Genetic Transformation And Functional Identification Of Chalcone Isomerase Gene From Solenostemon Scutellarioides

Flavonoids are pliant-specific secondary metabolites derived from the phenylpropanoid pathway,they always been called Vitamin P and they are accompany with Vitamin C.They are the natural pigment in vegetable,fruit,flower and corn,because they always assume yellow,so they been called bioflavonoids. They are involved in various biological processes during plant growth and development,including flower pigmentation,protection against UV irradiation,defense,pollen development,male fertility,cell cycle regulation,and auxin transport.They also play key roles in symbiotic interactions between plants and microbes.There are about 4000 kinds of flavonoid have been aparted until now.A lot of researches have indicated that flavonoids have multi-biologically active in our body,including anti-oxidation characteristic,clearing free radicals,resisting virus,resisting inflammation,vein expanding and so on,in some special time they can chelated metal hydronium.Chalcone isomerase(CHI,EC 5.5.1.6)is an important enzyme in the phenylpropanoid pathway.CHI is involved in a very early step of the flavonoid and isoflavonoid pathway,it can be found in most plants by monomer and the three-dimensional collapse structure of albumen is exclusive and it has been as proper gene badge in plants.So it has an important signification for learning isoflavonoid biosynthesis characteristic of leguminous plants,increasing the flavonoid biosynthesis of officinal plants and the molecular mechanism of flower colour.1,Construction of plant expressional vector of sense and antisense CHI genep^UCm-CHIand p^TriplEx2digested with X-ba I and Sma I was constructed for mediated vector p(T-CHI)in which has correct direction,p^T-CHIand p^{C2301-35S-Nos}digested with X-ba I and Sma I was Constructed for plant expressional vector p^C2301-CHIs,and p^C2301-CHIaof sense and antisense CHI gene.p^C2301-CHIsand p^C2301-CHIawere transferred into LBA4404,respectively.2,Introduction of CHI gene into Petunia mediated by Agrobacterium tumejaeknsPut Agrobaterium that preserved in -80℃refrigeratory in substrate YEB+Km 50mg/L+Sm125mg/L, and convert-cultivate at 27-28℃for 2-3 days.Choose single tribe into liquid substrate of YEB for surging at 180rpm until OD₆₀₀adjusted to 0.6-0.8,and then take 1-2μl Agrobaterium liquid into 20-25ml YEB, convert-cultivate until OD₆₀₀adjusted to 0.8 for use.Young leaves of were excised from sterile seedlings of tobacco,cut into 5×5 mm²,dipped into Agrobaterium(OD adjusted to 0.2)for 15min.Patted dry on the filter paper,transferred to MS+2.0mg/L 6-BA+0.5mg/L NAA medium and co-cultured for 2-3 days in darkness.After co-cultivation,transferred to MS+2.0mg/L 6-BA+0.5mg/L IAA+200mg/L Km+500mg/L Cef for selection culture.The resistant shoots were rooted on 1/2MS+2.0mg/L 6-BA+0.5mg/L IAA+100mg/L Km+200mg/L Cef and then transferred the plantlets with normal roots to field for further analysis..The tobacco transformed by p^C2301-CHIswas named W+ and the tobacco transformed by p^C2301-CHIawas named W^-.Untransformed tobacco is control W⁰. Statistic results showed that the resistant shoots W+ has23 lines;W^- has 18 lines and W⁰ has 10 lines.3,The identification of transformed plants and the mensuration of flavonoids and anthocynnin in transgenic tobaccoPut the leaf of transformed tobaccos into GUS,and then identify transformed masculing tobaccos by common PCR.The PCR results suggested that transgenic W⁺ has 12 lines;transgenic W^- has 13 lines.The flavonoids and anthocyanin were exracted after transplant for 2 month ago of tramsforned plants which were PCR positive results.W⁰ are controls.The results showed that the flavonoids of W⁺ was more than W⁰,the highest is nearly up to 1.5 folds,but 2-3 transformed plant is lower than the controls.The flavonoids of W^- was lower than W⁰.However,the content of anthocynnin in transgenic tobacco leaves increase remarkably,the highest is up to 2 folds;the content of anthocynnin of W^-is lower than the controls.