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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24h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。
In order to isolate the Nilaparvata lugens gene of brown planthopper (Nilaparvata lugens) which is regulated by rice resistance, the 4th instar nymphal of the brown planthopper, Nilaparvata lugens, which was fed on the 2-leaf seedlings of susceptible rice Taichung 1 and high resistant rice B5 for 24 h, A forward-reverse subtraction cDNA library was constructed between the two populations. The cDNA clone representing the gene under the resistance of rice was screened from the subtractive library by dot blot hybridization, sequenced and functionally analyzed, and the functional genes were selected for Northern blotting. The results showed that 98 positive clones screened by dot blot hybridization represented 92 non-repetitive single genes. Among them, 25 showed high homology with animal known protein genes. Northern blotting showed that 11 of these 25 genes were up-regulated and 8 were down-regulated, suggesting that they may play an important role in the adaptation of N. lugens to resistant rice. The results of this study lay the foundation for cloning the full-length cDNA sequence of the above novel gene and further studying its function in the interaction between N. lugens and rice.