白菜生物钟相关基因BrPRR7的克隆及表达模式分析

Cloning and expression pattern analysis of circadian clock related gene BrPRR7 in Brassica rapa

白菜生物钟基因的识别及多拷贝间的分析将有助于解析多倍化基因组带来的更复杂生物钟调控机理。本研究通过同源克隆方法获得了位于大白菜A10染色体的BrPRR7a基因。生物信息学分析表明,该基因和位于A02染色体的另一个拷贝BrPRR7b基因的DNA序列主要差异存在于5’UTR和启动子区域。蛋白进化分析表明,BrPRR7a和BrPRR7b之间以及相对于拟南芥已经产生分化。烟草亚细胞定位结果显示,GFP-BrPRR7a融合蛋白的绿色荧光信号定位于细胞核,支持其作为转录因子的负调控能力。4个转录组数据集的分析表明,在BrPRR7基因对光暗导引和温度循环导引后的内源昼夜节律性方面,BrPRR7a比BrPRR7b到达转录高峰更早,且BrPRR7a比BrPRR7b的最大转录水平更高;BrPRR7a和BrPRR7b基因在轻度干旱处理下的最大转录水平有少量的上升,暗示了它们在干旱胁迫状态下对植物生命活动进行微调的潜在作用。综合以上结果,推测BrPRR7a和BrPRR7b基因在白菜中均保留了拟南芥生物钟AtPRR7基因的部分功能,此外,也具有潜在的进化出新功能的可能性。该研究为白菜生物钟基因的功能及分子调控机制研究奠定了一定的基础。

The identification of circadian clock genes and the analysis of multiple copies of circadian clock genes in Brassica rapa are helpful to reveal the complex circadian clock model related to polyploid genome. BrPRR7 a gene located on A10 chromosome of Brassica rapa was obtained by homologous cloning. Bioinformatics analysis showed that the DNA sequence difference between BrPRR7 a and BrPRR7 b was mainly located in 5’UTR and promoter region. Phylogenetic analysis showed that the sequences of BrPRR7 a and BrPRR7 b have differentiated. Subcellular localization of BrPRR7 a showed that the green fluorescent signal of GFP-BrPRR7 a fusion protein was in the nucleus, which supported its negative regulatory ability as a transcription factor. Four transcriptome data sets reflected the endogenous circadian rhythm of BrPRR7 gene after the entrainment of light/dark and temperature cycling. The transcription peak of BrPRR7 a was earlier than that of BrPRR7 b, and the maximum transcription level of BrPRR7 a was higher than that of BrPRR7 b. Under mild drought stress, the maximum transcriptional levels of BrPRR7 a and BrPRR7 b genes both were slightly higher than those under well-watered condition, suggesting their potential role in the fine-tuning of plant life activities under drought stress. Based on the above results, we speculate that BrPRR7 a and BrPRR7 b genes retain some functions of the Arabidopsis circadian clock At PRR7 gene in Brassica rapa, and they also have the potential of evolving new functions. This study lays the foundation for the research on the gene function and molecular regulation mechanism of clock genes in Brassica rapa.