Metabolic Engineering Of Isoflavone And Proanthocyanidin Biosynthesis In Medicago Truncatula And The Screening Of Isoflavone Transporter

Isoflavones and proanthocyanidins(PAs,also called condensed tannins)are active components of forage grass which play positive roles on ruminant nutrition and health.Isoflavones are phytoestrogens that show beneficial effects on animal's production performance by interacting with endocrines and improving immunity.PAs are polyphenols,which reduce protein degradation in silage and thus enhance ruminant nutrition.More importantly,modest foliage PAs can prevent lethal pasture bloat of ruminant animals.However,the contents of both isoflavones and PAs in leaves of Medicago species are severely limited.Engineering isoflavone and PA production in Medicago shoots has been earnestly pursued in the past decades.This dissertation work focused on both isoflavone and PA biosynthesis,and the transport mechanism of isoflavones.For the metabolic engineering of isoflavones and PAs,transgenic materials with elevated levels of both flavonoids were achieved by multigene pyramiding breeding.For studying the transport mechanism of isoflavones,a functional photoaffinity probe was synthesized for identifying isoflavone-binding proteins.Four key genes involved in the flavonoid biosynthesis were chosen for modification of isoflavone pathway in Medicago truncatula:soybean GmIFS1 was chosen to increase the potential for the accumulation of isoflavone;GmCHS7 and GmCHI1A,two upstream genes of GmIFS1,were used to enhance the chalcone biosynthesis;a RNA interference construct of MtF3H was aimed to reduce the competition between GmIFS1 and endogenous MtF3H.The intermediate vectors were assembled and forming four plant binary constructs and were introduced into M.truncatula 'R108' via Agrobacterium-mediated transformation.T3 generation lines with down-regulated MtF3H and highly expressed GmIFS1,GmCHS7 and GmCHI1A were successfully obtained through qRT-PCR and Western blotting screening.The leaves of the selected lines accumulated massive amount of isoflavones genistein,formononetin and biochanin A as well as newly presented daidzein when anylized by LC-UVS and LC-ESI-MS.The total isoflavone in the transgenic lines substantially increased and reached as high as 1962.95 ± 606.33 nmol/g FW,which was 27.5 times greater than wild-type.More importantly,coexpression of GmIFSl with GmCHS7 and GmCHI1A generally generated substantial amount of isoflavons along with elevated PA content,which reached 0.34 mg/g FW and 1.4 times higher than wild-type.The biosynthesis,transport and cellular deposition of isoflavones are mainly linked by membrane transporter-mediated transport,but how the isoflavones are transported remains largely unknown.This dissertation document the synthesis and application of an effective photoaffinity probe,PAD A,aiming at the identification of isoflavone-binding proteins and transporter(s).PADA is a biotin-and benzophenone-conjugated derivative of isoflavone daidzein.An(iso)flavonoid glycosyltransferase,UGT78G1,can be photolabeled by PADA after exposure to UV,and the reactive was competitively inhibited by daidzein,demonstrating that PADA is a sensitive and specific photoaffinity probe of isoflavone-binding protein.On the contrary,non-isoflavone-catalyzing enzymes GmCHI1A and MtF3H can't be labeled by PADA,suggesting that the photoaffinity labelling specificity of the probe essentially relies on the recognition bewteen the isoflavone moiety and its target.The combination of photoaffinity cross-linking and biotin-based affinity chromatography was proven to be a strategic approach for the purification of isoflavone-binding protein.By this approach,multiple proteins photolabeled with PADA were separation and preconcentration from membrane protein extracts of soybean roots.This dissertation contributes valuable researches on the metabolic engineering of flavonoids and the transport mechanism of isoflavones.Our unsterstanding on changes of flavonoid metabolic flow in transgenic lines help researchers develop new methods and strategies for the pathway engineering of isoflavones and other flavonoids in forage grass.The artificial photoaffinity probe,PADA,is an effective tool to identify isoflavone-binding proteins,and thereby screen the specific transporter(s)and elucidate the tranport mechanisms in legumes.