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通过基因特异引物扩增和免疫层析试纸法分别对转基因大豆中外源EPSPs基因和其编码的蛋白进行检测,结果表明,EPSPs基因不仅已整合到大豆基因组中,而且EPSPs蛋白可以正常表达。利用染色体步移法获得了转基因大豆插入位点的侧翼序列,序列比对表明35S上游的大豆DNA序列起始于Gm02:7912740,NOS下游的大豆DNA序列起始于Gm02:7777705。在本研究检测序列范围内发现插入位点两侧不同位置有3个不同的未知片段、2个大豆基因组序列倒位,同时发现2个大豆基因组片段丢失。外源基因不是以点插入方式整合,而是导致大豆基因组约135kb片段的移位;NOS下游大豆基因组序列发生重排,导致一个编码HEC1和HEATrepeat两个功能域的基因(Glyma02g09790)结构受到影响,首次发现该基因在ABA和PEG处理时下调表达,推测该基因通过ABA信号通路参与胁迫应答。本研究通过对抗除草剂EPSPs基因在大豆基因组中的插入位点分析,明确了外源EPSPs基因在大豆基因组中的整合、定位及其侧翼序列,为转基因大豆安全评价提供了依据。
The results showed that the EPSPs genes were not only integrated into the genome of soybean but also the EPSPs proteins could be expressed normally by gene-specific primer amplification and immunochromatographic test strips. The flanking sequences of transgenic soybean insertion sites were obtained by chromosome walking. The sequence alignment indicated that the soybean DNA sequence upstream of 35S started from Gm02: 7912740, and the soybean DNA sequence downstream of NOS started from Gm02: 7777705. Three different unknown fragments were found at different positions on both sides of the insertion site in the detection sequence of this study. Two soybean genomic sequences were inversed and two soybean genomic fragments were found missing. The exogenous gene was not integrated by point insertion but resulted in the translocation of about 135 kb fragment in the soybean genome. Rearrangement of the downstream soybean NOS genomic sequence resulted in a structurally impaired structure of a gene (Glyma02g09790) encoding both HEC1 and HEATrepeat functional domains, It was found for the first time that the gene was down-regulated when treated with ABA and PEG, suggesting that the gene is involved in the stress response through the ABA signaling pathway. In this study, we analyzed the integration of EPSPs gene in soybean genome and its flanking sequences by analyzing the insertion sites of EPSPs gene in soybean genome, which provided the basis for the safety evaluation of transgenic soybean.