‘云香’水仙NtWRKYY2基因的克隆及功能分析
发布时间：2018-09-10 09:25
【摘要】：以‘云香’水仙为材料,采用PCR技术分离中国水仙WRKY转录因子家族成员NtWRKYY2(GenBank登录号为KX056496),其开放阅读框(ORF)长度为867bp,编码289个氨基酸。氨基酸序列比对及系统进化树分析显示,NtWRKYY2编码蛋白含1个WRKY结构域和C2H2锌指结构(Csx4Cx23HxH),属于第Ⅱd类WRKY转录因子。实时荧光定量PCR(qRT-PCR)分析显示,NtWRKYY2在‘云香’水仙应对盐胁迫中显著上调表达。利用InFusion克隆技术成功构建过表达载体pMDC140-NtWRKYY2,并采用农杆菌介导叶盘法转化烟草,获得11株卡那霉素抗性植株,转化子进一步PCR检测结果显示,其中有8株目的基因已成功导入烟草基因组中,转化率为72%。盐胁迫处理和叶绿素荧光参数分析显示,盐胁迫处理后NtWRKYY2过表达的转基因烟草萎蔫和黄化程度小于野生型植株,Fv/Fm值下降幅度小于野生型植株。研究表明,NtWRKYY2过表达的转基因烟草具有抵抗盐胁迫的能力。该研究为水仙抗盐转基因育种提供备选目的基因。
[Abstract]:NtWRKYY2 (GenBank accession number: KX056496), a member of the WRKY transcription factor family of Narcissus sinensis, was isolated by PCR from Narcissus sinensis. The open reading frame (ORF) length was 867 BP, encoding 289 amino acids. Amino acid sequence alignment and phylogenetic tree analysis showed that NtWRKYY2 encoded protein contained a WRKY domain and C2H2 zinc finger structure (Csx4Cx23HxH), which belonged to the second day WRKY transcription factor. Real time fluorescence quantitative PCR (qRT-PCR) analysis showed that NtWRKYY2 significantly up-regulated the expression of Narcissus in response to salt stress. The overexpression vector pMDC140-NtWRKYY2, was successfully constructed by InFusion cloning technique and transformed into tobacco by Agrobacterium tumefaciens leaf disc method. Eleven kanamycin resistant plants were obtained. Further PCR detection of the transformants showed that the transgenic plants were resistant to kanamycin. Eight of them were successfully introduced into the tobacco genome, and the transformation rate was 72%. The results of salt stress and chlorophyll fluorescence analysis showed that the degree of wilting and yellowing of transgenic tobacco with NtWRKYY2 overexpression after salt stress was less than that of wild type plants. The results showed that transgenic tobacco with NtWRKYY2 overexpression had the ability to resist salt stress. The study provides alternative target genes for salt-tolerant transgenic breeding of narcissus.
【作者单位】： 福建农林大学园艺学院;福建农林大学园艺植物遗传育种研究所;
【基金】：福建省种业创新与产业化工程项目(K8114001B)
【分类号】：Q943.2;S682.21
本文编号：2234039
[Abstract]:NtWRKYY2 (GenBank accession number: KX056496), a member of the WRKY transcription factor family of Narcissus sinensis, was isolated by PCR from Narcissus sinensis. The open reading frame (ORF) length was 867 BP, encoding 289 amino acids. Amino acid sequence alignment and phylogenetic tree analysis showed that NtWRKYY2 encoded protein contained a WRKY domain and C2H2 zinc finger structure (Csx4Cx23HxH), which belonged to the second day WRKY transcription factor. Real time fluorescence quantitative PCR (qRT-PCR) analysis showed that NtWRKYY2 significantly up-regulated the expression of Narcissus in response to salt stress. The overexpression vector pMDC140-NtWRKYY2, was successfully constructed by InFusion cloning technique and transformed into tobacco by Agrobacterium tumefaciens leaf disc method. Eleven kanamycin resistant plants were obtained. Further PCR detection of the transformants showed that the transgenic plants were resistant to kanamycin. Eight of them were successfully introduced into the tobacco genome, and the transformation rate was 72%. The results of salt stress and chlorophyll fluorescence analysis showed that the degree of wilting and yellowing of transgenic tobacco with NtWRKYY2 overexpression after salt stress was less than that of wild type plants. The results showed that transgenic tobacco with NtWRKYY2 overexpression had the ability to resist salt stress. The study provides alternative target genes for salt-tolerant transgenic breeding of narcissus.
【作者单位】： 福建农林大学园艺学院;福建农林大学园艺植物遗传育种研究所;
【基金】：福建省种业创新与产业化工程项目(K8114001B)
【分类号】：Q943.2;S682.21
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