Study On Genetic Detection And Resistance Expression Of The Transgenic Populus × Euramericana ‘Neva’ With Multi-Gene

In order to achieve the new varieties of poplar with the characterization of Quality,resilience and fast-growing, thirteen 107 poplar lines with insect resistance and salt tolerance genes(p209-Cry1Ac-Cry3A-BADH) obtained by Agrobacterium mediated transformation method were studied in this paper. PCR detection proved that exogenous gene have successfully introduced into the 107 poplar genome; The exogenous gene expression was analyzed at the transcription level by fluorescence quantitative PCR and was analyzed at the translation level by ELISA analysis of toxic protein. The insects-resistance test and salt tolerance test were carried out. Finally, three lines with high resistance were obtained. The main findings were as follows:1.The 13 transgenic tissue culture seedlings were acclimatized and transplanted to the field. Then the field seedlings were obtained. The presence of the exogenous gene in these lines were tested by PCR detection with specific primers.Plasmid p209-Cry1Ac-Cry3A-BA DH were used as the positive control, and untransformed 107 poplar were used as the negative control. The 473 bp target bands of NPTⅡ gene can be amplificated in all the 13 transgenic lines and positive plasmid. The 546 bp target bands of Cry1 Ac gene can be amplificated in 10 transgenic lines and positive plasmid. The 667 bp target bands of Cry3 A gene can be amplificated in 5 transgenic lines and positive plasmid. The 507 bp target bands of BADH gene can be amplificated in 4 transgenic lines and positive plasmid. No target bands were detected in negative control. Because of the time and survival questions,8 transgenic lines were obtained for subsequent tests.2.The 8 transgenic lines and untransformed 107 poplar were detected by fluorescence quantitative PCR. The results showed that multiple exogenous genes were expressed in 3 lines(10、11、13), double Bt genes were expressed in 1 line(1), Cry1 Ac were expressed at the transcriptional level in 4 lines(3、4、6、8). The transcription kurtosis of Cry1 Ac gene was ranged from 3.127E+03 to 2.652E+05 in all the 8 lines; the transcriptional kurtosis of Cry3 A ranged from 2.916E+03 to 5.699E+04 in the 4 lines; the transcriptional kurtosis of BADH ranged from 1.910E+03 to 1.010E+04 in the 3 lines. No fluorescence signal coule be detected in CK.3.The Agdia ELISA kit was used for Cry1 Ac and Cry3 A toxic protein detection in 8transgenic lines and non-transgenic 107 poplar(CK). 3 transgenic lines with multiple genes(10、11、13), 1 transgenic line with double Bt genes(1),4 transgenic lines with Cry1 Ac gene(3、4、6、8)showed blue positive reaction of Cry1 Ac protein, the CK showed no color reaction. The protein contents ranged from 7.99 ng·g-1 to26.32 ng·g-1. The sequence with the content of Cry1 Ac toxic protein by 3 transgenic lines is 10 > 11 > 13.The content of Cry1 Ac toxic protein there were some differences but not significant between 10 and 11, 13. 4 transgenic lines with double Bt genes showed positive reaction to Cry3 A protein, the CK showed no color reaction. The protein contents ranged from2108.91 ng·g-1to2724.79 ng·g-1. The sequence with the content of Cry3 A toxic protein by 3transgenic lines is 10>11>13. The toxin protein expression of Cry3 A is much higher than Cry1 Ac.4. The 8 transgenic lines were screened by toxicity evaluation tests. Plagiodera versicolora highly resistant strains PB29, Hyphantria cunea highly resistant strains CC84 were used as positive control and non-transgenic 107 poplar as negative control. 3transpolygenic lines showed medium resistance to P. versicolora and high resistance to H.cunea. 1 line with double Bt genes showed medium resistance to both P. versicolora and H.cunea. 4 transpolygenic lines showed low resistance to P. versicolora. The sequence of the transgenic lines resistance of Cry1 Ac gene is 10 > 11 > 13, and the sequence of the transgenic lines resistance of Cry3 A gene is 10>11>13.5. The 3 transpolygenic lines with BADH gene were selected for the subsequent salt-tolerance research. transgenic lines showed salt tolerance in the morphological characteristics such as the survival rate, height measurement, diameter measurement, the number of leaves and others. And these lines showed resistant to salt stress in the determination of physiological characteristics such as net photosynthetic rate, chlorophyll fluorescence parameters, biomass, chlorophyll and others by salt stress. Proved that the three transgenic lines order of the resistance is 11>10>13.