Cloning and Expression Analysis of a Pathogenesis-related Protein 10 Gene from Juglans sigillata
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摘要: 病程相关蛋白(pathogenesis-related protein, PR) 10的激活与积累在植物抗逆境胁迫中有非常重要的作用。根据漾濞大泡核桃(Juglans sigillata)编码PR10的EST (expressed sequence tag)序列设计引物, 利用快速扩增cDNA末端技术, 克隆得到PR10基因的全长cDNA序列, 并命名为JsPR10-1。JsPR10-1全长cDNA为776 bp, 含有483 bp的开放阅读框、74 bp 5'-非编码区以及219 bp 3'-非编码区, 编码含有160个氨基酸的蛋白质。全长基因序列中含有1个124 bp的内含子。JsPR10-1编码的蛋白质与栎树(Quercus suber)、欧洲山毛榉(Fagus sylvatica)以及欧洲板栗(Castanea sativa)的PR10相似性较高, 并且在PR10的系统进化树中与双子叶植物聚为一支。qRT-PCR分析结果表明, 植物信号分子水杨酸、茉莉酸、乙烯以及过氧化氢处理均可诱导JsPR10-1表达。在接种胶孢炭疽菌(Colletotrichum gloeosporioides)后, JsPR10-1的表达量迅速上升并在接种8 h时达到最大值, 表明JsPR10-1参与漾濞大泡核桃对胶孢炭疽菌的防卫反应。本研究为揭示漾濞大泡核桃抗性机制奠定了理论依据。Abstract: The activation and accumulation of pathogenesis-related protein(PR)10 play predominant roles in the defense response against pathogens. Based on a Juglans sigillata expressed sequence tag (EST) encoding PR10, gene-specific primers were designed and used to amplify the full-length cDNA of a novel PR10 gene by rapid amplification of cDNA ends (RACE). This gene was named as JsPR10-1. JsPR10-1 was 776 bp in length with an open reading frame (ORF) of 483 bp, a 5'-untranslated region (UTR) of 74 bp, and a 3'-UTR of 219 bp, and the ORF encoded a protein with 160 amino acid residues. There was an intron of 124 bp in the genomic sequence of JsPR10-1. Homology analysis indicated that JsPR10-1 was homologous with PR10s from Quercus suber,Fagus sylvatica and Castanea sativa; moreover, JsPR10-1 clustered together with the PR10s from dicots in the phylogenetic tree. qRT-PCR analysis showed that the expression levels of JsPR10-1 were induced by four different plant signaling molecules, including salicylic acid, jasmonic acid, ethylene, and H2O2. In addition, after inoculation with Colletotrichum gloeosporioides, JsPR10-1 was sharply up-regulated with the highest expression level at 8 h. The JsPR10-1 gene was involved in the defense response against C. gloeosporioides. This experiment lays a theoretical foundation for revealing the mechanism of resistance in J. sigillata.
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