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以‘玉露’桃叶片基因组ＤＮＡ为模板，用ＡＣＣ氧化酶基因特异寡聚核苷酸为引物进行ＰＣＲ扩增，得到１．３ｋｂ左右的扩增产物，将其克隆到ｐＵＣ１９加Ｔ载体上，组建亚克隆并进行序列测定，结果表明，该基因全长有１２８８个碱基，由４个外显子和３个内含子组成。外显子由９５７碱基组成，编码３１９个氨基酸。该基因与桃、番茄、矮牵牛、康乃馨、苹果ＡＣＣ氧化酶ｃＤＮＡ氨基酸序列同源性分别为９９．３％，８３．０％，７６．８％，７４．０％，７５．０％。ＲＮＡ点杂交表明，该基因在未成熟果实检测不到杂交信号，随着成熟度增加，硬度下降，基因表达增强。将ＡＣＣ氧化酶基因分别正向和反向克隆到植物表达载体ｐＢＩ１２１中，并通过酶切分析，ＰＣＲ和点杂交鉴定重组质粒正确。将重组表达质粒导入根癌农杆菌中，经ＰＣＲ和Ｓｏｕｔｈｅｒｎ杂交鉴定证实质粒已被导入。
The genomic DNA of ’Jade and Peach’ was used as template to amplify the amplified product of about 1.3kb with ACC oxidase gene-specific oligonucleotide as a primer. The amplified product was cloned into pUC19 plus T vector to construct Subcloned and sequenced, the results showed that the full length of the gene has 1288 bases, composed of four exons and three introns. The exon consists of 957 bases and encodes 319 amino acids. The deduced amino acid sequence of this gene was 99.3%, 83.0%, 76.8%, 74.0% and 75.0%, respectively, with the ACC oxidase cDNA of peach, tomato, petunia, carnation and apple. RNA dot blot showed that the gene could not detect the hybridization signal in immature fruit, with the increase of maturity, the hardness decreased and the gene expression increased. The ACC oxidase gene was cloned into the plant expression vector pBI121 in forward and reverse directions, respectively. The recombinant plasmids were identified by restriction enzyme digestion and dot blot hybridization. The recombinant expression plasmid was introduced into Agrobacterium tumefaciens and confirmed by PCR and Southern blotting that the plasmid had been introduced.