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苹果脂氧合酶基因 MdLOX1a 的同源克隆与功能验证
岳璇璇, 王庆鹏, 房鸿成, 胡甲飞, 苏梦雨, 张宗营, 王楠, , 陈学森
0
(山东农业大学园艺科学与工程学院/作物生物学国家重点实验室)
摘要:
脂氧合酶是脂肪酸代谢途径中的关键酶,在植物生长发育、响应环境胁迫、合成香气物质等方面起着重要作用。 本试验以‘紫红 3 号’叶片诱导出的红色愈伤组织为试材,对 MdLOX1a 基因及其部分启动子序列进行扩增。测序发现 MdLOX1a 的开放阅读框为 2592 bp,编码 863 个氨基酸,预测其蛋白分子量为 97.69 KDa,等电点为 5.14。MdLOX1a 定位于苹果基因组 的第 9 号染色体,由 8 个外显子与 7 个内含子构成。氨基酸序列进化树分析表明,MdLOX1a 与 Pb9S-LOX5 在同一进化枝。此 外,克隆获得 MdLOX1a 启动子序列 1058 bp,启动子元件预测表明该启动子包括响应多种胁迫的顺式作用元件。亚细胞定位发 现,MdLOX1a 在细胞膜与细胞核上均有定位。组织差异分析表明,MdLOX1a 基因在果皮中的表达量最高,在花和果肉中次之; 同时随着果实成熟 MdLOX1a 基因的表达量上调并且持续光照能够诱导 MdLOX1a 基因表达,低温(16 ℃)以及不同浓度的外源 ABA 处理后,MdLOX1a 基因的表达量均明显降低。过表达 MdLOX1a 验证发现较于对照组,实验组香气各成分有不同程度增加, 说 明 MdLOX1a 与香气物质的合成有关。
关键词:  苹果  MdLOX1a  表达分析  亚细胞定位  功能验证
DOI:10.13430/j.cnki.jpgr.20190731001
投稿时间:2019-07-31修订日期:2019-10-09
基金项目:国家自然科学基金(31730080,31701892);国家重点研究计划项目(2016YFC0501505)
Homologous Cloning and Expression Analysis of Apple Lipoxygenase Gene MdLOX1a
YUE Xuan-xuan, WANG Qing-peng, FANG Hong-cheng, HU Jia-fei, SU Meng-yu, ZHANG Zong-ying, WANG Nan, CHEN Xue-sen
(College of Horticulture Science and Engineering, Shandong Agricultural University / State Key Laboratory of Crop Biology)
Abstract:
Lipoxygenase is a key enzyme involved in the pathway of fatty acid metabolism, and plays an important role in the growth and development of plants, responses to environmental stresses and the synthesis of aroma components. In this study, the red callus from the leaves of ''Zihong 3'' was used for PCR amplification of the MdLOX1a gene and its partial promoter sequence. Sequencing analysis revealed that MdLOX1a has an open reading frame of 2592 bp encoding for 863 deduced amino acids, with a predicted protein molecular weight of 97.69 KDa and the isoelectric point of 5.14. The MdLOX1a gene was found to be localized on chromosome 9 of the apple genome, consisting of 8 exons and 7 introns. Phylogenetic tree analysis with amino acid sequence indicated that MdLOX1a and Pb9S-LOX5 were assigned within the same branch. In addition, we obtained the MdLOX1a promoter sequence with a length of 1058 bp, which contained several cis-acting elements in response to various stresses. Subcellular localization revealed that MdLOX1a was localized on both the cell membrane and the nucleus. The tissue disparity analysis showed that the expression level of MdLOX1a gene was abundant in the pericarp, followed by flower and pulp. The expression of MdLOX1a gene gradually increased with fruit ripening, and its transcription has been induced under continuous light treatment. The expression of MdLOX1a gene was significantly reduced after low temperature (16 ℃) and exogenous ABA treatment at different concentrations. The overexpression of MdLOX1a showed that the transgenic lines showed the induction of the aromatic components in relative to the control lines, suggesting that MdLOX1a might associate with the synthesis of aroma components.
Key words:  apple  MdLOX1a  expression analysis  subcellular localization  functional verification

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