珍珠黄杨ITS-PCR体系的建立与优化

Establishment and Optimization for ITS-PCR System of Buxus sinica var. parvifolia

本研究利用改良CTAB法从珍珠黄杨叶片中提取基因组DNA作为模板,在TaqDNA聚合酶量不变的基础上,利用正交设计L9(34)对4个因素(模板DNA、Mg2+、dNTP和引物)在3个水平上对珍珠黄杨ITS-PCR反应体系进行优化。实验结果表明,在总体积50μL的反应体系中,建立了最佳ITS-PCR扩增条件:Mg2+浓度2.0mmol/L、引物浓度0.3μmol/L、dNTP浓度0.3mmol/L、DNA模板浓度240ng/50μL、TaqDNA聚合酶的用量1.75U/50μL和退火温度56℃,该优化体系保证了珍珠黄杨ITS-PCR产物的纯度和质量要求。珍珠黄杨ITS片段克隆测序后获得的序列长度为642bp,其系统学信息将为珍珠黄杨的起源进化提供有力的分子水平证据。

In this research, we extracted the genomic DNA from the Buxus sinica var. parvifolia leaves by using the modified CTAB approach and then used as the template DNA. Base on the invariable quantity of TaqDNA, we att empted to establish and optimize ITS-PCR amplification system for Buxus sinica var. parvifolia by employing L9（34） orthogonal design with four factors, i.e. template DNA, Mg^2＋, dNTPs and primer. The result showed that we have established a ITS-PCR system most suitable for Buxus sinica var. parvifolia, which came out total volume 50μL reaction system, was as follows： 2.0 mmol/L Mg^2＋, 0.3 μmol/L primer, 0.3 mmol/L dNTP, 240 ng/50μL DNA template, 1.75 U/50 μL TaqDNA polymerase and the anneal temperature with 56℃.This optimized ITS-PCR system might ensure the purity and quality oflTS-PCR production. In this study, the length of the ITS fiagment ofBuxus sinica var. parvifolia was 642 bp by cloning PCR production and sequencing, and its phylogenetic information might facilitate to provide the direct molecular evidence to clarify its origion and evolution for Buxus sinica var. parvifolia.