Purification Of Relative Synthases In The Carotenoid Pathway And Effects Of UV-B Radiation On Expression Of Gene Ggps In Tobacco

Carotenoids are a group of natural pigments with yellow, orange and red colors, which distribute widely in all kinds of plants, animals and microorganisms.Carotenoids are the essential components of the photosynthetic membranes in pants and microorganism, and they can protect photosynthetic systems from high light oxidation destroying. Many carotenoids are strong antioxidants, and the source of the VA, which might play an important role in preventing cancers. Although carotenoids and their synthases are enriched in plants and microorganisms, but it is difficult to extract single pigment and purify the synthase complex. Many genes and their coding enzymes responsible for carotenoid synthesis in higher plants and microorganisms have been identified, which offers a convenience to study the synthase complex and provide a possibility to produce carotenoids by fermentation using engineering bacteria. Tobacco is a kind of important economic crop which is affected by environmental conditions and its leaves are the primary products. Therefore, it is necessary to study the physiological and biochemical changes such as caroteneoids synthesis and expression of carotenoid snythases in tobacco leaves under ultraviolet stress.In this study, four genes crtE, crtB, crtI and crtY related to carotenoids synthesis in Erwinia uredovora were cloned and used to construct engineering bacteria accumulating lycopene andβ-carotene, respectively. The two engineering bacteria culturing conditions suitable for carotenoids accumulation were also investigated at the same time. The early synthase complexes of the carotenoid pathway were purified from the engineering bacterium accomulating lycopene. Then the content of the photosynthetic pigment including caroteneoids and the expression level of GGPP snythase were meassured in tobacco leaves under ultraviolet stress. The main results were shown as follows:1. Purification of the polyenzyme complex for the carotenoids synthesis in early periodThree genes crtE, crtB, crtI related to lycopene synthesis in Erwinia uredovora were cloned into pET-15b to construct expressing vector pET-15bEIB, and crtY was firstly cloned into pET-15b to construct pET-15bcrtY, then crtY under the control of T7 promoter as well as T7 terminator were cut down from pET-15bcrtY and cloned into pACYC-184 to construct pACYC-184crtY. The expressing vector pET-15bEIB was introduced into E.coli BL21(DE3) to construct engineering bacterium. Co-expression of crtE, crtI, crtB, and crtY was achieved in the engineering bacterium induced by IPTG. Complexes with molecular weight of 209KD, 105KD and 70KD for the carotenoids synthesis in early period were purified through affinity chramotography on nickel resin column followed by gel filtration chromatography on Superdex-75 column. By using the same methods, polyenzyme complexes of 209KD, 173KD and 70KD for the carotenoids synthesis in early period were also purified from the mixture of recombinant GGPP synthase and phyteone synthase expressed in the engineering bacteria containing the plasmid pET-15bcrtE and pET-15bcrtIB, respectiviely.Through analyzing results of SDS-PAGE, Western blot and together with the molecular weights, we suspected that the main polyenzyme complexe for the carotenoids synthesis in early period contained four copies of GGPP synthase and two copies of phyteone, with a relative molecular weight of 209KD. Recombinant GGPP synthase existed in dimer in experimental conditions.2. Studies of Escherichia coli accumulating lycopene and its culturing conditionsThe engineering bacteria containing pET-15bEIB could accumulate red pigment when induced by IPTG. The red pigment was lycopene as judged by absorbance spectra analysis and HPLC analysis. The engineering bacteria could grow to 3.45gDCW/L and accumulate lycopene to 5.8mg/gDCW under optimal culturing conditions. The culturing conditions used were as follows: the medium was modified LB broth(Trypton 10g/L, Yeast Extract 5g/L, sodium citrate 5g/L, MgCl2 0.1g/L,NaCl 10g/L), the engineering bacteria were first shake-cultured at 37℃to OD600 of 0.6, and then IPTG was added to a final concentration of 0.5mmol/L, the engineering bacteria was further cultured for 14h at 30℃.3. Construction of engineering bacterium accumulatingβ-carotene and its culturing conditionsTwo expressing vectors, pET-15bcrtEIB and pACYC-184crtY, were introduced into E.coli BL21(DE3) simultaneously to construct engineering bacterium. The engineering bacterium could accumulate orange-colored pigment when induced by IPTG. This pigment wasβ-carotene as judged by absorbance spectra analysis. The culturing conditions used were as follows: the medium was modified LB broth (Trypton 10g/L,Yeast Extract 10g/L, soluble starch 5g/L, MgCl2 0.04g/L, FeCl30.01g/L , NaCl 10g/L, pH5.8), in the light, the engineering bacteria were first shake-cultured at 37℃to OD600 of 0.6, and then IPTG was added to a final concentration of 0.5mmol/L, the engineering bacteria was further cultured for 14h at 30℃. The engineering bacteria could grow to 6.53 gDW/L in LB medium and accumulateβ-carotene to 3.5 mg/gDW under culturing conditions.4. Effects of enhanced UV-B radiation on photosynthetic pigments and some enzymes of TobaccoEffects of enhanced UV-B radiation on physiological and biochemical characters of tobacco were investigated. The results show that the photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) increased clearly in tobacco leaves under enhanced UV-B compared with those in leaves growing under natural light. The three pigments increased 21%, 10% and 27%, respectively. The content of soluble proteins increased at the beginning of radiation and decreased later. The activity of peroxidase increased distinctly at the same time, and a new peroxidase isozyme appeared under UV-B radiation stress. The expression level of GGPs increased significantly as judged by Western blot analysis.