文心兰 PAD4 基因克隆及表达分析

为了研究文心兰（Oncidium hybridum）抗病相关基因 PAD4 在文心兰抗病种质资源培育中的功能，采用 RT-PCR 结合 RACE 法，从巧克力文心兰(Oncidium Sharry Baby ‘Sweet Fragrance’)中克隆得到一条全长 2 214 bp 的基因的 cDNA 序列，开放阅读框长 1 894 bp，预测可编码 647 个氨基酸，5′UTR 长度为 65 bp，3′UTR 长度为 255 bp。生物信息学分 析表明：PAD4 编码的蛋白属于水解酶超家族，有 6 个跨膜螺旋，无信号肽，亚细胞定位预测其主要定位于细胞质膜上。 系统进化树表明，文心兰 PAD4 与同为兰科植物的小兰屿蝴蝶兰（Phalaenopsis equestris）、铁皮石斛（Dendrobium catenatum）、深圳拟兰（Apostasia shenzhenica）亲缘关系最近。同源分析结果显示，与铁皮石斛的 PAD4 蛋白同源性最 高（78.08%）。实时荧光定量 PCR 分析显示，PAD4 在文心兰不同组织部位中都有表达，在假鳞茎中的表达量最高，其 次是下端叶，在根中的表达量最低。接种软腐病病原菌显示，PAD4 在感病文心兰中表达量上调，侵染后 4 h 时快速响 应并达到最高值。SA（salicylic acid）和 CaCl2 都能够诱导 PAD4 的表达，都在 24 h 时达到最高峰。研究表明，PAD4 基因可能在文心兰抗病过程中有重要作用。

Cloning and Expression Analysis of PAD4 Gene in Oncidium hybridum

The RT-PCR combined with RACE method was used to clone the complete cDNA sequence of PAD4 gene from Oncidium Sharry Baby ‘Sweet Fragrance’ in order to explore the function of PAD4 gene in the cultivation of disease resistant germplasm resources of Oncidium. The complete cDNA sequence of PAD4 was 2 214 bp and the ORF was 1 894, encoding 647 amino acids, including 65 bp 5′UTR, 255 bp 3′UTR. Bioinformatics analysis showed that PAD4 protein belonged to alpha/beta hydrolase superfamily, which was a transmembrane protein with 6 strong transmembrane helices and without signal peptide. Subcellular localization predicted that PAD4 was located in plasma membrane. The phylogenetic tree analysis showed that the protein had closer relationship with Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica which belong to Orchidaceae. Blastp analysis showed that PAD4 shared the highest similarity with D. catenatum (78.08%). qRT-PCR results showed that PAD4 expressed in all tissues and organs in O. hybridum and the highest expression was found in the pseudobulb, followed by the leaf and the lowest expression was found in the root. The expression of PAD4 accumulated quickly and after pathogen infection, and up-regulation was found in the pseudobulb at only 4 hours. The expression of PAD4 was also induced by SA (salicylic acid) and CaCl2, and the highest expression was found at 24 hours. The result suggested that the PAD4 may play an important role in Oncidium disease resistance.