The Recombinant Expression And Functional Analysis Of Organophosphorous Hydrolase In Tomato And Cucumber

The application of chemical pesticides,especially the high-toxic organophosphorous insecticides and herbicides,has brought great economic benefits for agricultural production and a wide range of pollution to the environment simultaneously.Recently a large number of studies focused on the bioremediation of pesticide residues from water or soil by plants,but paid little attention to decrease the enrichment of pesticide residues in agricultural products which will be ingested into food chain.Based on this situation,we attempted to enhance the degrading ability of genetically modified(GM) plants on pesticide residues.The following results were obtained.(1) Construction of expressing vectors and transient expressing analysisWe constructed two expression vectors of pSOP and pSE8OP for the organophosphorus pesticide degrading(OPD) gene under the control of the CaMV 35S promoter and E8 promoter respectively.Then they were transformed into tomato fruit using an agro-infiltration transient expression system.The results of GUS staining,RT-PCR,wavelength scanning and fluorescent reaction showed that the agro-infiltrated tomato expressed OPH with the maximum hydrolysis activity of about 11.59 U/mg total soluble protein.(2) Analysis of OPH expressed in transgenic cucumberThe optimized culture media for bud differentiation of cucumber were MS +4 mg/L 6-BA,and 1/2MS for rooting,MS +30 mg/L Kan for transgenic seedlings selecting.Then the 35S-OPD gene was transformed into cucumber mediated by Agrobacterium.The transgenic seedlings could degrade coumaphos with the maximum activity up to 7.82μmol/mg.min and the minimum activity of about 3.8μmol/mg·min.(3) Analysis of OPH expressed in transgenic tomatoThe optimized conditions for Micro-Tom tomato were that the bud differentiation medium was MS +2 mg/L 6-BA,the rooting medium was MS +0.3 mg/L IAA and the selecting medium for transgenic seedlings was MS+100 mg/L Kan. Then 35S-OPD and E8-OPD was transformed into Micro-Tom tomato respectively. The results of GUS staining,PCR,Southern blotting and RT-PCR indicated that the E8 promoter could not drive the expression of OPD in leaves of transgenic tomato seedlings and the expression of OPD was not interfered by the CaMV 35S promoter at upstream about 2 kb from OPD.The activity of OPH in transgenic tomato was about 43.8±4.5μmol/mg·min for the fruit at immature phase(IMG) and 101.0±4.3μmol/mg·min for the fruit at break phase(B).The maximum reaction velocity and K_m for OPH were 2.73μmol/mg·min and 4.04μmol/L respectively.With the further maturation of fruit,the activity of OPH became decrease.(4) Analysis of the characters of recombinant OPH expressed in tomatoThe optimum temperature and pH for recombinant OPH expressed in transgenic tomato were 30℃and 9.0 respectively.The temperature stability of it was high for 54%whole enzyme activity when stayed at 70℃for 2 hours.Its enzyme activity would be inhibited by EDTA,SDS,β-ME,TritonX-100,DTT and be activated two times by Co2+ than natural Zn2+ condition.The pesticides degrading capability was increased two times on parathion and three to seven times on chlorpyrifos by tomato fruit in which OPH expressed,and with no obvious affection on dimethoate.The maximum velocity in degrading parathion and chlorpyrifos by recombinant OPH was 1.17μmol/mg·min and 8.80μmol/mg·min,and the Km was 11.15μmol/L and 5.91μmol/L respectively.OPH was located at plasma membrane directed by signal peptide after translation in tomato,and was phosphorylated at eight serine sites,six threonine sites and two tyrosine sites.These amino acid sites are usually the enzyme active centre,so its phosphorylation would affect the functional structure and metabolism process to display different characters from bacterial enzyme.(5) Assessment of potential allergenicity for OPH,GUS and NptⅡIn accordance with the FAO/WHO suggestions and Codex requirements,the potential food allergenicity of OPH,GUS and NptⅡwas assessed in three website which is established for allergens prediction.Results of Blast or Fasta alignment online with full sequence or 80 aa window indicated that the sequence of OPH,GUS and NptⅡdid not exist allergenicity.However OPH has one 6 aa segment in the same with the sequence of profilin in soybeans,wheat and rubber tree,and another 6 aa segment match the sequence of allergen-related protein in German cockroach.GUS has one 6 aa segment match the allergen "Aed a2" in salivary glands of Aedes aegypti. NptⅡhas one 6 aa segment match the hemoglobin of Chironomus thummi,but these segments did not exist antigenicity,so it may be false positive for the matches.(6) Isolation and characterization of a dimethoate degrading fungusTwo fungal strains with dimethoate degrading capability were isolated from soil and named as F1 and F2.The strain F1 exhibited biodegrading activity more than five times as high as strain F2 in 100 mg/L dimethoate and nineteen times in 200 mg/L dimethoate,but less tolerant than strain F2 in 400 mg/L dimethoate.The strain F1 was capable of degrading 100 mg/L dimethoate completely in 7 days without any other carbon source supplements,but the degrading capability was weaken with the decrease of complexity of carbon source supplements.This strain was characterized and identified as Fusarium sp.by morphological characters and rDNA ITS sequence analysis.