长春花萜类吲哚生物碱生物合成途径中重要酶(DXR、SLS、G10H、STR)基因的克隆与表达

Cloning and Expression of cDNA Encoding Key Enzymes(DXR,SLS,G10H and STR) in Terpene Indole Alkaloids Biosynthesis Pathway from Catharanthus roseus

以分离提取的2周龄长春花植物幼苗总RNA为模板,经两步Gateway-PCR反应扩增得到长春花萜类吲哚生物碱合成途径中编码重要酶:5—磷酸脱氧木酮糖还原酶(DXR)、次番木鳖苷合成酶(SLS)、=牛儿醇-10羟化酶(G10H)和异胡豆苷合成酶(STR)的cDNA基因片段。利用BP重组酶将扩增片段克隆到Gateway化的入门载体pDONR201中,并进行测序验证。测序后的基因通过LR重组酶的作用转移到带有HIS标签的目标载体pETG10A中,构成重组表达质粒,转化大肠杆菌BL21(DE3),经IPTG诱导得到了高效表达,通过N i-TED树脂纯化方法得到了高纯度的蛋白。

cDNAs encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase （ DXR）, Secologanin synthase （SLS）, Geraniol 10-hydroxylase（G10H） and Strictosidine synthase （STR） were obtained from the leaves of Catharanthus roseus with 2-step gateway PCR. These cDNA clones were inserted into the GATEWAY donor vector pDONR201 at the corresponding sites by BP clonase and followed by sequencing analysis, respectively. Thereafter,the entry clones were introduced into the GATEWAY destination vector pETG10A fused to 6xHIS-tag by LR clonase. Overexpression of recombinant fusion proteins DXR,SLS and STR were initiated after IPTG induction. These recombinant fusion proteins are now available for developing the antisera.