武军艳,马骊,方彦,张亚宏,马学才,刘丽君,牛早霞,李学才,李爱国,孙万仓.白菜型冬油菜NCED3基因、启动子的克隆及其表达分析[J].干旱地区农业研究,2020,38(5):64~70
白菜型冬油菜NCED3基因、启动子的克隆及其表达分析
Gene and promoter cloning and gene expression analysis of NCED3 from winter rapeseed (Brassica rapa L.)
  
DOI：10.7606/j.issn.1000-7601.2020.05.10
中文关键词:  NCED3基因  白菜型冬油菜  抗寒性  启动子克隆  序列分析  胁迫  基因相对表达量
英文关键词:NCED3 gene  winter rapeseed  cold resistance  promoter cloning  sequence analysis  stress  relative expression
基金项目:甘肃省自然科学基金(17JR5RA149)；国家现代农业产业技术体系（CARS-12）；甘肃省重大专项( 17ZD2NA016-4)；甘肃省现代农业产业技术体系( GARS-TSZ-1)
作者单位
武军艳 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
马骊 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
方彦 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
张亚宏 甘肃天水市农业科学研究所，甘肃 天水 741001 
马学才 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
刘丽君 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
牛早霞 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
李学才 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
李爱国 河北省农作物抗旱研究实验室，河北 衡水 053000 
孙万仓 甘肃农业大学农学院/甘肃省油菜工程技术研究中心，甘肃 兰州 730070 
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中文摘要:
      从2个不同抗寒性的冬油菜品种中克隆了NCED3基因及其启动子序列，并分析其在叶片和根中的表达，研究NCED3基因在冬油菜中的作用机理。结果表明，陇油6号的NCED3基因开放阅读框(ORF)长度为1 794 bp，编码597个氨基酸，分子量65.74 kD，理论等电点为5.81；天油2号的ORF与其长度相同，分子量为65.78 kD，理论等电点为5.94。2个蛋白都是亲水蛋白，具有疏水峰。根据预测，启动子具有生物过程中常见的顺式作用元件如CAAT-box等、分生组织表达CAT-box相关的顺式作用元件、-30 TATA box附近的核心启动子元件等，还有ABRE、TGA-element、CGTCA-motif、TGACG-element等激素响应元件，此外，还鉴定出低温响应元件（LTR）。2个品种启动子序列的相似度为99.38%，只有1个不同的顺式作用元件，即circadian,推测其与昼夜节律相关。低温、PEG及ABA处理后NCED3在叶片和根中的表达均高于对照，增加范围为0.07~6.48，且均于8 h达到峰值。与根系的表达特性相比，胁迫处理后叶片的基因表达均在2 h显著升高，天油2号的升高幅度（1.54~6.00）均大于陇油6号（0.04~1.95）。
英文摘要:
      For studying the role of NCED3 gene in winter rapeseed (Brassica rapa L.), we cloned NCED3 from two different cold\|tolerant winter rapeseeds cultivars, aligned the sequences, and identified its expression in leaves and roots. Results showed that the open reading frame (ORF) of NCED3 had 1 794 bp encoding 597 amino acids. NCED3 from Longyou 6 had a calculated molecular mass of 65.74 kD and theoretical isoelectric point of 5.81 while it from Tianyou 2 was 65.78 kD and 5.94. The two proteins were both hydrophilic proteins with a hydrophobic peak. Compared with NCED3 from Brassica napus, some bases changed and resulted in the changes of amino acids. Results showed that promoters had such hormone\|responsive elements as ABRE, TGA\|element, CGTCA\|motif, and TGACG\|element. In addition, low\|temperature responsiveness element (LTR), defense and stress responsiveness element (TC\|rich repeats) were identified. The similarity of the two promoters was 99.38% and there was only one different cis acting element, circadian, was speculated to be related to circadian rhythm in them. The expression of NCED3 in leaves and roots after low temperature, PEG, and ABA was higher than that in the control group with an increase range of 0.07~6.48, and reached a peak at 8 h. Compared with the expression characteristics in roots, the expression in leaves significantly increased at 2 h after stress treatment, and the increase range in Tianyou 2 (1.54~6.00) was greater than that in Longyou 6 (0.04~1.95).
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