Genetic transformation and expression of Cry1Ac–Cry3A–NTHK1 genes in Populus × euramericana “Neva”

Abstract

To realize the genetic transformation of Cry1Ac, Cry3A, and NTHK1 genes in Populus × euramericana “Neva” and obtain transgenic plants, the plant transformation vector p09687199–Cry1Ac–Cry3A–NTHK1 was transformed in Populus × euramericana “Neva” through Agrobacterium-mediated transformation. The complete regeneration of the transformed plant was screened with kanamycin. Resistant roots of the transformed plants were detected through PCR, fluorescence quantitative PCR, and ELISA analysis of the toxic protein. The insect resistance and salt tolerance of transgenic plants were determined based on the findings of these methods. PCR detection showed that among six lines, five lines indicated the presence of three target genes, Cry1Ac and Cry3A genes were detected in one line. Fluorescence quantitative PCR detection showed that the transcript abundance of the Cry1Ac gene was within 1.13E+3 to 3.17E+4, that of Cry3A was within 4.40E+6 to 1.97E+7, and that of NTHK1 was within 2.48E+3 to 8.17E+3. Bt toxic protein detection showed that the content of the Cry1Ac gene was from 1.08 to 22.99 ng·g−1, and that of Cry3A gene was from 146.12 to 17027.96 ng·g−1. The expression of the Cry3A gene was significantly higher than that of the Cry1Ac gene. The insect resistance test demonstrated that the mortality of transgenic plants on Hyphantria cunea first instar larva reached 68.89 %. The mortality on Plagiodera versicolora first and second instar larvae reached 100 %, whereas that on the third instar larva was as high as 81.71 %. Four lines were selected to detect potting salt tolerance, and only the No. 2 line showed strong salt tolerance.