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N⁶-甲基腺苷（m⁶A）作为真核生物RNA中最丰富的表观转录修饰类型，通过动态的“写入-擦除-阅读”机制调控植物生长发育及逆境响应。ECT家族蛋白作为m⁶A阅读结合蛋白，通过识别m⁶A修饰位点参与RNA代谢调控。为明晰小麦TaECT3作为m⁶A结合蛋白响应逆境胁迫的机理，从小麦材料Fielder中克隆到TaECT3基因CDS全长，对其进行生物信息学分析。结果表明,TaECT3基因的编码区（CDS）长度为1 854 bp，编码617个氨基酸，其编码蛋白含有一个ECT家族蛋白典型的YTH保守结构域。TaECT3蛋白分子量为66.93 kDa，等电点为6.48，是亲水性稳定蛋白。其二级结构以无规则卷曲（79.74%）和α-螺旋（13.45%）为主，三级结构模拟显示该蛋白具有m⁶A结合口袋。经系统进化分析，小麦TaECT3与大麦HvECT3、高粱SbECT3和短柄草BdDF3A之间有较近的亲缘关系，暗示它们之间可能功能保守。EMSA试验显示TaECT3重组蛋白特异性结合m⁶A修饰RNA；亚细胞定位其可能位于细胞膜； qRT-PCR分析表明，高温（37 ℃，6 h）和PEG8000模拟干旱（15%，6 h）处理后，小麦Fielder中TaECT3的表达显著上调和下调。以上结果说明小麦TaECT3作为m⁶A结合蛋白可能参与高温与干旱胁迫响应。

英文摘要:

N⁶-methyladenosine(m⁶A), the most abundant epitranscriptomic modification in eukaryotic RNA, regulates plant growth, development, and stress responses through a dynamic “writer-eraser-reader” regulatory mechanism. Among these regulators, ECT family proteins act as m⁶A reader by recognizing m⁶A-modified sites to participate in RNA metabolism regulation. To investigate whether TaECT3 acts as an m⁶A reader protein in stress responses, this study cloned the coding sequence(CDS) of TaECT3 gene from a wheat cultivar Fielder and conducted comprehensive bioinformatics analyses. Results revealed that the CDS of TaECT3 spans 1 854 bp, encoding a 617-amino acid protein containing the characteristic YTH domain conserved in ECT family members. The TaECT3 protein exhibits a molecular weight of 66.93 kDa and a predicted isoelectric point of 6.48, demonstrating intrinsic hydrophilicity and thermodynamic stability. Secondary structure prediction revealed a predominance of random coils(79.74%) and α-helices(13.45%), while tertiary structure modeling confirmed the presence of a typical m⁶A-binding pocket. Phylogenetic analysis revealed that TaECT3 exhibits close evolutionary relationship to the ECT proteins of HvECT3, SbECT3, and BdDF3A from barley, sorghum, and brachypodium, respectively. Electrophoretic mobility shift assay(EMSA) confirmed that recombinant TaECT3 specifically binds m⁶A-modified RNA. Subcellular localization analysis suggested its potential membrane localization. qRT-PCR revealed significant regulation of TaECT3 expression in wheat cultivar Fielder under high-temperature(37 ℃, 6 h) and PEG8000-simulated drought(15%, 6 h) stress conditions, compared to the control. This study demonstrates TaECT3 functions as an m⁶A reader and participates in response to heat and drought stresses in wheat.

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