Cloning Of NtUGT1 In Tobacco And Its Functional Study In Synthesis Of Rutin

Tobacco is an important cash crop. Secondary metabolites such as flavonoids can affect its flavor character, especially the rutin that has been proved as flavor component. The past research on tobacco flavor focused on relationship between flavor component and environmental factors, lacking mechanism studied on biosynthesis and metabolism of flavor component. We obtained the coding region of NtUGTl gene by homology-based cloning and constructed overexpression vector. After resistance screening, totally 30 transgenic lines were selected for realtime PCR analysis. The three lines of GT3, GT18 and GT27 were chosen for further study, which showed 10 to 150 folds of NtUGTl expression higher than vector control. Coloning and characterization of NtUGTl gene enhanced the understanding of tobacco flavonoids pathway and provided the information for tobacco flavor improvement.NtUGT1 gene overexpression inhibited the expression of structure gene chalcone synthase NtCHS, Chalcone isomerase NtCHI and Flavanone 3- hydroxylase NtF3H, and promoted the expression of dihydroflavonol - 4 - reductase NtDFRl and anthocyanidin synthase NtANS1. Enzyme NtCHS, NtCHI and NtF3H can catalyze coumaric acid Co A to dihydrokaempferol. NtDFR1 and NtANS1 are the key enzymes of anthocyanin synthesis pathway. Flavonoid pathway genes expression of transgenic leaf were also analyzed using realtime PCR. Three genes which are responsible for dihydrokaempferol biosynthesis from coumaroyl-CoA showed reduced expression level, including NtCHS, NtCHI and NtF3H. This demonstrate that upstream gene could’t be induced by NtUGT1 overexpression because of the complicated regulaton of flavonoid pathway. The NtFLS gene expression was enhanced in transgenic leaf indicating that decreased flavanone content could stimulate flavanone synthase (NtFLS) gene expression. Furthermore, enhanced flavonone biosynthesis branch pathway may cause reduced metabolic flux towards anthocyanidin production, which induced key genes expression of anthocyanidin pathway, such as NtDFR1 and NtANS1.Flavonoids content changes of transgenic tobacco leaf and flower were significantly different. All of flavonoids contents in leaf were declinng. There were differences about flavonoids content in flower. Flavonoids content inleaf and flower were investigated using LC-MS. Most of detected metabolites showed descending level in leaf, including dihydrokaempferol, kaempferol rutinoside, dihydroquercetin andquercetin rutinoside (rutin). However, the content change in flower is different. Overexpression of NtUGTl resulted in around 1.2-fold increases of dihydrokaempferol, kaempferol and kaempferol rutinoside, but dihydrokaempferol-3-glucoside, kaempferol-3-glucoside and quercetin were decreased by 58%, 63% and 30%, respectively. Interestingly, the content of rutin in flower was increasedf. This variation between leaf and flower demonstrated the difference of flavonoids pathway in two organisms. This was also supported by metabolite profiling. For example, kaempferol, quercetin, hyperoside and luteolin could only be detected in flower and quercetin-3-glucoside only found in leaf. Most of flavonoids in flower were higher than in leaf according to the peak area, indicating more active flavonoids pathway in flower.Overexpression of glycosyltransferase gene NtUGTl in tobacco influenced gene expression of flavonoids synthesis pathways, such as inhibiting NtCHS, NtCHI and NtF3H expression and promoting NtDFRl and NtANSl expression, which led to decline of dihydrokaempferol, kaempferol rutinoside, dihydroquercetin and quercetin rutinoside (rutin) in tobacco leaf, and increase of dihydrokaempferol, kaempferol, kaempferol rutinoside and rutin in tobacco flower.