Cloning And Prokaryotic Expression Analysis Of Flavonoids O-methyltransferase Gene In Qingke(Hulless Barley)

Qingke (Hordeum vulgare L. var. nudum Hook. F.) also called hulless barley and can effectively improve and balance human nutrition, prevent of colon cancer, diabetes mellitus, cardiovascular disease and other diseases because it has a unique nutrition and health care ingredients. Methoxyflavonoids in plants, a sort of more importantly secondary metabolites, can improve the solubility of flavonoids, enhance metabolic stability and reduce toxic side effect, except for having the action of antibacterial, anticancer and antiobesity. Therefore, the green food with richer methoxyflavonoids has been more valued by people. But the wide application of methoxyflavonoids in grains is limited because of the lower methoxyflavonoids content in cereal and no clear mechanism for the O-methyltransferase enzyme reaction. In this study, the hulless barley line "94-19-1" with higher flavonoids content in grains was used to clone HvQK-OMT1 gene, which was transferred into E.coli for further prokaryotic expression and purification of recombinant protein. The objective of this study was to provide the theoretical basis for the enzyme functional verification and catalytic mechanism, and also lay an important foundation for further excavating the health care function of qingke and developing more excellent cultivars with richlier functional components in grains of qingke.1. The ORF sequence with the length of 1071 bp for the HvQK-OMT1 gene was cloned from hulless barley using homology-based cloning technology. ProtParam online analysis showed that HvQK-OMT1 gene encoded 356 amino acids, the reative molecular mass of the enzyme was 38.65 KDa, with isoelectric point of 5.33, and also the molecular formula of C1729H2700N448O506S21, indicating that it was a acid protein. Gene sequence contains five O-methyltransferase conserved sequences in plants, the sequences of conserved region I and II were completely conserved, and while region Ⅲ, Ⅳ,and Ⅴ sequences were different from conserved sequences in other plant about O-methyltransferase. NCBI-BLAST comparison showed that the protease belonged to class II of flavonoid OMTs.2. The NJ phylogenetic tree, which was constructed based on amino acid sequence of the O-methyltransferase in plant,was clustered into two categories. Of which 16 kinds of dicotyledon having closer genetic relationship were clustered into one group and 15 kinds of monocots were clustered into another. It showed that HvQK-OMT1 from hulless barley and HvOMT1 from barley had the closest phylogenetic relationship, while common wheat, maize, ryegrass, wild rice, brachypodium distachyon and other monocots grassy O-methyltransferase clustered together had closer homology. In evolution, hulless barley, barley, common wheat and brachypodium distachyon in flavonoids O-methyltransferase were existed high homology with caffeic acid O-methyltransferase. Phylogenetic analysis of the results are basically consistent with the BLAST results.3. HvQK-OMT1-pET recombinant plasmid was constructed and transformed into E.coil BL21(DE3) for induction expression. The results showed that the soluble fusion protein, which was about 58 KDa, could be optimally expression under the final concentration of 0.5mM for inducing 8 h at 37℃for this protein.4. The purified fusion protein HvQK-OMT1 was detected according to gradient elution Ni-NTA affinity chromatography by SDS-PAGE method, indicating that the inducedly expression fusion protein can be effectively eluted at the imidazole concentration 100 mmol·L-1.