The Study Of The Expressional Vector Harboring With The Novel Selectable Marker Gene (Alad) And The Reporter Gene (CobA)Transformed Into Plants

The tetrapyrrole molecules in higher plant playe important role in the normal operation of plant photosynthesis system, including chlorophylls, haem and siroheme, etc. δ-aminolaevulinic acid dehydratase (ALAD) and uroporphyrinogen III methyltransferase (UPMT) are two key enzymes in the upstream biosynthesis pathway of tetrapyrroles. ALAD catalyzes the asymmetric condensation of two molecules of ALA to form the monopyrrole, prophobilinogen (PBG), the precursor of heme, chlorophylls, and cobalamins. Uroporphyrinogen III is the substrate of UPMT and synthesized by prophobilinogen synthase (PBGS). There are many results as follows:1. Separately, ALADs from different origins with His tag at N reminal were expressed and purified by Ni-NTA in E. coli. ALADs from Bos taurus, which was suitable to be expressed in the chloroplast under the optium pH8.0, had ability to be more resistant to succunyl acetone, and had higher enzyme activity un than the ALADs from Bacillus subtilis and Rhodopseudomonas sphaeoides.2. Uroporphyrinogen III methyltransferase (UPMT), being encoded by the cobA gene, displayed strong red fluorescence under the UV light illumination in E. coli. And the main pigments were trimethylpyrrocorphin at354nm and sirohydrochlorin at378nm analyzed by UV-UIS and Fluoroescence Scanning Spectrometer.3. In E. coli, co-expression of ALAD and UPMT had synergistic effect on enhanceing strongly compared with single UPMT expression.4. The vector pC1301Ubi-ZmBtpA-pUP harboring with the alad gene and the cobA gene was transformed instantaneously to tobacco. The red pigments during over-expressing UPMT were mainly accumulated in the chloroplasts by fluorescence microscope.5. The vector pC1301Ubi-ZmBtpA-pUP was separately transformed to Arabidopsis thaliana Col-0and the rice (O. Sativa L. spp. japonica). The transformants were selected by different concentrations of Succinyl acetone (SA), the optimum of which was0.1μmol/L in Arabidopsis and0.3μmol/L in Rice. The results diaplayed SA had high sensitivity and toxicity to inhibit root development under nmol/L concentration of SA. On the other hand, The Arabidopsis T1generation, selected by SA and transplanted in the nutritional soil, had higher height and bigger leaves than the wild type. However, fermination and development of rice tranformants was inhibited by SA.