Cloning And Genetic Transformation Of Phenylalanine Ammonia-lyase Gene In Rhodiola Sachalinensis

Rhodiola sachalinensis (A.Bor), a kind of perennial herbal plant of Rhodiola genus, has been widely applied to many fields, such as foods, health protection and medicines. It's a prospecting rare and medicinal plant. Studies showes that it has remarkable functions on anti-oxygen deficiency, anti-radiation, anti-fatigue, anti-virus, anti-aging and so on. There are several factors leading to Rhodiola sachalinensis' low production, for example, demanding living environment, no special pollinating insect, low efficiency of pollination, reproductive barrier caused by physiological nature, and so on. The over utilization these years caused the destroy of it's ecology environment and it's natural resources was decreased sharply.Salidroside, and other secondary metabolites are main functional components of Rhodiola sachalinensis. Tyrosol is not only the effective and active monosomic of Rhodiola genus but also the direct substrate of Salidroside . At present, tyrosol synthesized in vivo can only be judged by its molecule structure and chemecial property. As tyrosol belongs to derivates of aromatic amino acid and is the typical simple phenolic compounds. Phen-propane' metabolic pathway is tyrosol's most probable biosynthetic pathway according to the commen rule of botan's secondary metabolic pathway. L-Phe ammom'a-lyase(PAL, EC 4.3.1.4) , which link primary metabolism and the first step of phen-propane' metabolism together, is a key enzyme and rate-limiting enzyme of this metabolic pathway and is a enzyme studied the most in phen-propane' metabolic pathway.A cDNA clone of PAL gene, named PALcll, which probably has some close relations to the biosynthetic of tyrosol was isolated from Rhodiola sachalinensis (GenBank accession number: AY879309), by RACE method. The sequence analysisrevealed that the lenth of PALcll cDNA is 2407bp, containing a complete open reading frame (ORF). It is predicted that PALcll encode a polypeptide of 77.45 kDa, consist of 710 amino acid residues, with its pi 6.50. There is a typical PAL structural domain between 55-586. Phylogenetic tree analysis showed that PALcll had a high sequence homology with other plant Phenylalanine ammonia-lyase, between 67%-82%. Studies on bioinformatics indicated that PALcll was a new member of PAL family. Studies on transmembrane domain indicated that there was no transmembrane domain in PALclP amino acid sequence and that PALcll was a typical matrix protein. Southern blot showed that there were many copies of PALcll gene in the genome of Sachalin rhodiola rhizome. Northern blot analysis showed that the PALcll was expressed more in callus than in leaves at transcriptional level at normal growth conditions. It's transcriptional level was improved after inducing by UV and was higher in callus than in leaves. Result of HPLC showed the correlation between transcription level and salidroside content in the callus and leaves was positively.On the basis above, to further investigate the function of PALcll, we constructed pBSPALcll and transformed it into Rhodiola sachalinensis via Agrobacterium. PCR, PCR-Southern and RT-PCR showed that the PALcll gene was integrated into Rhodiola sachalinensis genome and that its expression level in transgenic callus was much higher than that in wide type control and induced by UV.This study provides a solid foundation and theoretical basis for further investigation on the metabolism and biosynthesis of salidroside and tyrosol and offers some help for improving gennplasm resource of Rhodiola sachalinensis by genetic engineering means and raising the yields of salidroside and tyrosol.