| Salvia miltiorrhiza Bunge is a perennial herbal plant in the genus Salvia of the Lamiaceae family. Its dried root and rhizome is traditional Chinese medicine with high medicinal value, and was recorded in first pharmaceutical monograph Shennong’s Classic of Materia Medica. S. miltiorrhiza has been cultivated throughout Eastern Asia and used to treat and prevent cardiovascular, cerebrovascular, hyperlipidemia and acute ischemic stroke diseases. The active ingredients in S.miltiorrhiza are considered to contain both hydrophilic and lipophilic components. S. miltiorrhiza is widely used in clinical practice lead to increased demand, people start looking for new ways to solve the shortage of resources. Today, the research of key enzymes cloning and genetic transformation in the secondary metabolic pathways can improve the production of secondary metabolites, and gradually become a hot topic of medicinal plant biotechnology. Establishment of efficient genetic transformation system is prerequisite for genetic engineering research. Agrobacterium-mediated transformation as the primary method of transgenosis, has been gradually optimized. The optimizing of hairy root genetic transformation system is more perfect because short growth cycle and easy cultured, however the study of plant genetic transformation system optimization is less. This study has established the efficient plant genetic transformation system of S. miltiorrhiza through screening the influence factors in the process of Agrobacterium-mediated transformation. It will lay the foundation for quality improvement of S. miltiorrhiza through further use of the genetic engineering technology.1. Establishment of genetic transformation systemAn efficient Agrobacterium-mediated genetic transformation method has been developed for the medicinal plant S.miltiorrhiza after leaf explants were infected with Agrobacterium tumefaciens harbouring the plasmid pCAMBIA2301containing gus gene. This study discussed the influence of Agrobacterium strains, co-cultivation duration, antibiotics and concentration on conversion rate by transient expression of gus gene detection. The results showed that they are important factors involved in Agrobacterium-mediated genetic transformation of S.miltiorrhiza. The optimized transformation method is:the sterile leaf0.5cm sections soaked for30min in the bacterial suspension which resuspended by liquid MS medium to adjust the optical density of approximately0.6at600nm (ODλ600=0.6), dried on sterile filter paper and placed on MS medium with6-Benzylaminopurine (1mg/L),2,4-Dichlorophe-noxyacetic acid (0.5mg/L) to co-cultured for2d. Leaf explants after co-cultured were washed with sterile deionized water five times, then dried on sterile filter paper and transfered on MS selection medium with6-Benzylaminopurine (1mg/L),2,4-Dichlorophenoxyacetic acid (0.5 mg/L),400mg/L timentin. Screened shoots by50mg/L Kanamycin inoculated onto rooting medium1/2MS medium with0.2mg/L IBA, rooting and promote growth.2. Over express transgenic research of SmGGPPS and SmKSL in S. miltiorrhizaAccording SmGGPPS and SmKSL sequence information at both ends of the primers designed to amplify genomic fragment from S. miltiorrhiza, connect to the pMD19-T vector. We double digest with Xbal/Sad for SmGGPPS and BamHI/Sad for SmKSL, then insert the plant expression vector pCAMBIA2301respectively. Finally ensure the fragments have inserted in plasmid with enzyme detection and sequencing. We transformed plasmid pCAMBIA2301containing target gene into plant by Agrobacterium-mediated transformation we have established. We conducted a preliminary study of SmGGPPS and SmKSL transgenic plants after domestication. |
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