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本研究以双单倍体马铃薯DM的突变体为材料,利用PCR-walking的方法扩增其突变体的侧翼序列。在试验中,以60个鉴定过的阳性突变体样品为材料扩增后,有28个样品扩增出有效条带。PCR胶回收后测序,有2个样品测序无信号,其他26个样品,将测序与载体序列和马铃薯基因组序列用BLASTN比对,发现有6个样品分别插入到了马铃薯1、3或7号染色体上。通过进一步比对,发现编号为TDM90的突变体中,载体插入了PREDICTED:Solanum tuberosum trans-resveratrol di-O-methyltransferase-like(LOC102590422),m RNA马铃薯反式白藜芦醇基因中。试验说明了PCR-walking法扩增马铃薯侧翼序列是可行的。且载体插入马铃薯反式白藜芦醇基因中对马铃薯白藜芦醇基因的表达有何影响,有进一步研究下去的重要意义。
In this study, the double haploid potato DM mutants as the material, using PCR-walking method to amplify the flanking sequence of the mutant. In the experiment, 60 validated positive bands were amplified after amplifying the samples from 60 positive mutants. After the gel was recovered by PCR, two samples were sequenced without signal. The other 26 samples were sequenced, compared with the vector sequence and the potato genome sequence by BLASTN, and 6 samples were inserted into the chromosomes 1, 3 or 7, respectively . By further comparison, we found that the vector was inserted with PREDICTED: Solanum tuberosum trans-resveratrol di-O-methyltransferase-like (LOC102590422), m RNA potato trans-resveratrol gene, in the mutant numbered TDM90. Experiments show that PCR-walking method is feasible to amplify potato flanking sequences. Furthermore, the insertion of vector into the trans-resveratrol gene of potato affects the expression of potato resveratrol gene, which has important significance for further study.