Cloning And Functional Characterization Of SmGRP1 From Salvia Miltiorrhiza

Salvia miltiorrhiza Bge.is a well-known medicinal plant.Its root(called danshen or tanshen in Chinese but better known in the west as Chinese sage or red sage root) contains two major groups of identified biologically active compounds,the hydrophilic caffeic-acid-derived phenolic acids,and various lipophilic tanshinones belonging to the diterpene quinines,and has been formulated and used clinically for the treatment of various diseases such as cardiovascular and cerebrovascular disease. With continued pharmacologic studies on its secondary metabolites,more and more biological activities including antioxidant,anti-thrombosis,anti-hypertension,antivirus and antitumor have been reported.In recent years,the wild resources of S.miltiorrhiza are destroyed severely with the constantly rising demand for its pharmacologic properties.Therefore,screening genes related to the eminent characters of S.miltiorrhiza and discussing its molecular mechanism are of great significance.Here,a novel unigene with high abundance in the EST database of S.miltiorrhiza was found and its expression pattern was also studied.Furthermore,the function of this gene was elucidated via studying the physiological changes of its "knock in" and "knock down" mutants.This study will provide information for further investigating and utilizing this special gene.The main results were as follows:1.A unigene composed by 11 ESTs were found by screening the EST library of S.miltiorrhiza. It showed no significant similarities with the sequences in NCBI "nr" database by blast x analysis, which implied that this putative gene may be a novel gene.Very interestingly,this novel gene encoded a peptide,glutamic acid residue content of which was as high as 33.77%.Based on this,the new gene was designated as SmGRP1(Salvia miltiorrhiza Glutamic acid-Rich Protein 1).2.The entire DNA and cDNA sequence of SmGRP1 were cloned using PCR and RT-PCR methods.Comparation of these two sequences suggested that SmGRP1 consisted of two extrons and one intron of 381 bp whose position was between Glu⁵ and Val⁶.Bioinformatics analysis showed that the molecular weight of SmGRP1 was 15.801 kDa,the theoretical pI was 3.79 and the instability index is 103.93,which implied that SmGRP1 was an unstable acidic protein.Interestingly,there were no aromatic amino acids such as Trp,Tyr and Cys in the protein.Also no signal peptide and transmembrane domain existed in SmGRP1.Prediction by ProtScale showed that SmGRP1 was a hydrophilic protein.Besides,alpha helix was the main secondary structure unit of the protein.3.Totally,1,932 bp-long 5' flanking region of SmGRP1 gene was obtained by DNA walking. Plant growth regulators responsive elements and biotic or abiotic responsive elements were found in this region using PLACE and PlantCARE softwares.Real time quantitative RT-PCR technique was performed to characterize the expression patterns of the gene.It showed that SmGRP1 expressed in all examined tissues of S.miltiorrhiza but most highly in leaves and stems.The expression level of SmGRP1 was prominently enhanced under ABA and dark treatments.Also the expression level can be induced by high temperature,NaCl,and dehydration treatments,while low temperature suppressed its expression.4.A recombinant vector pQE30-SmGRP1 was constructed and transferred into E.coli M15 for protein expression and purification.Stains-all staining implied that the protein may be a Ca²⁺ binding protein,but this binding activity could not arouse the conformation change of SmGRP1 as revealed by Ca²⁺-dependent mobility shift assay.5.According to cis-acting elements' localization predicted by bioinformatical tools,five 5' deleted promoter segments were obtained by PCR and cloned into pCAMBIA-1391z vector.All of the "Promoter-reporter" constructs were transformed into S.miltiorrhiza and a large number of positive transformants of each construct were obtained.Consistent with the result of real-time quantitative RT-PCR analysis,the reporter gene's expression could be induced by ABA,dark,high temperature,NaCl and low temperature treatments by GUS staining analysis.Moreover,the transformants of different 5'-deletion construct responsed differently under diverse treatments.This result implied that the ABA responsive elements were mainly located in -1,851- -760 bp,dark responsive elements mainly in -759- -252 bp region,high temperature responsive elements maily in -251- +81 bp region and NaCl responsive elements mainly in -1,851- -102 bp region.6.The "knock in" and "knock down" mutants of SmGRP1 were achieved by Agrobacterium tumefaciens-mediated gene transformation method.Results indicated that all the mutants showed no obvious phenotypic differences compared with the control plantlets,except that the stoma aperture of the "knock in" mutants were always smaller.While the epidermal strips of both mutants and control plantlets were treated by ABA,dark,NO and H₂O₂,the stoma of the "knock down" lines remained open.Besides,the water loss rate of "knock in" mutants was slower than control lines when exposed to air,while that of"knock down" mutants was much quicker.In conclusion,SmGRP1 expressed highly in leaves and stems of S.miltiorrhiza,and its expression can be induced by ABA,dark,dehydration,NaCl and some other stress conditions. SmGRP1 was presumed to involve in stomatal closure course as a Ca²⁺ binding protein and play important roles in plant stress resistance.