Expression Of Nitrilase Gene Mutated And Vitreoscilla Hemoglobin Gene In The Methylotrophic Yeast Pichia Pastoris And Tobacco

Weeds compete with crops for moisture, nourishment and light, moreover, theyare hostsof disease or the place where disease survives in the winter, which influences the output andquality of crops. It is indispensable for modern agriculture to control weeds by usingherbicide. But herbicide also injures crops while killing weeds. At present, cultivatingherbicide resistant crops by genetic engineering has solved this contradiction. The broad-spectrum ,nonselective contact herbicide bromoxynil can kill weeds whosemechanism is to blockade transmission of photoelectron and make the photosynthesisobstruct,thus caused death. A kind of bacterial (Klebsiella ozaenae) taking bromoxynil asnitrogen source has been separated from soil bacterium, it can produce specific bromoxynilhydrolase which can convert bromoxynil to 3,5-dibromo- 4- hydroxybenzoic acid and toxicityof losing. Bxn gene encoding nitrilase has been cloned and expressed triumphantly in otherplants.Bxn gene in plasmid RD27 has four mutant sites and so in this experiment, PCR isapplied to the correction of bxn gene,we use yeast expression system to check if bromoxynilhydrolase after correction has some activities. The Vitreoscilla hemoglobin gene(vgb) whose codon were optimized can improveeukaryotic organismoutput, increase their growth speed , so be used in this experiment. In yeast experiment,we constructed the recombination plasmid pPIC9K-vgbbxn with vgbgene intracellular expressed and bxn gene extracellular secretion. After transformed intoPichia pastoris GS115, The results of PCR and SDS-PAGE indicate that the vgb gene andbxn gene had integrated into the genome of Pichia pastoris GS115 and expressed in efficientlevel. Also, the enzyme activity of their products had been verified respectively. We found,inthe hypoxic habitats,vgb gene not only maintain yeast growth but also advance nitrilaseexpression. Next, by dint of pGΩ4A intercarrier we constructed two kind of plants expressioncarrier pBI121-vgbbxn(one is vgb & bxn gene started by 35S promotor;another is vgb genestarted by 35S promotor, bxn gene started by RuBP promotor). pBI121-vgbbxn wasconformed into tobacco genome by agroinfection. Identification of tobacco genome PCR andcheck of transgenic tobacco resistivity herbicide bromoxynil found: bxn gene had integratedinto the genome of tobacco and expressed triumphantly. Vgb gene was checked by the way ofmeasuring chlorophyll content.The way of tobacco protein degradation bromoxynil made sureRuBP promotor has a stronger ability to start gene expression in the plant leaves than 35S. In conclusion, this experiment construct a double-gene plant express carrier and a yeastexpress carrier that can solve the supply and demand contradiction of oxygen in the highdensity ferment and improve the output of albumen of other source secretion express gene.This for get new- type to resist hebicide transgenic tobacco and the industrialization thatPichia pastoris yeast high density ferments establish the foundation.