西红花酸合成酶(7,8-二加氧酶)片段的克隆及在大肠杆菌中的表达与检测

Cloning of the crocus zeaxanthin 7,8(7′,8′)-cleavage dioxygenase new fragment (CsZCD-NEW) and its expression in Escherichia coli

根据已发表西红花酸合成酶(7,8-二加氧酶)基因序列设计PCR特异性引物,从西红花柱头总RNA中扩增并克隆到一段长为1.2 kb的片段.测序结果表明该片段含有两个序列.一个与已知西红花酸合成酶基因序列高度同源(同源性99%),命名为CsZCD;另一个的同源性为96%,命名为CsZCD-NEW.将CsZCD-NEW片段克隆到表达载体pQE31上,得到重组质粒CsZCD-NEW-pQE31.经IPTG诱导,重组质粒在E.coliM15中表达.

A fragment of 1.2 kb was amplified from cDNA of the saffron stigma using two specific primers designed from the sequence of crocetin synthesis gene published. The sequence analysis showed that the 1.2 kb fragment contains two sequences. One named CsZCD is 99 % homology with the published gene and the other sequence named CsZCD-NEW showed 96% homology. CsZCD-NEW was cloned into expression vector pQE31 and the recombinant plasmid pQE31-CsZCD-NEW was formed. After induced with IPTG, the proteins were expressed in E. coli M15.