油茶两个响应干旱NAC转录因子的克隆、亚细胞定位及自激活检测

Cloning, subcellular localization, and self-activation detection of two NAC transcription factors in response to drought for Camellia oleifera

关键词:  克隆，干旱胁迫，亚细胞定位，酵母自激活，ABA途径

Key words:  cloning, drought stress, subcellular localization, yeast self-activation, ABA pathway

摘要:

干旱胁迫是影响油茶生长发育、产量和品质的一类主要的非生物胁迫。NAC转录因子在植物响应干旱、盐碱等非生物胁迫反应中具有重要的调控作用。为探究NAC转录因子在油茶响应干旱胁迫中的调控机制，以两年生油茶苗为材料，通过TA克隆得到CoNAC5与CoNAC79的CDS序列，对其进行生物信息学分析、亚细胞定位及自激活分析。采用qRT-PCR检测CoNAC5与CoNAC79基因表达的组织特异性及PEG模拟干旱和ABA处理下的表达模式。结果表明：(1)基因结构分析显示，CoNAC5与CoNAC79的CDS长分别为1 044 bp和990 bp，分别编码348和330个氨基酸，理论等电点分别为8.86和8.57，蛋白的不稳定系数分别为41.35和37.47，均无跨膜结构域，分别与柿子和荔枝的同源性最高。亚细胞定位显示CoNAC5与CoNAC79均定位在细胞核上。(2)酵母自激活检测显示，CoNAC5与CoNAC79全长蛋白和N端结构域无自激活活性，但C端结构域均具有自激活活性。(3)CoNAC5与CoNAC79表达具有明显的组织特异性，主要在根和种仁中高表达；PEG模拟干旱和外源施加ABA处理油茶苗发现，CoNAC5和CoNAC79表达量均显著高于对照；CoNAC79的表达量在ABA处理48 h后下降，而在PEG处理下显著高于对照。综上认为CoNAC5与CoNAC79其N端可能存在抑制区域，从而阻碍了全长序列的转录；油茶两个NAC基因可能通过ABA合成途径间接参与干旱胁迫响应过程；在胁迫持续发生时，CoNAC79还可能通过其他途径直接参与干旱胁迫响应过程。该研究的结果为进一步探究NAC在油茶响应干旱胁迫过程中的作用提供了科学依据。

Abstract:

Water deficiency was an important factor for the development, yield and quality of Camellia oleifera. NAC transcription factors are widely involved in drought and salt-stress induced signal transduction in plants. Exploreed the role of NAC transcription factors in the drought stress responsed of C. oleifera uses two-year oil tea seedlings as materialls. The CDS sequences of CoNAC5 and CoNAC79 were obtained from through TA cloning. Bioinformatics, subcellular localization and self-activation were performed. qRT-PCR was used to determine the tissue specificity of CoNAC5 and CoNAC79 gene expression and the expression PEG and ABA at different treatment times. The results were as follows: (1) Gene structure analysis showed that CoNAC5 and CoNAC79 were 1 044 bp and 990 bp in length, respectively, encoding 348 and 330 amino acids. Their theoretical Isoelectric points are 8.86 and 8.57, respectively. The instability coefficients of the proteins were 41.35 and 37.47, respectively. No transmembrane domain was found between the two genes, the highest homology with persimmon and lychee respectively. Subcellular localization showed that both CoNAC5 and CoNAC79 were located in the nucleus. (2) Yeast transcriptional activation activity analysis revealed that CoNAC5 and CoNAC79 did not have self-activation activity in the full-length proteins and N-terminal domain. However, the C-terminal domain exhibited self-activating activity. (3) The expression of CoNAC5 and CoNAC79 has significant tissue specificity and mainly expressed in roots and kernels; When PEG simulated drought and exogenous ABA treated C. oleifera seedlings, the expression levels of CoNAC5 and CoNAC79 were significantly higher than the control; Furthermore, the expression level of CoNAC79 decreased after 48 h under ABA treatment, but significantly higher than the control under PEG treatment. In summary, it is believed that there may be an inhibitory region at the N-terminus of CoNAC5 and CoNAC79, which hinders the transcription of the full-length sequence; indicating that the two NAC genes in C. oleifera may be probably involved in ABA-mediated drought stress response; CoNAC79 can also participate in the drought stress response through ABA-independent signaling pathway. This study provided a scientific basis for further exploring the role of NAC in the response of C. oleifera to drought stress.