小麦抗白粉病相关基因GST克隆与表达

Cloning and Expression of Glutathione-S-transferase(GST) Gene Related to Powdery Mildew of Wheat

以从小麦抗白粉病相关基因差异表达分析中获得的EST-3(Genbank序列号EX567360)为标签,采用电子克隆的方法对其进行延伸,并对电子克隆结果进行半定量RT-PCR验证,最后对白粉菌不同侵染时间进行了表达分析。经RT-PCR扩增,EST-3表达的带型变化趋势与其在抑制性消减杂交SSH-cDNA的差异显示情况一致,且RT-PCR获得的序列与电子克隆的序列一致性达98%。生物信息学分析表明,该序列是由875bp核苷酸组成的,具有完整的开放阅读框架,编码蛋白为229个氨基酸,GenBank序列号JK841279,含有一个N端和C端谷胱甘肽硫转移酶结构域,该序列与小麦谷胱甘肽硫转移酶基因(GST)一致性较高,达97%。表达分析结果显示,白粉菌侵染24h表达受到抑制,48h开始表达,侵染72h表达最强,96h又开始下降,表明GST基因属于白粉菌诱导型相关基因,参与小麦对白粉病的应答反应。

The EST-3 related to the powdery mildew in the wheat was amplified by in silico cloning.And the cDNA sequence had been confirmed by RT-PCR.The result indicated that a new sequence with 875 bp length was cloned,which contained an integrated ORF,encoding 229 amino acids,GenBank accession number is JK841279.Based on domain structure,it has GST domain structure in N-terminal and C-terminal.The trend of EST-3 expression by RT-PCR was very identity to the expression by SSH-cDNA,and their sequences have 98% consistency.As showed that the result of in silico cloning was right.The amino acid sequence showed highly comparability with the glutathione-S-transferase（GST） from GenBank,and their sequences have 97% consistency.Expression analysis revealed that the expression of GST in wheat was inhibited after infection by powdery mildew for 24 h,then it began to express after infection for 48 h and increased gradually.It reached the highest level after infection for 72 h,and decreased after infection for 96 h.The result suggested that GST belong to the gene induced by powdery mildew,and it might be involved in powdery mildew response of wheat.