The Functional Study Of Laccases In Salvia Miltiorrhiaza Bge. And Transcriptional Factor WRKY34 In Isatis Indigotica Fort.

Rebuilding the metabolic flux by researching the reaction between intermediates,and clarifing the regulatory mechanism by researching the influence factors are two major parts in the analysis of nature products biosynthesis pathway.Both of the studies are based on the discovery of functional genes,however,the former means discovery of the key enzymes of ratelimiting steps in the biosynthesis pathway,the process is point to point.While the later means the discovery of influence factors which may regulate some one or more reactions in the pathway,the process is point to network.Out group work for the analysis of nature products biosynthesis pathway for a long time,and put forward two strategies for the discovery of the functional genes above,respectively.For mining of influence factors,building up gene function network and gene-product network using transcriptome and metabolome database by the alignment between two kinds of figures with distinctive phenotypes.According to this strategy,we group got the Ii WRKY34 as the key factors,which may regulate the lignan biosynthesis pathway in I.indigotica.For mining enzyme genes of reactions,make rational prediction of the reaction and the key enzymes according to the metabolic flux,then identify the target genes by combining the characters of distribution of product and the characters of expression of genes.According to this strategy,we analyzed the production of salvianolic acid B(SAB)in phenolic acid biosynthesis pathway in S.miltiorrhiza,made sure SAB is transformed from rosmarinic acid(RA)as the direct performer.And predicted that laccase might be the enzyme which catalyze the transformation.Considering the distribution of SAB in plant,pick out 5 target laccase from the family of laccase.This text would pick laccases as the research object to explore the catalytic functions from RA to SAB.And pick Ii WRKY34 as the research object to explore the regulation function of this transcriptional factors in lignan biosynthesis pathway I.indigotica.At the same time,to verify the feasibility of the two functional gene discovery strategies above.The main research contents:The functional study of laccases which participate in the biosynthesis of SAB.1.29 full-length sequence laccases were screened from the genomic database of S.miltiorrhiaza,based on the high content of SAB,which is response to Me JA,in root of S.miltiorrhiaza plant,5 target laccases,Sm LAC7,Sm LAC8,Sm LAC20,Sm LAC27,Sm LAC28 were determined by phylogenetic analysis.The 5 protein moleculars were modelled to do experiments.molecular docking with RA and 4 kinds of analogues,and the analysis of molecular dynamics were made to work out the binding capability between protein molecular and compounds,then find out the key residues.The results showed that,Sm LAC7,Sm LAC8 and Sm LAC28 have binding specificity with RA,in which Sm LAC7 showed the highest binding power.Gln,Phe and Arg might be the key residues in the binding process,which provided database to after2.For the functional study of target laccase in vivo,the expression of target laccase genes were decreased by using the gene editing system CRISPR/Cas 9,which could be used in plant got from the group of Zhu Jiankang,and the gene edited hairy-root was used to detect the laccase functions in vivo.5 kinds of phenolic acid compounds were detected,and the accumulation of L-phenylalanine,Coumaric acid and RA did not show obvious difference between transgenic lines and WT.Meanwhile,the content of SAB in group WT was higher than all of the gene edit groups,and the results of Sm LAC8 and Sm LAC28 gene editing lines showed significant difference.It means that,the de-expression of Sm LACs induced the decrease of SAB.At the same time,the content of SAA might be influenced by Sm LAC8.While,the biomass of the hairy-root of gene editing lines was smaller than that of WT;and vascular bundle in the gene editing groups were also been damaged.These results indicated that,Sm LACs might participated in the development of root in S.miltiorrhiaza.However,an interesting discover was gotten from the expression of these target genes detected in every group of hairy-root,Sm LAC7 would has more contactable for all of these target laccases.And the detection of Pro and oxidation resistance displayed the stress tolerance of the hairy-root,and the group of Sm LAC7 became the only target again.It could be a key point in the network of Sm LACs.These results showed that,Sm LACs could catalyze the transformation of SAB in vivo in S.miltiorrhiaza.3.For the functional study of target laccase in vitro,some species in Salvia family with higher content of RA and lower content of SAB relatively were found out,and the genetic transformation systems were induced by using Agrobacterium rhizogenes.As a result,2 of the Sm LAC7 transgenic hairy-root lines were induced successfully in one of sage species.The ratio concentration of SAB/RA in every line of transgenic hairy-root was higher than that in WT,which indicated that heterogeneous expression of Sm LAC7 might promote the biosynthesis of SAB in this sage species.These results showed that,Sm LACs(Sm LAC7)could catalyze the transformation of SAB in vitro in S.miltiorrhiaza.The functional study of transcriptional factor Ii WRKY34 in Isatis indigotiuca.Ii WRKY34 has a positive relationship with the accumulation of lignan products and the biomass of root.Display the function of this transcriptional factor in lignan biosynthesis pathway I.indigotica.overexpression hairy-root lines were induced and the content of 6 compounds were detected.In the overexpression hairy-root lines,the accumulation of 6 kinds of lignans increased,all of the contents show significant difference with WT,except pinoresinol 4-o-glucopyranoside.Further functional studies found that overexpression of Ii WRKY34 promoted root growth(increased biomass by 3.7-fold).While inhibiting the expression of Ii WRKY34 significantly reduces the content of lignins,the growth of roots was inhibited,the biomass is substantially reduced(approximately 70% reduction).The results showed that,the gene expression level of 1.For the study of influence by the gene expression variation of Ii WRKY34 on the development of plant and on the accumulation of lignan compounds,gene inhibition and2.For exploring the influence of expression variation of Ii WRKY34 on the ability of stress tolerance,the hairy-root lines were processed by different kinds of stress.The overexpression hairy-root lines increased tolerance to high salt and drought,while the gene expression inhibited lines showed high salt tolerance and low drought stress tolerance.This results provide that Ii WRKY34 participates in the progress of stress tolerance in I.indigotica,and the higher level of the gene expression will induce the stress tolerance of the plant.3.For exploring the regulation molecular mechanism,the transcriptome and metabolome analysis of the hairy roots are used to construct a network between Ii WRKY34-lignan biosynthesis related genes-lignans.Confirming the interaction between Ii WRKY34 and the key enzyme Ii4CL3 on the lignans synthesis pathway by EMSA.The results showed that,Ii WRKY34 regulate the accumulation of lignans by regulating the expression of key enzymes on the biosynthesis pathway,such as Ii4CL3.This research provided foundation for the deeper study on the molecular mechanism of regulation.In conclusion,the functional study of laccases in S.miltiorrhiaza and the transcription factor Ii WRKY34 in I.indigotica perfected the knowledge of the pathway of themselves,proved the discovery strategies of functional genes in nature products biosynthesis pathway put forward by our group.At the same time,provided methodological references to the future research of nature products biosynthesis pathway.