摘要
用Trizol试剂提取红莲型水稻杂种F1幼穗的总RNA,分离纯化mRNA后反转录并合成双链cDNA.经Sephrose CL-2B凝胶过滤柱层析后,收集大于400 bp以上的双链cDNA片段,将其连接到pTRG载体上,转化到XL1-Blue MRF′Kan感受态细胞后构建成红莲水稻幼穗细菌双杂交文库.经检测计算该原始文库容量为1.03×106pfu/mL,对随机挑取的100个克隆进行PCR鉴定显示重组率接近100%而且大于800 bp的插入片段达到97%.扩增后文库的容量达到1.05×1010pfu/mL.
The total RNA was extracted from the Young Panicle of F1 hybrid rice by Trizol reagent. The mRNA was purified from the total RNA by Poly-A Tract mRNA Isolation Kit and the double-stranded （ds） cDNA was synthesized after reverse transcription. The fragments longer than 400 bp in length were fractionated by Sephrose CL-2B Column, and then ligated into the pTRG vertor. The ligation product was transformed into XL1-Blue MRF Kan Library Pack Competent Cells to construct the bacterial two-hybrid system library. Analysis showed that the primary library contained 1.03 ×10^6 pfu/mL, and approximately 100% of the clones were positive recombinants and about 97 % of the cDNA fragments inserted were longer than 800 bp in length. The capacity of the amplified library was 1.05 ×10^10 pfu/mL.
出处
《武汉大学学报（理学版）》
CAS
CSCD
北大核心
2009年第3期365-369,共5页
Journal of Wuhan University:Natural Science Edition
基金
国家自然科学基金资助项目(30571144)
关键词
水稻
幼穗
细菌双杂交系统
文库构建
rice （Oryza sativa L. ）
young panicle
bacterial two-hybrid system
library construction
