摘要
为筛选东方山羊豆盐诱导差异性基因,以250 mmol/L NaCl处理的东方山羊豆cDNA为实验组,未经诱导刺激的为驱动组,利用抑制性消减杂交技术(SSH)构建消减文库并对其部分阳性克隆进行了ESTs序列分析。该消减文库克隆的重组率为92%,插入片段大部分集中在0.2～1kb之间。随机挑取500个克隆进行测序及同源性分析,获得258个cleanESTs,经聚类、拼接,去除冗余序列,共获得132个Unigene,其中含有32个contigs和100个singlets,该文库的冗余度为24%。对其进行功能预测及分类,得到大量参与信号转导、转录调控、渗透和代谢调节、机体防御以及光作用等过程的相关基因。随机选择非重复的4个差异表达的序列设计引物,以半定量PCR方法验证其消减效率,结果显示,诱导组的表达量显著高于非诱导组,表明该文库有较高质量,且所采用的技术手段有助于快速发现东方山羊豆新功能基因。
Subtractive cDNA library was constructed by suppression subtractive hybridization（SSH） using cDNA from Galega orientalis treated with（tester） or without（driver） 250 mmol/L NaCl.The library contained 92% recombinants with insert sizes ranging from 0.2 to 1 kb.Five hundred positive clones were randomly sequenced and analyzed,and 258 cleanESTs were obtained.By the aid of cluster and phrap software,132 Unigene were identified which consisted of 32 contigs and 100 singlets.The redundancy rate of the library was 24%.Function prediction analysis classified them into several sections,such as signal transduction,transcription regulation,osmotic and metabolic regulation,host defense,and the photosynthesis.The subtraction efficiency of the SSH cDNA library was confirmed by randomly testing 4 independent sequences using semi-quantitative RT-PCR,of which differential expression was detected.These results suggest that SSH is an effective technique to identify novel genes in Galega orientalis.
出处
《植物遗传资源学报》
CAS
CSCD
北大核心
2010年第6期777-783,共7页
Journal of Plant Genetic Resources
基金
"十一五"科技支撑(2008BADB3B01)
现代农业产业技术体系建设专项资金
关键词
东方山羊豆
盐诱导
抑制消减杂交
ESTS
RT-PCR
Galega orientalis
Salt treatment
Suppression subtractive hybridization（SSH）
Expressed sequence tags（ESTs）
RT-PCR
