›› 2009, Vol. 52 ›› Issue (10): 1059-1067.

• 研究论文 •    下一篇

利用抑制差减杂交技术分离受水稻抗性调控的褐飞虱基因

杨之帆,陈永勤,李春华,蒋思婧   

  1. (湖北大学生命科学学院, 武汉 430062)
  • 出版日期:2009-10-20 发布日期:2009-10-20
  • 通讯作者: 杨之帆

Application of suppression subtractive hybridization (SSH) to clone differentially expressed genes in Nilaparvata lugens (Homoptera: Delphacidae) responding to rice resistance

  • Online:2009-10-20 Published:2009-10-20

摘要: 为分离受水稻抗性调控的褐飞虱Nilaparvata lugens基因, 以取食感虫水稻台中1号和高抗水稻B5的2叶1芯秧苗24 h的褐飞虱4龄若虫为起始材料, 采用抑制差减杂交技术构建了两个群体间的正反向差减cDNA文库。通过斑点杂交从差减文库中筛选代表受水稻抗性调控的基因的cDNA克隆, 进行测序和功能分析, 挑选具功能的基因进行Northern杂交验证。结果表明, 通过斑点杂交筛选到的98个阳性克隆代表92个互不重复的单基因, 其中25个与动物的已知蛋白基因存在较高的同源性。Northern杂交表明, 这25个基因有11个表达上调, 8个表达下调, 提示它们可能在褐飞虱适应抗性水稻过程中发挥了重要作用。本研究结果为克隆上述新基因的全长cDNA序列及进一步研究其在褐飞虱与水稻互作中的功能奠定了基础。

关键词: 褐飞虱, 水稻品种抗性, 抑制差减杂交(SSH), 斑点杂交, 差异表达基因

Abstract: To isolate the differentially expressed genes in brown planthopper Nilaparvata lugens (Homoptera: Delphacidae) responding to host rice resistance, suppression subtractive hybridization (SSH) was carried out using the 4th instar larvae of brown planthopper feeding on the susceptible rice Taichung Native 1 (TN1) and the highly resistant rice B5 seedlings at the 3rd leaf stage for 24 h. A forward and a reverse subtracted cDNA libraries specific to host rice resistance were constructed, followed by differential screening by dot blot hybridization. The clones representing the regulated genes were sequenced and evaluated by nucleotide blast analysis in the GenBank. Expression of the clones highly homologous to the known genes in the database was further verified by Northern blot hybridization. The results showed that 92 out of 98 clones were identified as non-redundant expressed sequence tags (ESTs) by dot blot analysis, among which 25 ESTs had high homology (E value<0.01) to known genes in the GenBank. Northern blot hybridization analysis demonstrated that 11 of the 25 genes were induced and 8 were repressed by rice resistance, suggesting that these genes may play an important role in response of brown planthopper to resistant rice. The data obtained in this work will help to clone the full-length cDNA sequences of the novel genes and reveal their functions involved in rice resistance.

Key words: Brown planthopper (Nilaparvata lugens), host rice resistance, suppression subtractive hybridization (SSH), dot blot hybridization, differentially expressed gene