摘要
采用抑制消减杂交方法,以褐飞虱取食32h的水稻幼苗及未受褐飞虱取食的水稻幼苗为作为对比材料构建了消减cDNA文库,以分离水稻幼苗中褐飞虱应答基因。随机从消减cDNA文库中挑选16个白色菌落提取质粒,进行PCR扩增,发现插入片段的长度位于100～900bp之间。以在受褐飞虱取食的水稻幼苗中特异表达的基因(BpHi008A)为探针,通过斑点印迹分析发现在抑制消减后的cDNA池中,目的基因得到有效富集。利用反向总RNA斑点印迹分析和Northern杂交验证,从消减cDNA文库中筛选到了25个基因受褐飞虱取食的诱导。其中有17个克隆与编码已知功能蛋白的基因有显著的同源性,它们分别参与蛋白质的折叠与降解、蛋白质与蛋白质的相互作用及信号传递、脂类代谢、胁迫反应、物质运输和细胞生长等。总体上,参与胁迫反应和衰老的基因在褐飞虱取食后表达增强。
Suppression subtractive cDNA library was constructed by using rice seedlings exposed to BPH for 32 h and control plants as materials. Sixteen white bacterial colonies were randomly picked up from subtracted cDNA library,and analyzed by PCR method. The result showed they contained 100～900 bp inserts. To analyze the efficiency of subtraction,dot blot was conducted by using BpHi008A as probe,which was expressed only in BPH-infested rice seedlings. The result demonstrated that the differential expression genes were enriched in subtracted cDNA pool. 25 BPH-responsive genes in rice were isolated from the library by reverse total RNA dot blot analysis and confirmed by Northern blot analysis. Sequence similarity searches allowed putative functions to be assigned to 17 transcripts. These genes were placed into several functional categories including cell growth photosynthesis,macromolecule degradation,signaling,and a group responding to stress or invasion of pathogens. Overall,transcripts involved in senescence and stress in plants were induced.
出处
《广西植物》
CAS
CSCD
北大核心
2005年第3期237-240,i006,共5页
Guihaia
基金
国家自然科学基金资助项目(30170085)
关键词
抑制消减杂交
褐飞虱
水稻
suppression subtractive hybridization
brown planthopper
rice
