Cloning Of Salvia Miltiorrhiza SmMYB52 Gene And Preliminary Study On Its Function

The dried root of Salvia miltiorrhiza Bunge is a traditional Chinese medicinal material and has the function in promoting blood circulation and improving body functions.It has a long history in medicinal use because of the functions in anti-inflammatory,anti-oxidation,and inhibition of platelet aggregation,so it has been widely used to treat diabetes,angina pectoris,coronary heart disease,hyperlipidemia,cerebrovascular diseases,dysmenorrhea and amenorrhea.S.miltiorrhiza has been gradually developped as a model plant for studying the secondary metabolic regulation of medicinal plants due to its short generation cycle,small genome,low growth requirements,complete tissue culture and transformation systems.For S.miltiorrhiza,the active ingredient content and root yield are the main indicators of quality.At present,there have been many reports on regulating the content of active ingredients,but the research on the root growth and development has not been reported.The MYB family is one of the largest transcription factor families in the plant kingdom and plays an important role in plant growth and development,controlling cell fate,regulating metabolites,and responsing to biological stress.The R2R3-MYB transcription factor AtMYB93 is known to be a negative regulator for lateral root development in Arabidopsis.In this study,we analyzed the relationship between AtMYB93 and all members of the R2R3-MYB family in S.miltiorrhiza,and found that AtMYB93 showed the highest similarity with SmMYB52,which is thought to regulate the growth and development of root in S.miltiorrhiza.Based on this,we took SmMYB52 as the research object and conduct preliminary research on it.The main research contents and results are as follows:1.The full-length sequence of SmMYB52(GenBank accession number:KF059406)was cloned from the gDNA and cDNA,respectively.The gene sequence is 1041 bp in length,contains 2 intron sequences and 879 bp CDS,and encodes 292 amino acids.Bioinformatics analysis showed that the similarity between SmMYB52 and AtMYB93 reached to 46.03%.The relative molecular mass of SmMYB52 is 32.27 kD and the theoretical isoelectric point is 6.48;the predicted instability parameter is 52.47;and the average coefficient of the protein hydrophobicity is-0.416.Therefore,it is speculated that SmMYB52 is an acidic,hydrophilic,and unstable protein.SmMYB52 contains two MYB repeats,which are composed of 51 and 49 amino acid residues,respectively.2.The promoter region of SmMYB52 was cloned and analyzed.The promoter region of SmMYB52 was analyzed by software PlantCARE:this region contains abscisic acid response element,gibberellin response element,methyl jasmonate response element,light response element and other cis-acting elements.qRT-PCR determined the expression of SmMYB52 in various organs and the results showed that the gene expressed in roots,stems,leaves and flowers,with the highest expression in roots and the lowest in stems.The 60-day-old S.miltiorrhiza was treated with 0.1 mM GA3,0.1 mM IAA,0.1 mM ABA and 5 mM MeJA,respectively.The relative expression of SmMYB52 gene was detected by qRT-PCR,which showed that SmMYB52 responds to all four hormones.3.The online software WOLFPSORT predicted that SmMYB52 protein was localized in the nucleus.To study the subcellular localization of SmMYB52,we used Gateway technology to construct SmMYB52 in pEarleyGate103 and obtained the fusion protein expression vector pEarleyGate103-SmMYB52.Subcellular localization analysis by the transient expression experiment on onion epidermal cell showed that the SmMYB52-GFP fusion protein was distributed in the nucleus and cell membrane.4.Gateway technology was used to clone SmMYB52 into the yeast expression vector pGBKT7,co-transform with pGADT7 into the yeast AH 109 strain,and culture it on a SD/-Trp-Leu-His-Ade four-deficient solid medium.We found that the positive control grew on the four-deficient solid medium,and the negative control cells did not grow on it.The experimental group cells could also grow on the four-deficient solid medium.The experimental results showed that SmMYB52,as a transcription factor,has the self-activating activity.5.SmMYB52 over-expressing vector and interference vector were constructed,and was transformed in S.miltiorrhiza by Agrobacterium tumefaciens-mediated leaf disc method.After screened and verification at the DNA level,two over-expressing lines(OE-1 and OE-2)and three interference lines(myb52-1,myb52-2,and myb52-3)of SmMYB52 were obtained.qRT-PCR was used to detect the expression of SmMYB52 in the transgenic lines.The results showed that the expression of SmMYB52 in the over-expressing lines OE-1 and OE-2 was significantly increased,which were 252-fold and 551-fold respectively compared with the control;the expression levels of SmMYB52 in the interference line myb52-1,myb52-2 and myb52-3 were significantly reduced,which were 36%,53%and 29%of the control lines,respectively.6.The phynotypes of root in SmMYB52 over-expressing lines OE-1,OE-2 and interference lines myb52-1,myb52-2,myb52-3 were observed and biomass was weighed.Compared with the control,the roots of OE-1 and OE-2 are shorter,smaller,of which biomass dropped below 20%;while no obvious difference was observed between the interfering lines myb52-1,myb52-2,myb52-3 and control lines,with no significant increase in their biomass.Based on the above,we draw the following conclusion:SmMYB52,as a transcription factor in the R2R3-MYB family,has a regulatory effect on the growth and development of S.miltiorrhiza root,and it is a negative regulatory effect.It lays a foundation for further research on the specific functions of SmMYB52 and the mechanism of SmMYB52 in regulating the growth and development of root in S.miltiorrhiza.