摘要
从褐飞虱取食48h后的水稻幼苗中提取总RNA并纯化出mRNA,经反转录合成第一、二链cDNA,去除小于400bp的cDNA片断后,连接EcoRI人工接头,与去磷酸化的噬菌体长短臂连接,体外包装后感染,成功构建了含1.1×106重组子的褐飞虱诱导的水稻cDNA表达文库,重组子平均外源片断为1.5kb,扩增后的cDNA文库滴度为1.1×1011pfu/mL,适用于筛选低丰度mRNA的cDNA克隆。将已筛选得到的ESTBPHiw001为探针,在褐飞虱诱导的水稻cDNA文库中筛选得到1.4kb的全长锌指蛋白基因,命名为OsBi2。Northernblot分析说明此基因可能在水稻对褐飞虱的防卫反应中起作用。
Total RNA was exacted from rice shoots (B5) after 48 h fed by brown planthopper,mRNA was isolated and further purified.The first and second cDNA strands were synthesized by reverse transcription and inserted into phosphorylating Uni-ZAP XR vectors.The results revealed that cDNA library BPH induced contained 1.1×10~6 recombinants and the average length of inserts was about 1.5 kb.The tilter of the amplified cDNA library was 1.1×10^(11) pfu/mL.This library is suitable for screening genes in low abundance.Using the EST of zinc finger protein as probe to screen the cDNA library,a full length of dof zinc finger protein with 1.4 kb was isolated and named as OsBi2 .Northern blot analysis proved that OsBi2 was up-regulated in brown planthopper-fed rice.
出处
《武汉植物学研究》
CSCD
2004年第1期22-26,共5页
Journal of Wuhan Botanical Research
基金
国家自然科学基金资助项目(30170085)。
关键词
褐飞虱
CDNA文库
锌指蛋白
Nilaparvata lugens
cDNA library
Dof zinc finger protein
