猕猴桃ACC氧化酶cDNA克隆及全序列测定

Cloning and Sequencing of a cDNA Coding for 1 Aminocyclopropane 1 carboxylate Oxidase from Kiwifruit

从‘秦美’猕猴桃（ＡｃｔｉｎｉｄｉａｄｅｌｉｃｉｏｓａＣ．Ｆ．ＬｉａｎｇｅｔＡ．Ｒ．Ｆｅｒｇｕｓｏｎ．ｃｖ．Ｑｉｎｍｅｉ）呼吸高峰跃变初期的果实中提取总ＲＮＡ，然后纯化得ｍＲＮＡ，用Ｏｌｉｇｏ（ｄＴ）作为引物反转录合成ｃＤＮＡ第一链，以此为模板，用人工合成的寡聚核苷酸作为引物进行扩增，得到大小约０．９５Ｋｂ基因片段，并将其克隆到ｐＧＥＭ－５ｚｆ（＋）载体上。经限制性内切酶图谱分析，组建亚克隆并进行了全序列测定，在其开放读码框架内，由９５７个ｂｐ编码３１９个氨基酸组成，该ｃＤＮＡ与海沃德ＡＣＣ氧化酶ｃＤＮＡ的核苷酸和氨基酸残基同源率分别达９５．０１％和９５．６１％，证明己克隆到完整的秦美猕猴桃ＡＣＣ氧化酶基因编码区。

RNA was isolated and purified from mesocarp of preclimacteric kiwifruit. The first strand of cDNA was synthesized by using oligo (dT) as primer and a 0.95Kb DNA fragment was obtained with PCR technique. The restriction map of the DNA fragment was analysed and its whole nucleotide sequence was determined. The results showed that the entire cDNA encoding 1-aminocyclopropane-1-carboxylate oxidase has been cloned. The DNA sequence and deduced amino acid sequence reported here were compared with that of ‘Hayward’ and found 95.01% and 95.61% homologous respectively. The work of transforming this gene into kiwifruit plants in the form of antisense RNA is in progress.