Cloning And Functional Study Of Danshen Transcription Factors SmLBD23 And SmLBD16

Salvia miltiorrhiza Bunge,medicinal herb of Labiatae,used as its dry root and rhizome,and contains a variety of effective active ingredients such as tanshinone,cryptotanshinone,rosmarinic acid,danshensu,salvianolic acid B and so on,with antibacterial and anti-inflammatory,anti-oxidation,anti-hyperlipidemia,anti-liver damage,anti-fibrosis,anti-atherosclerosis,inhibition of cancer cell growth and other pharmacological activities,with blood stasis analgesia,pure heart upset effect,Attending Closed dysmenorrhea,dysentery accumulation,sore swelling,abdominal pain,upset,angina pectoris and other diseases,has a long history of clinical drug use.Since the traditional Chinese medicine is based on water decoction,the water-soluble salvianolic acid component is considered to be the main active substance for exerting the efficacy.Therefore,it is of great significance to use transgenic technology to increase the content of phenolic acids in Salvia miltiorrhiza.The phenolic acids as secondary metabolites of Salvia miltiorrhiza are affected by a variety of internal and external factors.Previous studies have shown that:1.The single-enzyme gene on the synthesis pathway of salvianolic acid was modified to increase the content of salvianolic acids.The purpose of the effect is not good.2.Some transcription factors such as MYB/bHLH can significantly increase secondary metabolite production by regulating multiple genes.LBD,as a transcription factor,also plays an important role in the regulation of plant growth and metabolism.So studying the secondary metabolism of Salvia miltiorrhiza LBD and Salvia miltiorrhiza is very significant.The main research contents and results are as follows:1.Two members of the LBD gene family were cloned from the genome library of Salvia miltiorrhiza.They were named SmLBD23 and SmLBD 16,respectively.SmLBD23 contained an open reading frame of 588bp and encoded 195 amino acids.SmLBD16 contained a length of 747bp.The open reading frame encodes 258 amino acids.Their encoded products contain a conserved LOB domain consisting of about 100 amino acid residues and belong to the DUF260 superfamily.Protein subcellular localization predictions revealed that both SmLBD23 and SmLBD16 are localized to the nucleus,with no transmembrane domain and no signal peptide.It shows that the two do not have the structural basis of directional transport,and are not exogenous proteins.As a transcription factor,they mainly play a role in the nucleus.In addition,analysis of the cis-acting elements in the promoter regions of these two genes revealed that they contained multiple signal-responsive elements,such as the light-responsive element Box 4,TCT-motif and TCCC-motif,and the GATC response element TATC-box.Methyl jasmonate response element TGACG-motif,salicylic acid response control element TCA-element.It also contains auxin response elements,low temperature response elements,and drought inducing elements.Among them,methyl jasmonate can significantly increase the secondary metabolites of Salvia miltiorrhiza.In addition,components such as MYC binding sites and MYB binding sites are also included.2.Separating and purifying Arabidopsis thaliana protoplasts with sufficient quantity and activity,and constructing subcellular localization vectors for SmLBD23 and SmLBD16,and transferring them into Arabidopsis thalianaplasma through PEG-mediated chemical transformation methods.In vivo,it was found that both SmLBD23 and SmLBD16 were localized in the nucleus,which was consistent with the prediction results,which was consistent with the characteristics that these two genes play a role as transcription factors in the nucleus.3.Overexpression vectors and interference vectors of SmLBD23 and SmLBD16 were constructed by Gateway and amiRNA technology,and transgenic plants were obtained through Agrobacterium tumefaciens-mediated transformation of leaves of Salvia miltiorrhiza.The process of obtaining transgenic plants was long and complicated.Finally,only SmLBD23 over-expressing plants and SmLBD16 interference plants(positive plants verified from both DNA and RNA levels)were obtained and the anthocyanins and total phenolic acids in the leaves and roots of the two transgenic plants were obtained.The content of total flavonoids was tested and it was found that:compared with the control,the anthocyanin content in the SmLBD23 overexpression line was reduced to varying degrees,the anthocyanin content in the SmLBD16 interference line was increased to varying degrees,SmLBD23 overexpression The content of total phenolic acids in the strain decreased to varying degrees,and the total phenolic acid content in the SmLBD16 interference strain increased to varying degrees.The content of total flavonoids in the SmLBD23 overexpression strain varied in different degrees in the leaves and roots.The general increase in leaves,generally reduced in the root.The content of total flavonoids in SmLBD16 interfering lines varied in different degrees in the leaves and roots of the plants,generally decreased in the leaves,and generally increased in the roots.Contrary to the result of overexpression of SmLBD23.4.The detection of tanshinones and salvianolic acids in the SmLBD23 overexpressing strain of Salvia miltiorrhiza and Salvia miltiorrhiza