枇杷果实中NAD-MDH基因克隆及植物表达载体构建

Cloning of NAD-MDH Gene in Loquat Fruit and the Construction of Plant Expression Vector

为探索枇杷果实中苹果酸的积累与降解机制,从枇杷果实转录组中得到NAD-MDH的开放阅读框的基因序列。以枇杷高酸品种解放钟和低酸品种白梨为材料,提取果实中总RNA,运用RT-PCR技术克隆出枇杷果实中苹果酸合成酶基因NAD-MDH,并进行荧光定量PCR分析。结果表明:枇杷果实中苹果酸合成酶基因NAD-MDH的开放阅读框(ORF)为996 bp,共编码了332个氨基酸。经荧光定量PCR分析,解放钟和白梨在花期后95 d时NAD-MDH基因表达量最高;花后80~105 d,NAD-MDH基因在高酸与低酸品种间存在显著差异。采用无缝克隆技术成功构建了植物表达载体EjNAD-MDH-PBI121,并导入根瘤农杆菌EHA105中得到植物转化工程菌,为后期利用转基因技术改良枇杷果实品质及遗传改良打下基础。

In order to explore the accumulation and degradation mechanism of malic acid in loquat fruit,the gene sequence of the open reading frame of NAD-MDH was obtained from the transcriptome of loquat fruit.By taking Jiefangzhong(the high-acid variety of loquat)and Baili(the low-acid variety of loquat)as the materials,the total RNA was extracted from the fruit,and the NAD-MDH gene of malate synthetase in loquat fruit was cloned by RT-PCR,and then the fluorogenic quantitative PCR analysis was conducted.The results showed that the open reading frame(ORF)of the NAD-MDH gene of malate synthetase in loquat fruit was 996 bp,encoding 332 amino acids.The fluorescence quantitative PCR analysis showed that the gene expression of NAD-MDH was the highest in Jiefangzhong and Baili at 95 d after flowering,while the NAD-MDH gene showed significant difference between the high-acid and low-acid varieties at 80-105 d after flowering.The plant expression vector EjNAD-MDH-PBI121 was successfully constructed by the seamless cloning technology,and then was introduced into the agrobacterium tumefaciens EHA105 to obtain the engineering bacteria for plant transformation,which laid a foundation for the later use of transgenic technology to improve the quality and genetic improvement of loquat fruit.