Biosynthesis Of Indole Glucosinolates From Turnip

Glucosinolate is a nitrogen-and sulfur-containing glycosidic natural compound with abundant species.It is not only the main source of flavor substances in cruciferous vegetables,but also has biological activities such as resisting insect pests or cancer.Brassica rapa var.rapa is a kind of crop widely used in medicine,food and feed in arctic-alpine area.Its root tuber contains various nutrients such as polysaccharide,flavone,glucosinolate and vitamin.The content and species of glucosinolates are superior to those of the other brassica vegetables and Arabidopsis thaliana,making it become high-quality materials for glucosinolate research.In this research,the genes involved in the indole glucosinolate biosynthesis pathway in Brassica rapa var.rapa are identified and their function are verified,use the Escherichia coli expression system to complete heterologous expression.The engineering bacterium for synthesizing indole glucosinolate is constructed by expression strain BL21(DE3)and expression vector pET-52b,and the induction conditions are optimized to increase the yield of indole-3-methanol glucosinolate(I3M).The major contents and results are as following:(1)Using the gene sequences involved in I3M biosynthesis in Arabidopsis thaliana,Brassica pekinensis and Brassica oleracea var.capitata,seven corresponding groups of genes were searched from genome of Brassica rapa var.rapa(CYP79B2;CYP83B1;GSTF9;GGP1;SUR1;UGT74B1;ST5a).In order to select genes with similar functions to reported genes in Arabidopsis thaliana more accurately,phylogenetic trees need to be constructed by MEGA software and the amino acid sequences of genes from the local blast results are subjected to multiple alignment by DNAMAN software.Comparing the homology and similarity of the search results with the gene sequence of Arabidopsis thaliana,seven target genes are finally identified.In addition,the chromosomal localization of seven groups of genes has been analyzed and the conservative motifs in their protein sequences are also identified.(2)The expression patterns of seven target genes in different parts of turnip at different growth stages before and after tuberous root development are analyzed.Meanwhile,the content of glucosinolate of different kinds in the corresponding period and part of turnip is also detected.In order to determine whether the target genes have the function of synthesizing I3M,firstly,detect the subcellular localization of seven target genes by laser confocal microscope,and then the gene function is verified by using tobacco transient expression system.The final result shows that compared with the control group,a large amount of I3M could be produced in the leaves injected with the mixed bacterial solution containing seven target genes.In summary,the co-expression of these genes could synthesize I3M.(3)After the functional verification of genes,in order to stabilize the expression in prokaryotic cells,the codons of the seven target genes need to be optimized,and the engineering bacterium for synthesizing I3M from scratch is constructed.Under the induction conditions of 18? and IPTG concentration of 0.5 mM,a spot of target product is detected,and the yield is about 1.89 mg/L.In order to promote the synthesis of the target product I3M,the induction conditions should be optimized.Finally,the most suitable IPTG concentration and temperature for inducing are determined to 0.5 mM and 25?.(4)After the most suitable induction conditions is determined,for further improvement of the yield,exogenous substances with the following concentrations are added to the original culture medium:0.5 g/L Trp,0.8 g/L Met,15 g/L Glucose and 0.05 M/L MgSO4·7H2O.The addition of MgSO4 inhibits the growth of some bacterial strains,the bacterial liquid can reach the standard OD600 value by appropriately prolonging the incubation time.The addition of exogenous substances can promote the synthesis of I3M and increase its yield.For further exploration of the culture medium formula which is more beneficial to the growth and protein expression of Escherichia coli expression strain,use TB medium to replace LB medium,and the same kinds ofexogenous substances with the same concentration are added,the yield of I3M is also increased.However,the addition of 0.8 g/L Met resulted in a significant decrease in I3M production.Therefore,when use TB medium to replace LB medium,the most suitable concentration and combination of exogenous substances obtained in this study is:0.5 g/L Trp,0.5 g/L Met,15 g/L Glucose and 0.05 M/L MgSO4·7H2O,the yield is about 3.6 mg/L in this condition.