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【目的】通过对棉花脱水素基因结构特征及其在低温胁迫下表达模式进行分析,探讨脱水素在棉花响应低温过程中的功能,为棉花抗冷育种提供理论基础。【方法】以棉花抗冷品种豫2067为试验材料,根据棉花陆地棉基因组序列查找已知脱水素(dehydrin,Dhn)基因的CDS序列,利用Primer5软件设计引物,克隆该基因,并命名为GhDHN1;采用生物信息学方法分析其蛋白质性质、氨基酸含量特征、功能结构域、系统进化树;选择XbaⅠ和SmaⅠ酶切位点对植物表达载体p BI121::GFP进行双酶切,采用In-Fusion连接技术构建融合蛋白瞬时表达载体p BI121-GhDHN1::GFP;分析其在洋葱表皮细胞中的瞬时表达,进行亚细胞定位;利用抗冷材料豫2067在三叶期对低温处理(4℃,24 h)前后的叶片和根系进行转录组测序,筛选差异表达基因;在三叶期时分别对豫2067(抗冷品种)和衡棉3号(冷敏感品种)进行低温(4℃,24 h)处理,利用实时荧光定量方法分别比较根、茎、叶中GhDHN1表达量,对该基因在2个抗冷差异材料叶中的表达量进行比较;对豫2067进行不同时间低温(4℃)处理,分析GhDHN1在叶片和根中的动态表达模式。【结果】该基因全长为726 bp,开放阅读编码框为636 bp,编码211个氨基酸,预测分子量为23.79 k D,等电点为5.04,富含谷氨酸(26.10%)和赖氨酸(19.40%),不含色氨酸,半衰期为30 h,蛋白呈酸性,带负带荷,带负电荷的残基总数为60%;GhDHN1的二级结构α螺旋(Alpha helix)包含116个氨基酸残基,占54.98%,组成该蛋白的主体结构,无规则卷曲(Random coil)的氨基酸残基有87个;GhDHN1位于陆地棉D亚组第9染色体(Dt_chr9)上,在cDNA的259—348位置上含有一个长度为90 bp的内含子,2个外显子长度分别为258和378 bp;SMART和CDD分析表明该氨基酸序列含有2个保守的富含赖氨酸的K片段和1个保守的富含丝氨酸S片段,具有亲水素蛋白结构域pfam00257,表明该蛋白为K_2S型脱水素;系统进化树分析表明,陆地棉亲水素GhDHN1与可可亲缘关系最近;洋葱表皮细胞中瞬时表达分析表明,GhDHN1蛋白主要定位在细胞膜附近。转录组分析表明,该基因在棉花三叶期叶片和根中,受低温处理后上调表达;荧光定量PCR分析表明,GhDHN1在4℃低温胁迫24 h后,在叶片、茎、根中均上调表达,在叶中上调表达倍数最大,在低温处理4 h和24 h时叶片中有2个表达高峰,在低温处理6 h和12 h时在根中有2个表达高峰,在抗冷材料叶片中的表达是冷敏感材料的2.47倍,说明该基因可能参与了棉花对低温的适应性调控。【结论】陆地棉GhDHN1属于典型的SK2型脱水素,与可可亲缘关系最近。该基因响应低温胁迫,在抗冷材料和冷敏感材料中表达差异显著,其表达量与棉花的抗冷性呈正相关,可以作为筛选不同抗冷材料的标记,同时可以作为重要的候选基因来培育棉花抗冷新材料。
【Objective】 The objective of this study was to investigate the structural characteristics of cotton dehydroxygenin gene and its expression pattern under low temperature stress, and to explore the function of dehumidification in response to low temperature of cotton, providing a theoretical basis for cold-resistant cotton breeding. 【Method】 The CDS sequence of known dehydrin (Dhn) gene was cloned from GenBank accession cotton (Upland cotton 2048) by using cold-tolerant cotton variety Yu 2067. The primer was cloned by Primer5 software and named GhDHN1. Bioinformatics methods were used to analyze the protein properties, amino acid content, functional domains, and phylogenetic tree. Double-digestion of the plant expression vector pBI121 :: GFP with XbaI and SmaI restriction sites was performed. In-Fusion The transient expression vector p BI121-GhDHN1 :: GFP was constructed and transiently expressed in onion epidermal cells for subcellular localization. The cold-resistant material 2067 was used to investigate the effect of low temperature treatment (4 ℃, 24 h) The leaves and roots were sequenced and the differentially expressed genes were screened. In the three-leaf stage, Yu 2067 (resistant to cold) and Hengmian 3 (cold sensitive) were treated at low temperature (4 ℃, 24 h) The expression of GhDHN1 in roots, stems and leaves was compared by real-time fluorescence quantitative analysis, and the expression of GhDHN1 was compared in leaves of two cold-tolerant materials. The treatment of Yu 2067 with low temperature (4 ℃) Dynamic expression patterns of HN1 in leaves and roots. 【Result】 The full length of this gene was 726 bp and the open reading frame was 636 bp, encoding 211 amino acids. The predicted molecular weight was 23.79 kD and the isoelectric point was 5.04. The gene was rich in glutamic acid (26.10%) and lysine (19.40%), no tryptophan, half-life of 30 h, the protein is acidic, negatively charged, the total number of negatively charged residues was 60%; GhDHN1 secondary helix (Alpha helix) contains 116 Amino acid residues accounted for 54.98%, which composed the main structure of the protein. There were 87 amino acid residues in random coil. GhDHN1 was located on chromosome 9 (Dt_chr9) 348 contains an intron of 90 bp in length with two exons 258 and 378 bp in length, respectively. SMART and CDD analysis revealed that this amino acid sequence contains two conserved lysine rich K fragments and 1 A conserved serine-rich S fragment with the pilin protein domain pfam00257, indicating that the protein was K_2S-type dehydratin. Phylogenetic tree analysis showed that GhDHN1 had the closest genetic relationship with cocoa; the transient expression in onion epidermal cells The results showed that the GhDHN1 protein mainly located near the cell membrane. Transcriptome analysis showed that the gene was upregulated after low temperature treatment in the leaves and roots at the third leaf stage. Fluorescent quantitative PCR analysis showed that GhDHN1 was up-regulated in leaves, stems and roots at 4 ℃ for 24 h , Up-regulated the expression fold in leaf, and reached its peak in leaves at 4 h and 24 h after cold treatment, and reached its peak in roots at 6 h and 12 h after cold treatment. Is 2.47 times of cold sensitive material, indicating that the gene may be involved in cotton’s adaptive regulation of low temperature. 【Conclusion】 Upland cotton GhDHN1 belongs to typical SK2-type dehydratin, and has the closest genetic relationship with cocoa. The gene was significantly different in cold-resistant material and cold-sensitive material in response to low temperature stress. Its expression level was positively correlated with cold resistance of cotton and could be used as a marker for screening different cold-tolerant materials and could be used as an important candidate gene to cultivate Cotton cold resistant new material.