Studies On Cloning And Expression Of CsZCD Gene,the Molecular Identification Of Crocus Sativus And The Pharmacology Of Crocin

Crocus sativus is a valuable plant of traditional Chinese medicine for many human diseases. Its stigmas contain not only expensive spice and pigment, but also bioactive components for therapeutic usages. Among them crocin is the most valuable component. The Crocus sativus characters of propagating only via annual corms, the very low yield and strict requirement for habitat result in its shortage for medicinal material and restrict its amplifications of in medicine. In order to exploit the resources of Crocus sativus and crocin further, the genetic transformation cloning and expression of CsZCD Gene the pharmacology of Crocus sativus and molecular labeling of Crocus sativus resources were studies in this dissertation.The gus gene {uid A) drived by CaM35S promoter was introduced into leaf, sheath and callus from bud and of Crocus sativus L. using Biolistic PDS-1000/He rnicrojectile bombardment system. Transient expression of gus gene was observed in sheath and callus by hischemical staining for 48 hours and even for 20 days after transformation. The highest transient GUS expression was obtained when calli incubated in a 0.5mol/L mannitol subculture liquid medium for 6 hour prior to bombardment in which the parameters were designed as helium of 1100psi and 9cm target distance. The concentration of kanamycin used in resistant calli selection was 500-800mg/L.The results suggest that gus gene can be used as a useful reporter gene in genetic engineering of saffron and the CaM35S promoter can be an effective promoter without tissue specificity. This is first report of particle bombardment on saffron.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 of the saffron stigma using two specific primers designed from the sequence of crocetin synthesis gene published. Several clones were selected for sequencing after this fragment was cloned to the pMD18-T Vector. The sequence analysis showed that this 1.2kb fragment contains two sequences. One named CsZCD is 99% homology with the published gene and another sequence named CsZCD-NEW showed 96% homology. After linking CsZCD to plasmid pBI121, the plant expression vector pBI121 -CsZCD was formed .CsZCD and CsZCD-NEW were cloned into expression vector pQE31 respectively, and formed into the recombinant plasmid pQE-31- CsZCD, pQE-31-CsZCD-NEW. After induction with IPTG, the proteins with 6>