Improving 2,4-D Resistance By Genetic Transformation With TfdA Gene From Cupriavidus Campinensis BJ71 In Zea Mays L.

Maize is an important raw material and food crop,and it occupies an important position in the national economy.One of the reasons decreasing the production of Maize is weed.It is reported that nearly half of maize fields has been affected by weeds in different levels,which can decrease production 30%~50% in severe cases.At present,spraying chemical herbicides is an indispensable method to control weeds in the modern agriculture.The advantage of the phenoxyalkanoic acid herbicides is low price,high efficiency,wide weeding and no residual,so that they are widely used in wheat fields,rice fields,corn fields,etc.2,4-dichlorophenoxyacetic acid(2,4-D)is a typical herbicide of phenoxycarboxylic acid.A low concentration could promote growth while an excessive concentration would play a weeding role that the stem tissue of weeds increases synthesis of protein and DNA,mature cells restore the ability to split so that it causes abnormal growth even death.In previous study,a strain Cupriavidus campinensis BJ71 having strong power to degrade was isolated from soil which sprayed 2,4-D herbicide.And TfdA gene which could degrade 2,4-D had been cloned from the strain.On this basis,this study constructed the monocotyledon expression vector pGM626-LF-TfdA which was driven by Actin promoters.Then we transferred TfdA genes into Maize via the Agrobacterium tumefaciens-mediated stem-tip transformation,and verified Tfd A's degradation ability,The aim is to supply the evidence for the functions of CcTfdA gene.The following findings were obtained:1.The degradation of 2,4-D gene TfdA was introduced into Zea mays L.by Agrobacterium-mediated transformation.174 strains resistant plants were screened,43 of whichwere positive via histochemical staining of GUS gene test.The results showed that there were transgenic plants with 801 bp target bands corresponding to the size of the positive plasmids while WT plants failed to amplify any bands,suggesting that 2,4-D degradation gene TfdA had been successfully integrated into the recipient plants.Five transgenic lines of T1,T2,T3,T4 and T5 with high resistance to 2,4-D were screened by applying herbicide.2.According to the spss analysis and LSD multiple comparison,the minimum concentration 20 mg·mL-1 of 2,4-D concentration,which had a significant effect on the plant,was selected as the susceptible screening concentration for the transgenic plants.After spraying 20mg·mL-1 2,4-D solution for 7 days,we found that 91% of wild-type maize plants developed damage symptoms of root and stem swelling deformities and lateral growth,leaf yellowing and so on while the transgenic plants grew well with little symptoms.3.Over time,the chlorophyll content in both transgenic and non-transgenic maize plants began to decrease gradually after 2,4-D treatment,but the decreasing amplitude of chlorophyll content in the latter was significantly(P <0.01)higher than that in the former.After 2,4-D treatment,the results of the H2O2-staining by diaminobenzidine(DAB)and malonaldehyde(MDA)content detection showed that there were significantly higher(P<0.01)H2O2 accumulation and MDA content in the leaves of wild-type plants compared with those in transgenic plants.The MDA content in non-transgenic and transgenic was 2.08 and 3.34 nmol·mgprot-1,respectively.At3 day after 2,4-D treatment,the maximum of enzyme activities of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)in transgenic maize plants were 34.88,4.10,31.56U·mgprot-1,which were 1.65,1.4 and 1.7 folds higher than these in wildtype ones,respectively.4.Cytological study of transgenic Zea mays L.with Tfd A gene.Based on the observation of paraffin section of Zea mays L.,there were no obvious difference among the epidermis,cortex,vascular cylinder,mesophyll and veins in root,stem and leaf between the TP and WT plants.However,bits of variations appeared in transgenic lines,such as the collenchyma of leaf vein,phloem parenchyma cells and medulla number,etc.The biomass at the early growth stage,leaf characteristics(length,blade width,area and shape index of leaf)field growing were also no obvious difference.Tfd A have effects on corn character and the output.