摘要
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(Phalaenopsisequestris)、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(salicylicacid)和CaCl2都能够诱导PAD4的表达,都在24h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。
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.
作者
郭艳芳
王丛巧
李蓉
陈裕坤
赖钟雄
王天池
GUO Yanfang;WANG Congqiao;LI Rong;CHEN Yukun;LAI Zhongxiong;WANG Tianchi(Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)
出处
《热带作物学报》
CSCD
北大核心
2018年第12期2436-2445,共10页
Chinese Journal of Tropical Crops
基金
福建省重大科技专项"福建特色园艺作物种质优异基因资源的挖掘与种质创新"(No.2015NZ-0002-1)
关键词
文心兰
抗病
PAD4
基因克隆
表达分析
Oncidium hybridum
disease resistance
PAD4
gene cloning
expression analysis
