Cloning, Expression of Crocus sativus Phytoene Desaturase Gene and Preparation of Anf iserum against It

摘要

A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L. ) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodesa polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET -21a( + ) and over-expressed in E. coli BL21 (DE3), The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain poly-clonal antiserum with titer of 1 X 10~5. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, andthe higher expression level of PDS in mature stigma than in young stigma.