为明确WRKY转录因子在云香水仙中的功能,该研究以云香水仙为材料,克隆了WRKY基因,命名为NtWRKYY1(GenBank登录号为KX0564; 95)。序列分析显示,NtWRKYY1基因开放阅读框(ORF)长度为510; bp,编码169个氨基酸。多序列比对和系统进化树分析显示,NtWRKYY1编码蛋白含1个WRKY结构域和C_2H_2锌指结构(Cx_4Cx_(2; 3)HxH),与AtWRKY57聚在一起,属于第Ⅱc类WRKY转录因子。组织表达和时空表达分析显示,NtWRKYY1基因在花中表达量高于根和叶,; 且在花瓣及副冠中的表达量随开花过程(花蕾期、始花期、盛花期、衰败期)呈上升趋势。植物激素和非生物胁迫分析显示,NtWRKYY1基因受脱落酸(AB; A)、高温、干旱和盐诱导,受茉莉酸甲酯(JA)抑制,表明NtWRKYY1基因可能在云香水仙花朵的衰老过程中起正调节作用,同时参与云香水仙ABA、; JA等激素信号转导及高温、干旱、盐碱等非生物胁迫过程的调控。利用In-Fusion克隆技术成功构建过表达载体pMDC140-NtWRKYY1,并; 采用农杆菌介导叶盘法转化烟草。RT-PCR和GUS染色结果显示,目的基因已成功导入烟草基因组中。WRKY transcription factors play an important adjusting role in the; process of plant growth and development,hormone signal transduction and; abiotic stress response.To clarify the gene Narcissus tazetea; var.Yunxiang,we cloned the NtWRKYY1gene(GenBank accession; No.KX056495)based on Yunxiang,analyzed the gene sequence; features,evolutionary relationship and expression characteristics,; constructed the expression vector before transformed into; tobacco.Sequence analysis revealed that the length of NtWRKYY1gene open; reading frame(ORF)is 510bp,encoding apolypeptide of 169amino; acids.Multiple alignments and phylogenetic analysis showed that; NtWRKYY1protein containing one WRKY consecutive structural domain and; C_2H_2type zinc finger(Cx_4Cx_(23)HxH)belong toⅡc sub-group of WRKY; transcription factor together with Arabidopsis AtWRKY57.Tissue-specific; expression and temporal and spatial expression showed that; NtWRKYY1inYunxianghad a much higher expression in flowers than that in; roots and leaves,and a rising express trend in petal and corona of; alabastrum stage,early flowering stage,full bloom stage and faded; stage.NtWRKYY1can be induced by abscisic acid(ABA), high; temperature,drought and saline,and restrained by methyl; jasmonate(JA)through hormones and abiotic stresses analyzing.We can; concluded that NtWRKYY1gene may play a regulating role during the flower; senescence process inYunxiang,involve in the hormone signal transduction; of ABA,JA and the abiotic stress regulation of high temperature,drought; and saline at the same time.In addition,we constructed overexpressing; vector pMDC140-NtWRKYY1using In-Fusion cloning technique,transformed; into tobacco by the method of Agrobacteriumthough leaf disc; transformation.The carrier of PCR and GUS staining results indicated the; resistant plantlets were positive.This study will make a good foundation; for further exploring the function of the WRKY transcription factor; inYunxiang.福建省种业创新与产业化工程项
