象草Dof转录因子的全基因组分析及其对低温胁迫的响应

doi:10.13560/j.cnki.biotech.bull.1985.2025-1105

摘要/Abstract

摘要：

目的解析象草（Cenchrus purpureus）Dof转录因子家族特征及低温胁迫响应机制，对CpDof基因进行全基因组鉴定，为挖掘象草耐寒候选基因提供支撑。方法基于象草基因组数据，通过保守结构域检索鉴定CpDof基因，结合生物信息学方法分析其理化性质、染色体定位、系统发育及顺式作用元件特征；以‘川畜4号’象草为材料，经4 ℃低温胁迫处理后，通过转录组测序和荧光定量PCR分析基因表达特征，克隆关键基因并构建酵母表达载体，开展异源表达耐寒功能验证。结果在象草的基因组鉴定到68个CpDof基因，不均匀分布于14条染色体，共线性分析发现，CpDof间存在51对片段重复和2对串联重复，亚细胞定位预测结果显示，几乎所有的CpDof家族成员定位于细胞核。低温胁迫诱导下，共有28个CpDof基因显著上调表达，其中CpDof17和CpDof28的相对表达量较为突出，分别在根和茎中高表达，且低温可增强二者在根及叶片中的表达。酵母异源表达实验显示，过表达CpDof28基因可显著提升酿酒酵母的低温耐受性，CpDof17无明显效应。结论 CpDof28可能作为关键调控因子在象草低温胁迫响应中发挥核心作用，明确了象草Dof基因家族核心特征，筛选出耐寒候选基因CpDof28，为象草抗寒分子育种及品种改良提供重要基因资源与理论依据。

关键词: 象草, Dof, 低温胁迫, 转录因子, 表达分析, CpDof28, 酵母异源表达, 低温耐受性

Abstract:

Objective To analyze the characteristics of the Dof transcription factor family in elephant grass (Cenchrus purpureus), and its response mechanism to cold stress, and conduct genome-wide identification of CpDof genes, and providing support for mining cold-tolerant candidate genes in elephant grass. Method Based on the elephant grass genome data, CpDof genes were identified through conserved domain search. Bioinformatics methods were used to analyze their physicochemical properties, chromosome localization, phylogeny, and cis-acting element characteristics. Taking ‘Chuanxu 4’ elephant grass as the material, after the treatment by 4 ℃ cold stress, transcriptome sequencing and quantitative real-time PCR were used to analyze gene expression characteristics. Key genes were cloned to construct yeast expression vectors, and heterologous expression experiments were conducted to verify cold tolerance function. Result A total of 68 CpDof genes were identified in the elephant grass genome, unevenly distributed on 14 chromosomes. Collinearity analysis revealed 51 pairs of segmental duplications and 2 pairs of tandem duplications among CpDof genes. Subcellular localization prediction showed that almost all CpDof family members were localized in the nucleus. Under cold stress induction, 28 CpDof genes were significantly upregulated, among which CpDof17 and CpDof28 had relatively prominent expressions. They were highly expressed in the roots and stems, respectively, and cold stress enhanced their expression in the roots and leaves. Yeast heterologous expression experiments showed that the overexpression of CpDof28 significantly improved the cold tolerance of Saccharomyces cerevisiae, while CpDof17 had no obvious effect. Conclusion CpDof28 may act as a key regulatory factor playing a core role in the response of elephant grass to cold stress. This study clarifies the core characteristics of the Dof gene family in elephant grass and screenes the cold-tolerant candidate gene CpDof28, providing important genetic resources and theoretical basis for cold-resistant molecular breeding and variety improvement of elephant grass.

Key words: Cenchrus purpureus, Dof, cold stress, transcription factor, expression analysis, CpDof28, heterologous expression in yeast, cold tolerance

张雅宁, 毛春力, 胡志勇, 杨丹, 吴佳海, 王碧娴, 黄琳凯. 象草Dof转录因子的全基因组分析及其对低温胁迫的响应[J]. 生物技术通报, 2026, 42(6): 267-278.

ZHANG Ya-ning, MAO Chun-li, HU Zhi-yong, YANG Dan, WU Jia-hai, WANG Bi-xian, HUANG Lin-kai. Genome-wide Analysis of Dof Transcription Factors in Elephant Grass and Their Response to Cold Stress[J]. Biotechnology Bulletin, 2026, 42(6): 267-278.

图/表 10

图1 CpDof基因染色体分布垂直条表示染色体密度，左侧刻度表示染色体长度

Fig. 1 Chromosomal distribution of CpDof genesVertical bars indicate chromosome density, and the scale on the left indicates chromosome length

图2 Dof在象草基因组中的分布和共线性分析

Fig. 2 Distribution and collinearity analysis of Dof in the genome of elephant grass (Cenchrus purpureus)

图3 象草、拟南芥和水稻Dof基因的共线性分析

Fig. 3 Synteny analysis of Dof genes between elephant grass and A. thaliana and O. sativa

图4 象草、水稻和拟南芥中Dof成员的系统发育树

Fig. 4 Phylogenetic tree of Dof members in elephant grass, rice (O. sativa) and A. thaliana

图5 CpDof顺式作用元件（CRE）分析

Fig. 5 Analysis of cis-acting elements (CREs) of CpDof

图6 CpDof家族成员的系统发育树、保守基序、保守结构域和基因结构

Fig. 6 Phylogenetic tree, conserved motifs, conserved domains, and gene structures of CpDof family members

图7 ‘川畜4号’象草基因组Dof基因在低温胁迫条件下的表达水平

Fig. 7 Expressions of Dof genes in the ‘Chuanxchu No. 4’ elephant grass genome under low-temperature stress conditions

图8 10个CpDof基因在低温（4 ℃）处理下的表达水平所有实验均独立重复3次，误差线代表3次重复实验的标准差。*表示与空白对照组（0 h）相比转录水平的显著性差异（* P0.05，** P0.01，*** P0.001，**** P0.000 1）

Fig. 8 Expressions of 10 CpDof genes under low temperature (4 ℃) treatmentAll experiments were independently repeated three times. Error bars indicate the standard deviation of three replicates. * indicate significant differences in transcript levels compared to the blank control (0 h) (* P0.05, ** P0.01, *** P0.001, **** P0.000 1)

图9 10个CpDof基因在不同组织中的表达水平所有实验均独立进行3次，误差线表示3个重复的标准差。不同字母表示根据Tukey检验得出的统计学显著差异（P0.05）

Fig. 9 Expressions of 10 CpDof genes in different tissuesAll experiments were independently repeated three times. Different letters indicate statistically significant differences (P0.05) according to Tukey’s test

图10 过表达CpDof17和CpDof28的酵母细胞对低温胁迫的耐受性A、B：经冷处理或未经冷处理的酵母转化子连续稀释液滴；C、D：经冷处理的转基因酵母与空白对照组酵母菌株在液体培养基中的生长曲线

Fig. 10 The tolerance of yeast cells overexpressing CpDof17 and CpDof28 to low-temperature stressA, B: Serially diluted drops of yeast transformants without or with cold treatment. C, D: Growth curves of cold-treated transgenic yeast and blank control group yeast strains in liquid medium

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