title: The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis.
abstract: Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.
authors.affiliation: Development and Utilization Key Laboratory of Northeast Plant Materials, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors.forename: Rui-Fang
authors.initials: RF
authors.lastname: Ma
authors.affiliation: College of Pharmacy, Jiamusi University, Jiamusi 154007, China.
authors.forename: Qian-Zi
authors.initials: QZ
authors.lastname: Liu
authors.affiliation: Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Department of Pharmaceutical Botany, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: College of Pharmacy, Jiamusi University, Jiamusi 154007, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Development and Utilization Key Laboratory of Northeast Plant Materials, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. Electronic address: yinjun2002@yahoo.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.affiliation: Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China. Electronic address: chenwanshengsmmu@aliyun.com.
authors.forename: Wan-Sheng
authors.initials: WS
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D031219:Isatis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D010940:Plant Proteins; D010650:Phenylalanine Ammonia-Lyase
keywords: Correlation analysis; Isatis indigotica Fort; Lignan; Phenolic compounds; Phenylalanine ammonia-lyase