橡胶树转基因植株Southern杂交体系的优化  

Optimization of Digoxigenin Based Southern Blot for Transgenic Hevea brasiliensis Analysis

作　　者：李季[1] 鲁旭[1,2] 黄天带[1] 华玉伟[1] 黄华孙[1] 

机构地区：[1]中国热带农业科学院橡胶研究所农业部橡胶树生物学重点开放实验室,儋州571737 [2]海南大学农学院,海口570228

出　　处：《生物技术通报》2014年第8期76-81,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31200503);现代农业产业技术体系建设专项(NYCYTX-34)

摘　　要：以地高辛标记的Southern杂交技术已广泛应用于多个物种中。选择橡胶树为材料,就基因组DNA的提取、探针标记方法的选择以及具体的杂交操作过程等方面对该体系进行了优化。结果表明,至少40μg高质量的DNA在300μL的大酶切体系中,酶切12 h可获得良好效果;PCR法标记的探针杂交条带清晰、背景浅,其效率明显强于随机引物法标记的探针。本研究优化的体系信号强、背景浅和灵敏度高,为橡胶树Southern杂交鉴定分析提供参考。Southern blot technology based on the DIG-chemiluminescent detection has been widely used in numerous species. Using Hevea brasiliensis as materials, the DIG-labeled Southern blot analysis was improved through optimizing several key steps, including extraction of genome DNA, usage of DNA samples amount, digestion system, the comparison between PCR and random-primed labeled method and a serial of procedure in the process of the hybrid. The results showed that desirable digestion result could be achieved when at least 40μg DNA samples with high quality were enzymed in 300μL reaction system for 12 hours. The efficiency of probe labeled with the method of PCR was higher than that of probe labeled with random primer obviously, while the Southern blot result of PCR labeled was clear, and the background was lightly, which was conducive to read the copy number accurately. Finally, Southern blot with DIG-labeled was optimized, and good result with high sensitivity and low background was achieved.

关 键 词：橡胶树 转基因 SOUTHERN BLOT 地高辛 PCR法标记 

分 类 号：S794.1[农业科学—林木遗传育种]