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目的:建立一种有效区分Glu-1D等位基因的Multiplex-PCR体系,快速检测转1Dx5小麦植株。方法:根据1Dx5和1Dx2亚基基因的区别设计特异的PCR引物,通过多重PCR技术检测花粉管通道法转化1Dx5核心片段的转基因T1代植株。结果:Multiplex-PCR能够在转基因T1代材料中扩增Glu-1D等位基因的特征条带,分别为343bp、320bp的1Dx5基因特异片段以及361bp的1Dx2基因特异片段,与预期结果一致。结论:该技术能够检测多个靶基因,有效地区分转基因Glu-1D上的等位基因,证实外源1Dx5基因已整合到受体基因组中,对检测基因组庞大、外源基因序列GC含量高且与内源基因同源性高的转基因小麦十分有效。
OBJECTIVE: To establish a Multiplex-PCR system that effectively distinguishes Glu-1D alleles and rapidly detect 1Dx5 wheat plants. Methods: According to the difference between 1Dx5 and 1Dx2 subunit genes, specific PCR primers were designed and the pollen tube pathway was used to detect the transgenic T1 generation of 1Dx5 core fragment by multiplex PCR. Results: Multiplex-PCR was able to amplify the characteristic bands of Glu-1D allele in transgenic T1 generation materials, which were 343 bp, 320 bp of 1Dx5 gene-specific fragment and 361 bp of 1Dx2 gene-specific fragment, respectively, consistent with the expected results. Conclusion: This technique can detect multiple target genes and effectively differentiate the alleles on the transgene Glu-1D, confirming that the exogenous 1Dx5 gene has been integrated into the recipient genome and that the detection genome is large and the GC content of the exogenous gene sequence is high Transgenic wheat with high homology to endogenous genes is very effective.