摘要
茶叶是世界上最流行的无酒精健康饮品之一，含有许多有价值的次生代谢产物，如类黄酮、咖啡碱等。其中类黄酮是由类苯基丙烷等前体缩合而成一组天然化合物，它们在植物的生长、发育和防御疾病等方面有着重要作用。而且，这些化合物具有较强的抗氧化活性，对于改善人体健康的效果也非常突出。因此，分离克隆茶树类黄酮生物合成途径中相关酶的基因具有重要的理论意义和实践价值。主要研究结果如下：
1．在原有茶树EST基础上，利用T_4RNA连接酶介导的5′RACE技术获得了茶树查尔酮异构酶(CHI)基因的全长序列，其在GenBank的登录号是DQ904329，其序列全长1163bp，其中开放阅读框长723bp，编码240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26.4kD，pI为5.19。序列分析表明它在GenBank已登录的植物中与番茄CHI基因序列的亲缘关系比较近。
2．根据原有EST设计引物，利用RT-PCR技术获得了茶树黄酮醇合成酶(FLS)基因全长序列，在GenBank登录号为EF205150，其序列全长1317bp，其中开放阅读框长996bp，编码331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37.5kD，pI为5.80。序列分析表明它在GenBank已登录的植物中与葡萄FLS基因序列的亲缘关系比较近。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，结果表明茶树黄酮醇合成酶基因能在大肠杆菌BL21中表达，电泳检测到一条大约61kD的外源蛋白，与预测的融合蛋白分子量相符。
3．利用3′RACE技术获得了茶树无色花色素还原酶(LAR)的3′端，将其与原有EST片段拼接，得到了LAR基因的全长序列，其在GenBank的登录号为EF205148，其序列全长1301bp，其中开放阅读框长1029bp，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37.5kD，pI为5.81。同源性分析表明得到的LAR序列与亚洲棉(Gossypium arboreum)、洋莓(Fragaria ananassa)和葡萄(Vitis vinifera)的氨基酸序列相似性分别为70％、68％、71％。十种植物的联配表明其氨基酸序列较为保守。
4．利用半定量PCR技术检测了类黄酮的主要成分——儿茶素含量不同4个茶树品种中与类黄酮合成相关的查尔酮合成酶(CHS)、黄烷酮3-羟化酶(F3H)、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)、花色素还原酶(ANR)和花青素合成酶(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儿茶素含量呈一定的相关性，而其它基因则与其相关性不大。
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.
引文
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