Cloning And Prokaryotic Expression Of Cytochrome P450Gene From Fagopyrum Tataticum, And Its Expression Level In Adversity At Sprout Stage

Cytochrome P450(CYP450), the largest family of enzyme in plant, could widely participate in secondary metabolites synthesis and degrade toxic substance in cell. It has become a spotlight in the field of plant secondary metabolism and stress-resistance physiology. As an excellent minor cereal with medicinal value, tartary buckwheat (Fagopyrum tatarium) shows the strong barren resistance and stress resistance. More importanly, it possesses not only abundant nutrients, but also plenty of flavonoids such as rutin. Flavonols, the secondary metabolites with many medical and health-care functions, are generally invoved in many physiological processes in plant such as development and stress resistance, and their biosynthesis need CYP450monooxygenase’s participation usually. In this study, we focus on the novel CYP450family gene FtP450-R4cloned from tartary buckwheat. Through prokaryotic expression and enzyme activity analysis, catalytic property of FtP450-R4was understood basically. By semi-quantitative RT-PCR and flavonoids detection, we analyzed the effects of stress conditions on FtP450-R4gene expression and flavonoids accumulation.1. Using homology-based cloning and RACE technology, a C YP450family gene ORF in length of1527bp was cloned from tartary buckwheat, and named as FtP450-R4. ProtParam online analysis showed that FtP450-R4encodes508amino acids, reative molecular mass is58.4KDa, the theory pI is7.12, and the molecular formula is C2652H4129N703O731S25. Phylogenetic tree based on CYP450amino acid sequence in plant showed that FtP450-R4is classified into the same group with CYP45081from Arabidopsis thaliana, Vitis vinifera, and Glycine max, and their sequence homology is79%-63%. Moreover, secondary structure prediction showed that it contains50%alpha helix,12.2%extended strand,3.54%beta turn and34.25%random coil.2. pET30b(+)-FtP450-R4recombinant plasmid was constructed and transformed into E.coil BL21(DE3). After induction expression, the enzyme activity was identified. The result showed that soluble FtP450-R4protein was expressed in E. coli, and the molecular weight was consistent wih the prediction about55KDa. The FtP450-R4activity depended on NADPH, and its enzyme activity and specific activity was18.85x10-3IU/ml enzyme solution and1.61x10-3IU/mg, respectively. Meanwhile, TLC proved a new unknown product was checked from reaction solution.3. Under the condition of UV-B light, cold and drought stress, FtP450-R4gene expression level was analyzed by semi-quantitative RT-PCR, and total flavonoids content was measured by AICl3method. The result showed that FtP450-R4expression level droped slightiy in cotyledon after UV-B and drought treatment, then arrived at the top of95.8%and98.4%, and finally decreased to27%and13.9%sharply, respectively. Through cold stress, FtP450-R4expression level droped slowly at2h after cold treatment, then kept between63%-67%. Under all the stresses, although FtP450-R4expression level showed a slight fluctuation in hypocotyl, it held on37.6%-43.1%finally. Statistical analysis results showed that, FtP450-R4gene expression level in UV-B and drought groups had highly significant difference from that in control group (P<0.01) in cotyledons, and it had significant difference (P<0.05) under cold stress. However, FtP450-R4expression level in all the treatment groups had no significant difference from that in control group (P>0.05) in hypocotyls. It is thus clear that FtP450-R4expression had tissue specificity, and different responses under different stress treatment. Total flavonoids detection showed that it in cotyledons was2-6times more than that in hypocotyls when it increased in varying degrees in both of tissues under stress treatment. Statistical analysis results indicated that total flavonoids content in both of tissues had significant difference from that in control group (P<0.05) after2h treatment time under UV-B light. However, under cold stress, total flavonoids content in cotyledons and in hypocotyls had significant difference from that in control group (P<0.05) after8h.treatment and after2h treatment, resptectively. Furthermore, under drought stress, total flavonoids in cotyledons showed no significant difference from that in control group(P>0.05) when it showed significant difference from that in control group (P<0.05) in hypocotyls after4h treatment.