采用EST测序技术和3′RACE技术,获得了茶树儿茶素代谢途径中的一个重要酶—无色花色素还原酶的全长基因,在GenBank的登录号为EF205148,序列全长1 301 bp,其中开放阅读框长1 029 bp,编码342个氨基酸,3′端有一个明显的多聚腺苷酸加尾信号,推测的蛋白分子量约为37.5 kD,理论等电点为5.81。将该基因重组到表达载体pET-32a(+)中进行原核表达,经IPTG诱导、SDS-PAGE检测,结果表明茶树无色花色素还原酶基因能在大肠杆菌BL21中表达,电泳检测到一条大约60 kD的外源蛋白,与预测的融合蛋白分子量相符。同源性分析表明茶树无色花色素还原酶基因编码的氨基酸序列与其他植物具有较高的相似性,例如与亚洲棉、草莓和葡萄的相似性分别为70%、68%、71%。利用半定量PCR技术检测总儿茶素含量不同的4个茶树品种中与类黄酮合成相关的黄酮醇合成酶(FLS)、二氢黄酮醇-4-还原酶(DFR)、无色花色素还原酶(LAR)、花色素合成酶(ANS)等7个基因的表达情况,结果表明DFR和LAR基因的表达量与茶树中总儿茶素含量呈一定的相关性,而其他基因则与其相关性不大。
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.
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