Cloning Of The Crocus Zeaxanthin 7, 8(7', 8')-cleavage Dioxygenase Gene And Its Expression In Escherichia Coli

Saffron (Crocus sativus L.), a perennial herbage of Iridaceae, is a preciousherbal medicine and widely used in the world. Its stigmas contain.not only expensivespice and pigment, but also bioactive components for therapeutic usages. Amongthem crocin is the most valuable component. In order to exploit the resources ofCrocus sativus and crocin further, molecular biological research of saffron wasstudied in the thesis. The Crocus zeaxanthin 7, 8(7', 8')-cleavage dioxygenase gene(CsZCD) was cloned and then expressed in E.coli. The product was purified and thepurified protein was used to produce specific antibody.The crocetin synthase is a key enzyme in the biosynthesis pathway of Crocetinglycoside in the stigma of saffron. A fragment of 1.2kb was amplified from cDNA ofthe saffron stigma using two specific primers designed from the sequence of crocetinsynthesis gene published. Several clones were selected for sequencing after thefragment was cloned to the pMD18-T vector. The sequence analysis showed that the1.2kb fragment contains two sequences. One named CsZCD was 99% homologywith the published gene and the other sequence named CsZCD-new showed 96%homology. Southern hybridization showed 2 bands.CsZCD and CsZCD-new genes were cloned into expression vector pQE31 respectively, and formed into the recombinant plasmid pQE31-CsZCD, pQE31-CsZCD-new. After induction with IPTG, the proteins with 6×His fused tothe N-terminal were expressed in E.coli strait M15 transformed by gene CsZCD, CsZCD-new, respectively. The fused proteins accounted for 13.7% and 14.4% of thebacterial total proteins, respectively. Detection of SDS-PAGE and Western blotindicated that the fusion proteins with 49kD and 52kD were expressed stably in theform of insoluble inclusion bodies after IPTG induction. After denaturizing, therecombinant proteins were purified by His Trap TM HP chromatography column. Thepurified recombinant proetins were used to prepare antibodies.The purified recombinant proteins were used to immunize rabbits to prepareantiserum, respectively. The titers of both antisera were 1:12800. Western blotanalysis confirmed that the antisera only recognized His6-CsZCD andHis6-CsZCD-new recombinant proteins, respectively.Both of the antisera were used to detect the total proteins of gardenia immaturefruit. However, the result showed none band.The work provide a basis to study the function and difference of CsZCD andCsZCD-new at the protein level and expand the source of crocin.