4-ceoumarate:CoA Ligase Gene’s Cloning And Its Expression Analysis In Qingke(Hulless Barley)

Flavonoids, including flavones, flavonols, flavanones, aurones, anthocyanins, etc, widely exist in plants. Long being famous for potential pharmacological activities in anti-aging, anti-virus, anti-carcinoma, high blood-lipidsprevention,cardiovascular protection, etc, these compounds have became the hot-spots of the current research in plants and medicine. As an excellent resource of both food and medicine, Qingke (hulless barey,Hordeum vulgare L. var. nudum Hook. F)is not only rich in nutrition, but also rich in flavonols,(catechin, myricetin, quercetin and kaempferol in specific). As one of the key enzymes involved in the synthesis of flavonoids, the corresponding ester acyl-CoA were produced from4-coumaric acid catalyzed by4-ceoumarate:CoA ligase (4CL).This study mainly focuses on the cloning of4CL gene in Qingke and its tissue-specific expression during endosperm development, therefore to further reveal relationship between4-ceoumarate:CoA ligase and biological synthesis of flavonoids in Qingke. Main results of this study are as follows:1. A pair of degenerate primers was designed based on the conserved regions of reported4CL amino acid sequences to amplify conserved fragment of4CL gene from Qingke through RT-PCR. Sequence analysis revealed that the300bp fragment is a part of the4CL gene. RACE was then performed to obtain cDNA sequence of4CL gene in full length. Sequence analysis indicated that the full-length of cDNA was2107bp,containing156bp5’-untranslated region,289bp3’-untranslated region[excluding poly(A)] and1662bp open reading frame(ORF), which encode553amino acid residues.2. Alignment analysis showed that the amino acid sequences of Hv4CL gene in Qingke were96%,90%,87%,87%,88%,88%,86%,96%,96%identical to that from wheat, Brachypodium distachyon, perennial ryegrass, switchgrass, rice, Cenchrus calyculatus and maize, respectively. The predicted amino acid sequence exhibits significant homology with those of other cloned4CL genes, contain two conserved boxes, the AMP binding box (box I):SSGTTGLPKGV and the boxâ…¡:GEICIRG.3. Using the housekeeping genes β-actinå'ŒÎ±-tubulin from Qingke as reference gene, the expression of4CL gene was analyzed in different tissues and different developmental stages by real-time quantitative PCR. Result indicated that the4CL gene expressed in all these organs, but with different expression. The highest expression was detected in stems, while the least in seeds.4CL gene in grains showed the highest expression value during the early stage of endosperm development, and then gradually reduced. The expression of4CL gene peaked in the stems at16days after anthesis. In leaves, a progressive increase in the expression levels of the4CL gene between12and16days after anthesis was observed. These results indicated that the4CL gene existed the variable expression in mRNA levels of different tissues during barley endosperm development.