SmLBD16 was performed by HPLC.Compared with the control,SmLBD23 overexpression lines 1-4,1-5 were detected.The content of rosmarinic acid in the leaves and roots of 1-6 lines was decreased to varying degrees,while salvianolic acid B,cryptotanshinone,and tanshinone IIA were not significantly changed in the leaves and roots of the SmLBD23 overexpression line.The content of rosmarinic acid in the leaves and roots of SmLBD16 strain 6-9,6-13,6-14 strains increased to varying degrees,while salvianolic acid B,cryptotanshinone,and tanshinone IIA in SmBD16 interference strains.There was no significant change in the leaves and roots.From the results of HPLC detection of secondary metabolites in Salvia miltiorrhiza,the rosmarinic acid levels in Salvia miltiorrhiza SmLBD23 and SmLBD16 transgenic lines were varied to varying degrees.In addition,the anthocyanin content was in the transgenic lines of Salvia miltiorrhiza SmLBD23 and SmLBD16.The strains also have varying degrees of variation.To understand how the transcription factors SmLBD23 and SmLBD16 alter the content of rosmarinic acid and anthocyanins,we selected 18 enzyme genes in the synthetic pathway of phenolic acids and The three enzyme genes synthesized by anthocyanins,in addition to a MYB transcription factor SMPAP1,its overexpression will cause an increase in anthocyanin content.The synthesis of anthocyanins and the synthesis of phenolic acids share the phenylpropanoid metabolic pathway.If the enzyme genes in the shared pathway are changed,they may cause a common change in these two substances.The results showed that the expression of the key enzyme genes in the salvianolic acid biosynthetic pathway was altered to varying degrees in the transgenic lines of Salvia miltiorrhiza SmLBD23.Compared to the control,TAT1,TAT2,TAT3,PAL2,C4H2,4CL1,HCT1 and The expression level of HCT2 was significantly decreased,and there was no significant change in other enzyme genes.PAL2,C4H2 and 4CL1 belong to the phenylpropanoid pathway and are the common upstream pathways of anthocyanin synthesis and phenolic acid synthesis.The expression levels of key enzyme genes CHS,F3'5'H and FLS in the anthocyanin synthesis pathway were significantly reduced,and the expression level of the transcription factor SMPAP1 was not significantly changed.In the interference strain of SmLBD16,the expression levels of HPPR1,PAL1,PAL3,C4H1,4CL2,and RAS were significantly increased,and there was no significant change in other enzyme genes.PAL1,PAL3,C4H1 and 4CL2 belong to the phenylpropanoid pathway and are the common upstream pathways of anthocyanin synthesis and phenolic acid synthesis.There were no significant changes in the expression levels of the key enzyme genes CHS,F3'5'H and FLS in the anthocyanin synthesis pathway,and the expression level of the transcription factor SmPAPl was significantly increased.It is speculated that changes in the anthocyanin content may be caused by changes in the enzyme genes involved in the metabolism of phenylpropanoids upstream of the anthocyanin synthesis pathway.Moreover,this change is likely to be related to the regulation of the transcription factor SmPAP1.5.SmLBD23 and SmLBD16 were expressed in root,stem and leaf of Salvia miltiorrhiza,but the expression level was significantly different.The expression level was the highest in leaves,followed by roots,and the expression levels in stems and flowers were relatively low.The genes may play an important role in the leaves,which is consistent with the role of the two genes in the synthesis of phenolic acids.After Salvia miltiorrhiza was treated with MeJA,the expression levels of SmLBD23 and SmLBD16 varied to varying degrees.Among them,the expression level of SmLBD23 was highest at 1 h and then decreased gradually.The expression level of SmLBD16 gene had two peaks,which were at 1 h and 4h.The results showed that SmLBD23 and SmLBD16 responded to jasmonate signal in Salvia miltiorrhiza and participated in the JA signal pathway.Compared with the control,the expression levels of SmLBD23 and SmLBD16 in the overexpressing SmMYC2 overexpressing strains were significantly increased,indicating that these two genes are related to SmMYC2,while SmMYC2 plays a role in the JA signaling pathway.,It is speculated that SmLBD23 and SmLBD16 also play a role.The expression level of SmLBD23 was increased in the strain overexpressing SmAZ1,decreased in the strain overexpressing SmJAZ8,and increased in the interference strain of the Jas conserved domain.The expression level of SmLBD16 was increased in the strain overexpressing SmJAZ1,decreased in the strain overexpressing SmJAZ8,and decreased in the interference strain of the Jas conserved domain.JAZ protein plays an important role in the negative regulatory protein in the jasmonate signaling pathway,and the expression of SmLBD23 and SmBD16 is affected by JAZ expression,indicating that they are related to each other.It is likely that they participate in the jasmonate signaling pathway and jointly regulate the secondary metabolism of Salvia miltiorrhiza.way.Yeast double-heterologous results showed that SmLBD23 and SmJAZl,SmMYB36,SmbHlH37,SmMYB97,and SmSmMYC2b all had protein interactions,indicating that they may work together in the Salvia miltiorrhiza plant or act in the form of protein complexes,requiring further experimental validation.