Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmPAL1) from Salvia miltiorrhiza.
Phenylalanine ammonia-lyase (PAL) is one of the branch point enzymes between primary and secondary metabolism. It plays an important role during plant development and defense. A PAL gene designated as SmPAL1 was cloned from Salvia miltiorrhiza using genome walking technology. The full-length SmPAL1 was 2,827 bp in size and consisted of an intron and two extrons encoding a 711-amino-acid polypeptide. Sequence alignment revealed that SmPAL1 shared more than 80% identity with the PAL sequences reported in Arabidopsis thaliana and other plants. The 5' flanking sequence of SmPAL1 was also cloned, and a group of putative cis-acting elements such as TATA box, CAAT box, G box and TC-rich repeats were identified. Transcription pattern analysis indicated that SmPAL1 expressed in all tissues examined, but more highly in leaf. Besides, expression of SmPAL1 was found to be induced by various treatments including ABA, wounding, and dehydration. To further confirm its function, SmPAL1 was expressed in Escherichia coli strain M15 with pQE-30 vector. The recombinant protein exhibited high PAL activity and could catalyze the conversion of L: -Phe to trans-cinnamic acid. This study will enable us to further understand the role SmPAL1 plays in the synthesis of active pharmaceutical compounds in S. miltiorrhiza at molecular level.
Song
Jie
J
Key Laboratory of Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, People's Republic of China.
Wang
Zhezhi
Z
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D027561:Salvia miltiorrhiza / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase