‘云香’水仙ACC氧化酶基因克隆及遗传转化

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。

Cloning of ACC Oxidase Gene from Narcissus tazetta var. 'Yunxiang' and Its Transformation

ACC oxidase (ACO) is a key enzyme in the biosynthesis pathway of ethylene.In order to explore the characteristics and function of the ACO gene in narcissus,the following work has been done in this study.Petals of Narcissus tazetta var.'Yunxiang'were used as experimental materials,a full-length 1 162 bp sequence was obtained by RT-PCR and named as NtACO1 (GenBank KX082935).The open reading frame (ORF) length of NtACO1 is 939 bp,which encodes 312 amino acids.Amino acid domain analysis showed that this gene has DIOX_N and 20G-Fe Ⅱ_Oxy domains and was identified as a member of the ACO protein family.Molecular phylogenetic tree analysis showed that ACO gene of'Yunxiang'was closely related to that of Narcissus tazetta var.chinensis,qRT-PCR results showed that the expression of NtACO1 increased gradually with Narcissus flower development and senescence,and the expression level in the petals were higher than that in contemporaneous coronas,indicating that the NtACO1 gene may be involved in the flower development and senescence of'Yunxiang'We obtained several NtACO1 overexpression transgenic tobacco plants using the Agrobacterium mediated.Compared with wild type,the vegetative growth stage of transgenic tobaccos was reduced,as well as the transgenic tobaccos showed early flowering and less leaves.This study lays an experimental foundation for further research on the function of ACO gene and provide a theoretical basis for the molecular breeding in prolonging plant florescence of Narcissus.