关键词: 欧洲黑杨, 抗虫基因, BtCry1Ac, 转基因, hiTAIL-PCR, 侧翼序列, 事件特异性检测

Abstract:

Objective: In order to further improve the background information and promote the safety evaluation and application of transgenic insect-resistant Populus nigra, flanking sequence of T-DNA insertion sites were isolated in two lines (n12 and n222 with BtCry1Ac). Method: The flanking sequences of exogenous gene insertion sites were separated by hiTAIL-PCR(high-efficiency thermal asymmetric interlaced PCR), and the insertion sites were determined by comparing the genomic sequences of P. trichocarpa. Two pairs of specific PCR primers were designed according to the flanking sequence of insertion sites, and a specific PCR method was established to detect the expression of genes around insertion sites in transgenic poplar with BtCry1Ac. Quantitative real-time reverse-transcription PCR (qRT-PCR) was used to analyze the expression of genes around insertion sites. Result: PCR and semi-quantitative PCR showed that the BtCry1Ac was stable in the transgenic materials. By comparing the genome sequence of P. trichocarpa, the T-DNA of transgenic poplar n12 was inserted into the second intron of Potri.015G076600 at 10162773 locus on Chr15 and the nucleotide composition was 65% for AT content. The integration locus of n222 was 41596184 in the gene spacer of Chr01 and the nucleotide composition was 69% for AT content. Specific PCR detection showed that n12 could amplify 709 bp (transgenic) and 1 159 bp (non-transgenic) specific bands, n222 and its hybrid offspring 1 265 bp (transgenic) and 1 827 bp (non-transgenic) specific bands, while the control could only amplify non-transgenic specific bands. The expression of Potri.015G076600 (serine-protein kinase, SPK) at n12 insertion site was up-regulated 4.3 times and that of nearby Potri.015G076700 (ataxia telangiectasia mutated family protein, ATM) was down-regulated 20 times, those may be involved in the regulation of poplar growth rate. Potri.001G395700 (isoflavone-7-O-β-glucoside 6″-O-malonyltransferase, IBG) and Potri.001G395800 (PIF1-like helicase, PIF) were up-regulated in n222 and its hybrids with P. deltoides 'Danhong'. Conclusion: The T-DNA of n12 and n222 preferentially inserted into AT rich regions, while the deletion of T-DNA vector boundary sequence caused the change of genes expression near the insertion site. And the specific detection method provides a basis for the management and monitoring of transgenic P. nigra with BtCry1Ac.

Key words: Populus nigra, insect-resistant gene, BtCry1Ac, transgenic, hiTAIL-PCR, flanking sequencing, event-specific detection