| Populus species are one of the most valuable woody plants in the world.However,the situation of poplars hurted by pathogen has become the main reason which limits its cultiviation.The development of plant genetic transformation system provides strategies strongly to increase resistance of poplars to different pathogen.According to previous studies,flavonoids such as anthocyanins and tannins play a very important adjunct role in the defense mechanisms of plants.Further studies have shown that when overexpressed two key transcription factors Del/Ros from snapdragon,plant accumulated anthocyanins at levels considerably higher than previously reported for the efforts to engineer anthocyanin accumulation,and the disease resistance of plant was increased with the promotion of the anthocyanin accumulation.Based on these studies,we overexpressed Del/Ros in P.alba to enhance the hydrophilic antioxidant capacity and increase its resistance to pathogen by promoting bioactive compounds such as anthocyanins.The detail results are as follows.1.The relative expression level of the P.alba and P.euphratica,as well as the anthocyanin content of P.alba and P.euphratica were studied.The results showed they are both higher in P.euphratica and lower in P.alba.The resistance of each poplar to Dothiorella gregaria was found to be the consistant with the content.2.We cloned the homologous sequence of Del/ Ros of Houttuynia cordata in P.alba and then assembled into the 35 s promoter and Nos terminator at pCAMBIA1305.Transferred the recombinant expression vector to P.alba with plant transgenic technology.After detection with RT-qPCR we obtained two highly expression lines Del/Ros-2 and Del/Ros-5.3.The phenotypes analysis of wild type and two overexoression lines.Their leaf type,fresh weight and plant height were measured.The results showed that there is no difference bewteen them after comparing the overexpression plants and wild type plant.The overexpression plants do not affect the growth of P.alba.The results showed that the content of tannin in Del/Ros-2 and Del/Ros-5 leaves was 4 ~ 5 times higher than that of wild type,and the results of DMACA staining showed that the content of flavonoids in leaves was higher than that in wild type.The tannin content in the overexpressed lines was significantly higher than that in the wild type.Anthocyanin content is consistant with tannins,2 to 4 times higher than the wild type.Quercetin and kaempferol and other flavonoids are also consistent with tannin,anthocyanin,1 to 1.5 times higher than the wild type.The total amount of flavonoids containing phenol rings in the leaves,that is,the total phenol content,is 1.6 to 2 times higher than that of the wild type.Interestingly,Del was expressed in Del/Ros-5 overexpressing lines as high as 1400 times the wild type,whereas Del/Ros-2 was more than 130 times that of the wild type.This difference also affected the accumulation of anthocyanins,tannins and other substances in the leaves of the two overexpressing lines.The content of flavonoids in Del/Ros-5 is 0.2 to 0.5 times higher than that in Del/Ros-2.This may be due to the dose effect of Del gene expression.4.The compartion of the resistance of Del/Ros-2,Del/Ros-5 and wild type to pathogen.The infection area of different pathogen was measured after the lesion of Dothiorella gregaria and Botrytis cinerea for 5~8 days.The results showed that whether it is Dothiorella gregaria or Botrytis cinerea,the wild-type lesion area is 2 times bigger than the overexpression plants.This indicated that Del/Ros-2 and Del/Ros-5 had stronger resistance to two fungal diseases than wild type.However,there was no significant difference in the lesion area between Del/Ros-2 and Del/Ros-5.We speculated that although the content of Del was different in the two lines,the content of flavonoids was different too,but the disease resistance was no more difference after reaching a certain extent. |
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