The Heterologous Expression Of Flavonoid Specific Regulatory Factor AtMYB12Gene And The Contributions To Plant Resistance

One subclass of plant polyphenols, bioflavonoids, are ubiquitous in the plant kingdomand have many diverse health-promoting functions including anti-allergic, anti-cancer,antioxidant, anti-inflammatory and anti-viral properties when consumed in foods of plantorigin. Currently, the extract pathway of flavonoids to satisfy consume is dependent onchemical methods to extract from ginkgo biloba leaves, onion and lemon etc.In this study, by combination fruit specific promoter E4, PG,2A12with Arabidopsisthaliana flavonol-specific transcription factor AtMYB12gene, we got many tomato transgeniclines enriched flavonoids, including rutin, kaempferol rutinoside and naringenin chalcone.The results further confirmed that transcription factor AtMYB12gene could regulate theflavonoids synthesis, and compared the differences of flavonoids accumulation controlled bydifferent fruit specific promoters. The studies of transgenic tomato provide references forproducing flavonoids using tomato as bioreactors.Firstly, we constructed the plant expression vector pX6-E4::AtMYB12,pX6-PG::AtMYB12and pX6-2A12::AtMYB12by replacing the GFP fragment with DNAfragment either of E4, PG or2A12promoters and AtMYB12in pX6-GFP vector respectively.They were all introduced into Agrobacterium strain AGL1and transformed into two cherrytomato cultivars, Qiaomeiren and Shengnvguo via leaf disc method. After identified by PCR,we got the positive transgenic tomato lines. We detected flavonoids in all three transgenictomato fruits peels by HPLC, and the content of naringenin chalcone was shown todramatically increase among the transgenic tomato fruits peel. Three different tomato fruitspecific promoters regulate AtMYB12gene expression in different positions, and induce thechanges of flavonoids content. The promoter of E4controls the expression of AtMYB12mainly in tomato fresh, and up-regulated expression of related genes involved in flavonoidssynthesis, such as CHS gene (chalcone synthase). The promoters PG and2A12control theexpression of AtMYB12in tomato peel, and induce the up-regulated expression of relatedgenes involved in flavonoids synthesis, such as CHS gene, PAL gene (phenylalanineammonium lyase) and GT gene (flavonol-3-glucosyltransferase).Second, we have charactered the functions for some other regulator genes for flavonol orcaffeoyl quinic acid synthesis. AtMYB11gene, one of the homologous gene of AtMYB12,was over-expressed in Samsun tobacco, leading to high-level accumulation of polyphenoliccompounds, especially flavonols. The same effect, AtMYB11gene failed to induce theaccumulation of caffeoyl quinic acid (CQA). Over-expression of the genes NtHCT, NtHQTand NtC3H respectively, which prdicated to participate in CQA synthesis, all failed to inducethe accumulation of CQA. To our supprise, over-expression of NtHQT reduced the content ofrutin which presumed to synthesized some novel contents.Further, a set of pathogenic assay have been tested on transgenic Samsun tobacco carryingAtMYB12dedrived under35S promoter. The results was shown tha the AtMYB12-expressiontransgenic tobacco lines enhance the plant defense responses against pathogens: bacteria suchas Ralstonia solanacearum; fungi such as Colletotrichum destructivum and Alternariaalternate; and insects such as aphid and whitefly because of the high-level accumulation ofthe flavanol rutin, but more sensitivity to potato virus Y (PVY). The enhancement of defenseresponses in transgenic tobacco plants is most attributed to the up-regulated expression ofresistance related genes. The result is further confirmed by the plate culture plused withtransgenic tobacco leaves extracts. The transgenic tomato fruits also enhance the defenseresponse against Botrtytis cinerea because of the accumulation of rutin in tomato fruits, andthe plate culture with different contents of rutin further confirms the result.Bioflavonoids is a large kind of plant metabolism productions ubiquitous in the plantkingdom, flavonoids play a critical role in preventing human for disease and have evolved asa protective mechanism for a variety of different plants. We use small-fruit-type tomato andmajor economic crop tobacco as bioreactor to product bioflavonoids, to get marker-freetomato and tobacco lines enriched flavonoids and CQA. At the same time, rutin as a proudmember of the flavonoid family, could be an activator to enhance the reaction of plant diseaseresistance. These results are formed a good foundation to support the further study oncultivating high yield and good quality of tobacco variety, especially for disease resistanceplant. Aslo it also open the opportunity to develop the bio-pesticide by combining thebioreactor to product flavonol and adjusted with additive pesticide compounding in industry.