Abstract: In order to explore Dof genes related to low nitrogen stress in oat, we obtained a differentially expressed AsDof6.5 gene from strong tolerance to the low N stress variety ‘Jiayan 2’ and poor tolerance to the low N stress variety ‘Qingyongjiu 016’ through the transcriptional sequencing in low nitrogen stress. Promoters and coding regions of AsDof6.5 gene were amplified using sequencing PCR. Bioinformatics analysis showed that the length of the AsDof6.5 gene contained an 858 bp open reading frame, encoded 285 amino acids, had a theoretical isoelectric point of 8.63 and a predicted molecular weight of 29 550.01 Da. The results showed that the AsDof6.5 promoter region contained cis-acting elements which were related to adversity stress, such as abscisic acid, jasmonic acid, and defense against stress response. Protein sequence alignment showed that AsDof6.5 was highly conserved structural domains of zf-Dof, belonging to Dof gene family. Phylogenetic tree and protein sequence alignment analysis showed that the AsDof6.5 protein was relatively the closest to LrDof4 and LpDof4, and the homology comparison concordance with other Poaceae Dof proteins was 73.99%, but the concordance of the zf-Dof structural domain was as high as 99.50%, suggesting that it might have a similar function. Subcellular localization results showed that AsDof6.5 was localized in the nucleus and cell membrane. Quantitative PCR results showed that the expression of AsDof6.5 gene significantly decreased （P<0.01） under low-nitrogen stress in strong tolerance to the low N stress variety ‘Jiayan 2’ and poor tolerance to the low N stress variety ‘Qingyongjiu 016’, it was almost not expressed in roots, and the expression in leaves was about 100 times higher than that in roots. Therefore, it hypothesized that AsDof6.5 gene might play an important role in the cell nucleus and cell membrane during the process of low nitrogen tolerance in oats, which laid a foundation for exploring the function and regulatory mechanism of AsDof6.5 gene.

Key words: Oat, Low nitrogen stress, AsDof6.5, Gene expression

摘要： 为筛选与燕麦（Avena sativa L.）耐低氮相关的Dof类基因,本研究以燕麦耐低氮品种‘加燕2号’和不耐低氮品种‘青永久016’为试验材料,在低氮胁迫下进行转录组测序获得一个与耐低氮相关的差异表达基因AsDof6.5。通过PCR扩增获得AsDof6.5基因编码区无内含子、开放阅读框含有858 bp和编码285个氨基酸,理论等电点为8.63和蛋白分子量为29 550.01 Da,并获得AsDof6.5基因启动子区域序列2227 bp。启动子分析预测表明,该区段包含脱落酸、茉莉酸以及防御应激反应等与逆境胁迫相关的多个顺式作用元件。蛋白质序列分析表明,AsDof6.5具有高度保守的zf-Dof结构域,属于AsDof基因家族成员。系统进化树及同源比对表明,燕麦AsDof6.5蛋白与黑麦草（Lolium rigidum L.）和多年生黑麦草（Lolium perenne L.）同源蛋白亲缘关系最近,且与其他禾本科植物Dof蛋白同源比较一致性为73.99%,但zf-Dof结构域一致性高达99.50%,具有高度保守性,说明可能具有类似的功能。亚细胞定位结果显示AsDof6.5蛋白定位在细胞核和细胞膜中。荧光定量PCR（RT-qPCR）结果表明,低氮胁迫下AsDof6.5基因的表达量极显著下降（P<0.01）,且根中几乎不表达,叶中的表达量是根中的100倍左右。因此推测,在燕麦耐低氮过程中,AsDof6.5基因可能在细胞核和细胞膜中发挥重要作用,为探索AsDof6.5基因的功能及调控机制奠定基础。

关键词: 燕麦, 低氮胁迫, AsDof6.5, 基因表达