番茄LeDREB1转录因子基因RNAi载体的构建和鉴定

Identification and Construction of RNA Interfere Vector on Tomato Transcription Factor Gene LeDREB1

利用rd29A基因启动子的DRE元件从番茄(丽春)cDNA文库中通过酵母单杂交技术筛选得到转录因子基因LeDREB1。核苷酸序列测定结果表明,该基因片段全长1 782 bp,具有900 bp的cDNA开放阅读框序列,编码300个氨基酸。该基因属于AP2/EREBP家族[1],可能调控许多逆境应答基因的表达。运用RNA干扰的思路,设计特异性引物,扩增正向和反向基因片段。将LeDREB1正向+反向基因片段插入到pCAMBIA2300-OCS的35s启动子下游,构建干扰载体pCAM-RNAi-LeDREB1,通过酶切和测序鉴定证明目的片段与载体片段连接正确。这一载体的成功构建为进一步研究LeDREB1的功能和调控作用机制提供有效的材料与技术支撑。

Transcription factor LeDREB1,was isolated by yeast one-hybrid system through the DRE cis-acting element in the promoter of rd29A gene hybridizing a cDNA expression library of tomato seedlings.DNA sequence analysis showed that the cloned fragment consisted of 1 782 bp with an open reading frame of 900 bp encoding 300 amino acids.LeDREB1 belongs to AP/EREBP family which might control the expression of stress response genes.Based on the idea of RNA interference,a PCR fragment including sense and antisense gene was obtained using the specific primers by PCR,and constructed the recombinant plasmid pCAM-RNAi-LeDREB1,the fragment of anti-sense gene＋plus-sense gene was inserted to the downstream of 35S promoter in the binary vector pCAMBIA2300-OCS.It was proved that the connection between the goal fragment and the vector fragment was correct by identification of enzyme-cutting and sequencing.Successful construction of pCAM-RNAi-LeDREB1 would provide effective supports for research materials on function and mechanism of gene LeDREB1 in the future.