Cloning And Functional Analysis Of SmSnRK2s And SmAREB1 Genes In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge,one of the traditional bulk medicinal materials,has a wide range of pharmacological effects.At present,the ever-increasing demand for S.miltiorrhiza has outstripped the productivity of wild resources.As one of the main active ingredients,phenolic acids have attracted considerable attention,due to their important medicinal effects as well as convenient water decoction as their main mode of application in traditional Chinese medicine.Researchers have made great efforts to increase the contents of phenolic acids of S.miltiorrhiza by means of genetic engineering.However,the water-soluble phenolic acids of S.miltiorrhiza are secondary metabolites and their quality and quantity strongly depend on environmental stresses,such as drought,cold,and salinity,which results in lower yield of S.miltiorrhiza.To our knowledge,few studies have been performed for improving the stress tolerance of S.miltiorrhiza by using transgenic methods.So,more genes should be explored via genetic engineering methods to breed new varieties of S.miltiorrhiza with stronger resistance to stresses and higher contents of active ingredients.Sucrose non-fermenting 1(SNF1)-related protein kinase 2s(SnRK2s)play a central role in ABA signaling pathways and in multiple environmental stress responses,they also participate in the regulation of carbon metabolism.In this study,based on the S.miltiorrhiza transcriptome database,we isolated SmSnRK2.3,SmSnRK2.4,SmSnRK2.6 and SmAREB1 from S.miltiorrhiza hairy roots.SmSnRK2.3,SmSnRK2.6,and SmAREB1 genes,strongly induced by exogenous ABA treatment,were chosed as the main subjects,we analyzed the effects SmSnRK2.3,SmSnRK2.6,and SmAREB1 overexpression on the content of phenolic acids,and investigated the interaction between SmSnRK2.3/2.6 and SmAREB1.The main contents and results were as follows:1.The cDNA sequences of SmSnRK2.3?SmSnRK2.4?SmSnRK2.6,and SmAREB1 genes of S.miltiorrhiza were cloned by RT-PCR.Sequence analysis showed that these four genes harboured open reading frames(ORFs)with length of 1,068 bp,1,068 bp,1,098 bp and 1,272 bp,and coded 355,355,365 and 423 amino acids,respectively.Combined with their DNA sequences,the results of GSDS2.0 online software analysis revealed that SmSnRK2.3 contained seven introns and eight exons,SmSnRK2.4 and SmSnRK2.6 contained eight introns and nine exons,while SmAREB1 contained three introns and four exons.Phylogenetic analysis showed that SmSnRK2.4 belonged to subclass I of SnRK2 s,while SmSnRK2.3 and SmSnRK2.6 were clustered within subclass III of SnRK2 s,and SmAREB1 shared the same branch with AtAREB1,AtAREB2,and AtABF3,which were stongly induced by ABA and osmotic stresses.2.Based on the S.miltiorrhiza transcriptome database,the 2,048 bp 5' flanking sequence of SmSnRK2.3,3,000 bp of SmSnRK2.4,1,865 bp of SmSnRK2.6,and 1,911 bp of SmAREB1 were defined via cloning and sequencing.The results of the analysis via PlantCARE showed that they all contained several phytohormone response elements,abiotic stress-related elements,and plant development-related elements,with the exception of core cis-acting elements,such as TATA box and CAAT box.3.Quantitative real-time PCR(qRT-PCR)was performed to determine the expression patterns of SmSnRK2.3,SmSnRK2.4,SmSnRK2.6,and SmAREB1 in different tissues of S.miltiorrhiza.Results showed that there four genes were ubiquitously expressed in roots,stems,and leaves of S.miltiorrhiza.No obvious differences were found in the expression level of SmSnRK2.3 among tissues;the expression level of SmSnRK2.4 in roots was much higher than that in stems and leaves,and the expression levels of SmSnRK2.4 in stem and leaves were almost identical;the expression levels of SmSnRK2.6 and SmAREB1 in leaves were significantly higher than those in the roots and stems,while that in the roots and stems were very similar.Additionally,qRT-PCR analysis was performed to investigate the responses of SmSnRK2.3,SmSnRK2.4,SmSnRK2.6,and SmAREB1 to exogenous ABA treatment,Results revealed that expression of SmSnRK2.4 was weakly induced by ABA,while the other genes were all strongly induced by ABA.4.Prokaryotic expression vector of the four target genes were constructed and the recombinant plasmids were introduced into prokaryotic expression strains E.coli Rosetta.After induced with IPTG,the SDS-PAGE was used to detect the expression of protein.SDS-PAGE results revealed that these four target genes induced a protein band that is consistent with the predicted protein size,respectively.Through induced expression and ultrasonic breaking of E.coli cells,purified proteins of SmSnRK2.3,SmSnRK2.6,and SmAREB1 were obtained under native conditions without denaturation.Then,the purified proteins were verified via Western blotting.Furthermore,in this study,we identified 3 non-redundant phosphorylation sites of SmSnRK2.3,while 14 non-redundant phosphorylation sites of SmSnRK2.6,via liquid chromatography tandem MS analysis.5.To examine the subcellular localization of SmSnRK2.3,SmSnRK2.6,and SmAREB1,we constructed the subcellular localization analysis vectors of them,respectively.A transient expression assay was performed to ascertain the subcellular localization of SmSnRK2.3 and SmSnRK2.6,Gene gun bombardment onion epidermal cell method was used to examine the subcellular localization of SmAREB1.Results indicate that SmSnRK2.3 and SmSnRK2.6 were located in the cell membrane,cytoplasm,and nucleus,and that SmAREB1 was located in the nucleus.6.The coding regions of SmSnRK2.3,SmSnRK2.6,and SmAREB1 were amplified and cloned into pCAMBIA1304 binary vector,under the control of the CaMV35 S promoter,respectively.The recombinant plasmids SmSnRK2.3-1304,SmSnRK2.6-1304,SmAREB1-1304 and empty pCAMBIA1304 vector were transformed into A.rhizogenes ATCC15834,and then infected leaf explants from sterile plantlets of S.miltiorrhiza to generate corresponding transgenic hairy roots(SmSnRK2.3-OEs,SmSnRK2.6-OEs,SmAREB1-OEs,and EVs).Compared with the EVs,overexpressing SmSnRK2.3 did not significantly promote accumulation of rosmarinic acid(RA)and salvianolic acid B(Sal B)in transgenic S.miltiorrhiza hairy roots,while overexpressing SmSnRK2.6 and SmAREB1 improved the contents of RA and Sal B.Further qRT-PCR analysis showed that overexpressing SmSnRK2.6 and SmAREB1 promoted greater metabolic flux to the phenolic acid-branched pathway,resulting in improving the expression levels of structural genes involved in phenolic acid synthesis pathway.7.Transcriptional activation analysis of SmAREB1 indicated that SmAREB1 did not activate transcription in AH109 yeast cells.The interaction between SmSnRK2.3/2.6 protein and SmAREB1 transcription factor was verified by yeast two-hybrid assay and Bimolecular fluorescence complementation analysis.