鹅掌楸AOX家族基因克隆与组织表达分析

Molecular isolation and tissue expression analysis of alternative oxidase family genes in Liriodendron chinensis

关键词:  鹅掌楸, 交替氧化酶, 基因克隆, 生物信息学分析, 组织表达

Key words:  Liriodendron chinensis, alternative oxidase, molecular isolation, bioinformatics analysis, tissue expression

摘要:

鹅掌楸(Liriodendron chinense)为重要的珍贵用材及园林观赏树种,开展抗逆基因的研究,对于提高鹅掌楸适应性有重要意义。该文以鹅掌楸为研究对象,通过采用RT-qPCR与RACE相结合的方法克隆获得3个AOX基因,其ORF长分别为858、1 032、1 044 bp,相应编码氨基酸数为285、343、347 aa,分别命名为LcAOX1a、LcAOX1b和LcAOX2。蛋白同源性分析发现鹅掌楸AOX家族蛋白序列高度保守,尤其在C端保守性极高,且均含有“EXXH”、“EEE-Y” 铁离子结合保守结构域。亚细胞定位分析结果显示LcAOX1a蛋白定位于线粒体及叶绿体之外的其他位置,LcAOX1b蛋白在叶绿体和线粒体中均有定位,LcAOX2蛋白定位于线粒体基质。采用RT-qPCR方法研究AOX基因在鹅掌楸茎、叶片、叶芽、花芽、花萼、花瓣、雄蕊、雌蕊8个不同组织中的表达模式,分析发现鹅掌楸AOX基因在花器官中表达量明显高于营养器官,LcAOX1a与LcAOX1b基因在雄蕊中表达量最高,特别是LcAOX1a基因在雄蕊中特异性表达,其表达量远远高于其他组织; LcAOX2基因在花瓣中表达量最高。该研究克隆3个鹅掌楸AOX基因并进行相关分析,为进一步研究其生物学功能奠定了基础。

Abstract:

Alternative oxidase(AOX), a terminal oxidase located in respiration electron-transport pathway, which is widely existed in higher plants and closely related to plant respiration. It has shown that AOX played a substantial role in plant growth, seedling morphogenesis and environmental adaptability in recent researches. Liriodendron chinensis is an excellent tree species for garden ornaments and timber use, while the adaptive capacity prevents the expansion of cultivated area. As a result, searching for resistant genes of L. chinensis and uncovering the mechanism of its stress-defence ability are great urgency. Three AOX genes were isolated from L. chinensis by RT-qPCR and RACE, and then sequence analysis was carried out in silico, including analysis of open reading frames, encoded amino acid sequences, protein domains, secondary structures and so on. The open reading frame length of AOX genes were 858, 1 032 and 1 044 bp, which encoded 285, 343 and 347 amino acids, respectively, and then we named the genes as LcAOX1a, LcAOX1b and LcAOX2. Protein homology and phylogenesis analysis revealed that the AOX family protein sequences of L. chinense were highly conserved, especially at the C-terminus, and all the three AOX genes contained “EXXH”, “EEE-Y” iron-binding conserved domains, those may have activities in combination with iron ions. Subcellular localization analysis showed that LcAOX1a protein was localized in other places outside the chloroplast and mitochondria. LcAOX1b protein was localized in chloroplast and mitochondria, while LcAOX2 protein was localized in mitochondrial matrix. The expression patterns of AOX genes were examined by using eight tissues, including stem, leaf, leaf bud, flower bud, calyx, petal, stamens and pistil. The result of RT-qPCR indicated that relative quantity(RQ)of LcAOX1a, LcAOX1b and LcAOX2 genes in floral organs was significantly greater than that in vegetative organs. The RQ value of LcAOX1a and LcAOX1b was the highest in the stamens, especially the LcAOX1a, the expression in the stamens was much higher than other tissues. The LcAOX2 gene has the highest RQ value in the petals. This study cloned three LcAOX genes and performed bioinformatics analysis, subcellular localization analysis and expression patterns analysis to provide a reference for further study of their biological functions.