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该研究以美国南部重要的造纸工业用材树种火炬松为材料 ,利用Transgenomic公司最近推出的Wave DNA片段分析系统 ,快速检测肉桂醇脱氢酶 (CAD)基因在个体间的单核苷酸多态性 ,获得了令人满意的结果 .首先利用Oligo5 .1软件对火炬松CAD基因全序列分区域进行引物设计 ,当设定每一区域能扩增出 10 0～ 2 0 0bp产物时 ,共在 8个碱基区域 (S1～S8)分别设计出了最佳正向引物和反向引物 ;以来自火炬松 12株优树自由授粉种子的胚乳中提取的DNA为材料 ,对CAD基因 8个碱基区域分别进行PCR扩增 ,有 5个碱基区域 (S3 ,S5 ,S6 ,S7,S8)得到了唯一的并且与预期碱基长度基本一致的PCR扩增产物 ,对其余 3个碱基区域进一步进行FailSafeTM PCR扩增 ,它们均在某些PCR缓冲液中得到了唯一的扩增产物 ;利用Wave DNA片段分析系统 ,对 6个碱基区域 (S2 ,S3,S5 ,S6 ,S7,S8)进行单核苷酸多态性检测 ,结果表明来自优树 2 2的种子与其它 11株优树的种子之间存在碱基序列变异 ,其中在S5 ,S6区域检测到了较大的单核苷酸多态性 ,这两个区域分别位于CAD基因的第 2和第 3内含子 (Intron2 ,Intron3) .
In this study, we use the recently introduced Wave DNA fragment analysis system from Transgenomic to rapidly detect single nucleotide polymorphisms in the cinnamyl alcohol dehydrogenase (CAD) gene among individual individuals in the United States, an important material for the paper industry in the United States. , Obtained satisfactory results.Firstly, Oligo5.1 software was used to design primers for the entire sequence of flax pine cDNA sequences. When setting 10 ~ 200 bp products in each region, a total of 8 A base region (S1 ~ S8) were designed the best forward primer and reverse primer respectively. The DNA extracted from the endosperm of 12 seeds of Pinus elliottii pollinated freely was used as material, The PCR products were amplified by PCR. Five of the 5 base regions (S3, S5, S6, S7, S8) obtained the only PCR amplification products that were consistent with the expected base length, and the other 3 base regions FailSafe PCR amplification was performed, and they all gave the only amplification products in some PCR buffers. The 6 base regions (S2, S3, S5, S6, S7, S8) were subjected to a Wave DNA fragment analysis system Single nucleotide polymorphism detection, the results show that from There was a base sequence variation between the seeds of tree 22 and the seeds of the other 11 superior trees, of which large single nucleotide polymorphisms were detected in S5 and S6 regions, which were located in the 2 and the third intron (Intron2, Intron3).