Research On Underlying Molecular Mechanism Of Biosynthesis Of Tanshinones Response To Salt Stress

Saline soil environment has became a big problem to affect the medicinal plant production,salt stress break the regular of plant physiology,affect plant growth and yield.Salvia miltiorrhiza is a traditional Chinese medicine,the medicinal active ingredients tanshinone are related to the stress of secondary metabolites.Tanshinone synthesis under salt stress directly regulated by the expression of genes and enzymes,and transcription factors plays an important role in salvia miltiorrhiza stress response,secondary metabolites synthesis.Aiming at the unique saline soil environment of Tianjin,the dynmic monitor of the plant physiological and biochemical indexes of salvia miltiorrhiza under salt stress,further study was made to explore the under mechanism of biosynthesis and accumulation of tanshinone under salt stress,and screening transcription factors related to the salt stress response.The main research results are as follows:(1)Salt stress influence the palnt physiology of salvia miltiorrhiza.The physiology indexes including cell membrane peroxidation product MDA,antioxidant enzymes SOD,CAT,POD,the non-enzymes antioxidant AsA and GSH,and organic osmotic regulation molecular proline.The MDA contents in salvia miltiorrhiza were increased as the extension of processing time.The SOD,POD,CAT enzyme activities have rosed shows that antioxidant enzymes was important in the removal of reactive oxygen,the POD enzyme activity under salt stress were increased first and then decrased suggests that this enezymes were mainly reacted at the early stage of stress.The AsA and GSH content increased first and then decreased as the extension of stress processing time,the antioxidant function of non-enezymes antioxidants mainly paly important roles in the early stage of stress.Proline content increased as salt stress treatment increased with the extension of time.(2)Mechanism of tanshinone biosynthesis and accumulation under salt stress.The changes of expression level of key enezymes genes and the activities of key enzyme of tanshinone biosythsis pathway were detected,and the contents of tanshinone under salt stress were determinded.We found that the expression of seven key enzyme gene SmDXS,SmCMK,SmAACT,SmHMGR,SmFPPS,Sm GGPPS and SmCPS were induced by salt stress,the expression of SmIPPI gene were inhibited by salt stress.The activities of DXS,HMGR enzyme change were consistented with SmDXS,SmHMGR gene expression.The results of HPLC quantitative analysis shows that low concentration of salt stress promote the synthesis and accumulation of tanshinone I,and high concentration of salt stress inhibits the biosynthesis and accumulationof tanshinone I,appropriate salt stress promote the biosynthesis and accumulation of tanshinone IIA and cryptotanshinone.(3)Explore of the function of transcription factor.Three transcription factors SmAP1,SmAP2 and SmERF2 were founded by BLAST with salvia miltiorrhiza EST database of NCBI.The properties of their amino acid sequence,secondary structure and tertiary structure were predicted by using bioinformatics websites.RT-PCR reveals that salt stress can promote the expression of transcription factor SmAP1,SmAP2,SmERF2.