| Chlorophenols(CPs)are a kind of common persistent organic compounds in the environment,with stable structure and arcinogenicity,teratogenicty,mutagenicity.They are difficult to be degraded in the environment.Moreover,it has bioaccumulation and long-distance migration,which is very harmful to both organisms and ecological environment.As early as last century it has been included in the list of priority pollutants around the world.Although these compounds have been discontinued,but the environment pollution still exists.At present,the methods for CPs remediation are mainly physical and chemical remediation technologies.However these technologies have some defects.The phytoremediation technology in bioremediation has becomed the most promising environmental remediation technology because of its low cost,environmental friendliness and permanent high efficiency.Therefore,perennial and deep-rooted alfalfas were selected as materials to transfer 2,4-dichlorophenol hydroxylase(TfdB)gene into Alfalfa to increase plant tolerance and bioavailability of CPS.(1)Using agrobacterium-mediated transformation,the constructed plant expression vector pCAMBIA3300N+TfdB was transfered into wild-type alfalfas,30 lines of transgenic alfalfas were obtained through Phosphinothricin resistance screening.(2)The transgenic alfalfa genomic DNA was extracted,through PCR testing,the results show that TfdB gene has been integrated into the alfalfa lines.The total proteins of transgenic alfalfa were extracted and analyzed by ELISA.The result showed that the TfdB gene could be expressed in transgenic plants and expressing TfdB protein content of different lines were different.Enzyme activity analysis of each positive strain showed that the enzyme activities in different plants were still different.The enzyme activity of wild type alfalfa was 0.0078 U/mg,and the transgenic plants ranged from 0.0084 U/mg to 0.0499 U/mg.T13 lines with the highest enzyme activity(0.0499 U/mg)were selected for cutting and propagation for subsequent use.(3)Using hydroponics,phytoremediation efficiency of transgenic alfalfas and Arabidopsis to 2,4-DCP and 2,4,6-TCP was studied.Hydroponics results showed that there were significant differences in tolerance of 2,4-DCP and 2,4,6-TCP between transgenic plants and wild-type plants.In the present of 40 mg/L 2,4-DCP,the removal rate of 2,4-DCP by transgenic alfalfas was about 1.8 times that of wild type alfalfas,and the tolerance index of 2,4-DCP of transgenic TfdB alfalfa was 76.92% compared with wild type alfalfa(-9.09%).Tolerance index was increased by 86%.When the concentration of 2,4-DCP is 10 mg/L and 30 mg/L respectively,the removal rate of 2,4-DCP by transgenic Arabidopsis was about 0.98 and 1.9 times respectively that of wild type Arabidopsis,and the tolerance index of transgenic TfdB Arabidopsis was-9.68% and-22.58% respectively,and increased by 95.58% and 66.89% respectively compared with wild type Arabidopsis(-105.26% and-89.47% respectively).In the medium containing 30 mg/L 2,4,6-TCP,the removal rate of 2,4,6-TCP by transgenic alfalfa was about 1.5 times that of wild type alfalfa,and the tolerance index of 2,4,6-TCP of transgenic TfdB alfalfa was-8.33% and increased by 18.94% compared with wild type alfalfa(-27.27%).When the concentration of 2,4,6-TCP is 10 mg/L and 20 mg/L respectively,the removal rate of 2,4,6-TCP by transgenic Arabidopsis was about 1.4 and 2.3 times respectively that of wild type Arabidopsis,and the tolerance index of 2,4,6-TCP of transgenic TfdB Arabidopsis was 7.89% and-23.68% respectively,and increased by 31.57% and 26.32% respectively compared with wild type Arabidopsis(-23.68% and-50% respectively).These results showed that expressing 2,4-dichlorophenol-hydroxylase(TfdB)gene in plants could effectively improve the tolerance of 2,4-DCP and 2,4,6-TCP in plants,so as to significantly improve the removal rate of 2,4-DCP and 2,4,6-TCP in water. |
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