| Salvia miltiorrhiza Bunge is a dicotyledonous lamiaceae plant.As a traditional Chinese medicinal material,its dried rhizomes are widely used in the treatment of many common diseases,such as cardiovascular and cerebrovascular diseases,liver and kidney dysfunction.Salvianolic acid is the main active ingredient of Salvia miltiorrhiza.The study found that the use of SA（Salicylic acid,SA）induction can increase the synthesis and accumulation of phenolic acid components,accompanied by the phenomenon of cytoplasmic calcium ion burst,Ca2+may be mediated by SA An important signal molecule for the biosynthesis of salvianolic acid compounds.ANNEXINs are calcium-dependent phospholipid membrane-binding proteins and may play an important role in calcium-mediated signal transduction pathways,but their specific functions are unclear.In this study,bioinformatics was used to analyze the sequence characteristics of Sm ANNAs（ANNEXINs）.After preliminary screening,molecular biotechnology was used to clone SmANN1 from Salvia miltiorrhiza,and its function was studied.Further,the hairy roots of Salvia miltiorrhiza overexpressing SmANN1 were obtained by induction Its role and regulation mechanism in salvia miltiorrhiza acid synthesis pathway.This study theoretically provides a basis for revealing the molecular mechanism of calcium signal regulating the secondary metabolism of Salvia miltiorrhiza,and lays a foundation for the quality control of Salvia miltiorrhiza in production.The main results obtained are as follows:1. Bioinformatics analysis of Sm ANNs.The homology,conserved domains,functional sites,transmembrane domains and hydrophobic properties of Sm ANNs were analyzed by bioinformatics.Sm ANNs are non-transmembrane protein molecules with 2-4 repeating ANNEXIN conserved domains.Functional sites include calcium ion binding sites,GTP binding sites,peroxidase residues,and actin residues.2. Expression analysis of Sm ANNs.Real-time quantitative PCR was used to analyze the transcription level of Sm ANNs in different tissues of Salvia miltiorrhiza,and it was found that Sm ANNs were expressed in different tissues of roots,stems,leaves,and flowers.SmANN1 had the highest expression level in leaves and the least in roots.Further,the vector HBT-SmANN1-TOMATO-HA with the target gene was transferred into Arabidopsis protoplasts to observe the localization of SmANN1,and the red fluorescence of SmANN1 was found in the cell membrane and cytoplasm.The vectors HBT-SmANN1-TOMATO-HA and GCa MP6 were co-transformed into Arabidopsis protoplasts,and it was found that SmANN1 completely coincided with the intracellular calcium ion distribution area.3. Overexpression of SmANN1 promotes the synthesis of rosmarinic acid and salvianolic acid B.The SmANN1 gene was cloned,a vector was constructed,and the hairy root of SmANN1 Salvia miltiorrhiza was overexpressed by Agrobacterium transformation.RT-q PCR was used to detect the transcription level of key enzyme genes in the salvianolic acid synthesis pathway in hairy roots,and high-performance liquid chromatography was used to detect the content of salvianolic acid in hairy roots.Compared with wild-type hairy roots,overexpression of SmANN1 increased the transcriptional expression levels of Sm C4H1（cinnamic acid-4-hydroxylase）in the phenylpropane pathway and Sm TAT1（tyrosine ketogiutarate transaminase）genes in the tyrosine-derived pathway.Elevated,thereby promoting the accumulation of rosmarinic acid,salvianolic acid B.4. The expression of SmANN1 is directly or indirectly affected by intracellular calcium and ROS.The NMT system was used to detect the calcium ion flow direction and flow rate of Salvia miltiorrhiza hairy root meristem after treatment with calcium ion treatment agent.At the same time,the expression of SmANN1 under the treatment agent was analyzed by RT-q PCR analysis.The expression of SmANN1 was significantly increased under the treatment of La Cl3,2-APB and EGTA,and the expression of SmANN1 decreased under the treatment of NADPH oxidase inhibitor（DPI）,indicating that the expression of SmANN1 Directly or indirectly affected by intracellular calcium ions and ROS. |
