• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

牡丹花青素合成基因PqMYB113启动子克隆及上游调控因子筛选

Cloning and Upstream Regulatory Factors of Anthocyanin Synthesis PqMYB113 Promoter in Paeonia qiui

  • 摘要:
    目的 PqMYB113转录因子是促进牡丹(Paeonia qiui)叶片花青素的合成、使牡丹叶片变红的关键基因,但其上游调控机制尚不明确,筛选调控PqMYB113的转录因子对卵叶牡丹叶色的调控机理的研究至关重要。
    方法 以卵叶牡丹叶片为试验材料,通过Gateway技术构建酵母单杂交cDNA文库,克隆PqMYB113基因的启动子,在氨基酸缺陷培养基中添加3-氨基-1,2,4-三唑(3-amino-1,2,4-triazole,3-AT)对启动子进行自激活验证和浓度筛选,利用酵母单杂交技术筛选与该基因启动子结合的上游调控因子。
    结果 PqMYB113的自激活抑制浓度为90 mM 3-AT,构建的cDNA表达文库库容5.5×106 cfu·mL−1,总克隆数为1.1×107 cfu,通过酵母单杂实验筛选到93个PqMYB113的上游调控因子,包含2个转录因子,1个染色质重塑因子互作因子,1个染色质重塑因子,发现其启动子中含有光、胁迫等环境响应元件和脱落酸、茉莉酸、乙烯等激素应答元件,并包含MYB、MYC等多个转录因子的结合位点。
    结论 该研究构建了卵叶牡丹酵母单杂cDNA文库,通过对PqMYB113的上游调控因子进行筛选,并预测其启动子上的作用元件,初步解析了PqMYB113的上游调控机制,为进一步研究牡丹花青素合成的调控机理和优良牡丹品种育种提供分子基础。

     

    Abstract:
    Objective PqMYB113 transcription factor, a key gene promoting anthocyanin synthesis that gives leaves the red color, and the upstream regulatory mechanism of Paeonia qiui were studied.
    Methods A yeast one-hybrid cDNA library was constructed using the Gateway technology. Promoter of PqMYB113 of P. qiui leaves was cloned and verified. 3-amino-1,2,4-triazole (3-AT) was added to the amino acid-deficient medium to self-activate and screen concentration of the promoter. Upstream regulatory factors binding on the promoter were identified using yeast one-hybrid technology.
    Results The self-activation inhibition concentration of PqMYB113 was 90mmol·L−1 3-AT. The constructed cDNA expression library had a capacity of 5.5×106 cfu·mL−1 and a total clone number of 1.1×107 cfu. Through the yeast one-hybrid experiments, 93 upstream regulatory factors of PqMYB113 were screened. They included two transcription factors, one chromatin remodeling factor interaction factor, and one chromatin remodeling factor. The promoter contained environmental response elements on light and stress, hormone response elements on abscisic acid, jasmonic acid, and ethylene, and multiple binding sites for transcription factors, such as MYB and MYC.
    Conclusion A yeast one-hybrid cDNA library for P. qiui was constructed. The functional elements on the promoter in the identified upstream regulatory factors of PqMYB113 were predicted. The results provided for further studies on the regulatory mechanism of anthocyanin synthesis and breeding of superior P. qiui varieties.

     

/

返回文章
返回