A Preliminary Study On The Function Of The Transcription Factor SmSPL7 Of Salvia Miltiorrhiza

Salvia miltiorrhiza belongs to the genus Salvia in the Labiatae family.Its dried roots and rhizomes have been used to treat various diseases,such as anti-atherosclerosis,anti-inflammatory,anti-tumor and alleviating diabetes.Previous studies have shown that 201 compounds have been identified from Salvia miltiorrhiza,including lipophilic diterpenoids,water-soluble phenolic acids and other ingredients,which have a variety of pharmacological activities.Plant specific transcription factor SPL plays an important regulatory role in plant growth and development.Although the function of SPL transcription factor has been identified in Arabidopsis and rice,the function of SPL in S.miltiorrhiza has not been reported.Therefore,the SmSPL7 gene and its promoter sequence were obtained from S.miltiorrhiza,and its function was preliminarily explored.The main contents and results of this research are as follows:1.The SmSPL7 open reading frame with a size of 2325bp was cloned from S.miltiorrhiza.The expression level of SmSPL7 in different tissues of S.miltiorrhiza was analyzed.And the results of real-time fluorescence quantitative PCR showed that SmSPL7 was expressed in the main root,lateral root,stem,leaves,corolla,calyx,pistil and stamen of S.miltiorrhiza with the highest expression in the leaves.2.The 1048bp sequence of the 5' promoter region of the SmSPL7 gene was cloned.The analysis of cis-acting elements showed that the SmSPL7 promoter contains abscisic acid,auxin,gibberellin,defense and stress response and anaerobic-induced cis-acting elements.Then,the expression pattern of SmSPL7 was analyzed under the following four hormone treatments:abscisic acid,auxin,gibberellin,and jasmonic acid.The results suggested that ABA treatment and MeJA treatment can exert a stable inhibitory effect on the expression of SmSPL7.3.The subcellular localization vector pEarleyGate 103-SmSPL7 and the yeast expression vector pGBKT7-SmSPL7 were constructed.The results of subcellular localization showed that SmSPL7 was localized in the nucleus;transcriptional autoactivation experiments showed that SmSPL7 had transcriptional autoactivation activity.4.The SmSPL7 promoter sequence of SmSPL7 was constructed on the pCAMBIA13 91Z vector to obtain the recombinant plasmid proSmSPL 7::G US.Agrobacterium-mediated inflorescence dip method was used to obtain the proSmSPL7::GUS transgenic Arabidopsis,and the T3 generation line was screened for GUS histochemistry dyeing.The results of the experiments showed that strong GUS signals can be detected in all stages of transgenic Arabidopsis.5.SmSPL7 was constructed into the overexpression vector pEarleyGate202(688)using Gateway technology and the recombinant vector 688-SmSPL7 was obtained.Then,688-SmSPL7 was transformed into S.miltiorrhiza by Agrobacterium dipping method,and finally an SmSPL7-overexpression(OE)S.miltiorrhiza lines was obtained.After the DNA and RNA levels of the obtained transgenic lines were tested,a total of 10 SmSPL7-OE lines were screened.Among them,the expression levels of SmSPL7 in OE-3 and OE-6 were relatively higher,which were 9.60 times and 10.06 times that of the control lines,respectively.The phenotypes of the overexpression lines OE-3 and OE-6 were observed,and it was found that the growth of the OE-3 and OE-6 lines was inhibited compared with the control,with specific manifestation of smaller plants,less root biomass,and purple leaves.6.The content of total phenolic acid,total flavonoid and anthocyanin in SmSPL7 overexpression line was determined.It was showed that the content of total phenolic acid in the SmSPL7-OE line was not significantly different from that of the control line;the content of total flavonoids in OE-3 line and OE-6 line were 1.30 times and 1.25 times that of the control group,respectively;the content of anthocyanin was 2.31 times and 3.30 times that of the control line.The contents of salvianolic acid B and rosmarinic acid in the SmSPL7-OE lines and the control line were determined by HPLC.The results showed that the content of salvianolic acid B was significantly reduced after overexpression of SmSPL 7,and the content of rosmarinic acid also decreased.7.Transcriptome sequencing analysis was performed on OE-6 and the control.Compared with the control line,there appeared 1154 differentially expressed genes in OE-6 line,of which 408 genes were up-regulated while 746 genes were down-regulated.We found that 22 DEGs in SmSPL7-OE were distributed in plant hormone signal transduction pathway,accounting for 12.57%of all differentially expressed genes;15 DEGs were distributed in phenylpropanoid biosynthesis pathway,accounting for 8.57%of all DEGs;in addition,many differentially expressed genes were enriched in plant pathogen interaction pathway.Totally 6 differentially expressed genes(SmTAT1,SmPAL3,Sm4CL9,Sm4CL10,SmC4Hl and SmRAS4)were found in the phenolic acid synthesis pathway,and their expression levels were 43.86%,34.13%,46.95%,42.55%,41.15%and 30.77%of the control,respectively.Four differentially expressed genes(CHS,F3H,DFR,and ANS)were found in the anthocyanin biosynthesis pathway,and their expressions were up-regulated by 3.43 times,4.08 times,2.93 times,and 2.73 times respectively,compared with that of control line.Real-time fluorescent quantitative PCR was used to verify the expression of enzyme genes in the biosynthetic pathway of phenolic acid and anthocyanin.The quantitative results were largely consistent with the transcriptome results,indicating that the transcriptome data was valid.8.The promoter sequences of key enzyme genes Sm4CL9 and SmTAT1 in the biosynthetic pathway of salvianolic acid B were analyzed.These sequences all contained GTAC motifs specifically bound to SBP protein.Yeast one-hybrid experiments and luciferase reporter gene experiments showed that SmSPL7 directly binds and inhibits the expression of the target genes Sm4CL9 and SmTAT1,thereby negatively regulating the accumulation of salvianolic acids.