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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。
In order to screen for the genes induced by soymilk in oriental goat, cDNAs of oriental goat beans treated with 250 mmol / L NaCl were used as experimental group. The stimulated subtractive hybridization (SSH) A part of positive clones were analyzed by ESTs. The recombination rate of the subtractive library clone was 92%, most of the inserted fragments concentrated in the range of 0.2 ~ 1kb. A total of 500 clones were randomly selected for sequencing and homology analysis. 258 clean ESTs were obtained. After clustering, splicing and removing redundant sequences, a total of 132 Unigenes were obtained, including 32 contigs and 100 singlets. The margin is 24%. Predict and classify them, and get a lot of related genes involved in signal transduction, transcriptional regulation, infiltration and metabolism regulation, body defense and light action. Randomly selected non-repetitive four differentially expressed sequences designed primers to semi-quantitative PCR method to verify the abatement efficiency, the results showed that induction group was significantly higher than the non-induction group, indicating that the library has a higher quality, and the use of The technical means help to quickly discover new gene of oriental goat bean.