Molecular Cloning And Prokaryotic Expression Analysis Of Phenylalanine Ammonia-lyase Gene(TaPAL) In Pinus Tabulaeformis

The status of Pinus tabulaeformis is very prominent in Pinus, it is the pioneer tree speciesfor barren hill afforestation in northwest and northeast of China because of its widedistribution range and strong ability to adapt. Pinus tabulaeformis plays an important role inair purification、soil and water conservation in northern area. The Pinus tabulaeformis hasbeen invading from pets and diseases for a long time, which restricts expansion of the area ofbarren hill vegetation in a certain time. Phenylalanine ammonia-lyase is a key andrate-limiting enzyme in phenylpropanoid pathway, and it’s metabolite take part in synthesis offlavone、lignine、phytoalexins and so on, which have an important part in plant growth、stressresistance、 pets and disease resistance. In this syudy, the gene of phenylalanineammonia-lyase(TaPAL) was cloned from Pinus tabulaeformis, and the recombinant proteinwas successfully expressed in BL21(DE3), the study provided some theoretical foundation forgenetic engineering of Pinus tabulaeformis and established the molecular basis for furtherstudy on the structure and function of TaPAL. The results were as followed:1.Molecular clone and bioinformatics analysis of PAL Gene(CDS) from Pinustabulaeformis. The DNA and cDNA sequences of PAL were obtained by the method ofhomology cloning. Bioinformatic analysis showed the cDNA sequence was2157bp in length,containing a complete open reading frame and718amino acids were encoded, designated asTapal (Accession number：JX280460). The molecular weight was predicted to be78.24Kdwith an isoelectric point at6.04, and the GRAVY value was-1.171, which was supposed to bethe hydrophilic protein. Secondary structure of TaPAL amino sequence was predicted usingSOPMA showed that: the sequence contained57.1%of α-helix,5.57%of β-fold,29.39%ofrandom coil and7.94%extended strands. Singnal3.0analysis indicated there was no signalpeptide in the sequence, so the protein would not be secreted to the outside of the cell.TMPred analysis suggested that a transmembrane domains from inside to outside might beexisted in261-286amino acid. The3D modeling of TaPAL protein was calculated takingadvantage of Swissmodel based on the model of Parsley PAL’s protein. The domination sites and catalytic active sites appeared in PAL protein of Oryza and Zea mays were also found inthat of inferred protein sequence. The TaPAL gene sequences was highly homologous to otherPALs’ from the result of multiple comparisons of nucleotide sequence and was99%homologous with Pinus massoninana’s PAL. Phylogenetic tree analysis suggested that all thePAL of Pinaceae clustered into a major category. Moreover, this gene was found to beintronless because the cDNA and genomic DNA sequence of TaPAL were completely thesame.2. Construction and expression analysis of prokaryotic expression vector of TaPAL gene.The recombinant expression vector pET-28a-TAL was constructed by recombinant-DNAtechnique using the CDS fragment (2157bp) of TaPAL and transformed E.coli BL21(DE3) forfusion expression.It turns out: SDS-PAGE Gel electrophoresis indicated that an anticipatedprotein molecular weight of about80KD was detected. The best expression conditions is28℃、1.5mmol·L-1and3h. Most of pET-28a-TAL protein exists in the form of inclusion bodyat the optimal condition. The specific activity of crude enzyme(20℃、1mmol·L-1IPTG、10h)is55.3μmol/min﹒g pro, the date was slightly larger than Parsley’s(53.99μmol/min﹒g) butlower than soybean’s(211.7μmol/min﹒g).