茶树黄酮醇合成酶基因的克隆与原核表达

Cloning and Prokaryotic Expression of Flavonol Synthase Gene from Tea Plant

本研究采用EST测序技术和RT-PCR技术,获得了一个茶树茶多酚代谢中的重要基因——黄酮醇合成酶(FLS)基因,在GenBank登录(GenBank accessionNo.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,理论等电点为5.80。序列分析表明它与葡萄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。用Ni-NTA亲和层析柱对融合蛋白进行纯化,得到了纯度在90%以上的纯化蛋白,为进一步研究PET-FLS融合蛋白的活性及功能奠定了基础。

The flavonol synthase gene, which was an important functional gene of catechins biosynthesis pathway, was cloned from tea plant by using EST sequencing and RT-PCR approaches. The full-length cDNA of flavonol synthase gene is 1 317 bp （GenBank accession No. EF205150）, containing a 996 bp open reading frame （ORF） encoding a 331 amino acid 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 the that of Vitis vinifera. The gene was then constructed into expression vector pET-32a（＋） for over expression in prokaryotic ceils. 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. The products were purified with Ni-NTA purification system, and the purity of target proteins was more than 90%. This study establishes a basis for further study of PET-FLS fusion proteins＇ natural activity and function.