从刺梨（Rosa roxburghii Tratt）中提取高质量RNA并构建抑制性消减杂交（SSH）文库。

Extraction of high quality of RNA and construction of a suppression subtractive hybridization (SSH) library from chestnut rose (Rosa roxburghii Tratt).

作者信息

Xu Qiang, Wen Xiaopeng, Tao Nengguo, Hu Zhiyong, Yue Hailin, Deng Xiuxin

机构信息

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, PR China.

出版信息

Biotechnol Lett. 2006 Apr;28(8):587-91. doi: 10.1007/s10529-006-0020-9.

DOI:10.1007/s10529-006-0020-9

PMID:16614897

Abstract

Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and beta-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 microg RNA g(-1) fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes.

摘要

刺梨（Rosa roxburghii Tratt）是一种珍稀水果作物，在中国水果和观赏植物开发方面具有重要的经济价值。由于刺梨中多酚、多糖和其他化合物含量较高，从刺梨中分离高质量RNA较为困难，但一种改良的不含苯酚的CTAB提取方法取得了令人满意的结果。提取缓冲液中使用了高浓度的聚乙烯吡咯烷酮（PVP，2%，w/v）、十六烷基三甲基溴化铵（CTAB，2%，w/v）和β-巯基乙醇（4%，v/v）以提高RNA质量。平均产量约为200微克RNA/克鲜叶。分离得到的RNA质量足以构建抑制性消减杂交（SSH）文库，该文库有助于分离多个病原体诱导的防御基因。