Isolation And Expression Analysis Of Genes Involved In Glucosinolate Biosynthesis And Metabolism In Radish(Raphanus Sativus L.)

Radish (Raphanus sativus L.) is one of the most important cruciferous vegetables with high nutrition and medical value. The anticancer activity is most due to the activity of the isothiocyanates sulforaphane (SF) derived from glucoraphanin (RAA). Glucosinolates (GS) are one of the most important secondary metabolites. Glucosinolates and their degradation products play very important roles in pathogen and insect interactions as well as in human health. In recent decades, most of the related genes involved in the glucosinolate biosynthesis pathway have been identified and functionally characterized in Arabidopsis thaliana and Brassica rapa, however, detailed analyses of the relevant genes in radish were still lacking. The objective of this work was to clone and analyze these genes including5’-flanking region. Furthermore, we also studied the expression characterization of these genes. These results would help to provide a useful base for producing new varieties of radish cultivation which have higher glucosinolate contents by means of genetically modified engineering or expression regulation means. The main results of this study are as follows:1. The full-length cDNA and genomic DNA sequences of RsBCAT4, RsUGT74Bl and partial genomic DNA sequences of RsGS-OX1have been isolated from radish. By compared the deduced amino acids with the GenBank corresponding sequence and then made phylogenetic trees. Furthermore, the differential expression of these genes in leaf, stem and root at various developmental stages were measured by means of RT-PCR and qRT-PCR. In addition, expression of these genes was also studied after treated with methyl jasmonate (MeJA) and wounding (WO) treatment. These results showed that RsBCAT4expressed in leaf, stem and root at seeding stage, but slightly expressed in root at taproot thickening stage and mature stage; RsUGT74B1and RsGS-OX1were slightly expressed during all the stages tested, while the expression level of these genes was significantly increased after WO and MeJA treatment. 2. The full-length cDNA and genomic DNA sequences of RsCYP79Fl, RsCYP83A1, RsSURl and RsSOT18have been isolated from radish. Then, the5’-flanking region of these genes have also been cloned by genomic walking method. Promoter sequences analyzed by PLACE and PlantCARE showed that it contents TATA-box, CAAT-box and some cis-acting element such as MYB binding site, cis-acting regulatory element involved in ABA response, cis-acting regulatory element involved in GA response, cis-acting regulatory element involved in MeJA response, some light-induced responsive elements and other teanscription factor-binding. These results showed that these factors may be involved in these gene expression and regulation.3. The full-length cDNA and genomic DNA sequences of RsMyrl and RsMyr2were isolated from radish. The5’-flanking region of RsMyr2have also been cloned by genomic walking method. Promoter sequences analyzed by PLACE and PlantCARE showed that it contents TATA-box, CAAT-box and some cis-acting element. These results showed that these factors may be involved in RsMyr2expression and regulation. To express RsMyrl and RsMyr2protein in E. coli, the full-length coding region for the mature protein without the signal peptide was ligated into the expression pET-30a (+) vector predigested with the same restriction enzymes. The recombiant plasmid were transformed into E. coli BL21and induced by IPTG. After induction, the cultures with pET-RsMyrl and pET-RsMyr2showed protein band about65kDa which was approximately8%higher than the deduced molecular weight because of His-tag fusion. Finally, the over-expression vector of RsMyr2was also successfully constructed. These results provided insight into elucidating the molecular characterization and biological function of myrosinase in radish.