Codon Usage, Synthesis And Expression Of The Vitreoscilla Hemoglobin Gene In The Tobacco

There is a sensitive system controlling and responding to the level of oxygen in the plants. Thus, it is important to maintain a steady oxygen level to avoid the unnecessary loss energy and to ensure survival. A novel genetic response to enhance oxygen metabolism has been observed in the microbial world: The obligate aerobic, Gram-negative bacterium Vitreoscilla synthesizes elevated quantities of a novel homodimeric hemoglobin (VHb) in oxygen-limited cultivations. Expression of VHb in Escherichia coli. increases specific growth rate, final cell density, and cloned reporter enzyme expression in oxygen-limited cultures. Therefore, we transfer the gene of VHb into tobacco and study the influence to plant metabolic level and growth, This work is helpful for further obtaining transgenic crops with improved yield.According to the Vitreoscilla hemoglobin (VHb) amino acid sequence and plant preference codon usage, vgbM gene was designed and synthesized by way of annealing 22 synthetic fragments together, although three big fragments were formed and cloned respectively first. After recombination, we obtained the plasmid pGSVHB harboring the vgbM gene which is 450 base pairs.The vgbM gene with a Nco I site at 5'terminal obtained by PCR amplification was cloned into the plasmid pBV221 and the recombinent plasmid was named pBV221SVHB. The gene was driven by PR?L promoter. The transformants were obtained after pBV221SVHB was introduced into E. coli. BL21 strain. The temperature induction expression of the recombinant E. coli. was carried out and VHb protein of 16kD was expressed. We demonstrated the VHb biological activity by CO difference spectrum. The growing graphs of E. coli BL2I containing pBV221 and pBV221SVHB according the OD60o values shown that the E. coli vgb* grew faster than the control experiment.The vgbM gene was inserted into the middle vector pG4AB and we got the middle vector pG4ASVHB which contains a 35S promoter, a NOS terminator, sequence and Kozak sequence. Then, the whole expressing box was cloned into the plant expression vector pBI121.1, so we finally obtained the plant high-efficiency expression plasmid pGBI4ASVHB.The tobacco leave discs were transformed with Agrobacterium tumefaciens carrying the plant expression plasmid pGBI4ASVHB. We got sixty transgenic tobacco plants containing the vgbM gene after kanamycin screening, PCR detection, Southern blot analysis. The expression of 16kD VHb protein in transgenic plants was proved by Western blot. Transgenic tobacco plants expressing VHb exhibited enhanced growth, on average 266-530% more flesh weight after 60 days of growth compared to wild-type controls. Furthermore, transgenic plants contained, on average, 20.3-23.4% more chlorophyll and 117-277% flowering rates.