| 周彩霞,李文砚,卢美瑛,周之珞,颜桢灵,韦雪英,卓福昌,韦优,周婧.蛋黄果SCoT-PCR反应体系的建立与优化[J].中国南方果树,2024,53(2): |
| 蛋黄果SCoT-PCR反应体系的建立与优化 |
| Establishment and Optimization of SCoT-PCR System in Lucuma nervosa A.DC |
| 投稿时间：2023-01-01 修订日期：2023-02-15 |
| DOI： |
| 中文关键词: 蛋黄果 SCoT-PCR 正交试验 |
| 英文关键词:Lucuma nervosa A.DC SCoT-PCR Orthogonal experiment |
| 基金项目:广西农业科学院科技发展基金项目（桂农科 2021JM131）；崇左市科技计划项目（崇科攻2021 ZC20）；广西壮族自治区农业科学院基本科研业务专项资助项目（桂农科2021YT165）；广西农业科学院基本科研项目（桂农科 2023YM36） |
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| 中文摘要: |
| 为建立和优化蛋黄果SCoT-PCR反应体系，本研究先以蛋黄果“仙桃1号”为试材，SCoT1为引物，采用L16(45)正交试验，对影响蛋黄果SCoT-PCR反应的DNA、Mg2+、dNTPs、引物以及Taq聚合酶等因素进行优化。正交试验结果表明，不同因素对蛋黄果SCoT-PCR反应体系的影响存在差异，但不显著，其中最大的影响因素是Taq 聚合酶的含量，其次是dNTPs、模板DNA、引物和Mg2+的浓度。最终确立20μL的蛋黄果SCoT-PCR最佳反应体为：模板DNA 80 ng，Mg2+浓度3 mmol·L-1，dNTPs 浓度0.25 mmol·L-1，引物浓度0.4 μmol·L-1，Taq聚合酶含量 2 U。选用3份地理位置相距较远的蛋黄果种质为模板，对优化的反应体系进行验证，结果表明，在该体系下，3份种质均能稳定扩增出清晰明亮、数目丰富且稳定性高的条带，说明优化的蛋黄果SCoT-PCR反应体系适用于蛋黄果SCoT分子标记。 |
| 英文摘要: |
| The study aimed to establish and optimize the SCoT-PCR reaction system of Lucuma nervosa A.DC. Extracting DNA from L. nervosa A.DC (Xiantao No. 1) as research material and SCoT1 as primer, the orthogonal experiment of L16(45) was applied to establish the SCoT-PCR reaction system and optimize the factors such as template DNA, Mg2+, dNTPs, primers and Taq DNA polymerases. Orthogonal results showed that the effects of various factors on the SCoT-PCR reaction system of L. nervosa A.DC were different, but not significant. The most important factor was the content of Taq DNA, followed by the concentration of dNTPs, template DNA, primers and Mg2+. The SCoT-PCR reaction system of L. nervosa A.DC was established and optimized (20μL), i.e.template DNA 80 ng, Mg2+ 3 mmol·L-1, dNTPs 0.25 mmol·L-1, primer 0.4 μmol·L-1, Taq DNA polymerases 2 U. And then three geographically distant(Guangxi, Yunnan, Panzhihua) germplasm resources were used as templates and SCoT1-SCoT10 as primers to validate the optimized reaction system. The experimental results showed that all three germplasms could amplify clear, bright, abundant and stable bands under this system. It indicated that the optimized SCoT-PCR reaction system was suitable for L. nervosa A.DC SCoT molecular marker. |
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