Salvia Miltiorrhiza Hairy Root System Of The Transcription Factor Conversion And Fructose Phosphatase Gene Cloning Research

Objective: The transcriptional factor AtMYB4 gene was transformed into the hair roots of Salvia miltiorrhiza Bunge to investigate the regulation of AtMYB4 on the metabolism of water-soluble components in S. miltiorrhiza. A novel fructose-bisphosphate aldolase gene was cloned from S. miltiorrhiza by using of rapid amplification of cDNA ends and bioinformatics analysis was performenced.Methods: RT-PCR was carried out to abtain AtMYB4, and the plant expression vector AtMYB4 + pBI121 and AtMYB4 + pCAMBIA1304+ were constructed then. Mature leaves of S. miltiorrhiza were used as explants and Agrobacterium-mediated transformated by strain C58C1, followed by co-culture, removing bacteria, liquid ultimately sterile culture of transgenic hairy roots. Applying real-time PCR technique to detect the expression level of the key genes in the rosmarinic acid metabolic pathway and inspect AtMYB4 related metabolic pathways and regulation of gene expression level. By using of rapid amplification of cDNA ends to clone the novel fructose phosphatase gene, and analyzed with the software.Results: Danshen phenolic acids are mainly rosmarinic acid and its derivatives as lithospermate B. Two paralle branches initialed the biosynthetic pathway. Integrating AtMYB4 into S. miltiorrhiza hairy root could active the expression of key enzymes in the pathway respectively, promoting endogenous genes PAL, 4CL1, 4CL2, TAT, RAS increased 1.91, 2.04, 2.11, 1.67 and 1.81 times respectively compared with control, and repressed the by path, reducing the expression of C4H, HPPD, HPPR by 48.5%, 5.9% and 73.5%.We cloned novel fructose-bisphosphate aldolase gene (designated as SmFBA, GenBank accession number FJ 540907) cloned from S. miltiorrhiza firstly. Its full-length cDNA was 1kb and 390 bp. Both the open reading frame was 1,065 bp and encoded 355 amino acid residues of a protein. The deduced protein had isoelectric point (pI) of 5.60 and a calculated molecular weight of about 37.78 kDa. SmFBA protein had high homology and identity with other plant FBAs. The SmFBA genomic DNA sequence was also obtained, revealing SmFBA had three exons and two introns. Real-time quantitative PCR analysis showed that SmFBA expressed constitutively in all tested organs with the highest expression level in roots.Conclusion: The cloning and functional analysis of fructose-bisphosphate aldolase gene will help us to transfer the interrelated genes into S. miltiorrhiza or other model plants and comprehend the metabolic flux in transgenic plant in future. This study helped us to understand EMP pathway, because SmFBA was the key gene in EMP pathway. It could give us a theory and foundation for next study in future.