| Flavonoids exist widely in plants, such as flavonols, flavones, chalcones, flavanones, anthocyanins and etc. Many researches have showed that most of them have the pharmacological activities such as antopxidation, anticancer, antivirus. Therefore, it has been one of most extensively explored areas of plant secondary metabolitic engineering in recent years. Tartary buckwheat (Fagopyrum tataricum) belongs to Polygonaceae family. It can be used as medicine and food because it is abundant of nutrients and flavonoids. The main kind of flavonoids in Tartary buckwheat is rutin. Flavonoids biosynthesis is controlled by a few key enzymes and transcription factors, and also affected by environmental factors. Phenylalnine ammonialyase (PAL) is the first key enzyme of phenylalnine pathway. Chalcone isomerase (CHI) is a rate-limiting enzyme in flavonoids pathways. Flavonol synthase (FLS) is the first key enzyme of flavonol biosynthesis pathway. MYB transcription factors play an important role to affect flavonoids biosynthesis through regulating the transcription of key enzyme genes. In this study, the total flavonoids contents were determined during the germination processes in hypocotyl and cotyledon, and the expression levels of Pal, Chi, Fls and other three MYB transcription factor genes FtMyb1, FtMyb2, FtMyb3 were also measured, respectively. The results would be helpful to understand the molecular mechanism of the accumulation of flavonoids during buckwheat germination. The main results are concluded as follow.1. Total flavonoids in cotyledon and hypocotyl of F. tataricum during 6-10 days after germination were extracted by the methanol extraction method, and were determined by aluminum chloride spectrophotometry. The results showed that the total flavonoids content in cotyledon and hypocotyl was significantly different (P< 0.01). The total flavonoids content in cotyledon ranged from 4.58% to 5.54%, and it increased with the progress of germination. While the total amount of flavonoids in hypocotyl decreased from 0.93% to 0.68% during the 6th to the 7th day after germination, and then stabilized at about 0.7%.2. Semi-quantitative RT-PCR was used to determine the expression level of the Pal, Chi and Fls gene in the cotyledon and hypocotyl during the seed germination processes of F.tataricum while the housekeeping gene H3 was used as a control gene. The results showed that the expression level of Pal and Chi in the cotyledon reached the first peak on the 7th day about 140% and 120%, and then sharply decreased to 20% and 25% respectively. The expression level of Fls increased gradually from 150% to the highest 375% determined at the 8th day; and subsequently it decreased gradually to 130%. The expression level of Pal in hypocotyl was similar with that in cotyledon, but the highest was 210% and the lowest was about 110%. The expression level of Chi was relative stable between 40% and 70% in hypocotyl. The expression level of Fls in hypocotyl was stable at about 220% on the 6th and 7th days, but decreased drastically down to 75% on the 8th day with a final amount 50% at the 10th day. From all the results above, we could conclude that different expression level of these key enzyme genes existed in different organizations.3. Semi-quantitative RT-PCR was also used to determine the expression level of FtMyb1, FtMyb2 and FtMyb3 genes. In cotyledon, FtMyb1 expression was lowest on the 8th day, and subsequently increased; FtMyb2 expression was stable at 40% during the 6-8th days, but dropped down to 5% in the 10thday; the expression level of FtMyb3 was the lowest with 40% on the 7th day, and then subsequently increased up to 90%. In hypocotyl, the expression level of FtMyb1 deceased gradually to 7.5% and then increased up to 27% at the 9th day but decreased to 5% at the 10th day; the expression level of FtMyb2 was decreased to 5% at the 8th day, but increased gradually to 70%; FtMyb3 expression was decreased slightly during the 6th to 7th day, but subsequently increased gradually to 95%. These results also showed that these MYB transcription genes had different expression level at different organizations.4. The expression of the six genes mentioned above in cotyledon and hypocotyl was statistically analysed with SPSS 13.0 statistical software one-factor ANOVA and Duncan multiple comparison. The results showed that, the expression of Fls gene was significantly different with the other five genes (P<0.05), which had no significant difference between each other (P>0.05) in cotyledon. In hypocotyl the expression levels of Pal was significantly different with any one of Chi, FtMyb1, FtMyb2 and FtMyb3 (P <0.05); and there was significantly different only between the expression level of Fls and FtMyb1 (P<0.05), but other genes had no significan differences with each other (P> 0.05).5. The relative expression data of the six genes and total flavonoid content was processed by Matlab7.0 software. Then, the correlationships among the key enzyme genes and accumulation of flavonoids were analysed. When the correlation coefficient absolute value is greater than 0.75, it indicates significant correlation. The results showed that, in cotyledons, the total flavonoids content was significantly and positively associated with FtMyb3 (0.9625), and negatively with FtMyb2 (-0.8572); Chi was significantly and positively associated with FtMyb2 (0.8468), and negatively with FtMyb3(-0.8010); the three key enzyme genes were significantly and positively correlated to each other with 0.9119,0.8920 and 0.7584, respectively. In hypocotyls, the total flavonoids content was significantly and negatively associated with Chi (-0.8989), while Fls was significantly negative associated with Chi (-0.7498). Visiblely, the regulation of flavonoid biosynthesis was rather complex during germination of F.tataricum, but some genes's expression was still significantly associated with each other. |
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