Molecular Cloning And Prokaryotic Expression Of Chalcone Synthase Gene In Qingke(Hulless Barley)

Qingke (Hordeum vulgare L. var. nudum Hook. F). also called hulless barley, is a general term of multiple-row’s hulless barley on the Qinghai Tibetan Plateau region of China. Qingke not only belongs to the "three high and two low" food with high protein, high fiber, high vitamin, low fat and low sugar, and also contains rich flavonoids, gamma-aminobutyric acid, phenolic compounds, B-vitamins, and beta glucan, etc.. These nutrients have better healthy function for human. With the improvement of people’s living standard, these will be more and more attented for the nutritional value of qingke.Flavonoids, a kind of the importantly secondary metabolites in plant, is the ideal material for people’s health because it can resist oxidant, scaveng free radicals, enhance immunity, prevent osteoporosis, protect the liver and other pharmacological effects. In plant, the natural flavonoids mainly includes more than ten kinds of flavonoids such as flavone, flavonol, flavanone and isoflavone, etc., while in barley flavonol is dominant. Chalcone synthase(CHS).widely distributed in the plant kingdom, and also the first key enzyme in the metabolic pathway of flavonoids, plays an important role in plant growth and adaptation of plant to the environment, and has also become one hot site of research in molecular biology and plant physiology in recent years.In order to understand the role of enzyme genes related to flavonoids metabolism, the qingke line "94-19-1" was used in this study to clone the full length CDS of chalcone synthase gene by homology cloning method. And the carrier pET-32a-HvCHS was also built to obtain fusion protein for prokaryotic expression.The main results are as follows:1. The full length CDS of chalcone synthase gene was cloned by homology cloning method,the full length CDS of the gene HvCHS was 1351 bp including open reading frame(ORF) of 1197 bp encoding 398 amino acid. The sequence analysis showed that the emzyme protein HvCHS was a stable, hydrophobic and acid protein with the molecular weight of 43.4 KDa. and theoretical pI of 5.92; it belonged to the unstable protein with instable coefficient of 40.10 (>40). meanwhile it was hydrophilic protein with the average hydrophilic coefficient of-0.090, and has the half-life of 30 h by prediction.2. According to sequences alignment in blast, the similarity degree between HvCHS gene and CHS gene of Hordeum vulgare、Triticum aestivum、Oryza sativa and Zea mays amounted high to 99%,94%,88% and 85%, respectively, while Sorghum bicolor、Vitis vinifera、Petunia x hybrid、Olea europaea、Solanum tuberosum Solanum lycopersicum amounted up to 65%-78%. The further phylogenetic tree analysis showed that qingke relatively had the closest distance from Triticum aestivum and Hordeum vulgare in the monocotyledon, while far away from Solanum lycopersicum in dicotyledonous plants.3. That the secondary structure of the enzyme protein HvCHS were mainly composed of 41% of alpha helix,12% of random coil and 15% of beta angle was discovered through the prediction of the secondary structure for the enzyme protein HvCHS by using the online software PHYRE 2 Server combining with the SOPMA software. The prediction for protein phosphorylation of N-glycosylation and N-forecasting of the ezyme protein HvCHS indicated that one glycosylation site was not found except that richer phosphorylation sites located on 9 Ser,6 Thr and 2 Tyr were existed.4. Prokaryotic expression of pET-32a-HvCHS, which was formed from the expressive vector pET-32a linked with the encoding sequence of HvCHS gene in qingke, was carried out in E.coli BL21. The fusion protein with 64 KDa was taken shape from the protein encoded by the gene HvCHS and the tag protein of expressive vector pET-32a. The research about optimizing the expressive conditions of fusion protein showed that the maximum expression for fusion protein was reached at the induced concentration of 1.0 mM for IPTG and the third hour after being induced.