The Identification Of Salvia Miltiorrhiza Laccase Gene Family And The Function Study Of SmLAC3

Salvia miltiorrhiza,a perennial medicinal plant,belongs to the Labiatae family.Its dry roots and rhizomes were used as Traditional Chinese Medicine.The main active ingredients of S.miltiorrhiza are liposoluble tanshinones and water-soluble phenolic acids.Most of the key enzyme genes in the biosynthesis pathway of phenolic acids have been identified;however,the pathway from rosmarinic acid(RA)to salvianolic acid B(SalB)remains unknown.It is currently speculated that Laccase may catalyze the polymerization of RA to form SalB.Laccase(EC 1.10.3.2)is a copper-containing glycoprotein oxidase in polycopper oxidase.It is mainly involved in lignin synthesis,wound healing,response to stress,and maintenance of cell wall structure and integrity in plants.The plant laccase gene families have been identified in Arabidopsis,rice,Brachypodium,poplar,sorghum,cotton and many more plants.At present,the research on laccase gene in S.miltiorrhiza is very limited,and its functional research has not been reported yet.Based on this,based on the whole genome sequence of S.miltiorrhiza,the identification and expression analysis of laccase gene family members of S.miltiorrhiza were carried out by bioinformatics.Four laccase genes which may catalyze the polymerization of rosmarinic acid to form salvianolic acid B in S.miltiorrhiza were screened and cloned,and one of the genes was functionally studied.The main results are as follows:1.33 laccase genes were identified in S.miltiorrhiza.The phylogenetic tree analysis indicated that these gene members could be divided into 7 subfamilies,with 4 pairs of paralogous genes and 10 conserved motifs,of which ? subfamily the laccase gene are unique in S.miltiorrhiza;adaptive evolution analysis indicated that the laccase gene family mainly experienced positive selection under the locus model;promoter analysis indicated that the promoter region of S.milliorrhiza laccase gene contain different hormone response elements and stress response elements,and its expression may be induced by different hormones and stress treatment;gene expression pattern analysis revealed that members of the S.miltiorrhiza laccase gene family were expressed in different tissues of S.milliorrhiza,and 8 of them were specific in roots;the results of the transcriptome analysis of methyl jasmonate and salicylic acid treatment showed that there were 8 genes that responded significantly to methyl jasmonate treatment and 9 genes that responded significantly to salicylic acid treatment.There are 4 genes that respond to both hormone treatments(SmLAC14,SmLAC28,SmLAC30 and SmLAC31).2.Four salvia miltiorrhiza laccase genes were cloned by PCR,which were SmLAC2,SmLAC3,SmLAC20 and SmLAC21.The transmembrane domain and signal peptide analysis indicated that SmLAC2,SmLAC3 and SmLAC20 belonged to non-transmembrane secreted proteins,while SmLAC21 is a transmembrane secreted protein;their ORF were constructed into the prokaryotic expression vector pET28a by restriction enzyme ligase method,and the target protein was mainly detected by SDS-PAGE after induction of 0.5 mM IPTG at 37 C for 6 h.The expression of the inactive inclusion body was expressed,and when the induction temperature was lowered at 30 C,the protein of interest was also expressed in the supernatant.3.The SmLAC3 overexpression vector was constructed by Gateway technology,it was successfully transformed into Salvia miltiorrhiza by the Agrobacterium tumefaciens-mediated leaf disc transformation method,and 20 positive transgenic lines were obtained by DNA level;four transgenic lines with high expression of SmLAC3 were screened by RNA level:OE-2,OE-10,OE-15,OE-17;HPLC method was used to detect the contents of rosmarinic acid and salvianolic acid B in these four lines and two control lines,the contents of rosmarinic acid in the roots of OE-2,OE-10,OE-15,and OE-1 7 transgenic lines were increased by 2.44,2.93,3.48,and 2.62 times,respectively,compared with the control line,and the contents of salvianolic acid B were increased by 1.82,2.79,3.42 and 2.13 times compared with the EV2 line;the content of total phenolic acid and total flavonoids in these strains was detected,the total phenolic acid contents in the roots of the transgenic lines was increased by 1.57,2.01,2.51 and 1.73 times,respectively,compared with the EV2 lines,and the contents of total flavonoids in the roots of OE-2,OE-10,OE-15 and OE-17 transgenic lines were increased by 1.71,1.84,2.06 and 1.69 times compared with the EV2 line;the real-time quantitative technique was used to detect the expression levels of the 9 enzyme genes in the salvianolic acid synthesis pathway and the 2 enzyme genes in the lignin synthesis pathway,the 9 enzyme genes of the phenolic acid synthesis pathway were significantly up-regulated,and the expression level of SmTAT1 in the roots of OE-2,OE-10,OE-15 and OE-17 transgenic lines was increased by 4.53.6.35,7.92 and 3.33 times compared with the EV2 strain,respectively.The expression level of Sm4CL1 in the roots of OE-2,OE-10,OE-15 and OE-17 transgenic lines was increased by 4.81,6.12,6.40 and 5.41 times,respectively,compared with EV2 strain.The expression level of Sm4CL2 in the roots of OE-2,OE-10,OE-15 and OE-17 transgenic lines were increased by 5.09,6.15,5.63 and 6.34 times,respectively,compared to the EV2 lines.While the expression level of two enzyme genes of the lignin synthesis pathway is also significantly improved.The lignin in the roots of each strain was stained by Safranin staining,and it was found that the amount of lignin in the transgenic lines was reduced compared with the control line.The results of this study enriched the understanding of the laccase family in Salvia miltiorrhiza and laid the foundation for the analysis of the biosynthesis pathway of salvianolic acid B.