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Genetic Transformation of Tobacco and Populus×Euramericana 'Neva' by Multi-gene Plant Transformation Vector

Author: RenYaChao
Tutor: YangMinSheng
School: Agricultural University of Hebei
Course: Tree Genetics and Breeding
Keywords: Multi-gene Plant Transformation Vector Tobacco Populus×euramericana’Neva’ Genetic Transformation
CLC: S572
Type: Master's thesis
Year: 2013
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Abstract


In order to achieve multi-gene genetic transformation of tobacco and poplar, in thisstudy, the multi-gene plant transformation vector p209-BtCry1Ac-BADH andp209-BtCry1Ac-BtCry3A-BADH were introduced into Agrobacterium tumefaciehs, andtobacco (wisconsin35) was transformed by Agrobacterium-mediated leaf disc method, thetransgenic plants were obtained by PCR and fluorescence quantitative PCR detection.Toxic protein detection and resistance test showed that after transforming, the target geneswere able to express in tobacco while showing resistance to some extent. Then Populus×euramericana ’Neva’ was transformed by the two vector as well, and transgenic lines wereobtained through initial detection by PCR. The main findings were as follows:1. The genetic transformation and expression detection of tobacco by vectorp209-BtCry1Ac-BADH.The screening concentration of Kanamycin was50mg·L-1in the differentiationmedium in the genetic transformation of tobacco, and75mg·L-1in the rooting medium.The concentration of Cefotaxime Sodium was400mg·L-1in the screening process.After transformation of tobacco by Agrobacterium-mediated, the completeregenerated plants were obtained by screening of Kanamycin.4transgenic lines thatdetected BtCry1Ac and BADH were obtained after PCR detection,2lines were onlydetected BtCry1Ac.Fluorescence quantitative PCR indicated that two target genes were expressed at thetranscriptional level in3lines that detected all the target genes, and there were somedifference among these lines. However, a line hadn’t been detected the expression ofBADH.2lines which were detected BtCry1Ac were detected the expression of BtCry1Ac.ELISA detection of toxic protein showed that3lines screened by fluorescencequantitative PCR were detected the expression of toxic protein. The content of toxicprotein was up to414.63ng·g-1. And the line detected BtCry1Ac was detected theexpression of toxic protein.The indoor insects-resistance test showed that only2lines in the3transgenic linesshowed resistance against Prodenia litura (Fabricius), the corrected mortality was up to38.2%. The others did not show obvious resistance to the larve.2lines were selected forplantlets salt-tolerance test, the results revealed that there were no significant differencesbetween the transgenic lines and the control. 2. The genetic transformation and expression detection of tobacco by vectorp209-BtCry1Ac-BtCry3A-BADH.After transformation of tobacco by Agrobacterium-mediated, the completeregenerated plants were obtained through screening of Kanamycin.7transgenic lines thatdetected BtCry1Ac, BtCry3A and BADH were obtained by PCR detection, a line wasdetected BtCry1Ac and BtCry3A, and a line was only detected BtCry1Ac.Fluorescence quantitative PCR showed three target genes were expressed at thetranscriptional level in4lines with differential expression, and3lines weren’t detectedexpression of BADH. A line detected BtCry1Ac and BtCry3A were detected expression oftarget genes. A line detected BtCry1Ac was detected expression of the target gene.ELISA analysis of toxic protein revealed that4lines were detected the expression ofBtCry1Ac and BtCry3A toxic protein. The content of BtCry1Ac toxic protein was up to267.90ng·g-1. The content of BtCry3A toxic protein was up to13749.30ng·g-1. Thecontent of BtCry3A toxic protein was significantly higher than that of BtCry1Ac toxicprotein. The other3lines which were detected expression of BtCry1Ac and BtCry3A weredetected BtCry1Ac toxic protein, the content of BtCry1Ac toxic protein was up to369.92ng·g-1. Only2lines were detected BtCry3A toxic protein, the content of BtCry3A toxicprotein was up to3262.86ng·g-1. The parts of other lines were also detected toxic protein.The indoor insects-resistance test showed that7lines that were detected all targetgenes showed resistance against Prodenia litura (Fabricius) larve, the corrected mortalitywas up to69.5%. The rest lines detected BtCry1Ac also showed resistance to the larve, thecorrected mortality was up to59.3%.2lines were selected for further salt-toleranceresearch, and the results showed there were no significant differences between thetransgenic lines and the control.3. Genetic transformation of Populus×euramericana ’Neva’ by multi-gene planttransformation vectors.The sterile plantlets of Populus×euramericana ’Neva’ were obtained by explantculture, and plantlets grew well through differentiation and rooting cultivation alternately.The screening concentration of Kanamycin was30mg·L-1in the differentiation medium,and50mg·L-1in the rooting medium. The concentration of Cefotaxime Sodium was400mg·L-1in screening process. Populus×euramericana ’Neva’ was transformed by the twoplant transformation vectors by Agrobacterium-mediated, and achieving the completeregenerated plants by screening of Kanamycin. Finally,10transgenic lines transformed byp209-BtCry1Ac-BADH and4transgenic lines transformed byp209-BtCry1Ac-BtCry3A-BADH were obtained after PCR detection.

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CLC: > Agricultural Sciences > Crop > Economic crops > Tobacco ( tobacco )
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