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Journal of Tea Science ›› 2010, Vol. 30 ›› Issue (1): 27-36.doi: 10.13305/j.cnki.jts.2010.01.005

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Cloning and Expression Analysis of Leucoanthocyantin Reducase Gene of Tea Plant (Camellia sinensis)

MA Chun-lei, QIAO Xiao-yan, CHEN Liang*   

  1. Research Center for Tea Germplasm and Improvement, Tea Research Institute Chinese Academy of Agricultural Sciences/National Center for Tea Improvement, Hangzhou 310008, China
  • Received:2009-07-06 Revised:2009-08-18 Published:2019-09-10

Abstract: The leucoanthocyantin reducase gene (LAR), which was an important functional gene of catechins biosynthesis pathway, was cloned from tea plant by EST sequencing and rapid amplification of cDNA ends (RACE). The full-length cDNA of LAR is 1 301 bp (GenBank accession No. EF205148), containing a 1 029 bp ORF encoding a 342 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 5.81. 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 leucoanthocyantin reducase proteins was expressed in Escherichia coli BL21, and its molecular weigh was found to be about 60 kD by checking with SDS-PAGE, nearly equal to the predicted. The deduced amino acid sequence of LAR 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. Four different catechin content cultivars were selected from our tea germplasm appraisal database to assay the gene expression level of the seven genes, flavonol synthase, dihydroflavonol-4-reductase, leucoanthocyanidin reductase, anthocyanidin synthase, etc, 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 contents. Nevertheless, the others did not show this tendency clearly.

Key words: tea plant (Camellia sinensis), leucoanthocyantin reducase, gene cloning, sequence analysis

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