| Salvia miltiorrhiza Bunge is a perennial medicinal plant of the genus Salvia belonging to the Lamiaceae family.Its dried roots and rhizomes used as medicine,called Danshen in Chinese.The main active ingredients of Danshen can be divided into two groups:fat-soluble tanshinones and water-soluble salvianolic acids.With the gradual enrichment of S.miltiorrhiza genome,transcriptome,metabolome,and proteomic data,the research on S.miltiorrhiza is becoming deeper and deeper.Increasing the content of active ingredients in S.miltiorrhiza and the yield of its roots is the ultimate goal for Danshen breeding,and also is the key link to improve the quality of S.miltiorrhiza medicinal materials.The SPL(SQUAMOSA promoter-binding protein-Like)gene family encodes a kind of plant specific transcription factor,which plays important roles in the process of plant nutritional phase transition,signal transduction,and stress response.The SPL gene family has been identified in a variety of plants,such as Arabidopsis,Oraza sativa,Zea mays,Betula platyphylla and Solanum lycopersicum.At present,15 SPL genes have been identified in S.miltiorrhiza,but their specific function in S.miltiorrhiza has never been investigated.Based on the S.miltiorrhiza genome database,we cloned the SmSPL6 gene and its promoter region,and constructed them into different vectors to investigatate the expression patterns of SmSPL6 and its regulation mechanism on the biosynthesis of salvianolic acids and root development.This research provides information for increasing the content of salvianolic acids and improving the root quality of S.miltiorrhiza.According to the results of phylogenetic tree analysis,we found that A.thaliana AtSPL9 and S.miltiorrhiza SmSPL6 clustered in the same subfamily,and a large number of literature reports indicated that AtSPL9 had an influence on the secondary metabolism of plants and affect the growth of lateral roots.Based on this,this study chose S.miltiorrhiza SmSPL6 as the research object,in order to explore whether SmSPL6 also involves in regulating the secondary metabolism of S.miltiorrhiza,and affects their lateral root development.The main results are as follows:1.The SmSPL6 of S.miltiorrhiza contains a 1083 bp open reading frame,which encodes 360 amino acids and contains SBP conserved domains.And the 3D structure of its amino acid sequence is in line with the typical SPL transcription factor feature.qRT-PCR results showed that SmSPL6 was expressed in the roots,stems,flowers,leaves of different ages,and other different tissues of S.miltiorrhiza,with the highest expression level in the upper leaves.The expression level of SmSPL6 was detected by qRT-PCR when the S.miltiorrhiza seedlings were treated with exogenous hormone MeJA,IAA,GA,or ABA.The results showed that SmSPL6 was responsive to multiple hormones,among which MeJA and IAA could significantly inhibited its expression.2.The 862 bp promoter region at the 5' end of the SmSPL6 was cloned.The promoter analysis by PlantCARE showed that some cis-acting elements responding to plant hormone such as gibberellin,auxin,and abscisic acid existed in the promoter region.The SmSPL6 promoter was constructed into the pCAMBIA1391Z vector containing the GUS protein to obtain the expression vector proSmSPL6::GUS,and the GV3101-mediated Arabidopsis thaliana inflorescence dip method was used to obtain transgenic Arabidopsis.The positive transgenic plants were selected by hygromycin to the T2 generation.GUS staining showed that GUS signal was strong at different growth stages and different tissue of transgenic Arabidopsis expressing proSmSPL6::GUS.3.SmSPL6 was constructed into the subcellular localization vector pEarlygate103 containing green fluorescent protein and the yeast bait expression vector pGBKT7-GW through Gateway technology.The subcellular localization experiment of SmSPL6 was performed by GV3101-mediated infecting onion epidermal cells.and the result showed that SmSPL6 located in the nucleus.The yeast strain AH109-mediated transcriptional autoactivation experiment indicated that SmSPL6 had transcriptional autoactivation activity.4.The overexpression vector of SmSPL6 was constructed,and transgenic S.miltiorrhiza plants were obtained by EHA105-mediated leaf disc transformation