Abstract:

The ACC synthase is the key enzyme in ethylene biosynthesis and fruit ripening. To study the mechanism of ACC synthase in peach ( Prunus persica (L.) Batsch) fruit ripening, we cloned a full length cDNA of ACC synthase pacs from peach using 5′/3′ RACE PCR. The nucleic acid sequence of pacs was 1848 bp, containing 177bp of 5′untranslated sequence, 1449 bp of an open reading frame, and 219 bp of 3′untranslated sequence (excluding the stop codon TAA). The pacs open reading frame encoded a 483 amino acid polypeptide with a predicted size of 54 kD and a calculated PI of 6.43. The deduced protein from ACC synthase cDNA pacs had 65%, 70%, 75%, and 90% homology with the other deduced proteins from tomato (S19677), plum (AB031026), papaya (U68216) and apple (AB034993), which contained the active site of ACC synthase SLSKDMGFPGFR conserved among these plant ACC synthases. RNA based PCR amplification combined with hybridization analysis with pacs and another ACC synthase cDNA pacs12 (AF467782) cloned by us before as probes, indicated that expression patterns of both clones were very similar. mRNAs of both clones expressed in the alabastrum and petal, and were induced after ethylene treatment. Wounding and IAA treatments could induce ACC synthase expression of both clones in the leaves. However, the wounding treatment of leaves has induced more abundant pacs ACC synthase expression than that of pacs12 . Pacs mRNA expressed in both green mature and ripening fruit, while pacs12 mRNA was little or undetectable in green mature fruit, but apparent in ripening fruit. Both clone mRNAs accumulated more in leaves (following wounding and IAA treatments) and flowers than in fruits.

桃ACC合酶基因的分离及其差异表达
金勇丰¹ 朱立成¹  张耀洲¹  张上隆²

（1. 浙江大学生物化学研究所；2. 浙江大学园艺系，杭州310029）

摘要： 利用 5′/ 3′RACEPCR技术 ,从桃 (Prunuspersica (L .)Batsch)果实中克隆了植物乙烯生物合成的关键酶———ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明 ,该基因全长 184 8个碱基 ,编码区为 14 4 9个碱基 ,5′端有 177个碱基的非编码区序列 ,3′端有 2 19个碱基的非编码区序列 (不包括终止密码子TAA)。pacs基因编码区共编码 4 83个氨基酸 ,蛋白质大小为 5 4kD ,等电点为 6 .4 3。pacs与番茄 (S196 77)、梅 (AB0 310 2 6 )、番木瓜 (U6 82 16 )、苹果(AB0 34993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为 6 5 %、70 %、75 %、90 % ,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR。RT-PCR结合杂交分析表明 ,pacs和我们以前克隆的桃ACC合酶cDNApacs12 (AF4 6 7782 )在叶片和花中基因表达模式基本一致 ,伤处理和IAA均能诱导叶片pacs和pacs12基因的表达 ,但pacs在伤处理叶片的表达水平比pacs12高 ;pacs和pacs12基因在果实表达有所不同 ,pacs在绿熟和成熟果实中均有表达 ,而pacs12在绿熟果实中基本检测不到 ,在成熟果实中才有表达 ,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低。

关键词： 桃ACC合酶；基因分离；差异表达

Key words: Prunus persica, ACC synthase, cloning, differential expression