Gene Cloning And Expression Of The Phenylalanine Ammonia-lyase In Poinsettia

Phenylalanine ammonia-lyase (PAL) is the key enzyme which catalyzes the spontaneous nonoxidative deamination of L-phenylalanine to trans-Cinnamic acid providing precursors for various phenypropanoid compounds such as flavanoids and anthocyanin.It plays a crucial role in the upper reaches of the anthocyanin synthesis pathway. The color of poinsettia bracts is mainly constituted by anthocyanin, So far, a few research about the relationship between PAL and anthocyanin synthesis of poinsettia was carried out. Here, the full-length PAL cDNA and genomic DNA of poinsettia were cloned using'Prestige'and'Early Velvet'as materials, and then,the expressions of PAL in different organ, in different leaves during developing stage and in bract at different time in asunny day were studied by RT-PCR and Real-time PCR .The results were as follows:1. The cDNA sequence of PAL gene fragment which was 500bp long was cloned from poinsettia bracts by using a pair of degenerate primers. According to this sequence fragment, the primers for 3'and 5'rapid-amplification of cDNA ends (RACE) were designed to clone the 3'and 5'end cDNA sequences of PAL gene by RACE method. The full-length PAL cDNA ( named EpPAL ) was got, through splicing, 2429 bp with poly(A) tailing and containing a 2166bp open reading frame(ORF)encoding a 721 amino acid protein. The GeneBank accession number was FJ594466. EpPAL was found to have extensive homology with those of other plants via multiple alignments. Phylogenetic tree analysis revealed that EpPAL had closest relationship with PALs from Jatropha curcas L. and Maninot esculenta crantz.2. The full-length genomic DNA sequence of EpPAL was cloned by PCR method through gene specific primer designed according to the full-length PAL cDNA sequence. Its length was 3315 bp and showed that transcript was divided into two exons by one intron. The intron was 886 bp long, located at 416 bp downstream initiation codon. 3. During the stages of bract growth and development, the expression levels of PAL gene were higher in roots and bracts, but lower in stem and green leaves. Meanwhile, the expression level of PAL gene increased in order from green leaves, turning color leaves to bracts, which was consistent to their anthocyanin content. the result above suggested that PAL gene may paly an important role in the anthocyanin synthesis pathway of poinsettia bract. The curve of diurnal variation of PAL gene expression level in bract was demonstrated two highest peaks at 9:00 and 18:00 respectively, and reaching the lowest peak at 12:00 in a clear day.With the maturation and senescence of bracts, the expression levels of PAL gene were gradually reduced in both green leaves and bracts, but decreased rapidly in bracts than green leaves.