质谱鉴定分析，通过RT-PCR和RACE技术从甘蔗品种新台糖22号（ROC22）中克隆PAL，以生物信息学方法对其序列进行预测分析，利用real-time PCR分析PAL在不同组织和不同胁迫条件下的表达特性。【结果】克隆获得甘蔗PAL，命名为ScPAL，GenBank登录号为KC172559。该cDNA全长2 590 bp，含有1个2 115 bp的完整开放阅读框（ORF），编码704个氨基酸。序列分析表明，其包含典型的PAL酶活性中心序列（GTITASGDLVPLSYIA），与其它植物的PAL蛋白有很高的相似性。系统进化树分析显示，甘蔗ScPAL与高粱的PAL蛋白亲缘关系较近。real-time PCR分析表明PAL为组成型表达，在根中的表达量最高，是叶中表达量的66倍。其在低温（4℃）、聚乙二醇（PEG）、NaCl和H2O2四种外源胁迫下均诱导表达，但表达模式不同。【结论】从甘蔗品种ROC22中克隆获得苯丙氨酸解氨酶基因（ScPAL），是典型的PAL家族成员，推测其参与了甘蔗抗黑穗病过程，且在甘蔗抗寒、抗旱和抗盐胁迫过程也起到某种作用。

关键词: 甘蔗 , 苯丙氨酸解氨酶 , 基因克隆 , 表达分析

Abstract: 【Objective】The aim of this study was to clone full-length cDNA of sugarcane phenylalanin ammonia-lyase (PAL) gene (ScPAL), a key enzyme gene related to phenylpropanoid metabolism in sugarcane, investigate its sequence characteristics, analyze its expressions in different organs and under five different stress conditions, thus providing a theoretical support for using this gene in sugarcane stress tolerance breeding. 【Method】 The differentially expressed protein was identified by MALDI TOF/TOF and analyzed using Applied Biosystems GPS Explorer software with Mascot analysis against the NCBI and Uniprot database using a combined peptide mass fingerprint and MS/MS search, then the full sequence of ScPAL was cloned from sugarcane variety ROC22 using RT-PCR and RACE techniques. The bioinformatics method was used to analyze the putative amino acid sequence, and real-time PCR method was used to analyze the expression of ScPAL in different tissues and under different stresses. 【Result】 The full-length cDNA of ScPAL (GenBank accession number: KC172559) in sugarcane was cloned. The sequence consists of 2590 bp with an intact open reading frame of 2115 bp, encoding a polypeptide of 704 amino acids. Sequence analysis showed that it contains the typical PAL enzyme active site sequence (GTITASGDLVPLSYIA). Homology analysis showed that the deduced ScPAL protein was highly homologous to other PAL proteins from different species. Phylogenetic tree analysis indicated that ScPAL was very closely related to PAL of sorghum. Real-time PCR results showed that the ScPAL expressed in root, stalk and leaf, respectively, and its expression was different among three organs. The mRNA of ScPAL in root was the highest among three organs and was about sixty-six times higher than that in leaf. Furthermore, ScPAL transcription level was induced under the treatment of low temperature, PEG, NaCl and H2O2 stresses, but the expression patterns were different. 【Conclusion】 The gene ScPAL which was firstly cloned and characterized from sugarcane (ROC22) is a member of PAL family typically. The results of study indicated that it participated in sugarcane resistance to smut, also played a role in the sugarcane resistance to chilling, drought and salt stress processes.

Key words: sugarcane , PAL , gene cloning , expression analysis

宋修鹏1, 黄杏2, 莫凤连1, 田丹丹3, 杨丽涛12, 李杨瑞12, 陈保善1. 甘蔗苯丙氨酸解氨酶基因（PAL）的克隆和表达分析[J]. 中国农业科学, 2013, 46(14): 2856-2868.

SONG Xiu-Peng-1, HUANG Xing-2, MO Feng-Lian-1, TIAN Dan-Dan-3, YANG Li-Tao-12, LI Yang-Rui-12, CHEN Bao-Shan-1. Cloning and Expression Analysis of Sugarcane Phenylalanin Ammonia-lyase (PAL) Gene[J]. Scientia Agricultura Sinica, 2013, 46(14): 2856-2868.

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甘蔗苯丙氨酸解氨酶基因（PAL）的克隆和表达分析

Cloning and Expression Analysis of Sugarcane Phenylalanin Ammonia-lyase (PAL) Gene

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