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新疆野苹果 MsICE1 和 MsCBF1 转录因子的克隆及功能验证
Thesis Advisor王玉成
Degree Grantor中国科学院大学
Place of Conferral新疆乌鲁木齐
Degree Discipline工程硕士
Keyword新疆野苹果 Msice1 转录因子 Mscbf1 转录因子 基因特性 胁迫抗性 Malus Sieversii Msice1 Mscbf1 Genetic Characteristics Stress Resistance

抗冷过程是一个由众多基因参与的复杂生物过程。目前,已经基本清楚,冷反应基因激活的一条主要途径是 ICE1-CBF 调节通路。在这个信号通路中,ICE1 是 CBFs 的上游关键的 bHLH 类调控因子,在低温条件下 ICE1 可以诱导CBFs 基因的表达,进而激活其下游 COR 靶基因的表达,增强植物的坑冷性。新疆野苹果是现代栽培苹果的祖先,能耐极端最低温-35°C~-30°C,耐旱力较强,并能耐瘠薄土壤,是非常珍贵的天然基因资源库。但是近年来由于人为及虫害的原因,新疆野苹果的面积急剧锐减,因此开展新疆野苹果抗逆机制的研究以及抗逆基因的深入挖掘尤为重要。本研究以新疆野苹果 RNA 为模板, RT-PCR 扩增出 ICE1、 CBF1 基因的cDNA 序列,分别命名为 MsICE1、 MsCBF1。 MsICE1 基因完整开放阅读框 ORF长 1 596bp,编码 531 位氨基酸,分子量为 57.29kD,理论等电点为 5.42。亚细胞定位分析显示, MsICE1 蛋白定位于细胞核。酵母自激活分析显示, MsICE1转录因子的激活活性区在 N 端第 46~133 位氨基酸之间。基因表达分析显示,MsICE1 基因在根、茎、叶中均有表达,且在叶中表达量最高。在干旱(300mmol.L-1 甘露醇)、盐(200 mmol.L-1 NaCl)、冷(4°C)等胁迫处理下, MsICE1基因的表达量均无明显变化。MsCBF1 基因完整开放阅读框 ORF 长 660bp,编码 219 位氨基酸,具有 CBFs类转录因子典型的 AP2 保守结构域。亚细胞定位显示 MsCBF1 蛋白作用于细胞核;转录自激活活性分析显示 MsCBF1 蛋白具有转录激活活性,其激活活性区域位于 C 端第 166~219 位氨基酸之间。基因表达分析显示, MsCBF1 基因在根、茎、叶中均有表达,且表达量相似。 MsCBF1 基因响应盐(200 mmol.L-1 NaCl)、冷(4°C)胁迫诱导,不响应干旱(300 mmol.L-1 甘露醇)胁迫诱导。

Other Abstract

In plant, the cold resistance process is a complex bioprocess involving a lot ofgenes. At present, it is almost certain that the main path of activatingcold-responsive genes is ICE1-CBF regulatory pathway. ICE1 is a bHLHtranscription factor that binds to the CBFs promoter and activates its expression.CBF proteins can then activate the expression of downstream COR (cold-responsive)genes and confer enhanced freezing tolerance in plant.Malus sieversii (Ledeb.) Roem in Xinjiang of China is the ancestor ofcultivated apples, showing a strong adaptability to abiotic stress, which is anextreme precious natural gene pool. However, in recent years, the area of M.sieversii in Xinjiang has been sharply reduced due to the influences of unreasonablehuman acitivity and pest and disease damage. Therefore, it is significance inunderstanding the resistance mechanism of M. sieversii and exploration of powerfulresistant genes.In this study the M.sieversii CBF1(MsCBF1) and ICE1(MsICE1)genes wereobtained by RT-PCR. The open reading frame (ORF) of MsICE1 is 1596bp in length,encoding an amino acid sequence of 531 residues. The result of subcellularlocalization indicated that MsICE1 was targeted to nucleus. The transcriptionalactivation activity analysis indicated that the N-terminal region (46-133) of MsICE1has transcriptional activation ability. The result of qRT-PCR showed that MsICE1gene was expressed in root, stem and leaf, and the highest expression was found inleaves. MsICE1 did not response to drought stress (300 mmol.L-1 Mannitol)、 saltstress (200 mmol.L-1 NaCl) and cold treatment(4℃).The open reading frame (ORF) of MsCBF1 is 660bp in length, encoding anamino acid sequence of 219 residues. The MsCBF1 protein contained a typical AP2domain. The result of subcellular localization indicated that MsCBF1 was targetedto nucleus. The transcriptional activation activity analysis indicated that theC-terminal region (166-219) of MsCBF1 has transcriptional activation ability. The result of qRT-PCR showed that MsCBF1 gene was expressed in root、 stem and leaf,and the expression was similar.MsCBF1 was up-regulated under salt stress (200mmol.L-1 NaCl) and cold treatment(4℃), but did not response to drought stress(300 mmol.L-1 Mannitol).

Subject Area基因组学
Document Type学位论文
Recommended Citation
GB/T 7714
张克闯. 新疆野苹果 MsICE1 和 MsCBF1 转录因子的克隆及功能验证[D]. 新疆乌鲁木齐. 中国科学院大学,2018.
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