小麦重要自噬相关基因ATG18的鉴定和表达分析

Identification of Wheat Key Autophagy-Related Factor ATG18 and Profiling of Their Expression Under Biotic and Abiotic Stresses

【目的】克隆小麦重要自噬相关基因ATG18,分析其编码产物的序列特征和高级结构,了解其在生物、非生物胁迫及激素处理条件下的表达模式,阐明其生物学功能。【方法】利用EST拼接和RT-PCR方法从白粉菌侵染的小麦叶片中克隆ATG18 cDNA序列,利用生物信息学方法进行基因的外显子-内含子结构分析、编码蛋白的结构域和保守氨基酸预测、高级结构分析和物种间同源蛋白的进化分析。采用实时荧光定量PCR方法研究基因表达对白粉菌侵染和外源激素处理的响应模式及对高盐、干旱、低温黑暗和缺氮培养等逆境胁迫处理的响应模式。【结果】获得了4个小麦ATG18家族成员(TaATG18a、TaATG18b、TaATG18c和TaATG18d)cDNA。4个基因高度相似,均含有1 158 bp开放阅读框(ORF),编码385个氨基酸的蛋白质。4个基因的编码蛋白在一级结构上均含有典型的WD-40结构域、磷脂酰肌醇-3-磷酸(PI3P)结合基序和保守的ATG2结合位点氨基酸残基。4个基因均具有2种转录后的可变剪接方式,2种剪接产物mRNA分别编码完整的有功能蛋白和N端截短导致结构域和功能位点缺失的无功能蛋白。TaATG18a蛋白在高级结构上折叠成与其他WD-40蛋白类似的β-推进器样构象,PI3P结合基序位于推进器第五叶片的折叠4和第五、六叶片的连接部分,ATG2结合位点位于连接第二叶片和第三叶片的loop上。TaATG18s能够被白粉菌侵染诱导表达,但具体的诱导表达模式在抗、感白粉病反应之间存在明显差异。在广谱抗白粉病基因Pm21和小种专一性抗白粉病基因Pm3f介导的抗病反应中,TaATG18s均呈现接种白粉菌后0—36 h期间的2次诱导表达模式,2次诱导表达时间与白粉菌侵染进程密切相关。在中感材料扬麦158遗传背景上的感病反应中,TaATG18s尽管也呈现2次诱导表达,但第一次诱导表达持续时间短且强度低于含Pm21的近等基因系上抗病反应中的表现,相反第二次诱导表达强度高于抗病反应中的表现。高感材料Chancellor遗传背景上的TaATG18s响应白粉菌侵染的表达波动较小。外源乙烯或SA处理对TaATG18s表达的调控作用在抗、感白粉病材料上明显不同,在感病材料上表现激活作用,而在抗病材料上表现抑制作用。激素处理对TaATG18s表达的调控不仅作用于转录水平,还作用于转录后的mRNA剪接环节。高盐、干旱、低温黑暗和缺氮培养等逆境处理也能够上调TaATG18s的表达。【结论】推测鉴定的4个TaATG18s编码蛋白具有通过结合PI3P定位于自噬膜表面和通过形成ATG18-ATG2复合物参与小麦自噬过程的功能。TaATG18s及其参与的自噬过程与小麦对白粉菌侵染的免疫反应密切相关,也与小麦响应非生物逆境胁迫环境相关。抗、感材料TaATG18s对同种激素信号的不同响应模式可能是导致抗、感白粉病表型差异的原因之一。

[Objective] One objective of this study is to identify wheat homologues of autophagy-related gene ATG18, to analyze the sequence characteristics and 3-D structures of their encoding proteins. The other objective is to profile their expression patterns under biotic and abiotic stresses and under phytohormone applications, thus laying a foundation for elucidating their biological functions. [Method] EST assembly and RT-PCR were carried out to clone ATG18 cDNA sequences from RNA pool generated from the powdery mildew （Pm） causal fungus Blumeria graminis f.sp. tritici （Bgt）-challenged wheat leaves. Bioinformatics tools were employed to analyze gene exon-intron models, to predict protein domains, conserved amino acids and 3-D structures and to analyze the phylogenetic relationships among plant orthologues. Real-time fluorescence quantitative PCR was used to investigate gene expression patterns in responses to Bgt infection, treatments of various phytohormones or exposure to conditions of high salinity, drought, low temperature and darkness or nitrogen deficiency. [Result] Four members （TaATG18a, 18b, 18c and 18d） of the wheat ATG18 family were identified in this study. They are of great similarity and each has an ORF of 1 158 bp encoding a protein of 385 amino acids. The deduced protein sequences of TaATG18s consist of two classic WD-40 domains, one phosphatidylinositol 3-phosphate （PI3P） binding motif and the conserved amino acids at the ATG2-binding site. The four TaATG18s all have two kinds of alternatively spliced mRNA isoforms, respectively encoding the complete functional protein form and the N-terminal truncated non-functional form missing the WD-40 domain and the conserved functional residues. TaATG18a was predicted to fold into a six-blade [3-propeller-like structure similar to other WD-40 proteins. The PI3P binding motif is located at the strand 4 of blade 5 and the loop connecting blades 5 and 6, and the ATG2 binding site is located at the loop connecting blades 2 and 3. Expression of TaATG18s can be upregulated by biotic stress of Bgt infection, however, the detailed regulation patterns by Bgt infection differed between wheat-Bgt compatible and incompatible reactions. In the two incompatible reactions triggered by the broad-spectrum Pm resistance gene Pro21 and the isolate-specific Pm resistance gene Pm3f two times of transcripts accumulation of TaATGISs were observed during 0-36 hours after inoculation of Bgt, which are closely related to the progression of Bgt infection. Although TaATG18s of the moderately susceptible line Yangmai 158 also showed a bimodal Bgt-induced expression profile, the first time of transcripts accumulation had a shorter duration and a lower peak than that observed in the incompatible reaction on Pm21-carrying Yangmai 158 isogenic line. Expression of TaATG18s of the highly susceptible line Chancellor fluctuated little in response to Bgt infection. Differences were also found between the resistant line and the susceptible line as to their TaATG18s expression responding to the same exogenously applied phytohormone salicylic acid （SA） or ethylene, which showed negative effects on the resistance line but positive effects on the susceptible line. Phytohormones regulated the expression of TaATG18s not only at the transcription level, but also at the process of mRNA splicing. Expression of TaATG18s can also be upregulated by abiotic stresses of high salinity, drought, low temperature and darkness or nitrogen deficiency. [Conclusion] Proteins encoded by TaATG18s identified here probably participate in the wheat autophagy process through their Pl3P-mediated autophagic membrane localization and the formation of ATG18-ATG2 complex. TaATG 18s and their involved autophagy are implicated in the wheat immune response to Bgt infection and in the wheat responses to various abiotic stresses. Different expression responses of TaATG18s to one kind of phytohormone SA or ethylene in the resistant and the susceptible lines may partially account for the different phenotype outcomes of Pm resistance or susceptibility.