Establishment Of Plant Genetic Transformation Selection System Based On Biuret And Its Metabolic Enzymes

In plant genetic transformation,only a small portion of cells can be transformed,so it is necessary to use efficient selection markers for screening the stable transformation events.There are mainly two kinds of frequently used selective marker genes,namely antibiotic-resistant genes and herbicide-resistant genes,because of their technical maturity and wide applicability.However,since these marker genes become dispensable after transformation and may cause a series of biosafety problems,it is always of particular interest for researchers to develop new safe selection marker systems.Currently,an approach based on phosphite(Phi)and its dehydrogenase(Ptx D)has attracted much attention,which integrates multiple roles of screening marker,phosphorus utilization,and weed control.This system enables transgenic plants to convert toxic Phi into utilizable nutrient phosphate(Pi),and can be screened out due to the growth advantages over non-transgenic individuals..Inspired by this insight and on account of two special notions(toxicity of biuret(BU)to plants,BU decomposition into usable ammonia by its metabolic enzymes),this work attempts to develop a new system of transformation selection marker/N utilization/weed control,from the viewpoint of the indirect provision of another essential macronutrient element N.Using BU as the selective agent,the BU hydrolase(BH)gene,either alone or together with the gene of allophanate hydrolase(AH,required for complete hydrolysis of BU)to form the BA2 H fusion,is introduced into tobacco as a selective marker.The feasibility of this technological system is evaluated by investigating the transformation efficiency and the growth profiles of transgenic plants resistant to BU stress.The main results obtained are as follows:1.Expression vector construction of the BA2 H and BH genesThe BA2 H gene,an in-frame fusion form of BH and AH,was synthesized according to the gene codon usage preference of tobacco,and then cloned into plasmid p BI121 by Xba I/Sac I digestions to obtain the plant expression vector p BI(BA2H).Meanwhile,the BA2 H fragment was inserted into p ET32a(+)to generate the prokaryotic expression vector p ET(BA2H)by using Nde I / Xho I.Likewise,the BH gene was re-amplified using BA2 H as template,and subcloned by restriction enzyme digestions to create plant expression vector p BI(BH)and prokaryotic expression vector p ET(BH),respectively.All newly constructed vectors were verified by sequencing.2.Obtainment and characterization of BA2 H and BH transgenic tobaccos(1)The resistance regeneration of wild-type(WT)tobacco leaf explants was evaluated on normal MS medium containing 0,0.5,1,2,5 m M BU,and it was found that the regenerated shoots from tobacco calli could emerge only on MS medium with a BU stress of less than 1 m M concentration.(2)Based on the results in(1),plant vectors p BI(BA2H)and p BI(BH)were transformed into tobacco leaf explants by Agrobacterium infiltration,and selected by0.5,0.8,1 m M BU,using 100 mg/L Kanamycin(Kan)as the control.Under Kan selection,BA2H(Kan)and BH(Kan)transgenic tobaccos could be obtained in about3 months,with positive rates of 66.7% and 87%,respectively.In contrast,on normal MS medium,BA2H(BU)transgenic tobacco could be screened by BU after 62-70 days and the highest positive rate was 80% under 1 m M BU.The highest positive rate of BH(BU)transgenic tobacco was 53% under 0.8 m M BU stress.However,on N-deficient MS medium with BU,the calli of tobacco were of worse growth,longer time and lower rate of regeneration.Nevertheless,the positive rate of transgenic tobacco was slightly higher than that from MS medium with the same BU concentration.In addition,RT-PCR results showed that BH and AH were expressed in the roots,stems and leaves of BA2H(BU)transgenic tobaccos,and BH expression was also of similar patterns in BH(BU)transgenic tobaccos.(3)Seed germination of BA2 H and BH transgenic tobaccos were conducted on 0,0.5,0.8,1 m M BU and Kan plates(MS and N-deficient MS medium),using WT as the control.In both types of MS medium,the seed germination and seedling growth of WT tobacco were inhibited by BU,and the degree of which increased with BU concentration.Contrastively,this inhibitory effect on BA2 H,BH transgenic tobacco was not significant,showing a strong resistance to BU.In addition,under each stress of the same BU concentration,the seed germination and seedling growth of tobacco on normal MS medium was significantly better than that on MS medium lacking N.(4)A seed mixture of WT tobacco,BA2 H or BH transgenic tobacco,and weeds(tall fescue and Bermudagrass)were sowed in greenhouse.The results showed that BA2 H and BH transgenic tobacco outperformed WT tobacco and weeds,obviously with a competitive growth under 206 mg/L BU irrigation.3.Recombinant expression and enzymatic activity analysis of BA2 H and BHVectors p ET(BA2H)and p ET(BH)were transformed into Escherichia coli BL21(DE3)for induced expression.The results showed that both BA2 H and BH had high expression levels and solubility.After a purification through His-tag affinity chromatography and an enzymatic assay via GDH method,BA2 H and BH were verified with an activity of hydrolyzing BU to ammonia,and the specific activity of BH was 12.436 U/mg and that of BA2 H was 6.859 U/mg.In conclusion,BA2 H and BH enzymes have the ability to hydrolyze BU into ammonia,and can be conjugated with BU(as selection agent)to develop such a plant transformation selective system called BU / BH or BU / BA2 H,which can reach the similar level of transformation efficiency of the commonly used NPT II(Aminoglycoside-3’-phosphotransferase II)/ Kanamycin(Kan)screening system,but with relatively shorter period.In addition,the expression of BA2 H and BH in transgenic tobaccos enables the plants to convert BU into utilizable N source,and meanwhile eliminates the toxicity of BU to a certain extent,thus responsible for their growth remarkably better than that of WT tobacco and weeds.Apparently,it is feasible to establish a new tertiary system based on BU / BH or BU / BA2 H,covering multiple roles in plant transformation selection marker /N utilization/weed control.This study can provide a new technical choice for plant genetic engineering,and seems reported firstly and not in the current literature.