Molecular Cloning And Expression Analysis Of A Phenylalanine Ammonia-lyase Gene From Dendrobium

Dendrobium Sw. is a species of perennial herb in the Orchidaceae family, whichwas first introduced in the book Shennong Bencao Jing and is well-known for itsvaluable medical uses. Dendrobium Sw. has been recognized as a medicinal herb becauseit can be used for maintaining gastric tonicity, enhancing the production of bodily fluids,and relieving symptoms such as dryness of the throat and thirst with blurred vision. Themain chemical compounds of Dendrobium Sw. include polysaccharides, alkaloids, andothers. Alkaloids are the most important secondary metabolite of Dendrobium Sw., as ithas many officinal effects, including anticancer, neuroprotective, anti-angiogenesis, andimmunomodulatory. Alkaloid synthesis is catalyzed by phenylalanine ammonia-lyase(PAL), a critical enzyme that controls the speed of the first step in the biosynthesis ofphenylpropanoid metabolites, i.e. In this study, DcPAL gene was cloned fromDendrobium candidum. Protein was sueeessfully expressed in Escherichia coli. Andfluorescent quantitative detection of the expression of the level of transcription indifferent tissues.The results we experiment as followed.1. Molecular Cloning and Sequence Analysis of DcPALIn this study, a PAL gene was cloned from Dendrobium candidum using homologycloning and RACE. The full-length sequence and catalytic active sites that appear in PALproteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D.candidum (designated DcPAL, GenBank No. JQ765748) has2,458bps and contains acomplete open reading frame (ORF) of2,142bps, which encodes713amino acidresidues. The amino acid sequence of DcPAL has more than80%sequence identity withthe PAL genes of other plants, as indicated by multiple alignments. The dominant sitesand catalytic active sites, which are similar to that showing in PAL proteins ofArabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL. Phylogenetic treeanalysis revealed that DcPAL is more closely related to PALs from orchidaceae plantsthan to those of other plants.2. Fluorescent quantitative detection of the expression of the DcPALThe expression level varied significantly. In the protocorm-like bodies and leaves,the expression level was very low, with little difference in expression between these plantparts. However, in the roots and stems, the expression level was very high. Therefore, theinfluence of PAL probably varies, depending on the biological process.3. Construction of prokaryotic expression vector We have constructed the expression vector, and it was espressed in E. colisuccessfully.