摘要：
In this research,a gene named NtACOY2 encoding a novel ACC Oxidase (ACO) protein was isolated using RT-PCR from 'Yunxiang' (Narcissus tazetta var.'Yunxiang').The full length cDNA of NtACOY2 consisted of an open reading frame (ORF) of 936 bp,and encoded a 35.83 kD protein with 311 amino acids.Bioinformatics analysis indicated that NtACOY2 contained a typical DIOX_N and 20G-Fe Ⅱ_ Oxy binding domain.Evolutionary analysis indicated that the amino acid sequence encoded by NtACOY2 was highly conserved.Real-time fluorescence quantitativePCR results revealed that the expression of NtA-COY2 in the petal and deputy crown is increasing with the senescence of 'Yunxiang'.The expression of NtACOY2 in petals in each period was higher than that in the deputy crown,and the highest expression in petals is at bud stage.So it is inferred that NtACOY2 may be involved in the development of 'Yunxiang' and related to flower senescence.The expression vector of NtACOY2 was successfully constructed by PCR and identified by restriction enzyme digestion reactions.And the sense plant express vector of NtACOY2 was used to transform the tobacco through Agrobacterium-mediated procedure.PCR detection showed that a total of 15 transformed plants were amplified fragments with the same size as the positive controls.The result of RT-PCR identification showed that 12 transgenic tobacco plants were obtained.Two randomly selected transgenic plants (Z1,Z2) and wild type (WT) exactly the same condition of transplanting under greenhouse cultivation,the results showed that the flowering time of Z1,Z2 were 8 d and 7 d earlier flow-ering compared with control plants,and the flower number of transgenic tobacco were more than the wild type,it is showed that NtACOY2 could normally expressed at the transcriptional level.The experimental results laid the foundation for further research on the function of NtACOY2 gene.%利用RT-PCR技术,从‘云香’水仙中克隆了1个新的ACO(ACC氧化酶)基因(NtACOY2).NtACOY2基因全长975 bp,开放阅读框(ORF)936 bp,编码311个氨基酸残基,蛋白分子量为35.83 kD.蛋白结构预测发现,该蛋白含有典型的ACO家族保守的DIOX_N和20G-FeⅡ_Oxy结构域.进化分析表明,NtACOY2编码的氨基酸序列高度保守.实时荧光定量PCR表明,NtACOY2在花瓣和副冠中的表达均随着‘云香,水仙花的衰老而逐渐上升,并且各个时期花瓣中的表达量均高于副冠,在花蕾期的花瓣中表达量达到最高,表明NtACOY2基因可能参与‘云香’水仙花的发育,并且与其花衰老密切相关.通过PCR和酶切反应鉴定,成功构建了NtACOY2基因的正反义植物表达载体,将正义载体经农杆菌介导转化烟草,PCR检测显示共有15株转化植株扩增出了与阳性对照大小相同的片段,进一步用RT-PCR鉴定阳性转化植株,最终获得了12株转基因烟草.随机选取2株转基因植株(Z1、Z2)与野生型(WT)完全相同条件下移栽温室培养,结果Z1、Z2分别比WT提前8和7d开花,且转基因烟草花朵开放数量均多于野生型,表明NtACOY2在转录水平能够正常表达.实验结果为进一步研究NtACOY2基因的功能奠定了基础.
