Cloning And Functional Characterization Of Anthocyanin Biosynthesis Genes In Saussurea Involucrate Kar. Et Kir.

Saussurea involucrate Kar. et Kir. is a medicinal plant known for its flavonoids,including anthocyanins, that have been shown to significantly inhibit tumorigenesis.The limited natural resources of S. involucrate can not satisy the demand of themarket. It is necessary to serve this problem use the morden molecular biologytechnology. The genes of key step in the biosynthesis of anthocyanins were clonedfrom S. involucrate. These genes of S. involucrate were put under the control of thecauliflower mosaic virus35S promoter, and homologous transformation used anAgrobacterium-mediated transformation system. For protect and sustainable thenature resources of S. involucrate through suspension culture the transformed callusto get more flavonoids.1. Cloning and Analysis of DFR and3GT cDNA from S. involucrateThe Dihydroflavonol-4-reductase (DFR) and Flavonoid-3-O-glucosyltransferas(3GT) are important to plants secondary metabolites and catalyze the key step in thebiosynthesis of anthocyanins. DFR gene (GenBank Accession JN092126) and3GTgene (GenBank Accession JN092127) were cloned from S. involucrate by RT-PCRand RACE. The cDNA sequence of DFR consisted of1029bp open reading frame(ORF) encoding343amino acids, this gene has high homology with Saussureamedusa showed97%identity. The cDNA sequence of3GT consisted of1548bpopen reading frame (ORF) encoding516amino acids, the deduced3GT proteinshared91%and89%identities with that of Fragaria x ananassa GT6, Manihotesculenta anthocyanidin3-O-glucosyltransferase. The transcripts of DFR and3GTmembers were found mainly in leaves and callus.2. Construction of Transformation System in S. involucrateThe effects of different antibiotics (kan, hyg and car) on induction anddifferentiation of callus (S. involucrate), was discussed in this article and S.involucrate leaves were used as explants. A doses strategy of selection and bacteriuminhibition during agrobacter mediated genetic transformation was established. Theresult showed that the kanamycin of40mg/L was significantly influenced on callusgrowth, when the concentration of kan was50mg/L, the growth of callus wasinhibited completely; the hygromycin of8mg/L was the best concentration forinhibiting the callus growth, the callus were geatly damaged and the chracters weresmall, browning and dying with the concentration of hygromycin up to20mg/L. Meanwhile, the low concentration (0.5mg/L-2mg/L) of hygromycin can enhancethe rate of callus differentiation. Carbenidillin is an antibacterial agent ofAgrobacterium, carbenidillin had apparently influnced on growth of callus atdifferent concentrations, and when the concentration of carbenidillin was greater than400mg/L, the growth of callus was effected observably. The plant transformationvectors pMON530-DFR and pCAMBIA1305.1-3GT were constructed in respective,which contained cauliflower mosaic virus (CaMV)35S promoter, DFR gene and3GT gene. The result showed that10min and concentration of Agrobacterium withdifferent plasmids DNA at1:1were the best condition for transformation.3. Genetic Transformation of DFR and3GT in S. involucrateThe DFR and3GT of S. involucrate were transferred respctively andcollectively into S. involucrate used an Agrobacterium rihizogenes-mediatedtransformation system and a large amount of kanamycin-resistant and hygromycin-resistant callus were obtained. The transformation efficiencies of DFR gene was12.67%,3GT gene was9.33%and corotation of these two genes was2.63%. Theresults on UV spectrophotometry showed S. involucrate callus after suspensionculture that an average of total flavonoids on transgenic of DFR gene callus was1.86times higher than non-transgenic callus,3GT gene was2.06times higher thannon-transgenic callus, corotation of these two genes was3.61times higher thannon-transgenic callus.