普通小麦中一个类受体激酶基因的克隆、鉴定和表达分析

摘要

To investigate whether homologs of wheat(Triticum aestivum L.) LRK10 gene was expressed in powdery mildew-resistant lines after inoculation with Blumeria graminis f. sp. tritici, one degenerate primer for 5'-RACE was designed according to the 6th kinase subdomain of LRK10 and other plant kinases. 5'-RACE was performed with the template cDNA synthesized with RNA extracted from seedling leaves of a powdery mildew-resistant wheat line "99-2439" after inoculation with B. graminis. One 1 551 bp cDNA fragment representing a protein kinase gene was obtained (S 1125, GenBank accession number: AY584533). Subsequently, a 2 255-bp full-length cDNA clone with a complete encoding region (open reading frame, ORF) was obtained by RACE. The clone encodes a polypeptide consisting of637 amino acid residues. The result of homology search showed that it belongs to a receptor-like kinase gene family in wheat, which was named as wlrk (wheat leaf rust kinase) previously. Similar to LRK10, this protein kinase has five distinct domains: a hydrophobic signal sequence at the amino-terminus, a putative extracellular domain, a transmembrane domain, a highly charged sequence and a serine/threonine kinase domain at the carboxy-terminus, and thus it was named as TaLRK(Triticum aestivum LRK). The expression pattern of TaLRK at transcription level in leaves after B. graminis infection was investigated by semi-quantitative RT-PCR(semi-QRT-PCR), using wheat actin gene as a control.The result showed that the transcription of TaLRK was significantly enhanced by B. graminis infection. The expression pattern of TaLRK in different tissues showed that this new wheat RLK gene was expressed only in green parts of wheat. This study suggests that TaLRK may function in wheat powdery mildew resistance responses.%为研究抗白粉病小麦(Triticum aestivum L.)品系在小麦白粉病菌(Blumeria graminis f.sp.tritici)侵染后有无LRK10同源基因表达,依据小麦蛋白激酶LRK10和其它植物蛋白激酶第6亚结构域设计了一个5'-RACE兼并性引物.以接种小麦白粉病菌后的小麦抗白粉病品系"99-2439"幼苗叶片cDNA为模板进行5'-RACE扩增,获得了一个1551 bp长的蛋白激酶基因cDNA片段(S1125,GenBank登录号:AY584533).此后,通过RACE技术成功地获得了该基因的全长cDNA克隆.该克隆编码637个氨基酸组成的多肽.同源性查寻表明,该基因属于先前命名为wlrk(wheat leaf rust kinase)的小麦类受体蛋白激酶基因家族.与LRK10相似,这个新的小麦类受体蛋白激酶有5个明显的功能域:位于氨基端的疏水信号序列、推测的胞外结构域、跨膜域、高荷电序列和位于羧基端的丝氨酸/苏氨酸激酶域,因此被命名为TaLRK(Triticum aestivum LRK).以小麦肌动蛋白基因为对照,通过半定量反转录PCR(semi-QRT-PCR)技术对叶片中TaLRK基因在小麦白粉病菌接种后的转录水平表达谱进行了研究.结果表明,小麦白粉病菌的侵染使TaLRK基因的转录显著增强.组织特异性表达分析证明,这一基因仅在小麦的绿色部分表达.研究结果提示TaLRK可能参与了小麦的抗白粉病反应.