ACC氧化酶cDNA克隆及其对美洲黑杨体内乙烯产生的反义抑制

利用ＲＴ－ＰＣＲ技术克隆了番茄ＡＣＣ氧化酶基因ＬＥＥＴＨＹＢＲ编码区０．９ｋｂ的ｃＤＮＡ片段，经酶切图谱分析和序列分析鉴定后，反向插入到植物表达载体ｐＢｉｎ４３８中，构建了表达ＡＣＣ氧化酶反义ＲＮＡ的二元载体。用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检测筛选到１６株转基因杨树植株，Ｓｏｕｔｈｅｒｎｂｌｏｔ分析初步确证了外源基因是以单拷贝插入到杨树基因组中；对杨树幼苗乙烯释放量的测定结果表明转基因杨树的乙烯释放量为对照植株的２８％。

Cloning of ACC Oxidase cDNA and Its Inhibition of Ethylene Synthesis by Its Antisense RNA in Transgenic Populus deltoides

A 0.9 kb fragment of ACC oxidase cDNA fragment prepared from total tomato RNA was amplified by polymerase chain reaction (PCR) and cloned into pGEM R T vector.The cloned ACC oxidase gene was further inserted into a binary vector,pBin438,in an inverted orientation between the CaMV 35S promoter and Nos 3 termination sequence (pBACO).Transgenic poplar plants were obtained by regeneration of agrobacteriummediated transformed leaves.PCR and Southern blot analyses confirmed the integration of a single antisense ACC oxidase gene in transformed poplar genome.The results from RTPCR of RNAs isolated from transgenic poplar leaves confirmed that the antisense RNA of ACC oxidase presented in these transgenic plants.The amount of ethylene released from transgenic poplars was reduced significantly to about 28% of that released from nontransformed controls.