Gene Cloning And Expression Analysis Of Phenylalanine Ammonia-lyase And MYB Transcription Factor In Mulberry

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the first key enzyme of phenyl- propanoid metabolism in plant secondary metabolism and plays important roles in regulating plant development and defense. A PAL gene fragment of 360 bp was isolated from young mulberry leaf by RT-PCR using degenerated primers. Then its 3'and 5'flanking sequences were obtained using rapid amplification of cDNA ends (RACE) and got its full-length cDNA sequence, which we designated MaPAL (GenBank accession: HQ025956). Sequence analysis showed that MaPAL is 2615 bp in length and contained a complete open reading frame (ORF) of 2181bp encoding 726 amino acids. Amino acids sequence alignment revealed that MaPAL shared more than 82% identity with the PAL sequences reported in other plants. Phylogenetic tree analyses showed that MaPAL gene had a very close relationship with PAL gene from Rubus idaeus, Prunus avium and Pyrus communis. Transcription pattern of MaPAL at different parts of mulberry was investigated. The results showed that MaPAL transcripts expressed in all examined parts of mulberry, but are most abundantly expressed in radicle and mature purple fruit. Meanwhile, the expression level of MaPAL gene in different germplasm of mulberry was also different. Besides, expression of MaPAL gene was found to be induced by some treatments including silkworm fed, ultraviolet (200~275nm), wounding, low temperature and salt-stress compared to the normal growth environment, especially by ultraviolet.Regulation of gene expression at the level of transcription controls many crucial biological processes including growth and development, signal transduction and stress resistance. A number of different factors are required for the processes of transcription, in which the transcription factors play an important role. The MYB transcription factors comprise one of the largest families in plant transcription factors. It can specifically interact with MYB cis-acting elements and induce expression of multiple responsive genes under biotic and abiotic stresses. A MYB gene fragment of 250 bp was isolated from mulberry young leaf by RT-PCR using degenerated primers. Its 3'and 5'flanking sequences were also obtained using RACE. The full-length cDNA of MYB transcription factor gene was 954 bp in length and contained a complete open reading frame (ORF) of 783 bp encoding 261 amino acids, which was designated as MaMYB (GenBank accession: HQ025957). Amino acids sequence alignment revealed that MaMYB shared more than 70% identity with the MYB transcription factor reported in other plants. Phylogenetic tree analyses showed that MaMYB gene had a very close relationship with MYB transcription factor gene from Humulus lupulus, Populus trichocarpa and Ricinus communis. Transcription pattern of MaMYB analysis indicated that MaMYB expression level were similar in all examined tissues and in different germplasm of mulberry. MaMYB expression level was also not found to be induced by some treatments including silkworm fed, ultraviolet (200~275 nm), wounding. Besides, expression of MaMYB gene could be increased mildly under salt-stess, while decreased slightly under low temperature compared to the normal growth environment.