基于改进的Southern杂交法检测古树木端粒长度的研究  

Telomere Length Detection of Ancient Tree Based on a Modified Southern Hybridization Assay Method

作　　者：门璟煜 郑广顺[1] 孙丽春 孙玉萍[1] 杨颖 于婷乔[1] 刘海洋 陈玉珍[1] 卢存福[1] Men Jingyu;Zheng Guangshun;Sun Lichun;Sun Yuping;Yang Ying;Yu Tingqiao;Liu Haiyang;Chen Yuzhen;Lu Cunfu(College of Biological Sciences and Biotechnology/ National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing 100083)

机构地区：[1]北京林业大学生物科学与技术学院/林木育种国家工程实验室,北京100083

出　　处：《中国农学通报》2018年第13期24-30,共7页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金项目"沙冬青脱水素基因家族适应性进化研究"(31270737);高等学校学科创新引智计划项目"林木发育及逆境适应性的分子机制"(B13007);长江学者和创新团队发展计划项目"林木发育遗传调控与抗逆分子机制的研究"(IRT13047);北京市自然科学基金项目"常绿抗逆灌木沙冬青Am CIP基因功能研究"(6112016)

摘　　要：研究旨在利用DNA印迹法测量端粒长度从而预测植物的年龄,但木本植物体内富含黄酮类、酚类、多糖、萜类等次生代谢物质,严重影响了提取的基因组DNA产量和质量,以及后续的酶切及杂交实验结果。笔者以北京颐和园5种古树为实验材料,在传统CTAB法提取DNA基础上,不同树种分别添加2%~6%PVP,可有效去除次生代谢物质对DNA的干扰,可被限制性内切酶HinfⅠ酶切完全。同时,对DNA印迹法的其他实验参数如DNA量、曝光时间进行了优化,并进行了3种不同年龄古树端粒长度测定,结果显示古树的平均端粒长度要高于幼树,本研究建立的实验方法为多年生林木特别是古树端粒长度、树龄检测与寿命预测等提供了参考。Southern hybridization assay is reliable for detecting telomere length to predict plant age. However,secondary metabolites such as flavonoids, phenols, polysaccharides and terpenoids are rich in woody plants,which seriously affect the yield and quality of the extracted genomic DNA as well as subsequent results of enzyme digestion and hybridization. In this research, we modified the method of telomere length measuring by using 5 kinds of ancient trees collected from the Summer Palace in Beijing, on the basis of the conventional CTAB method, 2%-6% PVP（polyvinylpyrrolidone） was added to the DNA extracts of different tree species,which effectively removed the interference of secondary metabolites, therefore DNA could be digested completely by restriction enzyme HinfⅠ. At the same time, we also adjusted DNA content and the exposure time for the Southern hybridization assay, and detected telomere lengths of 3 kinds of trees. The results show that the average telomere length of ancient trees is longer than that of young trees. The improved Southern blotting assay is constructed and lays a foundation for predicting tree age related to telomere length.

关 键 词：古树 端粒长度 SOUTHERN杂交 PVP 

分 类 号：S79[农业科学—林木遗传育种]