Fuctional Cloning Of CMK Gene And HDS Gene From Dioscorea Zingiberensis C.H Wright

Diosgenin is an important intermediate for synthesis of numerous steroidal drugs.Dioscorea zingiberensis accumulate high level of diosgenin in rhizomes and has been the main raw material for production of diosgenin in Chian.So far,diosgenin biosynthesis pathway has not been fully elucidated.It has been beloved that in plants,isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP,the isomer of IPP)produced by the cytosolic mevalonate(MVA)pathway are the precursors for diosgenin biosynthesis.It is well known that there is another pathway for production of IPP and DMAPP in the plastids of plants,2-C-methyl-D-erythritol 4-phosphate(MEP)pathway.There are reports that cross-flow of IPP and DMAPP between MVA and MEP pathways happens in some plants.Whether this cross-flow exists in D.zingiberensis and the MEP pathway is involved in diosgenin biosynthesis remains unclear.In order to investigate these issues in future,the genes encoding 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol kinase(CMK,the fourth enzyme of MEP pathway)and 4-hydroxy-3-methyl-2-buten1 yl diphosphate synthase(HDS,the sixth enzyme of MEP pathway)were cloned from this species by RT-PCR and RACE,and their function was verified.The main results are followings:1.The full length cDNA encoding D.zingiberensis CMK(designated Dz CMK)is 1303 bp,having an open reading frame(ORF)of 1170 bp,a 5' untranslational region(UTR)of 60 bp and a 3' UTR of 73 bp.DzCMK encoding a protein(Dz CMK)of 389 amino acid residues with a calculated molecular mass 42.99 KD and an isoelectric point of 6.77.Bioinformatic analyses reveal that DzCMK is highly identical to other plant CMKs and shares 82% identity with Hevea brasiliensis CMK,and has a putative plastid transit peptide of 44 amino acid residues at N-terminus.2.The full length cDNA encoding D.zingiberensis HDS(designated DzHDS)is 2488 bp,having an ORF of 2250 bp,a 5' UTR of 60 bp and a 3' UTR of 178 bp.DzHDS encoding a protein(DzHDS)of 749 amino acid residues with a calculated molecular mass 82.7 KD and an isoelectric point of 6.08.Bioinformatic analyses indicate that Dz HDS is highly identical to other plant HDSs,too,sharing 88% identity with Ananas comosus HDS,and has a putative plastid transit peptide of 43 amino acid residues at N-terminus.3.Bacterial expression vectors of DzCMK and DzHDS,pTrc-DzCMK and pTrc-DzHDS,were constructed.Escherichia coli cells were co-transformed with pAC-BETA and pTrc-DzCMK or pTrc-DzHDS,respectively.The cells harboring pAC-BETA and pTrc-DzCMK or pAC-BETA and pTrc-DzHDS were much yellower than those containing pAC-BETA and pTrc,proving that expression of DzCMK or DzHDS enhanced ?-carotene production in E.coli cells,and therefore DzCMK and DzHDS are functional genes of MEP pathway.4.The nucleotide sequences encoding the plastid transit peptides of DzCMK and pTrc-DzHDS were fused with green fluorescent protein(GFP)gene,yielding in two fused genes,CMKP-GFP and HDSP-GFP,respectively.CMKP-GFP and HDSP-GFP were subcloned into plant binary vector pCAMBIA2×35S-Tnos,yielding in vectors pCAMBIA2×35S-CMKP-GFP-Tnos and pCAMBIA2×35S-HDSP-GFP-Tnos,respectively.These two vectors were introduced into Agrobacterium tumefaciens cells,respectively,and then transgenic tobacco plants were produced by Agrobacterium-mediation transformation.Protoplasts were isolated from leaves of the two type transgenic plants.It was observed under laser confocal microscope that both the fused proteins,CMKP-GFP and HDSP-GFP,located in chloroplasts in the protoplasts.These observations are consistent with the facts that both CMK and HDS are enzymes of plastidial MEP pathway.