紫心甘薯转录因子IbMYB1基因启动子的克隆及功能分析

Cloning and functional analysis of the promoter of gene encoding the transcription factor Ib MYB1 in purple-fleshed sweet potato

[目的]获得紫心甘薯Ib MYB1基因上游启动子序列,对其进行生物信息学分析和功能验证。[方法]利用hi Tail-PCR技术克隆获得Ib MYB1基因5’端上游的一段DNA序列,命名为PIb MYB1。使用Plant CARE和PLACE在线软件对其进行生物信息学分析,并通过瞬时转化烟草叶片和稳定转化拟南芥植株进行GUS组织化学活性检测,以验证该启动子的功能。[结果]成功克隆获得了长度为2183 bp的PIb MYB1片段,生物信息学分析结果表明该片段具有CAAT-BOX和TATA-BOX等启动子核心元件,还具有光、糖和赤霉素的响应元件以及MYB、b HLH等转录因子的结合位点。GUS组织化学活性检测结果表明,PIb MYB1(-2183~-1 bp)和5’缺失突变体PIb MYB1F1(-2000~-1 bp)、PIb MYB1F2(-1500~-1 bp)均能驱动GUS基因的表达。[结论]成功克隆获得了Ib MYB1基因的启动子序列,其驱动Ib MYB1基因表达的主要区域位于-2183~-1000 bp区段,该区域具有核心启动子元件、环境因子响应元件和MYB、b HLH等转录因子结合位点。

[Objective]To obtain the promoter of Ib MYB1 from purple-fleshed sweet potato,and its bioinformatics analysis and functional verification were carried out.[Method]The DNA sequence at the upstream of the 5’end of the Ib MYB1 was cloned by hi Tail-PCR,and therefore we named it PIb MYB1.Plant CARE and PLACE were used to predict the cis-acting elements of the promoter sequence.GUS assay was performed by transiently transforming tobacco leaves and stably transforming Arabidopsis to verify the function of the promoter.[Result]A promoter sequence with the length of 2183 bp was successfully cloned.Bioinformatics analysis showed that this sequence has core promoter elements CAAT-BOX and TATA-BOX,as well as response elements for light,sugar,gibberellin,and binding sites for transcription factors such as MYB and b HLH.The results from GUS assay demonstrated that PIb MYB1(-2183~-1 bp)and PIb MYB1 F1(-2000~-1 bp),PIb MYB1 F2(-1500~-1 bp)could drove the expression of GUS gene.[Conclusion]The promoter sequence of the Ib MYB1 was successfully cloned.The expression of Ib MYB1 was driven by PIb MYB1 and the core active region was mainly located in the range of-2183~-1000 bp,which contains core promoter elements,environmental factor response elements and binding sites of MYB,b HLH transcription factors.