Molecular Cloning And Expression Analysis Of Chalcone Isomerase, Flavonol Synthase And Leucoanthocyantin Reducase Genes Of Tea Plant (Camellia Sinensis)

Tea is the most popular non-alcoholic and healthy beverages in the world, which has plentiful secondary metabolic products, such as, flavonoids, purine, etc. Flavonoids are a diverse group of plant natural products synthesized from phenylpropanoid and acetate-derived precursors, which play important roles in plant growth, development, and defense against microorganisms and pests. These compounds often possess antioxidant activity, and the potential health benefits of tea might mostly be because of this property of flavonoids and other phytochemicals. Isolation and cloning of important functional genes of tea plant (Camellia sinensis), which involved in flavonoids biosynthesis pathway, has crucial significance for using biotechnology method to regulate the metabolism of tea plant. The main results are as follow:1. The chalcone isomerase (CHI) gene, which was an important functional gene of catechins biosynthesis pathway, was cloned from tea plant by using EST sequencing and RACE (rapid amplification of cDNA ends) approaches. The full-length cDNA of chalcone isomerase gene is 1 163bp (GenBank accession No. DQ904329), containing a 723bp open reading frame (ORF) encoding a 240 amino acids protein, and its 3' untranslated region has an obvious polyadenylation signal. The deduced protein molecular weight was 26.4 kD and its theoretical isoelectric point was 5.19. Sequence analysis result showed that it is closely related with that of Lycopersicon esculentum.2. The flavonol synthase (FLS) gene was cloned from tea plant by using RT-PCR approaches based on our previous EST sequencing project. The full-length cDNA of flavonol synthase gene is 1 317bp (GenBank accession No. EF205150), containing a 996bp ORF encoding a 331 amino acids protein, and its 3' untranslated region has an obvious polyadenylation signal. The deduced protein molecular weight was 37.5 kD and its theoretical isoelectric point was 5.80. Sequence analysis result showed that it is closely related with that of Vitis vinifera. The gene was then constructed into expression vector pET-32a (+) for over expression in prokaryotic cells. The SDS-PAGE showed that induced by IPTG, the flavonol synthase proteins was expressed in Escherichia coli BL21, and its molecular weigh was found to be about 61 kD by checking with SDS-PAGE, nearly equal to the predicted.3. The 3'-end fragment of leucoanthocyantin reducase (LAR) gene was amplified using 3' RACE PCR technology. Then complete LAR gene was obtained by BLAST comparison the 3'-end fragment and the other fragment that we have known, and splicing according to the overlapping region. The full-length cDNA of LAR gene is 1 301bp (GenBank accession No. EF205150), containing a 1 029bp ORF encoding a 342 amino acids protein, and its 3' untranslated region has an obvious polyadenylation signal. The deduced protein molecular weight was 37.5kD and its theoretical isoelectric point was 5.81. The deduced amino acid sequence of LAR gene from tea plant showed high identity with that of other plants, for instance 71%, 70% and 68% with Vitis vinifera, Gossypium arboretum and Fragaria ananassa, respectively.4. Four different catechin content cultivars were selected from our tea germplasm appraisal database to assay the gene expression level of the seven genes, chalcone synthase,flavanone 3-hydroxylase,flavonol synthase,dihydroflavonol 4-reductase,leucoanthocyanidin reductase,anthocyanidin reductase and anthocyanidin synthase, which were involved in the flavonoids biosynthesis. The result showed that, the DFR and LAR transcripts were expressed increased with the increasing of tea catechin content. Nevertheless, the others did not show this tendency clearly.