Cloning And Preliminary Functional Identification Of Phenylalanine Ammonia Lyase Gene Of Fritillaria Unibracteata

Fritillariae Cirrhosae Bulbus is the dry bulb of fritillaria cirrhosa、fritillaria unibracteata、fritillaria przewalkii、fritillaria delavayi、fritillaria taipaiensis or fritillaria unibracteata wabuensis.It has many functions,such as clearing heat,preventing phlegm,moistening lung,stopping coughing,relieving cough,dispelling masses and reducing swelling.According to their traits,the 2020 version of the Pharmacopoeia divides them into"songbei","green scallop","lube"and"cultivation".Fritillaria unibracteata is the main primordial plant of songbei.it has strict requirements on the growth environment and is mainly distributed in alpine regions with an altitude of 2800～4400 m,such as southern Gansu in Qinghai and northwestern Sichuan.Nowadays,the resources of fritillaria unibracteata are gradually depleted.As a result,fake pine shells are very common in the market.Research on the secondary metabolism and growth of fritillaria unibracteata can be used to solve the resources and medicinal ingredients of fritillaria unibracteata.Phenylalanine ammonia lyase is a key enzyme in the phenylpropane pathway,which catalyzes the production of cinnamic acid from phenylalanine,which protects plant growth and stress resistance.The study of fritillaria unibracteata PAL will help to understand the structural characteristics of fritillaria unibracteata PAL,and then understand the catalyzes molecular mechanism of fritillaria unibracteata PAL.It is of great significance to improve the secondary metabolism of fritillaria unibracteata,improve the resistance to increase the yield of fritillaria unibracteata.This project was successfully cloned full-length sequence of the PAL gene based on the transcriptome data of fritillaria unibracteata.We named it FuPAL.Bioinformatics analysis shows that FuPAL has 2169 bp and encodes 722 amino acids.the relative molecular mass of FuPAL protein is about 78 k Da,and the theoretical isoelectric point is 6.07.It contains 20amino acids,no obvious hydrophilicity and hydrophobicity.Without transmembrane domain and signal peptide,so speculated that FuPAL is not a secreted protein.This result was verified by transferring FuPAL into Nicotiana tabacum.Analyzing the different PAL amino acid sequences,it is found that the conserved region of FuPAL and Asparagus officinalis PAL is GTISSSGDLVPLSYIAG,which is converted into the active center by the self-cyclization of the SSG amino acid triplet.Plant PAL protein is highly conserved.The sequence similarity between FuPAL and Lilium hybrid division PAL can reach 92.34%,and the similarity of PAL between different species can reach 80%.The prediction of the secondary and tertiary structure of D32-C722 part shows that the protein is dominated byα-helix,and each monomer is composed of 3 parts,MIO domain（32D-268T）,core domain（269A-535E 657R-722C）and shielding domain（536E-656N）,four monomers gather to a tetramer.Quantitative PCR analysis of the expression of FuPAL gene in different parts of fritillaria unibracteata shows that the expression of FuPAL gene is tissue-specific.Among them,the expression level is the highest in leaves,followed by flowers,and the lowest in stems.Relevant studies have confirmed that PAL is highly expressed in the stems of Salvia sylvestris and the roots of Araceae heterophylla,Scutellaria baicalensis Georgi.It is speculated that the tissue-specific expression pattern of PAL is related to the existence of other PAL subtypes in plants.In this project,the FuPAL gene was transferred into E.coli and successfully obtain a recombinant protein of about 80 k Da,which was consistent with FuPAL relative molecular mass.The best conditions for FuPAL protein expression are OD₆₀₀ 0.6,0.2 m M IPTG,17℃,and induction for 12 hours.Further testing found that FuPAL has the activity of catalyzing phenylalanine and tyrosine.Subsequently,it was determined that the optimal catalytic temperature of FuPAL protein was 50℃,and the optimal p H was 7.9.Among the many metal ions,Zn²⁺had a greater inhibitory effect on FuPAL activity,and FuPAL protein had better thermal stability at 60℃.Through the analysis of the FuPAL enzyme structure,it is found that141F and 490E are related to the activity and are the core sites of FuPAL.These two sites are mutated and the enzyme activity of the mutant protein is detected.It is found that the catalytic activity of FuPAL-F141H on phenylalanine is reduced.The catalytic activity of tyrosine is increased,and the catalytic activity of FuPAL-E490N on phenylalanine is reduced,no catalytic activity of tyrosine.It is confirmed that 141F and 490E are the core sites of FuPAL protein。...