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为了建立适合南药益智的ITS-PCR体系来研究不同地理居群益智遗传多样性,本研究利用植物基因组试剂盒法提取益智基因组DNA为模板,采用单因素和正交试验对ITS-PCR过程中的关键影响因素进行优化,并对ITS-PCR产物进行测序鉴定。实验结果表明最佳ITS-PCR反应体系(25μL)为:Taq酶1.0 U,d NTPs 4 mmol/L,Mg2+0.5 mmol/L,引物2.0μmol/L,模板20 ng,10×PCR Buffer(不含Mg2+)2.5μL;最佳扩增程序为:94℃预变性2 min;94℃变性30 s,55℃退火30 s,72℃延伸1 min,共32个循环;最后72℃延伸5 min。采用该最佳体系对益智基因组DNA进行PCR扩增,获得扩增产物经单向测序获得了益智ITS部分序列。建立了稳定的ITS-PCR体系,为研究益智遗传多样性分析奠定了基础。
In order to establish ITS-PCR system suitable for Southern medicine puzzle to study the genetic diversity of different geographical populations, this study used genomic kit of plant genomic DNA to extract puzzle genomic DNA as template, using single factor and orthogonal test to ITS- The key influencing factors in PCR process were optimized, and the ITS-PCR products were sequenced and identified. The results showed that the optimum conditions of ITS-PCR reaction system (Taq polymerase 1.0 U, dNTPs 4 mmol / L, Mg2 + 0.5 mmol / L, primer 2.0 μmol / Containing Mg2 +) 2.5 μL. The optimal amplification procedure was as follows: pre-denaturation at 94 ° C for 2 min, denaturation at 94 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min for 32 cycles, and finally extension at 72 ° C for 5 min. The optimized system was used to amplify the genomic DNA of the puzzle, and the ITS sequence of the puzzle was obtained by one-way sequencing of the amplified product. The establishment of a stable ITS-PCR system laid the foundation for the study of genetic diversity of puzzle.