Abstract: BBX is a kind of important transcription factors which belongs to zinc finger proteins in plants. BBX regulate the expression of some genes involved in abiotic stress, and improve the resistance of plants to adversity stress. In order to explore the expression pattern of LaBBX gene responds to low temperature stress in Lepidium apetalum seedlings and analyse its codon bias. Based on the previous analysis of Lepidium apetalum transcriptone database BBX CDS sequence was cloned. The cDNA of this gene was 733 bp in length, encoding 242 amino acids, and was named as LaBBX. LaBBX contained two B-box zinc finger protein domains, its protein molecular weight was about 61.66 kDa and the theoretical isoelectric point was 5.09, with no signal peptide segments and no transmembrane region. The qRT-PCR analysis showed that LaBBX gene was rapidly and significantly up-regulated when seedlings of Lepidium apetalum were under low temperature stress, after 6 hours stress, the expression of LaBBX was 21 times as the control, and retained high level expression within 24 hours. The results suggest that LaBBX play an important role in regulating the resistance of low temperature stress in Lepidium apetalum seedling. Analysis of codon usage bias of the Lepidium apetalum LaBBX gene showed that: The ENc, CAI, and GC content of LaBBX were respectively 55.453%, 0.244%, and 46.78%, indicating that the codon bias level of LaBBX in Lepidium apetalum was low and biased toward the synonymous codons with A or T. According to the RSCU value, 8 codons showed hign-frequency. The CDS sequence and RSCU cluster analysis showed that the preference of LaBBX gene was closest to that of Brassicaceae burnett in dicotyledonous plants. Many factors play an important role in shaping codon usage bias of the Lepidium apetalum LaBBX gene such as mutation and selective pressure. The yeast expression system was found to be most suitable for heterologous expressing LaBBX gene, and Arabidopsis thaliana is the most ideal genetic transformation receptor of the LaBBX gene. This study provides important information for further studies on the heterologous expression and the function of the LaBBX gene in Lepidium apetalum.

Key words: Lepidium apetalum, BBX transcription factor, gene cloning, expression analysis, codon usage bias, cluster analysis

摘要： BBX是调控植物非生物胁迫相关基因表达,响应植物逆境胁迫的重要锌指蛋白类转录因子。为探究BBX在独行菜(Lepidium apetalum)幼苗耐受低温生长中的表达响应及其密码子偏好性,本研究基于独行菜转录组数据,筛选独行菜BBX编码基因的cDNA序列,并从独行菜幼苗中克隆获得全长733 bp的基因编码区序列,命名为LaBBX。序列分析表明,LaBBX由242个氨基酸组成,包含2个B-box锌指蛋白结构域,蛋白分子量为61.66 kDa,等电点为5.09,无信号肽和跨膜区。独行菜幼苗受低温胁迫时,LaBBX基因显著上调表达,6 h上调表达至21倍,且24 h内均呈现高水平表达,说明该转录因子可能在独行菜幼苗受低温胁迫时起重要作用。偏好性分析结果显示,独行菜LaBBX基因的ENc、CAI值和GC含量分别为55.453、0.244和46.78%,其密码子使用偏性较弱,相对偏好使用AT,高频密码子有8个,CDS序列及RSCU聚类分析中与十字花科植物最相近;其偏好性的形成与突变和自然选择等诸多因素相关,酵母菌表达系统更适合作为LaBBX基因的异源表达受体,拟南芥是适宜LaBBX基因的遗传转化受体。本研究为了解独行菜LaBBX基因的异源表达及基因功能提供了重要参考。

关键词: 独行菜, BBX转录因子, 基因克隆, 表达分析, 密码子偏性, 聚类分析