利用mRNA差别显示技术分离橡胶树死皮病相关cDNA

从中国热带农业科学院橡胶树早期预测试验地采集不同品系的橡胶树胶乳和树皮组织，分别提取纯化mRNA 合成cDNA第一链。通过4个简并锚定引物和10个随机引物进行PCR扩增，经6％变性聚丙烯酰胺凝胶电泳后发现了至少4个差异DNA片段只在健康树中表达而在死皮树中没有出现，这4个差异DNA片段分别命名为Hb1(725bp),Hb2,Hb3,Hb4。对Hb1进行了分离，纯化，经Northern杂交证实，Hb1在健康树胶乳中表达活跃，而在死皮树胶乳中表达受到强烈抑制，表明Hb1是与死皮病相关的cDNA片段，进一步将Hb1片段进行回收和克隆，并进行DNA序列分析，序列在GenBank中进行BLAST分析未发现相关同源片段，死皮病相关cDNA片段的获得，将分离全长死皮病相关基因，搞清该基因表达调控的机理提供条件，进而为从分子水平上阐明死皮病的致病机理打下了基础。

Isolation of the TPD cDNA in Hevea brasiliensis by mRNA Differential Display

RNAs of the barks and latexes from different rubber trees(Hevea brasiliensis)cultivated in the experimental fields of Chinese Academy of Tropical Agricultural Sciences were isolated for mRNA differential display analysis.Screened by4OligodT 12 (C,A,G)GC and ten26-oligonucleotide arbi trary primers,differential cDNA fragments-Hb 1(725bp),Hb 2,Hb 3and Hb 4,only appeared in the barks and latexes of healthy trees of three clones of Hevea brasiliensis(RRIM600,RY13-28,RY1-20).Hb 1was isolated and purified.It was confirmed by RNA dot hybridization that Hb 1was ex pressed in latexes of rubber trees,but its expression decreased significantly in latexes of TPD(tap ping panel dryness)trees,which showed that Hb 1was correlated with TPD.Hb 1was cloned and se quenced.After searched in Genbank,no similar sequences were reported.This cDNA may provide basis for obtaining integral genes associated with TPD and studying the molecular mechanism underlying TPD.