Genetic Transformation Of The Insect-resistant And Salt-tolerant Gene In Tobacco And Populus×euramericana‘Neva’ And The Expressin Analysis

Single expression vector fused multi-gene conversion was one of the focuses ofgenetic engineering research.In this study, by Agrobacterium-mediated method andkanamycin as a selectable marker, the plant expression vector p096899(containingBtCry1Ac and NTHK1),p09687199(containing BtCry1Ac, BtCry3A, and NTHK1) wereused for tobacco transformation, So, after one transformation,containing BtCry1Ac,BtCry3A and NTHK1tobacco clone lines were attained.DNA and protein levels weredetected to get of tobacco clone lines. Further, this plant expression vector was transformedinto populus×euramericana ‘Neva’ to get of containing BtCry1Ac, BtCry3Aa and NTHK1populus×euramericana ‘Neva’ clone lines.The main conclusions were as followed:1.Optimized tobacco genetic transformation regeneration system,it was determinedthat the kanamycin (kan) critical concentration was50mg·L-land200mg·L-lCefotaxime(CTX) was a suitable concentration to control the propagation of Agrobacteria.2.Based on Agrobacterium-mediated transformation method，using plant expressionvector p096899and p09687199,the BtCry1Ac, BtCry3A and NTHK1genes weretransformed into tobacco，the10clone lines of resistance stability were obtained. PCRdetections indicated that the target genes were integrated into the tobacco genome.3.Real-time PCR revealed that both BtCry1Ac gene and BtCry3A gene have integratedinto the tobacco genome. The transcription kurtosis of BtCry1Ac gene was ranged from1.08E+04to3.40E+05; the transcriptional kurtosis of BtCry3A ranged from3.23E+05to1.34E+06. Since the tobacco genome itself contain NTHK1gene, NTHK1gene cannot bequantitative detection.4.ELISA analysis showed that in the most of the transgenic tobacco clone lines theexpression levels of Bt1and Bt3toxic proteins were higher than the comparison, thehighest clone could be up to0.0287%and0.3792%of the leaf total solubleproteins,respectively. Feeding tests showed that lines with high Bt Toxin Protein content isalso high resistance of Spodoptera litura, after the7days,the highest mortality of one linereach99%, the control only41%.5.Differentiation medium containing different NaCl concentration were used for transgenic tobacco leaf discs culture in tissue culture conditions, The growth phenotype arebetter than the wild-type control plants, of the gene transferred to, The result indicated thatNTHK1transformation could improved the salt tolerance of tobacco to some extent.6.Determined the concentration of selection pressure in genetic transformationprocess of populus×euramericana ‘Neva’, The kanamycin (kan) critical concentration was30mg·L-land the antibiotic Cefotaxime (CTX)400mg·L-lwas a suitable concentration tocontrol the propagation of Agrobacteria.7.Based on Agrobacterium-mediated and kanamycin as selection marker, using plantexpression vector p096899and p09687199, the BtCry1Ac, BtCry3A and NTHK1geneswere transformed into populus×euramericana ‘Neva’， the8clone lines of resistancestability were obtained. PCR analysis indicated that the target genes were integrated intothe populus×euramericana ‘Neva’ genome.