烟草NtCBT基因启动子克隆及腺毛特异表达区域缺失分析

Cloning of NtCBT gene promoter and deletion analysis of glandular trichomes specific expression in Nicotiana tabacum

从栽培烟草(Nicotina tabacum)‘K326’中克隆了腺毛特异表达基因NtCBT 5′端上游序列,大小为990 bp,命名为pNtCBTa。经测序分析,pNtCBTa与Genbank中登录的林烟草NsCBT-2a基因启动子序列相似性为99.29%,启动子区顺式作用元件分析结果表明,pNtCBTa含有TATA-box、CAAT-box、ABRE、Box 4等调控元件以及MYC、MYB等转录因子结合位点;将克隆的启动子全长分别缺失360 bp(-479^-119)、160 bp(-279^-119)、6 bp(-198^-192)后与GUS基因融合,在转基因烟草T0代植株中,启动子三个片段的缺失均可驱动GUS基因在烟草腺毛及非腺毛组织中表达,推测腺毛特异表达基因NtCBT启动子中的6 bp-motif(-198^-192,CAGTTA)为决定NtCBT基因腺毛特异表达模式的核心区域之一,该6 bp序列的缺失可使NtCBT基因由腺毛特异表达变为组成性表达。

A 990 bp upstream sequence of glandular trichome specific expression gene NtCBT 5’-terminal was cloned from‘K326’(Nicotina tabacum)and named pNtCBTa.Sequence analysis showed that the sequence similarity between pNtCBTa and NsCBT-2a gene promoter was 99.29%similarity in Genbank.Analysis of the promoter’s cis-acting element showed that pNtCBTa contained MYC and MYB transcription factor binding sites,TATA-box,CAAT-box and other cis-acting elements.The full length of the promoter was deleted to 360 bp(-479^-119),160 bp(-279^-119),6 bp(-198^-192)respectively,and fused with GUS gene.In transgenic tobacco plants of T0 generation,the deletion of three promoter fragments could drive GUS gene expression in glandular trichomes and non glandular trichomes of tobacco.It is speculated that 6 bp-motif(-198^-192)in the promoter of glandular trichome specific expression gene NtCBT is one of the core regions to determine the expression pattern of NtCBT gene.The deletion of the 6 bp sequence can change the expression of NtCBT gene from glandular trichome specific expression to constitutive expression.