| Phenylalanine ammonia-lyase (PAL,EC4.3.1.5)mostly catalyzes the spontaneous nonoxidative deamination of L-phenylalanine ( L-Phe ) to trans-Cinnamic acid, which provides precursors for phenolics, flavanoids and isoflavanoids. It links primary metabolism to phenylpropanoid metabolism and it is the key enzyme and rate-limit enzyme in that it catalyses the first reaction in phenylpropanoid pathway which produces many secondary metabolites such as anthocyanins, lignins, phytoalexins etc.. These secondary metabolites play important roles in plant development,disease and adversity defense.In this research, the Phenylalanine Ammonia-lyase gene was cloned from Glycine max, and their structure and function were analyzed by bioinformatics.The results are as follows:1 GmPAL2 genomic DNA(GenBank accession number: GQ358921) was firstly isolated from Glycine max by PCR and its sequence was 4210bp in length and contained 2 exons and 1 introns. At one time, GmPAL1 genomic DNA(GenBank accession number: X52953) was cloned and its sequence was 3658bp in length and contained 2 exons and 1 introns.2 The known genes of GmPAL1 mRNA(GenBank accession number: X52953) and unknown GmPAL2mRNA (GenBank accession number: GQ220305) were cloned from Glycine max by RT-PCR. GmPAL1 cDNA sequences obtained were 2144bp in length and the cDNA encoded 712 amino acids and the amino acid sequences showed high identity with other plants PAL genes. GmPAL2cDNA sequences obtained were 2284bp in length, and the cDNA encoded 717 amino acids and the amino acid sequences showed high identity with other plant PAL genes. 3 GmPAL1 and GmPAL2 protein structure and function were predicted with different bioinformatic methods. The results of primary structure analysis showed that PI and Mw of GmPAL1 were 6.14 and 77.715kDa while that of GmPAL2 were 5.83 and 78.116kDa. The PAL amino acid sequences derived from Glycine max have extensive homology with PAL from other higher plants. The phylogenetic tree displayed by MEGA2.0 soft showed that the PAL from Glycine max had closer relationship with Pisum sativum and Phaseolus vulgaris than with other higher plant. Secondary structure analysis indicated that PAL belonged to mixed protein class. The alpha helix,extended strand and Random coil of GmPAL1 were about 24.72% and 16.01%, and that of PAL2 were about 24.13% and 16.01%.4 The Motiff analysis of online systems were used, obtained that the amino acid sequence region 194-210 of GmPAL1 and 199-215 of GmPAL2, which was the active site of PAL,which was the characteristics of plant PAL sequences,these further proving that the sequence obtained were PAL sequence.Promoter is the originate sequence of genetic transcription,it can discrimination and bind to the RNA polymerase.As an important regulatory element, the research of promoter has became the hot point of molecule biology. To study and research the function of promoter, having the important meaning for us to understand the growth and development growth,investage the fit mechanism of the biology and it habitat,controlling the expression of exogenous in the transgenic plant.The principle consequence of this research is just as follow:Using the RAIL—PCR, obtaining the GmPAL1 and GmPAL2 gene's promoter sequence,about 2509bp and 1384bp.To analysis the sequence, found many CAAT-boxes and TATA-boxes, TATA-box can lead the RNA polymerase II to the right transcriptional start site, CAAT-box mainly control the starting frequence of transcription, is an element of regulate transcription tarting,having no direct relationship for the specification of the promoter. It also found some potentional is-acting element, for example, the ABRE ,which is related to the ABA derivation; the MBS,which is related to drought induction; the TGACG-motif, which is related to the jasmonic methylester,and SpI.some photoresponse elemant, such as ACE,G-box,GTl-motif, I-box ,and the consensus of the putative "core" sequences of box-L-like sequences in carrot (D.c.) PAL1 promoter region. |
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