| Qingke(Hordeum vulgare L. var. nudum Hook. f) is also called hulless barley, which is rich in protein and dietary fiber, and also contains active functional substances like flavonoid and gamma-amino butyric acid. The role of qingke in health and medical care has been paid close attention by barley researchers. Flavonoid, which is a kind of ideal healthy substance for people, has various pharmacological functions, such as anti-oxidizing, anti-cancer, anti-carcinoma, anti-aging, protecting cardiovascular and so on. In plants, the natural flavonoids mainly include flavone, flavonol, flavanone and isoflavone, etc, while as in barley flavonol is dominant in flavonoids. The phenylpropanoid metabolism is one of the most important secondary metabolism pathways in plant, and the flavonoid biosynthesis pathway, which located to the downstream of the general phenylalanine pathway during phenylpropanoid metabolism, is a predominant way of flavonoids synthesis in plant.Cinnamate-4-hydroxylase(C4H), is a key enzyme in second-step of general phenylalanine pathway, which is also considered as a rate-limiting enzyme. C4H participates in phenylpropanoid metabolism as the first member of cytochrome P450 zymoprotein family, efficiently catalyzes the first oxidation reaction that trans-cinnamic acid is hydroxylated by C4H on C4 position in cyclobenzene and turns into p-counaric acid under oxygen and NADPH. For the sake of understanding the function of genes in flavonoid biosynthesis from the molecular level, the full length cDNA encoding of cinnamate-4-hydroxylase gene(HvC4H) was firstly cloned from leaves of the qingke line "94-19-1" by RT-PCR combining homologous clone and RACE technique, and the expression levels of HvC4H gene in different tissues(kernel, stem and leaf) during 5 periods of endosperm development was also analyzed by real-time-fluorescence quantitative PCR.The main results are as follows:1. According to C4H gene sequences in monocotyledon of gramineae reported in NCBI database, a pair of degenerate primers in the conserved domain were designed through sequence alignment and the conserved fragment of HvC4H was amplified by homologous cloning. Based on the conserved fragment, the sequences of 3’end and 5’end in HvC4H gene were amplified by RACE technique and a full length cDNA was also got relying on the sequences’ joint. The results showed, the full length cDNA of HvC4H was 1951bp, including 1518bp’s open reading frame(ORF) encoding 505 amino acids, and 124bp’s 5’-UTR as well as 309bp’s 3’-UTR with a tail of 27bp Poly-A bases.2. According to sequences alignment in blast, the similarity degree between HvC4H gene and C4H gene of Brachypodium distachyon amounted high up to 94%, while up to 92% between Bambusa oldhamii, Panicum virgatum, Neosinocalamus affinis and Setaria italica, and yet, up to 82% between Oryza sativa, Zea mays, Eucalyptus urophylla, Sorghum bicolor and so on. The amino-acid sequence of HvC4H contained known conserved domains and specific motifs of CYP450 zymoprotein family such as heme binding domain,5 substrate recognition sites and proline hinge. Meanwhile, the particular active amino-acid sites, which distinguished HvC4H from other CYP450 proteins, had been recognized.3. Using β-actin and α-tublin which expressed stably during endosperm development in barley as reference genes, the expression levels of HvC4H gene in 3 tissues(grain, stem and leaf) were analysed during 5 periods of endosperm development by real-time fluorescent quantitative PCR. The results showed that the expression levels of HvC4H gene existed obvious disparities in different tissues during each period of endosperm development. The expressions in stem were highest at the beginning and then reduced rapidly after 4 days. Expressions in leaf reached to the highest level on the 8th day. Expressions in grain kept a low level but disparities were tiny in those periods. Generally, during the endosperm developing, the expressions in stem were dominant and the expressions in grain kept in plateau of HvC4H gene. |
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