Artificial miRNA (amiRNA) is a powerful technology to silence genes of interest. It has a high efficiency and specificity that can be used to explore gene function through targeted gene regulation or to create new traits. To develop this gene regulation tool in apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone previously characterized (Md-miR156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (MdPDS in apple). Two pairs of miRNA:miRNA* regions were designed (named h and w). The monocistronic Md-miR156h with these MdPDS targets was placed under the control of the CaMV 35S promoter to generate the two plasmids: pAmiRNA156h-PDSh and pAmiRNA156h-PDSw. Two Agrobacterium-mediated transformation experiments were performed on the cultivar 'Gala'. A total of 11 independent transgenic clones were obtained in the first experiment and 5 in the second. Most transgenic lines had a typical albino and dwarf phenotype. However, six clones had a wild type green phenotype. Molecular analyses indicated clear relationships between the degree of albino phenotype, the level of MdPDS gene expression and the amount of mature amiRNAs. This study demonstrated for the first time in apple the functionality of an artificial miRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects.

中文翻译：

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人工miRNA作为沉默和探索苹果基因功能的新工具。

人工miRNA（amiRNA）是一种强大的技术，可以使目标基因沉默。它具有很高的效率和特异性，可用于通过有针对性的基因调控来探索基因功能或创造新的性状。为了在苹果中开发这种基因调控工具，我们基于先前表征的苹果内源性miRNA主链（Md-miR156h）设计了两个amiRNA构建体，并检查了它们在易于评分的标记基因上的效率：番茄红素去饱和酶基因（苹果中的MdPDS） 。设计了两对miRNA：miRNA *区域（分别命名为h和w）。将具有这些MdPDS靶标的单顺反子Md-miR156h置于CaMV 35S启动子的控制下，以生成两个质粒：pAmiRNA156h-PDSh和pAmiRNA156h-PDSw。在品种“ Gala”上进行了两个农杆菌介导的转化实验。在第一个实验中总共获得了11个独立的转基因克隆，在第二个实验中获得了5个。大多数转基因品系具有典型的白化病和矮化表型。然而，六个克隆具有野生型绿色表型。分子分析表明白化病表型程度，MdPDS基因表达水平和成熟amiRNA数量之间存在明确的关系。这项研究首次在苹果中证明了基于内源性miRNA骨架的人工miRNA的功能。它为苹果遗传功能研究以及苹果遗传改良项目提供了重要的机会。分子分析表明白化病表型程度，MdPDS基因表达水平和成熟amiRNA数量之间存在明确的关系。这项研究首次在苹果中证明了基于内源性miRNA骨架的人工miRNA的功能。它为苹果遗传功能研究以及苹果遗传改良项目提供了重要的机会。分子分析表明白化病表型程度，MdPDS基因表达水平和成熟amiRNA数量之间存在明确的关系。这项研究首次在苹果中证明了基于内源性miRNA骨架的人工miRNA的功能。它为苹果遗传功能研究以及苹果遗传改良项目提供了重要的机会。