| Fagopyrum tatacuricum is rich in flavonoids and has nutrition and healthy value for human being. The research on metabolic regulation of flavonoids has become a new hotspot. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of phenylpropanoid metabolic pathway, and the expression level of Pal can effectively influenced on the synthesis of flavonoids. As an important element of eukaryote gene expression regulation, the promoter and many of the cis-regulatory elements, which distribute in the promoter, regulate the gene expression via control the starting and frequency of and response to elicitor from outside. This research focuses on cloning, characterization, and identification Pal promoter sequence from Fagopyrum tatacuricum (named PFtPal).The results are as follow:1. A1702bp of Pal promoter sequence from Fagopyrum tatacuricum was obtained via the Tail-PCR method. Bioinformatics analysis results showed that this sequence contained four potential transcription starting sites; there were basic promoter sequence in-1463bp～-1414bp,-1353bp～-1304bp,-761bp～-712bp and-164bp～-115bp region (far from upstream side of translation starting sites), which had the respective possibility are0.92,0.98,0.88and0.99. The cis-regulatory element analysis showed that the sequence not only contained some essential elements like TATA-Box and CAAT-Box elements, it also contained many functional elements, which might response to jasmonic acid methyl ester, low temperature, and tissue-specific expression.2. A series of5’terminal deletion fragments were constructed into plant expression vector PBI121(named PFtPalDel-PFtPal De5), and then transformed into tobacco leaves by agrobacterium mediated method. By dyeing analysis of transgenic plants, the results suggested that all of5’terminal deletion segments were able to drive the expression of Gus gene, and its basic function core region located between-274bp to translation starting sites.3. Activity analysis of GUS enzyme in transgenic tobacco callus showed that there were no significantly difference among PFtPal and PFtPal De4(-505bp～-1bp) and P35S (P >0.05); the activity of PFtPal Del fragment (-1271bp～-1bp) significantly higher than other5’terminal deletion fragment and P355(P<0.05); PFtPalDe2(-945bp～-1bp) and PFtPalDe3(-696bp～-1bp) fragments were significantly lower than P35S (P<0.05); the activity of PFtPal De5(-274bp～-1bp) was the lowest.4. Transgenic tobacco callus were treated with jasmonic acid methyl ester (MeJA) for4h. The analysis of GUS enzyme activities showed that only PFtPalDe3(-696bp～-1bp) fragment’s ratio of GUS enzyme activity of MeJA treatment groups and the GUS enzyme activity of control group was greater than1, the others were less than1. The results showed that there were negative regulation elements responses to MeJA, which located in-945bp～-696bp and-505bp～-274bp region, and they might co-regulate the MeJA response, and a positive regulation element response to MeJA, which located in-696bp～-505bp region.5. Transgenic tobacco callus were treated with low temperature for24h. The analysis of GUS enzyme activity showed that the ratio of GUS enzyme activities of experimental groups and control group were decreasing, but all of them were greater than3. PFtPal might have several elements response to low temperature, when these elements were missing gradually, its abilities of response to low temperature also gradually reduce. |