method.The contents of total phenolic acids,total flavonoids,and total anthocyanins in SmSPL6 overexpression(OE)plants(OE5?OE6 and OE8)were determined.The total phenolic acid content increased significantly in the overexpression lines OE5 when compared with CK,and in OE5,OE6,OE8 were 1.36 times,1.18 times and 1.26 times higher than that of CK,respectively.The total flavonoid content in OE5 and OE6 were significantly increased,there are 2.11 times,1.42 times in OE5 and OE6 higher than that of CK,respectively,and in OE8 is 1.12 times higher than that of CK.The total anthocyanin content of SmSPL6 overexpression lines decreased,in OE5 and OE8 decreased significantly,which were 76.9%and 86.2%of CK,respectively,followed by OE6,which decreased to 93.4%of CK.It shows that SmSPL6 can promote the accumulation of total phenolic acids and total flavonoids of S.miltiorrhiza,while inhibiting the accumulation of total anthocyanins.5.The accumulation of salvianolic acids in SmSPL6-OE lines were measured by high performance liquid chromatography(HPLC).The results showed that the content of rosmarinic acid(RA)and salvianolic acid B(SaIB)were significantly increased in both the whole plantlets and root of SmSPL6-OE lines.Compared with the control(CK),the phenolic acid contents in OE lines significantly increased.The content of RA and SalB in the whole plantlets of OE5 is 1.39 times and 3.69 times that of CK,respectively.The content of RA and SalB in the root of OE5 is 2.36 times and 8.19 times that of CK,respectively.These results indicated that SmSPL6 positively regulates the biosynthesis of salvianolic acids.6.The morphological structure of the root system of SmSPL6-OE lines obviously changed,including root length,number of lateral roots,and fresh weight.The analysis results showed that overexpressing the SmSPL6 in S.miltiorrhiza significantly increased the length of adventitious root.Th e root lengths of OE5,OE6 and OE8 were 1.88 times,2.01 times and 2.36 times that of the CK,respectively.The number of lateral roots and the fresh roots weight were significantly reduced in transgenic lines when compared with CK.The fresh weight of the root of OE5,OE6 and OE8 were 80.75%,77.52%and 49.74%of the CK,respectively.The endogenous auxin content in the roots of the CK and the transgenic lines OE5,OE6 and OE8 was measured.The results showed that the auxin content in OE5 and OE6 was reduced to 84.9%and 79.3%of that of CK,respectively.Compared with CK,the auxin content in OE8 has no significant difference,but it also shows a downward trend,which is 95.9%of the CK.7.RNA-seqing was performed in the well-grown OE5 and CK,and the differential expression genes were analyzed.815 differentially expressed genes(DEGs)were identified in OE5 under the condition that FDR=0.01 and FC=2,of which 188 genes were up-regulated,and SmSPL6 increased highestly which was approximately 28 times,while the other 187 genes all were up-regulated less than 8 fold,and 627 genes were down-regulated,among which 18 genes were down-regulated extremely significant,approximately were 6%of that in CK.231 DEGs were related to plant metabolism,accounting for 63.99%of the annotated genes.We further analyzed the DEGs in the phenolic acid biosynthetic pathway and the auxin signaling pathway in OE5,which laid the foundation for the future research on the molecular mechanism of SmSPL6 in regulating the lateral roots development and the.salvianolic acids biosynthesis of S.miltiorrhiza.8.We determined the expression levels of 15 key enzyme genes in the pathway of salvianolic acid biosynthesis in the root of S.miltiorrhiza by qRT-PCR.The results showed that the overexpressing SmSPL6 activated the expression of the enzyme genes SmHPPRl,SmHPPR2,SmHPPR3,Sm4CL1,Sm4CL9,SmRAS2,SmRAS4 and SmCYP98A14.It shows that SmSPL6 promotes the accumulation of salvianolic acids by activating the expression of multiple enzyme genes in the biosynthesis pathway of salvianolic acids.9.The 2000 bp promoter regions of the 5' end of 815 DEGs were analyzed to search the numbers of GTAC motifs which were specifically bound by the SBP conserved domain,which provided theoretical reference for screening the target genes of SmSPL6 in the future. |
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