Establishment Of Plant Genetic Transformation Based On Phosphite And Bacterial Alkaline Phosphatase

Generally,selection markers are indispensable in plant genetic transformation.Therein,two major categories including antibiotic resistance genes and herbicide resistance genes are prevalently used at present.However,increasing concerns about the biosafety have already pointed to them,and thus the development of a new and safe selection marker is getting more and more attention.Phosphite dehydrogenase?PTDH?is an NAD⁺-dependent enzyme that oxidizes phosphite?Phi?to orthophosphate?Pi?.Phi can not be directly utilized in plants and is rather toxic.Due to these characters,the set of Phi/PTDH has already emerged as a new and multipurpose system having large application potential in the aspects of dominant selection marker/plant phosphorus utilization/weed control.In order to break the international patent barrier of this technological system,the key point is to identify the functional alternative of PTDH.Regarding that bacterial alkaline phosphatase?BAP?also can oxidize Phi into Pi without coenzyme dependence,herein we attempted to establish a similar system based on Phi/BAP combination,by introducing Escherichia coli BAP gene?EcBAP?into plants and using Phi as a selector to verify whether it can replace PTDH.The major results of this study are as follows:1.Gene cloning of EcBAP and construction of its expression vectorsUsing the total genomic DNA of E.coli DH5?as the template,the gene fragment EcBAP was amplified.After dual digestion with BamH I/Sac I or Nde I/Xho I,EcBAP was subcloned into vector pBI121 or pET32a?+?to create plant expression vector pBI?EcBAP?or prokaryotic expression vector pET?EcBAP?,respectively.2.Generation and characterization of EcBAP transgenic tobacco?1?In case of tobacco transformation of vector pBI?EcBAP?via Agrobacterium infiltration and kanamycin?Kan?selection,EcBAP?Kan?transgenic tobacco could be obtained after a period?85-90 d?of culturing on normal MS medium,with a positive transformation rate of 66.7%.?2?As observed from the resistance regeneration of leaf explants on Pi-deficient or normal MS medium containing different concentrations of Phi,EcBAP?Kan?transgenic tobacco showed significantly lower degree of chlorosis in almost all cases than WT tobacco,but could only have the resistant green buds regenerated from the callus on normal MS medium containing Phi of less than 3 mM.?3?According to above results of?2?,Agrobacterium-mediated tobacco transformation of pBI?EcBAP?was performed by culturing on normal MS medium containing the selection reagent of 2.5 mM Phi.After a cultivation time of about 68 d,EcBAP?Phi?transgenic tobacco could be obtained,with a positive transformation rate of 80%.In contrast,even lasting the selective cultivation on Pi-deficient MS medium for 2.5 months,the same transformation could only be seen with differentiated callus no matter what the concentration of Phi in the range of 0.5-2 mM.Additionally,RT-PCR results showed that the foreign gene EcBAP was expressed in roots,stems and leaves of EcBAP?Phi?transgenic tobacco.?4?Phi-screened EcBAP transgenic tobacco and WT tobacco were tested for seed germination on MS plate under Phi stress.Therein,the growth of WT tobacco seedlings was significantly inhibited by Phi in both Pi-deficient and normal MS media,but this phenomenon was not obvious in EcBAP transgenic tobacco.Moreover,under the same Phi stress,the seedling growth status of WT and transgenic tobacco on normal MS medium was remarkably better than that on Pi-deficient MS medium.?5?In greenhouse,Phi-screened EcBAP transgenic tobacco was sowed with WT tobacco and weeds?Cynodon dactylon?L.?Pers.and Festuca elata Keng ex E.Alexeev?in a matrix composed of vermiculite,perlite,and stone.The results showed that EcBAP transgenic tobacco could outcompete with clearly dominant growth over other tested plants in case of irrigation with ddH₂O containing 120 mg/L Phi.3.Recombinant expression,purification and enzymatic activity analysis of EcBAPThe vector pET?EcBAP?was expressed in E.coli BL21?DE3?by IPTG induction.The results showed that the solubility of recombinant protein EcBAP was very low under 37oC induction,but increased significantly under overnight induction at 25oC.Through analyses by His-tag affinity chromatography purification,gel activity staining and spectrometric determination of enzyme activity,the recombinant protein EcBAP was confirmed with an ability of oxidizing Phi to Pi,and its specific enzymatic activity was 8.463 U/mg on average.In conclusion,the above results demonstrate that E.coli BAP?EcBAP?has the ability to oxidize Phi to Pi,and can replace PTDH?as a selective marker?to couple with Phi?as a selector?for plant genetic transformation.Its efficiency can be comparable to or even better than that of the commonly used kanamycin selection system.EcBAP transgenic tobacco can convert Phi into utilizable phosphorus nutrition thus underlying the Phi resistance,and competitively processes dominant growth over WT tobacco and weeds under Phi stress.Therefore,this Phi/BAP-based new system,integrating all advantages for selection marker,plant phosphorus utilization and weed control,can provide a new technology choice for plant genetic engineering.