Gene Expression Of Key Enzyme In Salvianolic Acid B Biosynthesis During Shoot Redifferentiation Of Salvia Miltiorrhiza Leaf In Vitro

In this experiment,leaves of sterile seedlings of Salvia miltiorrhiza were used as explants.Tissue culture techniques were used to investigate the differentiation of Sm.leaves induced by plant growth regulator 6-BA and NAA.Established an efficient regeneration system for Sm.Based on this,molecular biology techniques were used to study the relationship between expression of key enzyme genes in the biosynthetic pathway of salvianolic acid and adventitious bud differentiation.Finally,agrobacterium-mediated genetic transformation system was established for Sm.tablets.This study lays the foundation for revealing the relationship between Sm.organ differentiation and salvianolic acid biosynthesis and using the overexpression and inhibiting expression to study the function of salvianolic acid biosynthesis related genes.the main research results include:1.Establishment of an effective regeneration system of Sm.The leaves of aseptic seedlings of Sm.were used as explants and were inoculated into MS basic medium supplemented with6-BA,NAA,found that 6-BA or NAA was used alone,has almost no differentiation of callus and tuft shoots.Combined use of two exogenous hormones can obtain good callus differentiation status,Callus induction rate is 100%,adventitious bud differentiation rate is14%-87%,but the excessive concentration of 6-BA inhibited the induction of callus,and high concentrations of NAA resulted in browning of callus.The best ratio of hormones was:MS+6-BA3.0 mg·L^-1+NAA 0.2 mg·L^-1.The callus induction rate can reach 100%,and the differentiation rate of adventitious buds can reach 80%;1/2MS+NAA 0.1mg·L^-11 is the best rooting medium,and the root induction rate is 100%.2.The changes of physiological and biochemical indexes in Sm.The contents of proline,soluble protein,soluble sugar,and phenylalanine ammonia-lyase in leaves,differentiated callus and hard-differentiated callus were determined.The contents of proline and soluble sugar in hard-differentiated callus were found.The highest,the lowest leaf;phenylalanine ammonia-lyase activity is difficult to differentiate callus the highest;the highest soluble protein content in leaves.3.The relationship between the different differentiation state of callus and the expression level of key enzyme genes in the biosynthesis pathway of salvianolic acid was studied.The relative expression levels of key enzymes involved in the biosynthesis of salvianolic acid biosynthesis in different differentiated callus and stage of Sm were determined.It was found that Sm4cl and Smhppr first decreased and then increased in the two calli.Smc4h showed a decreasing trend;At the 23 d of culture,the expression level of Smhppr in differentiated bud callus was 13%higher than that of poorly differentiated callus,At 7?15 and 23 d,Sm4cl was75%?32%?86%higher;Smc4h was 61%?47%?40%higher,Combined with the results of previous experiments,it was found that the accumulation of salvianolic acid was less in the process of differentiation and growth of difficult-to-differentiate callus,while the accumulation of salvianolic acid was increased in the callus of differentiated bud,indicating that the content of salvianolic acid and the expression of three genes is closely related.In roots,stems,and leaves,the expression of Smc4h was 85%,81%,and 80%higher than in inflorescence,Sm4cl was 87%,84%,and 82%higher than inflorescence,respectively;Smhppr was 74%,79%,and 18%higher than inflorescence.4.The gfp gene was used as an exogenous marker gene to establish agrobacterium mediate genetic transformation system of Sm.The optimal transformation conditions were preculture time 0 d,bacterial concentration OD₆₀₀=0.02,infestation time 5 min,co-culture time5 d,the highest transformation efficiency was up to 40%.