Study On The Physiological Response To Low Phosphate,and The Function Of Genes SmMYB98b And SmMYB9a In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is widely used in traditional Chinese medicine market because of its pharmacological action,the main active ingredients in S.miltirrhiza are tanshinones and phenolic acids,the content of these two kinds of substances are the qulity evaluation standard of various preparations.Therefore,improving the content and yield of tanshinones and phenolic acids in S.miltiorrhiza is important.Phosphate is an essential plant macronutrient that is often deficient,thereby limiting crop yield,the problem of phosphate deficiency also exists in S.miltiorrhiza planting.Studies show that phosphate deficiency could cause metabolism changes and increases the accumulation of secondary metabolism in plants.It would be considered as a strategy for saving Pi resources and increasing the yield of active substances in herbs.In the present study,we investigate the effects of pi deficiency on the growth and metabolic regulation of S.miltiorrhiza,and screen the optimum phosphate concentration for the accumulation of secondary metabolism.Then,hairy roots of S.miltiorrhiza and S.castanea were used to identify the differences and similarities of Pi deficiency response mechanisms between the two Salvia species.We cloned two R2R3 MYB TF-SmMYB9 a and SmMYB98 b,SmMYB9a and SmMYB98 b positive regulate the tanshinones biosynthesis and are involved in pi deficiency response.We also obtain two phosphate deficiency response genes-SmSPX2 and SmSPX4,and investigate the interaction between SmMYB9 a and SmSPX2/4.The main results and fundings were as follows:1.We investigated the effects of different phosphorus level on the growth and secondary metabolism in S.miltiorrhiza.200 mg/kg,100 mg/kg,10 mg/kg and 0 mg/kg were used to correspond to a control group,a slightly reduced level,a seriously reduced level and the complete absence of Pi,respectively.The biomass of roots,the content of phosphate and chlorophyll,the photosynthetic rate,several phytonormone,and important secondary metabolites were measured.Compared to control level,100 mg/kg and 10 mg/kg reduced the Pi content of plant.However,the biomass of roots were little reflected,the content of chlorophyll,the photosynthetic rate and the anthocyanin accumulation were promoted.The content of GA was reduced while IAA was increased.Accumulations of four kinds of tanshinones were increased with the decrease of Pi level.2.Hairy roots of S.miltiorrhiza and hairy roots of S.castanea were used to identify the differences and similarities of Pi deficiency response mechanisms of the two Salvia species.The results showed that phosphate deficiency increased the accumulation of specific secondary metabolites such as phenolic acids and tanshinones by promoting the expression of key enzyme genes,and antioxidant activity in the two Salvia species.These data demonstrated that Pi deficiency increased the quality of the medicinal material in the plant.S.castanea was minimally influenced by phosphate dosages compared to S.miltiorrhiza with respect to biomass inhibition,increased secondary metabolism and enhanced antioxidant activity.3.We cloned R2R3-MYB transcription factors-SmMYB98 b,the open reading frame of SmMYB98 b was 699 bp.It was belonged to subgroup 20,the same subgroup with AtMYB2 and AtMYB62 which were reported involved in Pi starvation.SmMYB98 b codes 232 amino acids,the molecular weight of SmMYB98 b was 27 KD,the isoelectric point was 7.58.SmMYB98 a is located in nucleus.The over-expression of SmMYB98 b in S.militorrhiza hairy roots stimulated tanshinone accumulation by 6-30 folds,structure genes’ expressions in tanshinones biosynthesis pathways were induced,while SmMYB98b-RNA interference(RNAi)transgenetic inhibited tanshinones biosynthesis.Functions of SmMYB98 b to tanshinone accumulation were affected by phosphate level.The OE-SmMYB98 b S.miltiorrhiza hairy roots showed phosphate starvation responses,such as decreased phosphate concentration and activated phosphate starvation-inducible genes.Interestingly,phosphate starvation responses induced by over-expression of SmMYB98 b were inhibited by low phosphate.The differences in tanshinones accumulation,phosphate concentration and expressions of starvation-inducible genes narrowed between transgenic and wild type hairy roots in low phosphate compared to high phosphate condition.4.We cloned R2R3-MYB transcription factor-SmMYB9 a,the open reading frame of SmMYB9 a was 786 bp.It was belonged to subgroup 20 also.SmMYB9 a codes 261 amino acids,the molecular weight of SmMYB9 a was 29.8KD and the isoelectric point is 6.4.SmMYB9 b located both in nucleus and cell membrane.Protein induced by using prokaryotic expression showed that the protein induced by IPTG is consistent with the predicted protein size.The over-expression of Sm MYB9 a promoted tanshinones accumulation by 8-35 folds while RNAi-SmMYB9 a transgenic hairy roots inhibited tanshinones biosynthesis and induced phenolic acids biosynthesis by 1.4-2.5 folds.Phosphate content in OE-Sm MYB9 a hairy roots were induced and PSI gene expression were promoted.However,phosphate deficiency weaken this phenomone5.We cloned two SPX genes in S.miltiorrhiza hairy roots-SmSPX2 and SmSPX4,the open reading frame were 855 bp and 882 bp respectively.SmMYB9 a could interact with SmSPX2 and SmSPX4.