Cloning and Expression Analysis of Phenylalanine Ammonialyase Gene AdPAL1 in Angelica dahurica cv. Yubaizhi

Abstract

【Objective】This study aimed to explore the correlation between the content of imperatorin and the expression of the phenylalanine ammonia-lyase gene (AdPAL1) of Angelica dahurica cv. Yubaizhi (YBZ). In addition, AdPAL1 was cloned and expressed in prokaryotes.【Method】The content of imperatorin in the roots and leaves of YBZ during its rapid growth stage and harvest stages was measured using high-performance liquid chromatography (HPLC), and the expression of the AdPAL1 gene in the roots and leaves during the same stages was detected using real-time fluorescent quantitative PCR (qRT-PCR) to observe whether there was a correlation between imperatorin content and the expression of the AdPAL1 gene. Furthermore, the AdPAL1 gene was cloned from the root of YBZ based on its transcriptome using reverse transcription-polymerase chain reaction (RT-PCR) technology, and it was expressed in Escherichia coli.【Result】During the rapid growth period of YBZ, the content of imperatorin in the roots is much higher than that in the leaves, and the expression level of AdPAL1 in the roots is also much higher than that in the leaves. Compared with the rapid growth stage, the content of imperatorin in the roots slightly decreases during the har vest stag e, and the expression level of AdPAL1 gene in the roots also decreases at the same time. These results indicate a correlation between the expression of AdPAL1 gene and the content of imperatorin. In addition, bioinformatics analysis show that the AdPAL1 gene (GenBank accession number: OQ822236) of YBZ has an open reading frame length of 1 764 bp and encods 557 amino acids. The relative molecular weight of AdPAL1 protein is 61.10 kD, and its isoelectric point is 5.92. It has a conserved domain of phenylalanine ammonia lyase (IPR005922, 1-547), which belongs to the PAL protein family and it is located in the cytoplasm and is mainly composed of α- helixes and irregular coils. AdPAL1 has the closest genetic relationship with the KpPAL2 protein in Kitagawia praeruptora, with a local sequence similarity of up to 99.28%. The relative molecular weight of the recombinant protein of AdPAL1 gene expressed in E. coli is about 84.00 kD, which is consistent with the expected protein size.【Conclusion】The correlation between the content of imperatorin in roots and leaves of YBZ and the expression of AdPAL1 gene was preliminarily determined, and the AdPAL1 gene was cloned and successfully expressed in E. coli. These results provide a reference for further research on the mechanism of AdPAL1 gene in imperatorin biosynthesis.

Keywords

• angelica dahurica cv. yubaizhi
• imperatorin
• phenylalanine ammonia-lyase
• tissue specific expression
• gene cloning
• bioinformatics analysis
• prokaryotic expression