Abstract: 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.