Cloning And Prokaryotic Expression Analysis Of HvCHI Gene In Qingke (Hulless Barley)

Qingke (Hordeum vulgare L. var. nudum Hook. F), also named hulless barley, which has the high nutritional value and health protection in medical treatment, had been being paid closer attention to by barley researchers, because the nutrition and functional components such as flavonoids etc. are richer in grains of qingke. Flavonoids, one of the polyphenolic secondary metabolites, which belongs to the derivatives of chromone or chromane, plays an important role in the physiological activities of plants, including the UV protection, flower coloration, regulate growth and anti-pathogenic microorganisms, as well as the induction of plants-microorganism interaction. And also flavonoids, which is thought to be closely linked with human health, processes more highly important medicine value in the elimination of freeradicals, the protection of the cardiovascular, anti-tumor, antioxidant and anti-inflammatory. Flavonoid metabolism, one of most importantly primary secondary metabolic pathways in plants, is an important way for flavonoid synthesis of plants.Chalcone isomerase (chalcone isomerase, CHI, EC5.5.1.6), the first one enzyme-related to flavonoid biosynthesis firstly recognized, and the key enzyme controlling the metabolism of flavonoid, plays a vital role in synthesis of flavonoids. It catalyzes the intramolecular cyclization reaction to convert the bicyclic chalcone into tricyclic two hydroxy flavanones with biological activity. In this study, the hulless barley line "94-19-1" with higer flavonoid content screened out from the qingke resources in our laboratory, was used to clone the full-length CDS of HvCHI gene by homologous cloning method. But sequences analysis by bioinformatics combining with phylogenetic tree method, and preliminarily prokaryotic expression analysis were also studied to provide a theory basis for futher studying the molecular mechanism of CHI gene in flavonoid metabolism of hulless barley, and guide hulless barley breeding with high flavonoids content. The main results are as follows:1. According to the CDS of CHI gene of barley reported in GenBank, a pair of primers were designed, the full length CDS fragment of HvCHI was amplified by homologous cloning and successfully sequenced. Sequence analysis showed full length of CDS district was 696 bp, encoding 231 amino acids. The molecular weight of the enzyme was 23.68 KDa with isoelectric point (PI) of 5.22, indicating that it was acid protein, and stable protein with the instability coefficient of 23.96. The amino acid composition analysis indicated that Ala was the main composition of the protein, and the content of Cys, Gin, and Trp were the least. The results of analysis by biology software displayed the deduced protein was hydrophilic for the average index of 0.299, and has five serine and one tyrosine active phosphorylation sites exsisted. The results for signal peptide prediction showed in the deduced protein was no signal peptide, and it secretory protein. Disulfide-prediction software analysis showed that disulfide bonds might be exsisted in the area of Cys113 and Cys183.2. The NJ phylogenetic tree was constructed based on the CHI gene by using 14 kinds of plants including hulless barley. The evolutionary tree was divided into two subgroups: monocots (Ⅱ) and dicots (Ⅰ). The category Ⅰ includes the petunia, grapes, lotus root, wild buckwheat, alfalfa, beans and other plants, while common wheat, corn, onions, barley, aegilops and other plants were clustered into category Ⅱ. The reults indicated qingke had the nearest genetic relationship with barley, Aegilops belonging to the monocotyledon while the farthest genetic relationship with citrus sinensis, petunia belonging to the dicotyledon.3. Prokaryotic expression of pET-32a-HvCHI was carried out in E.coli BL21. That the fusion protein with 44 KDa was formed from the protein encoded by HvCHl and the tag protein of expression vector pET-32a, agreed well with the predicted results. The induced concentration for prokaryotic expression was 0.5 mM, the expression of fusion protein reached the maximum at the fourth hour after being induced. Then the primarily purified fusion protein was obtained.