Cloning And Functional Analysis HDR1 Gene For Salvianolic Acid And Construction Of Plant Expression Vectors With PMI Gene As Selection Marker And Their Utilization In Transformation Of Salvia Miltiorrhiza Bge. F.Alba

The enzyme 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR) is a terminal-acting enzyme in the plastid MEP pathway, which produce isoprenoid precursors. The full-length c DNA of HDR, designated Sm HDR1(Genbank Accession Number JX516088), was isolated for the first time from S. miltiorrhiza Bge. f. alba. Sm HDR1 contains a 1,389-bp open reading frame encoding 463 amino acids. The deduced Sm HDR1 protein, which shows high identity to HDRs of other plant species, is predicted to possess a chloroplast transit peptide at the N-terminus and four conserved cysteine residues. Transcription pattern analysis revealed that Sm HDR1 has high levels transcription in leaves and low levels transcription in roots and stems. The expression of Sm HDR1 was induced by 0.1 m M methyl-jasmonate(Me JA) and salicylic acid(SA), but not by 0.1 m M abscisic acid(ABA), in the hairy roots of S. miltiorrhiza Bge. f. alba. Complementation of Sm HDR1 in the E. coli HDR mutant MG1655 ara<>isp H demonstrated the function of this enzyme. A functional color assay with E. coli showed that Sm HDR1 accelerates the biosynthesis of β-carotene, indicating that Sm HDR1 encodes a functional protein. Overexpression of Sm HDR1 enhanced the production of tanshinones in cultured hairy roots of S. miltiorrhiza Bge. f. alba. These results indicate that Sm HDR1 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza Bge. f. alba.In this srudy,we also constructed plant expression p CAMBIA1301-PMI successfully by substituting PMI for hygromycin resistance gene in p CAMBI A1301 and to obtain transgenic Salvia miltiorrhiza bge. f.alba using PMI/mann ose selection system.The 6-phosphomannose isomerase gene(PMI)of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on p CAMBIA1305, the plant expression p CAMBIA1305-PMI was constructed successfully by substituting with PMI for hygromycin resistance gene in p CAMBIA1305. p CAMBIA1305-PMI was transformed into Agrobacterium tume faciens)LBA4404, and then the leaves of Salvia miltiorrhiza bge. f.alba were inoculated in LBA4404 with p CAMBIA1305-PMI.Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic Salvia miltiorrhiza bge. f.alba plants was developed using PMI/mannose selection system.