Expansin (EXP) plays an important role in plant root formation. The EXP genes associated with chrysanthemum roots have not yet been reported. Here we isolated a root-specific EXP gene in chrysanthemum (Chrysanthemum morifolium), namely CmEXPA4. Bioinformatics analysis showed that CmEXPA4-encoded protein has a conserved DPPB (Double-Psi Beta-Barrel) domain in the N-terminal with a series of Cys residues, an HFD (His-Phe-Asp) motif in the central region, and a pollen allergen domain in the C-terminal. The protein also has a specific α-insertion of WCNP (Trp-Cys-Asn-Pro), which suggests that it belongs to the A-subgroup of the EXP family. In the present study, we cloned the 1,129 bp promoter region upstream of CmEXPA4, and the analysis revealed an abundance of cis-acting elements associated with hormones, light and stress-related responses, and some root-specific regulatory elements in particular. Subcellular localization results indicated that CmEXPA4 locates in the cell wall. Exogenous indole butyric acid induced the up-regulation of CmEXPA4 expression, whereas exogenous abscisic acid inhibited its expression. Tissue expression analysis showed that CmEXPA4 was preferentially expressed in the roots and was synchronized with the rapid emergence of the root. These results suggested that CmEXPA4 may act on the growth and development of chrysanthemum roots. The function of CmEXPA4 was further tested by virus-induced gene silencing, and the results showed that CmEXPA4 silencing inhibited the normal development of the chrysanthemum root system. The roots appeared thinner and shorter, and several important root parameters, including total length, average diameter, surface area, total volume, and root tip number, decreased significantly. The cortical cells of the transgenic plant roots were significantly smaller and shorter than those of the control. Collectively, our results demonstrated that CmEXPA4 gene plays a key role in the growth and development of chrysanthemum roots and affects the root system by acting on the individual cells.

中文翻译：

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CmEXPA4参与菊花根发育的表达和功能验证

扩展蛋白 (EXP) 在植物根系形成中起重要作用。与菊花根相关的EXP基因尚未见报道。在这里，我们在菊花（Chrysanthemum morifolium）中分离出一个根特异性EXP基因，即CmEXPA4。生物信息学分析表明，CmEXPA4 编码的蛋白质在 N 端有一个保守的 DPPB（双 Psi Beta-Barrel）结构域和一系列 Cys 残基，在中心区域有一个 HFD（His-Phe-Asp）基序和一个C-末端的花粉过敏原结构域。该蛋白质还具有 WCNP (Trp-Cys-Asn-Pro) 的特定 α 插入，这表明它属于 EXP 家族的 A 亚群。在本研究中，我们克隆了 CmEXPA4 上游的 1,129 bp 启动子区域，分析揭示了大量与激素、光和应激相关反应相关的顺式作用元件，尤其是一些特定于根的调节元素。亚细胞定位结果表明CmEXPA4定位于细胞壁。外源吲哚丁酸诱导 CmEXPA4 表达上调，而外源脱落酸抑制其表达。组织表达分析表明，CmEXPA4优先在根部表达，并与根部的快速出苗同步。这些结果表明CmEXPA4可能对菊花根的生长发育有影响。通过病毒诱导的基因沉默进一步测试了CmEXPA4的功能，结果表明CmEXPA4沉默抑制了菊花根系的正常发育。根显得更细更短，几个重要的根参数，包括总长度、平均直径、表面积、总体积和根尖数量显着减少。转基因植物根的皮质细胞明显小于对照的皮质细胞。总的来说，我们的结果表明 CmEXPA4 基因在菊花根的生长发育中起关键作用，并通过作用于单个细胞来影响根系。