ACC合酶cDNA克隆及其对美洲黑杨体内乙烯产生的反义抑制

CLONING OF ACC SYNTHASE cDNA AND ITS INHIBITION OF ETHYLENE SYNTHESIS BY ITS ANTISENSE RNA IN TRANSGENIC POPULUS DELTOIDES

利用ＲＴＰＣＲ技术克隆了大豆ＡＣＣ合酶基因ＧＭＣＡＣＣＳＩ编码区１５ｋｂ的ｃＤＮＡ片段，经酶切图谱分析和序列分析鉴定后，反向插入到２元载体ｐＢｉｎ４３８中，构建了表达ＡＣＣ合酶反义ＲＮＡ的植物表达载体，转化农杆菌后用该农杆菌侵染美洲黑杨叶片，在含卡那霉素的ＭＳ培养基上选择转化子和植株再生，通过ＰＣＲ检测从抗卡那植株中选到１５株转基因植株，Ｓｏｕｔｈｅｒｎ杂交分析初步确证了外源基因是以单拷贝插入到杨树基因组ＤＮＡ中；对杨树幼苗乙烯释放的测定结果表明转基因杨树幼苗的乙烯释放量为对照的２２％。

A 1.5kb fragment of ACC synthase cDNA fragment prepared from total RNA of soybean was amplified by polymerase chain reaction(PCR) and cloned into pGEM T vector.The cloned ACC synthase gene was further inserted into a binary vector,pBin438,in an inverted orientation between the CaMV 35S promoter and Nos 3′termination sequence (pBACS).Transgenic poplar plants were obtained by regeneration of Agrobacterium mediated transformation of leaves of Populus deltoides. PCR and Southern blotting analyses confirmed the integration of a single antisense ACC synthase gene in transformed poplar genome.The results form reverse transcription PCR (RT PCR) of RNAs isolated from transgenic poplar leaves confimed that the antisense RNA of ACC synthase presented in these transgenic plants.The amount of ethylene released from transgenic poplar was reduced significantly to about 22% of that released from non transformed control poplars.