| DNA methylation is an important epigenetic modification,which plays an essential role in plant growth,development and environmental response.The level of plant genomic DNA methylation is mainly controlled by DNA methyltransferases and DNA demethylases,which enable plants to exhibit dynamic DNA methylation levels at different developmental stages and under different environmental conditions.Several genes have been found involved in the DNA methylation and demethylation process in Arabidopsis Thaliana.Among them,DNA methyltransferase MET1(methyltransferase 1)maintains methylation of symmetric CG sites,while transglucosylase(DEMETER,DME)catalyzes DNA demethylation by excising5-methylcytosine(~mC).In this study,the in vitro leaves of Populus alba×P.glandulosa’84K’were used as experimental materials,and the chemically-induced promoter and Arabidopsis Thaliana methyltransferase gene At MET1 and demethylase gene At DME were introduced into84K genome,and the transgenic plants were obtained.The 17-β-estradiol induced expression of At MET1/At DME was detected by q RT-PCR.By different time of 17-β-estradiol treatment on explants of the transgenic clone,regenerants with various mutations was generated.The main results of the paper are as follows:1.Eighteen At MET1 transgenic P.alba×P.glandulosa’84K’clones were obtained.By agrobacterium-mediated genetic transformation,chemical inducible promoter and Arabidopsis thaliana methyltransferase gene At MET1 was transformed into the genome of 84K.A total of648 hygromycin-resistant buds were obtained and at last 18 transgenic plants were identified by PCR and DNA sequencing detection methods,which were labeled with AM-1～18#.The leaves of a randomly selected transgenic line were treated by chemical inducer 17-β-estradiol.Results of q RT-PCR showed that the expression of At MET1 in the transgenic clone leaf explants reached its highest for 3h by 17-β-estradiol treatment,and then decreased at 6h,increased at 12h and decreased again to less than half of the expression at 12h.These results indicated that the chemical inducer can efficiently and rapidly induce the expression of At MET1 in transgenic poplar plant that laid a solid foundation for further study of the role of MET1 in the regulation of methylation of poplar genome.2.Six At DME transgenic P.alba×P.glandulosa’84K’clones were obtained.Through agrobacterium-mediated genetic transformation,chemical inducible promoter and Arabidopsis thaliana methyltransferase gene At DME into the genome of P.alba×P.glandulosa’84K’.A total of 224 hygromycin-resistant buds were obtained from the leaf explants of 84K and finally six transgenic plants were identified by PCR and DNA sequencing detection methods,which were labeled with AD-1～6#.The leaf explants of a randomly selected transgenic line were treated by chemical inducer 17-β-estradiol.Results of q RT-PCR showed that the expression of At DME reached its highest level after 3h treatment of 17-β-estradiol,and its expression gradually decreased after 12 h’s treatment.The results indicated the chemical inducer17-β-estradiol can rapidly and efficiently induce the expression of At DME in transgenic poplar,which lays a solid foundation for further study on the mechanism of DME in the regulation of poplar genome methylation.It laid a solid foundation for the study of the chemical expression characteristics of poplar.3.Plants with various phenotypic variations were regenerated from leaf explants of a At MET1 transgenic clone on medium with 17-β-estradiol.A total of 140 regenerated shoots were obtained,and 88 plants were obtained after rooting.Among them,35,17,7,9,7,5,5,3plants were obtained from leaves of 17-β-estradiol treatment for 0 h,3h,6h,12h,24h,48h,96h and 144h.The regenerated plants obtained from 17-β-estradiol treatment for 3h,12h,24 h,as well as the uninduced transgenic controls and non-transgenic controls were transplanted into greenhouse and their physiological and biochemical traits were measured.Rresults showed that the plant height of clone 2#from 12 h treatment was significantly lower than that of the control;the leaf length-width ratio,Pn,Gs and soluble protein content of all plant of the three treatments were significantly lower than that of control;the Ci and soluble protein content of several clones from 12 h and 24 h treatments were significantly lower than that of controls,while the soluble sugar content was significantly higher than that of control.The Tr of clone 2#from 3 h treatment was significantly lower than that of control,WUE was significantly higher than that of control;the POD activity of clones from 3 h and 12 h treatments were significantly higher than that of control,but other differences between the regerants and control were not significant.4.Plants with various phenotypic variations were regenerated from leaf explants of a At DME transgenic clone on medium with 17-β-estradiol.A total of 188 regenerated shoots were obtained,and 141 regenerated plants were obtained after rooting.Among them,40,15,15,16,18,16,13,8 plants were obtained from leaves of 17-β-estradiol treatment for 0 h,3 h,6 h,12 h,24 h,48 h,96 h and 144 h.The regenerated plants obtained from induced 3,12,24 h leaf discs,and the uninduced transgenic controls,non-transgenic controls were transplanted into greenhouse and their growth,physiological and biochemical parameters were measured.Results showed that the leaf length-width ratio,Gs,Ci,Tr and soluble protein content of all theplants from the three treatments were significantly lower than those of the control,and the relative chlorophyll content,WUE and protective enzyme activity were significantly higher than those of the control.The soluble sugar content of all clones from 3 h and 24 h treatments was significantly higher than that of the control,but the other differences between the regenerated clones and the control were not significant. |
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