Screening Of Genes Regulating Secondary Metabolite Synthesis And Construction Of Genetic Transformation System In Bletilla Striata

Objective:To screen the regulatory genes related to the biosynthesis and accumulation of secondary metabolites like Coelonin,Dactylorhin A and Militarine in Bletilla striata(B.striata),to analyze the mechanism of 4-coumarate-Co A ligase(4CL)genes impacting on biosynthesis of secondary metabolites.And to explore the conditions of two genetic transformation systems through agrobacterium mediated into callus and through magnetic nano materials into protoplasts of B.striata.Methods:1.Candidate genes’screening and expression pattern profilingR language was used to screen candidate genes related to the synthesis of Coelonin,Militarine and Dactylorhin A in B.striata suspension cultured cells.q PCR was used to verify the expression number of important candidate genes in different stages of suspension culture and the expression pattern in different tissues and organs of B.striata.The candidate genes were cloned,and the overexpression recombinant plasmid with fusion expression of candidate genes and Green fluorescent protein(GFP)gene was constructed.2.Analysis of 4CL gene family regulating secondary metabolite synthesis of B.striataThe protein primary structure,secondary structure,motif and KEGG enrichment results of 4CL gene family of B.striata(Bs4CL)were analyzed by bioinformatics analysis method.The genetic polymorphism of Bs4CL in different B.striata strains was detected by EST-SSR.The regulatory role of Bs4CL gene in secondary metabolites was explored by correlation analysis,and using q PCR technology to analyzed the expression pattern of Bs4CL gene family.3.Construction of genetic transformation system of B.striata callusAdded 0,0.1,0.3 and 0.5 mg/L of Phosphinothricin(PPT)to the B.striata callus subculture medium to explore the optimal screening concentration of PPT.Then the expression vector p CB2004-35S-HDG11 was transformed into competent cells of EHA105,LBA4404 and GV3101 Agrobacterium inactivated cells,and the positive clonal bacteria was cultured to infect the callus.Then the Agrobacterium mediated genetic transformation system of B.striata callus was established through co-culture,sterilization,screening culture,callus proliferation culture,bud proliferation culture and rooting and strengthening seedling culture,and the DNA of PPT resistant B.striata seedling was extracted for PCR detection of HDG11 gene.4.Construction of genetic transformation system of B.striata protoplastsThrough the pretreatment of low-temperature darkness and mannitol,the enzymatic hydrolysis solution was added in the ratio of 10 mL/g and hydrolyzed in the dark at 25℃and 50 rpm for 4 hours.The purified protoplasts were obtained by filtration of 200 mesh cell sieve and cleaning with cleaning solution.Different concentrations of cellulose,pectinase and segregating enzyme were used to screen the ratio of enzymatic hydrolysate suitable for the separation of B.striata callus.Take the content as more than 5×10~5pieces/mL of 100μL B.striata protoplasts and 10μL magnetic nanoparticles with a mass ratio of 1:5 and the complex of p BI221-GFP expression vector were mixed evenly,and the transformation was induced by magnetic field standing for 30 minutes.Then,it was washed and dark cultured with KM8P medium for 48 hours to observe the expression of GFP gene.Results:1.Screening and analysis of regulatory genes in transcriptome databaseThrough the correlation analysis between the differential gene expression and the accumulation of secondary metabolites in different growth stages of B.striata suspension cultured cells,38 genes,29 transcription factor genes and 85 unknown genes in the known synthesis pathways related to the synthesis of Coelonin,Militarine and Dactylorhin A were screened.Some genes were associated with the synthesis of multiple secondary metabolites at the same time.Three of them,BsMYB,BsUP1 and BsUP2 gene were selected and cloned.The expression pattern analysis showed that there were significant differences in the expression of these three genes in B.striata different tissues.2.Identification and expression analysis of 4CL gene familiesBy analyzing the annotation information of the above 152 genes,it is found that the4CL gene family is closely related to the synthesis of secondary metabolites and the growth and development of cells.Through in-depth analysis,it is found that 21 4CL homologous genes in the transcriptome database based on B.striata suspension cultured cells.KEGG enrichment analysis showed that B.striata 4CL gene families were mainly involved in lipid metabolism,alpha-linoleic metabolism,amino acid metabolism,phenylpropanoid biosynthesis,and other secondary metabolites.Through EST-SSR detection,it was found that Bs4CL gene showed genetic polymorphism in three local varieties of B.striata.q PCR analysis found that the expression level of Bs4CL gene in B.striata different tissue parts had great differences,among which the expression of Bs4CL2 gene was the highest in B.striata root and tuber.Through the correlation analysis between the relative expression of Bs4CL gene and the contents of Coelonin,Dactylorhin A and Militarine,it is found that4CL gene plays an important regulatory role in the synthesis of Coelonin,Dactylorhin A and Militarine.3.Establishment of callus genetic transformation systemin B.striata.The establishment of B.striata genetic transformation system is helpful to further explore the function and mechanism of candidate genes.In this study,the genetic transformation system of B.striata callus based on Agrobacterium mediated method was constructed by p CB2004-35s-HDG11 expression vector,and then the HDG11 gene of PPT resistant genetic transformation seedlings was detected by PCR.It was found that the positive rates were 1.1%,14.4%and 14.4%respectively.4.Study on genetic transformation system of B.striata protoplasts.The isolation system of protoplasts(1.5%Cellulase R-10+0.4%Pectolyase Y-23+0.5%Macerozyme R-10)was established.The content of protoplasts was 6.33×10~5protoplasts/ml with activity rate of 86.99%.The genetic transformation of protoplasts was constructed by magnetic bead method,and the expression of GFP gene in protoplasts was realized.The transformation rate was 47.86%by microscopic observation.Conclusion:In this study,152 genes are involved in the regulation of the synthesis and accumulation of B.striata secondary metabolites Coelonin,Dactylorhin A and Militarine.Further analysis found that Bs4CL gene family was involved in the growth and development of B.striata secondary metabolites.The Agrobacterium mediated genetic transformation system of B.striata callus was constructed,and the protoplast separation system of B.striata suspension cultured cells and the protoplast genetic transformation system mediated by magnetic nanoparticles were preliminarily constructed.