| 桉树EST—PCR产物测序方案的优化 |
| |
| 引用本文： | 张晓红,李发根,王宇,徐立安,李梅,甘四明.桉树EST—PCR产物测序方案的优化[J].广西农业生物科学,2009(3):535-543. |
| |
| 作者姓名： | 张晓红 李发根 王宇 徐立安 李梅 甘四明 |
| |
| 作者单位： | 中国林业科学研究院热带林业研究所;南京林业大学森林资源与环境学院; |
| |
| 基金项目： | 国家863计划(2006AA100109);;国家自然科学基金(30671703);;中央级公益性科研院所基本科研业务费专项(RITF2007-6)共同资助 |
| |
| 摘 要： | EST—PCR产物测序是EST标记开发的重要步骤。本研究利用乙醇-醋酸钠法、清洁试剂盒、虾碱磷酸酶（SAP）-核酸外切酶Ⅰ（ExoⅠ）法和凝胶回收试剂盒4种方法，对6个桉树EST-PCR产物的纯化效果和测序结果进行了比较，推荐乙醇-醋酸钠法为首选的纯化方法；利用测序试剂盒Bigdye Terminator V3．1，对1／2、1／4、1／8、1／16和1／32标准用量（8．0μL）的测序结果的分析表明，1／16和1／32用量仍可有效测序，均可作为优先的测序试剂用量。同时，也发现EST-PCR扩增谱带单一、明亮的序列与设计的目标EST具有较好的同源性，开发SNP和Indel标记的潜力极大，但较弱的谱带则可能是非特异性扩增的产物。乙醇-醋酸钠法纯化和Bigdye Terminator1／16或1／32用量的测序方案，具有成本低和操作简便的优点，适合96孔板或384孔板大规模EST—PCR产物的测序。
|
| 关 键 词： | 桉树 表达序列标签 PCR产物纯化 重测序 |
An Optimized Protocol for Sequencing EST-PCR Products in Eucalyptus |
| |
| Zhang Xiaohong , Li Fagen Wang Yu , Xu Li'an Li Mei Gan Siming Research Institute of Tropical Forestry,Chinese Academy of Forestry,Guangzhou, College of Forest Resources , Environment,Nanjing Forestry University,Nanjing.An Optimized Protocol for Sequencing EST-PCR Products in Eucalyptus[J].Journal of Guangxi Agricultural and Biological Science,2009(3):535-543. |
| |
| Authors: | Zhang Xiaohong Li Fagen Wang Yu Xu Li'an Li Mei Gan Siming Research Institute of Tropical Forestry Chinese Academy of Forestry Guangzhou College of Forest Resources Environment Nanjing Forestry University Nanjing |
| |
| Institution: | Zhang Xiaohong 1,2 Li Fagen 1 Wang Yu 1,2 Xu Li'an 2 Li Mei 1 Gan Siming 1 1 Research Institute of Tropical Forestry,Chinese Academy of Forestry,Guangzhou,510520,2 College of Forest Resources , Environment,Nanjing Forestry University,Nanjing,210037 |
| |
| Abstract: | Sequencing of EST-PCR products is important for EST-based marker development. In this study, four procedures for purifying EST-PCR products, namely, ethanol-NaAc, cleanup kit, SAP （shrimp alkaline phosphatase）- Exo Ⅰ （exonuclease Ⅰ）, and gel extraction kit, were compared for their yield and effects on subsequent sequencing, from which ethanol-NaAc was selected as the sound choice. Further, five dosages of Bigdye Terminator V3.1, including 1/2, 1/4, 1/8, 1/16, and 1/32 of the manufacture＇s standard （0.8 μL）, were compared for their sequencing results, from which the last two were recommended in terms of reliability and cost control. In addition, the EST-PCR products, if bright and single in electrophoresis, were highly identical to the original target ESTs and had a great potential in developing SNP and Indel markers, whereas weak EST-PCR products might represent non-specific genomic region to the target EST sequences. The protocol optimized here is advantageous in low cost and technical simplicity and suitable for large-scale sequencing based on 96- or 384- well plates. |
| |
| Keywords: | Eucalyptus EST （expressed sequence tag） PCR product purification Re-sequencing |
| 本文献已被 CNKI 维普 等数据库收录！ |
|
