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枇杷果实EjNADP-ME2基因及启动子克隆与表达分析 被引量:1

Cloning and Expression Analysis of EjNADP-ME2 Gene and Its Promoter in Loquat Fruit
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摘要 以‘解放钟’和‘白梨’果实为材料,研究基于转录组筛选出的表达量较高且在2个品种中有差异的EjNADP-ME2(Unigene0001461)基因,采用RACE和RT-PCR技术成功克隆EjNADP-ME2的cDNA全长。系统进化树分析表明,EjNADP-ME2与苹果的NADP-ME基因关系最近。生物信息学预测结果显示,EjNADP-ME2蛋白应该位于细胞质中。用染色体步移法成功克隆了EjNADP-ME2的启动子序列,序列预测该基因启动子区域含有水杨酸、茉莉酸、脱落酸响应的顺式作用元件,此外还有厌氧诱导元件、MYB和MYC参与黄化和干旱诱导的结合位点MBS。荧光定量PCR分析表明,EjNADP-ME2基因的表达量变化趋势在2个品种中大致相同,均呈现出先降低后升高又降低的趋势,且该基因在低酸品种的表达量稍低于高酸品种。相关性分析结果显示,低酸品种EjNADP-ME2基因转录水平与苹果酸含量呈显著负相关。本研究的结果为进一步探究EjNADP-ME2基因在苹果酸代谢途径中的功能奠定坚实基础。 Fruits of a high-acid cultivar ’Jiefangzhong’ and a low-acid cultivar ‘Baili’ were used as the materials to study the EjNADP-ME2(Unigene0001461) gene whose expression level was relatively high yet different between the two cultivars as shown by transcriptomic data. Its full-length cDNA was successfully cloned by RACE and RT-PCR. Phylogenetic analysis showed that EjNADP-ME2 was closely related to the NADP-ME gene of apples. Bioinformatics analysis predicted that the EjNADP-ME2 protein should be located in the cytoplasm. The promoter of the gene was successfully cloned by chromosome walking and predicted that contained cis-acting elements responsive to salicylic acid, jasmonic acid, abscisic acid, anaerobic induction, and sites for MYB, MYC and MBS(yellowing and drought induction) binding. Real-time PCR analysis showed that the trends were similar in the expression of the gene between the two cultivars, showing a lower, higher and lower pattern at the earlier, middle and later stages of the fruit development, respectively. Correlation analysis suggested that EjNADP-ME2 expression was negatively correlated to malic acid content in the fruit of the low acid cultivar ’Baili’, but not such in the other cultivar. The results of this study would provide starting materials for further exploration of the function of the EjNADP-ME2 gene in the metabolism of malic acid in the fruit of loquat.
作者 杨俊 郑雪莲 高欢欢 寇燕 陈旭 郭傲 郑国华 YANG Jun;ZHENG Xuelian;GAO Huanhuan;KOU Yan;CHEN Xu;GUO Ao;ZHENG Guohua(College of Horticulture,Fujian Agriculture and Forestry University,Institute of Storage and Transport of Horticultural Products,Fuzhou,Fujian 350002,China)
出处 《热带作物学报》 CSCD 北大核心 2019年第3期490-498,共9页 Chinese Journal of Tropical Crops
基金 福建农林大学科技创新专项基金项目(No.KAF18081A) 国家重点研发计划"政府间国际科技创新合作"项目(No.YFE0127200)
关键词 枇杷 果实 细胞质型苹果酸酶 基因表达分析 启动子 Eriobotrya japonica fruit cytoplasmic malic enzyme gene expression analysis promoter
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