Study On The Exogenous Genes Expression Of The Insect-resistant Populus×euramericana Cv.“74/76”

In order to identifying and selecting bivalent genetic transformation（Bt Cry1Ac gene and API gene）Populus×euramericana cv.（Abbreviation:Transgenic 107 poplars）with high resistance against to Drury and Anachoreta,studying the effects of coming two insect-resistance genes in plants.Ten bivalent genetic transformation 107 poplars lines were selected as materials and non-transgenic 107 poplars as control.,comparative studies were determinnated of exogenous gene expression,the experiments of Insect resistance ability and the insertion site of transgenic lines.Preliminary detection by PCR,proved that bivalent genetic have integrated into the genome of 107 poplars.The main results are as follow:1.The amplificated of BtCry1Ac gene and API gene of bivalent genetic transformation107 poplars lines were derected by PCR analyses with special primers.The target electophoretic 546 bp bands of Bt Cry1Ac gene in those 107 poplars appeared as the plasmid positive template,The target electophoretic 500 bp bands of API gene in those 107poplars appeared as the plasmid positive template;non-transgenic 107 poplars was no targeted bands.2.The RNA were extracted from the transgenic 107 poplars and no-transgenic 107poplars lines,using m RNA as template,c DNA was synthesized by reverse transcription,then the Bt Cry1Ac gene was amplified by realtime fluores-cence quantitative PCR.The results show that in August Bt Cry1Ac gene was amplificated fluorescence signals in PB1、PB2、PB3、PB6、PB7、PB8、PB9 and PB10,the highest transcript abundance was10⁷-10⁹,and Upper>middle>lower,PB4 PB5 and CK107 were not detected the fluorescence signals.The detected of CK PB1 PB4 and PB8 in May June July September,only PB1 PB8 could detect the fluorescence signals,but the magnitude compared with August lower10²-10⁴,CK and PB4 no fluorescence signals.3.ELISA was used to detect the Bt Cry1Ac toxic protein expression of transgenic lines,the results show that in August,PB1,PB2,PB3,PB6,PB7,PB8,PB9 and PB10 could detect the toxin protein expression,the content of protein was 16.38-665.11ng/g,,and pper>middle>lower,CK107,PB4 and PB5 couldn’t detected the protein;The detected of CK PB1 PB4 and PB8 in May,June,July and September,only PB1 PB8 could detect the protein,but the content of protein was 5.191-20.692 ng/g,CK107 and PB4 couldn’t detected the protein.4.By TAIL-PCR to detect the insert position of the exogenous gene of PB1,PB2 and PB6,the sequencing analysis showed that PB1,PB2 and PB6 respectively inserted into the genome of 14,6 and 3 in the 107 Poplar genome and not the same strain.The DNA were extracted of PB1,PB2,PB6 for the flanking sequence comparison of TAIL-PCR,by the amplification of primer,plastic recycling,even vector and identification,the result show that the sequences analysised of those transgenic lines were inserted into the genomic DNA of different chromosomes,were not the same strain.5.To target pests with Drury and Anachoreta,used the 10 lines of transgenic 107poplar for insect feeding test,the results show that the insecticidal effect of transgenic lines is Drury higher than Anachoreta.Screened 8 strains（PB1,PB2,PB3,PB6,PB7,PB8,PB9and PB10）with high resistance to Drury,2 strains（PB4 and PB5）resistance low.The high lines were fed for the Hlyphantria cunea L₁-L₃ larvae,the mortality was above 80%at the2th-3th day and reached 100%at the fouth day.With the increase of age,larvae of the resistant also gradually improve,the mortality of L6 was above 76.67%at the 8th to 10th day and reached 100%at the fourteenth day.Screened 5 lines（PB1,PB2,PB6,PB9 and PB10）for Anachoreta larvae with high insect resistance,3 lines（PB3,PB7 and PB8）insect medium,two lines（PB4 and PB5）is a low resistance.The high lines had lethal effect to L₁-L₆ larvae;the medium lines had cytotoxic effect to L₁-L₂ larvae of Anachoreta,but had no cytotoxic effect to more than L₃ larvae;the lower lines had no cytotoxic effect to larvae,but those lines had some influence on the adult,such as little feet,little tentacles and so on.