Establishment Of Transgenic Tobacco Plants With Ppk Gene And Identification Of Its Function With The Bioremediation Of Eutrophicated Water

Dissoluble phosphate is a major pollution of water bodies'eutrophication. Excess soluble phosphate in the water will lead to phytoplankton, especially the toxic algae, grow rapidly, which could make the BOD&COD values increase sharply and dissolved oxygen decrease, and generate lots of microcystin. It would restrict the production and life of mankind seriously. Therefore, efficient and low-cost biological phosphorus removal method has been becoming global research hotspots.This paper was based on the plant expression vector (pCAMBIA2300), cloned gene (ppk ) and a promoter (pro) which could be expressed in stem of plant, and construct a vector containing these exogenous genes which called p23PPN. By using the technique of the Agrobacerium transform, tobacco was transformed containing the gene (ppk) and the promoter (pro). PCR and Southern blot analysis from regenerated plants indicate that ppk gene has been introduced into tobacco genome. PCR and Southern blot analysis from T1 generation indicate that it had the phenomenon of gene isolation.By using the technique of aquatic culture, changed the tobacco growth environment successfully until it can grow well in the fresh water with floating-bed. By measuring the POD,SOD and CAT, revealed that the tobacco have body stress response from land into water. At first stage (before the water root appear), POD,SOD and CAT activity increased significantly; after the water root appeared, the tobacco could adapt to the aquatic habitat. POD,SOD and CAT activity restored normal levels, and it could grow normally in the water condition.Used the technique of floating-bed, the phosphate removal experiments show that:in the high concentration of phosphate (32 mg·L-1) conditions, the non-transgenic tobacco and transgenic tobacco had obvious disparity, non-transgenic tobacco could remove the amount of phosphate 3.14 mg·L-1; But the transgenic tobacco could remove the amount of phosphate 4.74 mg·L-1, over the same period, soluble phosphate was removed 1.5 times more than non-transgenic tobacco. It indicated that the transgenic tobacco which contained ppk gene had a strong ability to remove phosphate. In the low concentration of phosphate (0.4mg·L-1) conditions, non-transgenic tobacco could remove the amount of phosphate 0.03mg·L-1;But the transgenic tobacco could remove the amount of phosphate 0.396mg·L-1, over the same period, soluble phosphate was removed 13 times more than non-transgenic tobacco. It indicated that there was a good result of phosphate removal under the low concentration of phosphate.In the experiment of single tobacco, the concentration of phosphate was 0.5mg·L-1. It indicated that transgenic tobacco plants could remove more phosphate compared control, but there was a significant difference in the capacity of phosphate removal among the transgenic tobacco. In the study period, the phosphate removal rate of control was 11.24%, the highest phosphate removal rate of transgenic tobacco was 65.74%, and the lowest phosphate removal rate of transgenic tobacco was 23.67%.