Culture Of Adventitious Roots And Cloning And Functional Identification Of Phenylalanine Ammonia-lyase (PAL) Gene Of Astragalus Membranaceus

Radix Astragali is one of the commonly used traditional Chinese medicines,it has been utilized in composite herbal medicines.To sustainable utilization of resources of Radix Astragali,the effect of several factors on adventitious roots of Astragalus membranaceus (Fisch.) Bge.during tissue culture was studied.The results showed that the optimal growth of adventitious roots was found in B₅ medium with 30g/L white sugar and 2mg/L IBA.During bioreactor culture,the suitable conditions were obtained by the 20g/L of inoculum size per 5 L airlift bioreactor(3 L medium),2 cm-long adventitious roots(none-cut),and 0.10 vvm of air volume.The results of analysis of effective component was showed that polysaccharides of adventitious roots was significantly higher than that of cultivated A.membranaceus,saponins and flavonoids of adventitious roots were no significant difference with those of cultivated three-year A.membranaceus,but,significantly higher than those of cultivated annual A. membranaceus.Phenylalanine ammonia-lyase(PAL;EC4.3.1.5) is the first and also a rate-limiting step in phenylpropanoid pathway,which supplies precursors for a variety of secondary metabolites including flavonoids.PAL as a link of the primary metabolism and phenylpropane secondary metabolism,not only has the very vital role regarding physiological metabolism and the resistance of adverse stress in plant,but also has the great practical significance and the application value in medical application and biochemical industry.In this study,using degenerate PCR primers that target evolutionarily conserved sequeces of PAL gene in plants,we have firsrt cloned full-length sequences of a novel PAL gene in A. membranaceus by the technique of RT-PCR and RACE,the PAL gene named as AmPAL and the genebank accsesion number is EF567076.The full-length of AmPAL cDNA was 2650bp, including a 2154 bp open reading frame(ORF).The analysis of bioinformatics showed that AmPAL was a new number of PAL family,the putative AmPAL protein consisted of 718 amino acids with prediated molecular weight of 78.05 and isoelectric point(pI) of 5.96,had the homology with PAL of known leguminous plants and shared above 80%identity of nucleotide and amino acid sequences.The analysis of Southern blot showed that the AmPAL was a singlecopy gene in A.membranaceus genome.In order to identify the function and activity of AmPAL,we constructed pQE30- AmPAL vector and transformed it into E.coli M15 to allow expression of PAL with the N-terminal six consecutive histidine residues.The PAL proteins were purified using Ni-NTA metalaffinity chromatography with molecular weight about 78 KDa and final yield of 45mg/g dry cells,specific activity was 6 500U/mg and the Km value was 3.35×10^-4mM/L for L-Phe. On the basis above,we constructed pBI-AmPAL vector and transformed it into Arabidopsis thaliana.PCR,Southern blot and Northern blot showed that the AmPAL gene was integrated into A.thalianas genome and that expressed in transcription level,enzyme activity of transformed A.thaliana was much higher than that of untransformed A.thaliana.This study provides a solid foundation and theoretical basis on improving contents of secondary metabolites of phenylpropanoid pathway by transgene(AmPAL) to other Chinise tratditional medicines.