tailieunhanh - BÁO CÁO NGHIÊN CỨU KHOA HỌC KỸ THUẬT: IDENTIFICATION BY METABOLIC SCREENING OF GENEES INVOLVED IN THE COUMARINS BIOSYNTHESIS PATHWAY OF Arabidopsis thaliana

Bộ môn Công Nghệ Sinh học, Đại học Nông Lâm , Việt Nam (**) Phòng thí nghiệm Agronomie environnement, ENSAIA-INPL Nancy, Pháp ABTRACT Coumarins là chất chuyển hóa thứ cấp liên quan đến cơ chế phản ứng với stress và sự thích nghi của các nhà máy để họ điều kiện môi trường. | 194 NGHIÊN CỨU KHOA HOC KY THUAT XAC ĐỊNH MOT SOGENE THAM GIA CON 8UONG SINH TONG HỌP CAC HỌP CHAT COUMARIN O CAY Arabidopsis thaliana BANG PHUONG PHAP chon LOC CHUYEN HOA IDENTIFICATION BY METABOLIC SCREENING OF GENEES INVOLVED IN THE CO UMARINS BIOSYNTHESIS PATHWAY OF Arabidopsis thaliana Nguyen Vu Phong Alain Hehn Frederic Bourgaud Bo mon Cong nghe Sinh hoc Đại hoc Nong Lam Viet Nam Laboratoire Agronomie Environnement ENSAIA-INPL Nancy Phap ABTRACT Coumarins are secondary metabolites involved in the mechanism of response to stress and adaptation of plants to their environmental conditions. Although their important roles the coumarin biosynthesis pathway is poorly understood. The objective of this work was to carry out metabolic profiles of Arabidopsis thaliana mutants insertional and to try to characterize the ortho-hydroxylation step at the molecular level. The HPLC high performance liquid chromatography analysis on 8 different mutants showed that 3 of them CYP78A6 CYP84A4 and CYP706A2 exhibit 5 fold weaver scopoletin content than the wild-type. In order to test the implication of these P450 in the biosynthesis pathway we cloned the genees and programmed to express the corresponding protein in a yeast expression system. As the genes seemed not to be constitutively expressed plants were treated to enhance the expression level. We could amplify and clone CYP84A4 from mRNA extracted from plants treated by wounding after 1 and 4h and by 2 4D after 24h. Yeast expression will now be performed for CYP84A4 and CYP706A2 and a metabolic screening will be realized. Key words Arabidopsis thaliana scopoletin biosynthesis of coumarins P450 cytochrome heterologue expression. MỞĐẦU Coumarins la nhom hợp chất thứ cấp co vai tro quan trong trong cợ chếphản ứng vôi stress va sứ thích ứng cua cay đối vôi moi trứông song. Sự sinh tong hợp cua cấc chất nay thứông đứợc kích thích bôi cấc stress do tấc nhan sinh hoc hoặc khong sinh hoc gay nen. Ben canh đo cấc hợp chất nay con