Prokaryotic Expression Of Saffron Carotenoid Genes

Saffron(Crocus sativus L.),a valuable Chinese medicine,is known as "plant gold" because of the rich and special carotenoids in its stigma.Saffron is only used as a medicine for stigma.People always hope to solve the resource bottleneck problem of its application in various ways.The saffron carotenoid synthesis pathway and related genes have been elucidated.On this basis,this thesis studies the synthesis of saffron carotenoids by cloning several key enzyme genes for carotenoid synthesis in the saffron stigma and constructing an expression system of E.coli.The genes related carotenoids synthesis of CsZCD(zeaxanthin cleavage dioxygenase gene),CsCCD2(carotene cleavage dioxygenase 2 gene)and aldehyde dehydrogenase(CsALDH)gene family(CsALDH2,CsALDH3,CsALDH5,CsALDH6)were cloned by reverse transcription and polymerase chain reaction.By homologous recombination,12 expression vectors,including pET28a-Crt-ZCD and pET28a-Crt-CCD2 zeaxanthin synthesis vector,pTRI-P-ALDH2,pTRI-P-ALDH3,pTRI-P-ALDH5,pTRI-P-ALDH6 induction type and constitutive type,were constructed.On this basis,the plasmids pET28a-Crt-ZCD or pET28a-Crt-CCD2 were transformed separately and co-transformed into Escherichia coli with the vectors pTRI-P-ALDH2,pTRI-P-ALDH3,pTRI-P-ALDH5 or pTRI-P-ALDH6 respectively,establishment of 29 E.coli carotenoids synthesis systems.The E.coli synthesis products was extracted and separated by organic solvent and analyzed by high-pressure liquid phase analysis.Three carotenoid substances with retention times of 25 min,32 min,and 33 min,which were higher than those of the control group,while with retention times of 16 min,19 min,and 21 min,22 min of the four carotenoid-producing substances currently not detected in the control.The system constructed in this experiment provides a certain theoretical research model and application basis for studying the synthesis of saffron carotenoids in Escherichia coli...