Structural Characterization of ACC Synthase Genes from Melon and Cucumber and their Promoter Activities Determined by GUS Transient Assay

In orader to clarify the differences in regulatory mechanism(s) of the expression of 1-aminocyclopropane-1-carboxylate(ACC) synthase(ACS)genes during ripening in climacteric melon fruit and non-climacteric cucumber fruit, two sets of their genomic DNA sequences, including ca. 2kb of the promoter regions were determined, using PCR-based methods. ACS genes from melon (CMe-ACS1,2) were structurally similar to their counterpart from cucumber (CS-ACS1,2) in terms of size and position of exons and introns, restriction map, and sequencd identity of exeons, introns, proximal 5'-flanking promoter regions and splice junction. Southern blot analysis indicated that each ACS gene is present as a single copy. Transient promoter activity was investigated with two constructs of promoter-β-glucuronidase (GUS) fusion, CMe-ACS1:GUS and CS-ACS1:GUS, in mature mesocarp tissue of the two fruits. In melon disks, GUS activities conferred by the promoters of both CS-ACS1 (-2098～+42) and CMe-ACS-1(-2187～+67) were detected, which were decreased by treatment with 1-methylcyclopropene(1-MCP), an ethylene action inhibitor. In cucumber disks, however, only CS-ACS1:GUS was expressed; the activity was decreased with 1-MCP, and it was not affected by propylene. These results suggest that the promoter of CS-ACS1 has a potential to be expressed in the mesocarp tissue of ripening melon fruit, and that the difference in ethylene biosynthesis between melon and cucumber during ripening may be due to the difference in capability of forming trans-acting factor(s), not due to their ACS1 promoter activities.

クライマクテリック型果実のメロンとノンクライマクテリック型果実のキュウリの果実追熟に伴う１-アミ ノシクロプロパン-１-カルボン酸（ACC）合成酵素（ACS）遺伝子の発現調節機構の相違を明らかにするた めに，それぞれ２種類のACSのゲムノDNA配列（約２kb）をPCR法を基にして決定した．メロンのACS 遺伝子は，エキソンとイントロンのサイズおよび位置，制限酵素地図，エキソン・イントロン・近位の５’上 流プロモーター領域・スプライシング部位の塩基配列において，それぞれ対応するキュウリのACS遺伝子 とよく似た構造をしていた．サザンブロック解析の結果，各ACS遺伝子はシングルコピーとして存在する と考えられた．CMe-ACS1とCS-ACS1の一過的プロモーター活性をβ-グルクロニダーゼ（GUS）をレポ ーター遺伝子として両果実の成熟果肉を用いて調べた．メロン切片では，CS-ACS1（－2098～＋42）なら びにCMe-ACS1（－2187～＋67）のプロモーター発現によるGUS活性が検出され，エチレン作用阻害剤 の１-メチルシクロプロペン（１-MCP）処理によって減少した．しかし，キュウリ切片においては，CS-ACS1： GUSのみでGUSが発現し，活性は１-MCP処理で減少し，プロピレン処理ではコントロールと同レベル であった．これらの結果より，CS-ACS1のプロモーターがメロン成熟果肉組織で発現する潜在能力をもつ こと，メロンとキュウリの成熟に伴うエチレン生合成の相違はACS1プロモーター活性によるものではなく， 両者のトランス因子合成能の相違によることが示唆された．