Cloning And Expression Of CsPDS Gene From Crocus Sativus And Studying On The Plant Resource Containing Crocin

Saffron (Crocus sativus), a corm herbage, is used as a source of food additives, colorants and flavoring agent because of its pungent colour and aromatic flavor. As a valuable traditional medicine, saffron is applied in controlling viral hepatitis, increasing immune system, preventing cardio-vascular disease and anti-cancer, etc. Crocin as a effective component in saffron is synthesized by carotenoids metabolic pathway. Phytoene desaturase (PDS) has recently been identified as an important enzyme in carotenoid metabolitic pathway.A new full-length cDNA clone encoding phytoene desaturase gene was isolated from stigma of saffron using RT-PCR and RACE techniques( (GenBank accession No. AY 183118) ). The cDNA is 2 149 bp long with an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids, preprotein of 62.3 kDa; 5' untranslated regions contains 59nt, 3' untranslated regions contains 391nt, a Poly (A) tail signal and long Poly (A) tail. Sequence analysis showed highly similarity to other plants such as Narcissus pseudonarcissus., and they all possess a short highly conserved N-terminal sequence encoding a dinucleotide cofactor binding site. Thus it is a new member of phytoene desaturase gene family. Southernanalysis showed that the CsPDS gene was a single copy in saffron. Northern blot analysis showed higher expression level of PDS gene in the stigma and anther than in the leaf and stem. The information we have got is very important for the further research of gene structure, expression and function of phytoene desaturase.For Prokaryotic expression, the coding region of the cDNA of the phytoene desaturase gene was cloned by PCR and inserted into a prokaryotic expression vector pET-21a(+). The gene was overexpressed in E. coli BL21 (DE3) and gave rise to a 63 KD mature protein in response to the IPTG induction. Its content was about 9.8% among total cell protein by Gene Genius Bio Imaging System. The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion proteins was obtained by His6 technique used to immunize rabbits to obtain polyclonal antiserum with titer of 1*105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in mature leaves and stamen, and the higher expression level of PDS in mutant stigma than in young stigma.The coding region of the cDNA of the phytoene desaturase gene was cloned by PCR and inserted into vector pBI121 containing intron-kanamycin. Plant expression vector pBIl21-CsPDS was constructed in which the expression of CsPDS gene was under the control of Camv35S promoter and T-nos terminator via genetic engineering. CsPDS gene was successfully introduced into tobacco plant by Agrobacteriwn-mediated (Agrobacterium Tumefaciens EHA105) transformation, and the transgenic plants were obtained and confirmed by PCR and PCR southern blot analysis. The transformation rate is about 68%.By using molecular genetic method, the spacer domains of 5S-rRNA were cloned and sequenced from the genomic DNAs that were isolated from Crocus sativus and C. asumcmiae. The fragments of 5S-rRNA gene spacer region in Crocus was about 550 bp, while three adulterants less than 300 bp. So Crocus can be detected by agarose gel electrophoresis or sequenced. Furtherore, a unique EcoR I site was used for the rapid identification of Crocus sativus from different specids of Crocus and adulterants.To explore the resources of crocin, the extraction method of crocin from flowers of Buddieia officinalis was studied by means of solvent-extraction process and ultraviolet-visible spectrophotometry. A HPLC method to determine the crocus I in Buddleia officinalis was established, and the results showed it is simple and sensitive, and accurate to deterine the content of crocus I.The callus is initiated in three media (B5, MS, White) from the buds, leaves and stem segments of Buddleja officinalis. The medium with B5+6-BA 0.5mg/L+2,4-D 0.5mg/L is selected as the best medium for callus cult...