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

【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 H2O2stresses,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.