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从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基因片段串联,构成全长1837bp的融合基因。将该融合基因以反义的方向插入植物双元载体pYPX145中番茄果实表达特异启动子下游,获得ACC氧化酶基因和ACC合成酶基因融合的植物双元载体pOSACC。该载体外源基因表达单元的两端含两个烟草SAR序列,利于转基因的稳定遗传。以番茄栽培品种合作903子叶和下胚轴为外植体,利用根癌农杆菌进行基因转化,通过200mg/L卡那霉素选择和GUS检测,获得了105株番茄GUS阳性植株,转基因番茄果实在当代表现明显耐贮特点。经过4代的耐贮和果实农艺性状的综合选择,获得了两个表现良好的株系DR-1和DR-2,两株系果实乙烯释放量显著下降,是未转基因材料的9.5%,番茄的贮存期在50天以上。
The tomato fruit-specific promoter 2A11 was cloned from the total DNA of a strong Mishousa variety of tomato, and the full-length ACC oxidase gene and the ACC synthase gene fragment of the tomato were cloned by RT-PCR using the RNA of a ripe tomato fruit as a template. After cloning and sequencing of the two genes, the 888bp tomato ACC oxidase gene and the 943bp ACC synthetase gene fragment were connected in series to form a full-length 1837bp fusion gene. The fusion gene was inserted into the plant binary vector pYPX145 in the antisense direction downstream of the tomato fruit-specific expression promoter to obtain the plant binary vector pOSACC in which the ACC oxidase gene and the ACC synthase gene were fused. The vector exogenous gene expression unit contains two tobacco SAR sequences at both ends, which is conducive to the stable inheritance of the transgene. Totally 903 cotyledons and hypocotyls of tomato cultivars were used as explants and Agrobacterium tumefaciens was used for gene transformation. 105 kumatozoa-positive plants were obtained from 200 mg / L kanamycin selection and GUS-positive plants. The transgenic tomato fruits In the contemporary performance of the obvious storage characteristics. Two generations of DR-1 and DR-2, which had good performance, were obtained after 4 generations of storability and fruit agronomic traits. The ethylene production of two lines decreased significantly, which was 9.5% of the non-GMO. The shelf life of more than 50 days.