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以‘云香’水仙花瓣为试验材料,采用RT-PCR技术克隆出乙烯生物合成途径中的关键酶ACC氧化酶(ACO)基因的全长序列1 162 bp,命名为NtACO1(GenBank登录号为KX082935)。其开放阅读框(ORF)长度为939 bp,编码312个氨基酸。NtACO1蛋白包括ACO家族保守的DIOX_N和20G-FeⅡ_Oxy结构域。分子进化树分析表明,NtACO1蛋白与‘金盏银台’水仙亲缘关系最近。通过qRT-PCR检测NtACO1在‘云香’水仙花朵开放过程中的表达模式,结果表明,NtACO1在花瓣和副冠中的表达量随着花朵发育衰老而逐渐上升,并且各个时期花瓣中的表达量都高于副冠,推测NtACO1可能参与了‘云香’花的发育和衰老。构建了NtACO1的正、反义植物表达载体,并以烟草叶片为外植体,通过根癌农杆菌介导的遗传转化体系,获得NtACO1过表达转基因植株。转基因烟草与野生型相比缩短了营养生长期,开花较早,叶片较少。该研究为进一步探究水仙花ACO基因功能,以及延长水仙花花期的分子育种奠定了试验基础。
A total of 1 162 bp full-length cDNA sequence encoding the key enzyme ACC oxidase (ACO) in ethylene biosynthesis pathway was cloned by RT-PCR using ’Yunxiang’ daffodil petals as experimental material, named as NtACO1 (GenBank accession number KX082935 ). Its open reading frame (ORF) length of 939 bp, encoding 312 amino acids. The NtACO1 protein includes the conserved DIOX_N and 20G-FeII_Oxy domains of the ACO family. Molecular phylogenetic tree analysis showed that NtACO1 protein has the closest genetic relationship with ’Cinnabar Yintai’ Narcissus. The expression pattern of NtACO1 in the opening of ’Narcissus’ (Cymbidium citrinopileatus) Narcissus was detected by qRT-PCR. The results showed that the expression of NtACO1 increased in the petals and the vice crown as the flower developed and senescent, and the expression of NtACO1 Are higher than the deputy crown, suggesting that NtACO1 may be involved in the development and aging of the flower. The antisense plant expression vectors of NtACO1 were constructed. Tobacco leaves were used as explants to obtain NtACO1 overexpression transgenic plants through Agrobacterium tumefaciens-mediated genetic transformation system. Transgenic tobacco shortens the vegetative phase compared to the wild type, with earlier flowering and fewer leaves. This study laid the foundation for further exploring the function of ACO gene in daffodil and extending the molecular breeding of daffodil in flowering stage.