Study On Construction Of Polyphosphate Transgenic Organisms And The Phosphorus Accumulating Characteristics

The eutrophication of freshwater bodies in China is very serious,61.6%of the 26 key national-controlled lakes and reservious are class V or worse than class V according to water quality standard in China.Eutrophication of freshwater bodies not only brings hazards and far-reaching impacts on economic loss,but also endangers human health.Therefore,keeping low level nutrients in freshwater bodies to improve water use efficiency benefits social and economic values.Phosphorus and nitrogen are the most important indicators for eutrophication controlling.As a result,on the one hand,decrease of exogenous pollutants input and reduce emissions of nitrogen and phosphorus into the water bodies are common approaches for eutrophication controlling;on the other,reduction of endogenous contaminants release from the sediments to water must be implemented to limit possibility of secondary pollution.Thus,combination of both aspects of the work,reducing of freshwater bodies eutrophication can be realized based on nuitrients especially nitrogen and phosphorus decreasing.And incidental water pollution disaster such as water blooms and related hazards can be avoided.Reducing of phosphorus in wasterwater was the key method for exogenous phosphorus controlling.Biological phosphorus removal is most widely used in wastewater treatments,the mechanism of which is transformation of phosphorus into polyphosphate(polyP)by a kind of bacteria exist in the wastewater system called phosphorus accumulation organism(PAO).PAO is removed in the form of activated sludge in which process phosphorus is reduced in the wastewater.Therefore,efficiency of biological phosphorus removal rate can be promoted effectively by improving the content of phosphorus accumulating in bacteria,improving phosphorus content in activated sludge in other words.Present studies find that,polyP is catalyzed by phosphate kinase expressing by phosphate kinase gene(ppk)which had been cloned into bacteria to over-express.The constructed gene engineering bacteria with high phosphorus accumulation can absorb phosphorus from the wastewater.However,these studies are currently limited in the labs.Here,we constructed a high phosphorus accumulating organism KTPPK with Pseudomonas as host which over-expresses ppk from bacteria in low-copy recombianant plasmids.KTPPK was studied in sequence batch biofilm reactor(SBBFR)with high COD and nitrogen removing rate used lava stones as carriers which could promoted 20%phosphorus removal rate(the concentration of PO43--P in the effluent was from 12 mg/L to 2 mg/L in the end of aerobic phase after 200 hrs operation)in comparison with that in the control reactors.As we know,there are physical,chemical and biological methods to reduce endogenous phosphorus in water bodies,among of which biological approach like phytoremediation is economic,environmental friendly and landscaping.But traditional phytoremidation focuses on finding tolerance and hypeaccumulating plants in wild but probably difficult to meet environmental restriction requirements.With high developed root system,cattail accumulated heavy metals in roots but bodies above the ground,which limits pollutants removal.Molecular biology provides methods to overcome such problems which also can promote tolerance and accumulation of pollutants for plants.Here,we introduced a high effective method of plant regeneration from root-culture-derived callus of T.latifolia(cattail).Root culture and subculture of cattail provided rich callus induction materials at any time,12.7 calli induced from root culture per germinated seed in comparison with 0.31 inuced from shoots.And laborious seeds collection and germination of cattail could be avoided in the process of callus induction from root-culture-derived callus.Auxins and cytokinins and the optimum ratios of which were used to obtain good regeneration rate and shoots and roots growth.This regeneration system could be used for rapid propagation and introducing foreign genes for cattail.Also,suspension culture of yellow flag was set up to promote callus production and regeneration.In addition plant transformation has its roots in the research on Agrobacterium which has achieved significant developments,but the difference in the competence of Agrobacterium to infect a specific tissue,genotype or species is still a major drawback in utilizing Agrobacterium routinely for the introduction of the gene(s)of interest in plants.Particle bombardment is widely applied in plant transformation which is species-independent and bypasses the complex interaction between bacterium and plant tissue,but the low efficiency of stable integration of DNA is about 10-100 times lower than that of Agrobacterium-mediated method.In present study,we reported the generation of transgenic rice,maize,and orchid,using Tn5 transposase-mediated transformation technique,which not only produced a larger proportion of transgenic plants but also increased the single copy insertions proportion by decreasing the amount of assembled transposon DNA.For example,assembled 20 ng DNA,the transformation rate of rice was 2 times higher that of traditional particle bombardment transformation;for maize and orchid the transformation rate was 10 times higher than traditional methods.The single copy inserions were 40%(rice),24%(maize)and 33.2%(orchid)respectively which were higher than that of traditional method.Based on the transformation method,a phosphorus accumulating rice was constructed by expressing ppk which promoted phosphorus absorption of rice from surroundings.In the same time,the improving of the ability of 1-5%phosphorus uptake by rice can reduce the use of phosphate fertilizer and phosphorus loss which benefits eutrophication controlling.