关键词: 转基因杨树, 多基因载体, 基因互作, 外源基因表达, Bt毒蛋白

Abstract: [Objective] This paper was aimed to explore the stability of existence and expression of the exogenous Bt gene in transgenic poplar 107 (Populus×euramericana ‘74/76’), and to explore the expression patterns of Bt toxin in different parts of transgenic poplar 107 at different times, and to investigate the effects of gene interaction and multi-gene vector structure on the stability and efficiency of foreign gene expression in polygenic transformation vectors.[Method] Three strains of one-year-old transgenic poplar 107 with genes of Cry1Ac-Cry3A-NTHK1(A1, A2, A3) and three strains with genes of Cry1Ac-Cry3A-BADH(B1, B2, B3) were selected to detect and validate foreign genes in the transgenic poplar trees using PCR and to detect transcript abundance of Bt gene usng fluorescence quantitative PCR, and to detect toxic protein contents in different parts of the transgenic poplar trees at different times using ELISA.[Result] The PCR detection showed that the transgenic poplar 107 could amplify a specific band at the same size as that of the positive control, and the negative control did not amplify the specific band, which proves that the foreign gene is stable in transgenic poplar 107; The fluorescence quantitative PCR showed that two Bt genes were stably expressed in each line, and the transcript abundance of Cry1Ac gene was ranged from 2.1×10⁴ to 5.1×10⁴, the transcriptional abundance of Cry3A gene ranged from 2.8×10⁶ to 5.6×10⁷. There was no correlation between transcript abundance of Cry1Ac gene and transcriptional abundance of Cry3A. The ELISA detection showed existence of both Cry1Ac and Cry3A toxic proteins in each line of the transgenic poplar 107. There was no correlation between transcript abundance and toxic protein content of the two Bt genes. But there was a positive correlation between toxic protein content of the two Bt genes. The content of two types of Bt toxic proteins was lower in June and July of the two different vectors, and increased sharply in August. The content of Cry1Ac toxic protein in each transgenic line reached the peak in September, and the content of Cry3A toxic protein reached its peak in August, then fell sharply in October. The content of Bt toxic protein in leaves and xylem of different parts (upper, middle and lower) did not show consistent pattern in August.[Conclusion] The expression abundance of each exogenous gene was significantly different, and the Cry3A gene was significantly higher than the Cry1Ac gene in transcriptional abundance. The content of Cry1Ac toxic protein in each line was extremely low, and the content of Cry3A toxic protein was extremely higher than that of Cry1Ac, which was consistent with the detection of transcriptional abundance. There was no significant difference in the transcriptional abundance, the expression pattern and expression level of toxic protein between the two vectors of Cry1Ac gene, neither of Cry3A gene. During the entire growing season of transgenic poplar 107, the contents of two Bt toxic proteins displayed a similar pattern of single peak.

Key words: transgenic poplar, multi-gene vector, genetic interaction, exogenous gene expression, Bt toxin