Cloning Of FtAH4L Gene From Tartary Buckwheat, And Its Expression In Adversity Stress In Sprout Stage

Flavonoids are a large class of important secondary metabolites in plant. With the help of many transportation proteins on biological membrane, flavonoids would be in intracellular transmembrane or long distances transportation after they are synthesized at multienzyme complex in cytoplasm. Particularly, H+-ATPase protein family on plasma membrane is involved in the transportation of proanthocyanidins and flavonols. Tartary buckwheat (Fagopyrum tataricum), the main cultivated species of buckwheat, is rich in flavonoids. However, the flavonoid transportation mechanism is still unclear in tartary buckwheat. In this study, an FtAH4L gene encoding P-type H+-ATPase was cloned from tartary buckwheat, and its expression and flavonoids content was analsized after tartary buckwheat was treated under different lights, cold stress and jasmine induction. This work could provide an important way to understand the mechanism of synthesis, transportation and location of flavonoids in tartary buckwheat. The main results are as follows:1. Through the homologous cloning method and RACE technique, an ATPase family gene was cloned from tartary buckwheat, and named as FtAH4L (autoinhibited H+-ATP ase isoform 4 like) gene. Its cDNA was 3398bp in full length, and it included an ORF with 2865bp encoding 965 amino acid residues. Its accession numbers is KF955601 on GenBank. Blast-P homology analysis showed that, there was the amino acid similarity from 85% to 89% between FtAH4L and ATPase4 proteins in other plants. A typical plasma membrane ATPase transmembrane region and the ATP-binding domain were located in FtAH4L which was classified into P-type ATPase family like protein AHA 10 related with flavonoid transporte in Arabidopsis.2. After tartary buckwheat sprouts were cultivated under white fluorescent, white LED light, red LED light, blue light and UV-B LED light, FtAH4L expression and flavonoids content were analysized in cotyledons and in hypocotyls. The results showed both of FtAH4L expression level and flavonoids content were no significant change (P>0.05) under all the lights in cotyledons, but they were increased significantly in hypocotyl (P<0.05). Particularly, FtAH4L expression level and flavonoids content were extremely significantly improved under LED blue and UV-B light (P<0.01).3. Under cold stress, FtAH4L expression level and flavonoid content were enhanced dramatically remarkably (P<0.01) both in hypocotyls and cotyledons of tartary buckwheat sprouts, and they showed the positive correlation.4. Under jasmonates induction, FtAH4L expression in cotyledons and hypocotyls of tartary buckwheat sprouts was increased firstly, and then decreased a little following with induction time. Furthermore, both of their maximum points were at induction 10-hour, which were sharply up-regulated (P<0.01). Meanwhile, flavonoids content was developed significantly from induction 4-hour (P<0.01), and it arrived at the top both in cotyledons and hypocotyls with 10-hour induction. Statistics analysis indicated FtAH4L expression and flavonoids content showed the positive correlation under jasmonates treatment.Under different light, cold stress and jasmonates induction, FtAH4L expression in cotyledon was significantly lower than that in hypocotyl (P<0.05). However, FtAH4L expression in cotyledon was remarkably higher than in hypocotyl as the response to coldness and jasmonates stimulation (P<0.05). Statistics analysis indicated there was no significant correlation between FtAH4L expression and flavonoids content in cotyledons, but a strong positive correlation in hypocotyls. The above shows FtAH4L would be involved in serial responses to light stimulation, cold stress and jasmonates induction, and it also mediate the accumulation process of flavonoids in tissues of tartary buckwheat. Moreover, the different light reaction and stress-resistance mechanisms are possibly contributed to hypocotyls and cotyledons of tartary buckwheat.