Molecular Cloning And Functional Analysis Of A Phenylalanine Ammonia-Lyase Gene From Pinellia Ternata

Pinellia ternate,a perennial medicinal herb plant in Araceae,the dried tubers of which are used as medicine.At present,many chemical compounds of P.ternata have been isolated:alkaloids,sterols,amino acids,organic acids,flavonoids and tannins.Alkaloids are the main medicinal compounds of P.ternata.Ephedrine is the main active ingredient in alkaloids of P.ternate,which belongs to phenylpropylamino alkaloids.Ephedrine has been used in clinical treatment for the bronchial asthma attacks,nasal mucosal congestion caused by various reasons,and nasal congestion caused by swelling.However,the content of ephedrine is very low in P.ternata,which seriously affects the quality of P.ternata.In order to improve the content of ephedrine,it is of great significance to use plant metabolic engineering to cultivate high-yield alkaloids for medicinal plants.Phenylalanine ammonia lyase?PAL?is the first key enzyme in phenylpropylamino alkaloid biosynthesis pathway in plants,which connects primary and secondary metabolism in plants.L-Phenylalanine is a direct precursor of phenylpropylamino alkaloid biosynthesis pathway.PAL catalyzes L-phenylalanine to trans-cinnamic acid?t-CA?,which is the first reaction in phenylpropylamino alkaloid biosynthesis pathway.In this study,P.ternate was used as the research material.This work screened a unigene candidate gene containing the complete reading frame from P.ternate transcriptome database,and the gene was named PtPAL.The ORF sequence of PtPAL gene was cloned by RT-PCR.In addition,bioinformatics analysis,tissue expression profiling and enzyme kinetic analysis were performed.The promoter sequence of PtPAL gene was cloned by FPNI-PCR,and the histochemical localization of GUS in transgenic Arabidopsis thaliana was analyzed.This work lays the foundation for further exploring ephedrine biosynthesis pathway of P.ternate.The main research methods and results are as follows:1.Cloning,bioinformatics and tissue expression analysis of PtPALThe complete open reading frame?ORF?sequence of PtPAL was cloned by RT-PCR.The ORF sequence of PtPAL contained 2289 bp encoding 762 amino acids,and the stop codon was TAA.Multiple amino acid sequences alignment analysis of PAL showed that PtPAL contained PAL-HAL,PLN02457,phe_am_lyase,Lyase_aromatic and HutH domains,and belonged to the family of Lyase_Ilike Superfamily.PtPAL had the highest similarity with Lilium regale,and the similarity reached 78%.Phylogenetic tree analysis indicated that PtPAL was concentrated in monocotyledon,which closely related to Lilium regale,Ananas comosus and Elaeis guineensis from monocotyledon.The expression of PtPAL in different tissues was analyzed by qPCR.The results showed that the expression of PtPAL was the highest in leaves,followed by tuber and root,and the expression of PtPAL was the least in flower.2.Prokaryotic expression,purification and activity assay of the recombinant PtPALThis work selected as a prokaryotic expression vector pET32a?+?,and the recombinant PtPAL was heterologously expressed in E.coli BL21?DE3?.The recombinant of His-tagged PtPAL was isolated and purified,which had a molecular weight?MV?of 102.3 kDa.It was identified that PtPAL had the function of catalyzing L-Phe to t-CA by LC-MS.However,PtPAL could not catalyze L-tyrosine to p-coumaric acid under the same catalytic conditions.The PtPAL activity was measured in different pH?7.0-9.8?and different temperatures?30-90°C?.The results showed that the optimum pH of PtPAL was 9.0,and the optimum temperature of PtPAL was 70°C.The enzymatic kinetics analysis indicated that the K_m and V_maxax of PtPALwere 0.89 mM and 63.96nkat·mg^-1,respectively.The K_catat of PtPAL was 6.56 s^-1,and the K_cat/K_m of PtPAL was7.37×10³ s^-1·M^-1.These results demonstrated that the recombinant PtPAL had a high catalytic efficiency for the L-Phe.The effect of metal ions on the activity of PtPAL was further tested.The results showed that Na⁺,K⁺,Ca²⁺had no significant influence on the catalytic activity of the recombinant PtPAL.Ba²⁺increased the PtPAL activity,while Mn²⁺,Co²⁺,Cu²⁺and Zn²⁺inhibited the PtPAL activity.3.cloning of the PtPAL promoter and GUS histochemical localizationBased on the coding region sequence?CDS?of PtPAL,the primers were designed.The promoter sequenceof PtPAL wasclonedby chromosomewalking?FPNI-PCR?,which had 1149 bp.Bioinformatics analysis indicated that the promoter sequence of PtPAL contained the major functional elements including TATA-box and CAAT-box,and it contianed the responsive elements such as light?G-box,GT1-motif?,participating in drought stress?MBS?,salicylic acid?TCA-element?and methyl jasmonate?CGTCA-motif?.The promoter of PtPAL was fused to the GUS reporter gene and transformed into Arabidopsis thaliana.GUS histochemical localization and the expression of GUS analysis showed that the promoter of PtPAL could drive the expression of GUS in transgenic Arabidopsis,and the GUS gene was expressed in the roots and leaves of transgenic Arabidopsis seedlings.With the growth of transgenic Arabidopsis seedlings,the GUS gene was expressed in inflorescence,flower,cauline leaf,rosette leaf,stem and root of mature tissues of transgenic Arabidopsis thaliana.In conclusion,the recombinant PtPAL could effectively catalyze L-phenylalanine to trans-cinnamic acid.The PtPAL gene could be expressed in roots,leaves,stems and flowers of Arabidopsis thaliana.Cloning and functional identification of PtPAL gene provided a new candidate gene for further exploring the biosynthetic pathway of ephedrine in Pinellia ternata.