番茄ACC合成酶cDNA克隆及其对果实成熟的反义抑制

Cloning of ACC Synthase cDNA and Its Inhibition of Fruit Ripening by Its Antisense RNA in Transgenic Tomato Plants

利用ＲＴＰＣＲ技术克隆了ＡＣＣ合成酶多基因家族成员之一ＬＥＡＣＣ２编码区约１７ｋｂ的ｃＤＮＡ，经酶切图谱和序列分析鉴定无误后，反向插入到植物表达载体ｐＢｉｎ４３７中，构建了表达ＡＣＣ合成酶反义ＲＮＡ的二元载体。经农杆菌途径转化番茄“丽春”品种后，通过ＰＣＲ检测从抗卡那霉素再生植株中筛选到６株转基因植株，Ｓｏｕｔｈｅｒｎ杂交确证了外源基因是以单拷贝插入到番茄染色体中；对果实乙烯释放的测定结果表明转基因番茄果实的乙烯释放量仅为对照的３０％左右，在室温下转基因番茄果实采后保存６０ｄ以上仍然没有变红、软化。以上结果表明其反义ＲＮＡ在转基因番茄中的表达能有效地抑制乙烯的生物合成从而延缓果实成熟，表现出良好的耐储保鲜特性。对转基因植株子一代（Ｔ１）的分析结果进一步表明反义ＡＣＣ合成酶基因以典型的单基因方式传到子代。通过对子二代的分析已初步筛选到一个耐储藏的转基因番茄纯合品系。

A 1.7kb fragment of ACC synthase cDNA,one of the ACC synthase multigene family,was amplified from total tomato cDNA through Polymerase Chain Reaction (PCR) and cloned.The cloned ACC synthase gene was then inserted into a binary vector,pBin437,in an inverted orientation between the CaMV 35S promoter and the Nos 3' termination sequence,to construct an expressing vector.Transgenic tomato plants were obtained by Agrobacterium mediated transformation of cotyledons.PCR detection and Southern blot analysis confirmed the integration of the antisense ACC gene in transformed tomato genome.The results from RT PCR of RNAs isolated from transgenic tomato leaves confirmed that antisense ACC RNA was synthesized in these transgenic plants.The amount of ethylene released from transgenic tomato fruits was reduced significantly to about 30% of that released by non transformed controls.The inhibition effect of antisense ACC RNA on fruit ripening was observed in the transgenic plants and their progenies (T1).The shelflife of transgenic tomato fruits was at least 60 days at room temperature without significant change in hardness and color.Upon 15～20days treatment of the transgenic fruits with ethylene,most of them could reach the ripe stage,turning red as that of the controls.The antisense ACC synthase gene was inherited as a single gene in the T1 progeny as determined by T1 progeny analysis,consistant with the results of Southern blot analysis.Transgenic homozygotes showing prolonged shelflife have been selected by genetic analysis of T2 progenies.