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为了探明类甜蛋白(thaumatin-like proteins,TLPs)基因与小麦抗叶锈病防御反应的相关性,本研究采用RT-PCR和电子克隆技术从叶锈菌侵染的TcLr19小麦叶片中获得了一个类甜蛋白基因,命名为TaLr19TLP1(GeneBank登录号:KJ764822)。生物信息学分析表明该基因包含一个长516 bp的开放阅读框(open reading frame,ORF),不含内含子,编码171个氨基酸残基,具有一个甜蛋白保守结构域(thaumatin,THN),氨基酸序列与其他物种类甜蛋白序列具有较高同源性。实时荧光定量PCR分析明确该基因表达受叶锈菌、脱落酸和水杨酸的诱导。接种叶锈菌后不同时间点,TaLr19TLP1基因在非亲和组合中表达早于亲和组合,且表达量明显高于亲和组合。另外,该基因表达具有组织特异性,在茎部表达量高于叶部和根部。Western杂交分析表明,TaLr19TLP1蛋白与小麦叶锈菌诱导具有相关性,与基因水平表达模式相一致。以上研究结果说明TaLr19TLP1基因参与小麦TcLr19小麦抗叶锈防御反应。
In order to find out the correlation between thapsin-like proteins (TLPs) genes and defensive responses to wheat leaf rust, we obtained one of TcLr19 wheat leaves infected with leaf rust by RT-PCR and electronic cloning techniques The thaumatin gene, designated TaLr19TLP1 (GeneBank Accession Number: KJ764822). Bioinformatics analysis showed that the gene contained a 516bp open reading frame (ORF) without introns, which encoded 171 amino acid residues and had a thaumatin (THN) Amino acid sequence and other species of sweet protein sequence has high homology. Real-time PCR analysis revealed that the gene expression was induced by leaf rust, abscisic acid and salicylic acid. TaLr19TLP1 gene was expressed earlier than the affinity combination in non-affinity combinations at different time points after inoculating the leaf rust, and the expression level was significantly higher than that of the affinity combination. In addition, the gene expression was tissue-specific and higher in the stem than in the leaf and root. Western blot analysis showed that the TaLr19TLP1 protein was associated with the induction of Puccinia triticina and was consistent with the gene expression pattern. The above results show that the TaLr19TLP1 gene is involved in the anti-leaf rust defense response of wheat TcLr19.