陆地棉HD-Zip家族全基因组鉴定及响应非生物胁迫的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0869

摘要/Abstract

摘要：

【目的】HD-Zip（Homeodomain and Leucine zipper）家族是植物特有的转录因子家族之一，在植物的生长发育、逆境响应中发挥重要作用。从陆地棉全基因组范围内鉴定GhHDZ基因家族成员，分析相关基因表达特点，为后续深入研究提供支撑。【方法】利用生物信息学方法从陆地棉TM-1基因组中鉴定出GhHDZ基因家族成员，并对其理化特性、系统进化关系、染色体定位、基因复制、基因结构和启动子区顺式作用元件等进行分析。利用转录组数据结合实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, RT-qPCR）分析GhHDZ家族基因在不同非生物胁迫下的表达模式。采用烟草瞬时转化法检测目标蛋白的亚细胞定位情况，通过转基因过表达方法验证目标基因功能。【结果】在陆地棉基因组中共鉴定到205个GhHDZ基因，系统发育分析将GhHDZ家族划分为4个亚组。片段复制是GhHDZ基因家族进化的主要原因，且该基因家族经历了强烈的纯化选择。在转录组分析基础上，对其中10个GhHDZ基因在4种非生物胁迫下的RT-qPCR鉴定分析表明，GhHDZ12、GhHDZ46、GhHDZ119正向响应冷胁迫，GhHDZ15、GhHDZ188负向响应冷胁迫；GhHDZ15、GhHDZ46、GhHDZ50、GhHDZ76、GhHDZ116、GhHDZ146、GhHDZ176、GhHDZ188正向响应热胁迫；GhHDZ15、GhHDZ50、GhHDZ76、GhHDZ116、GhHDZ119、GhHDZ146、GhHDZ176、GhHDZ188正向响应盐胁迫；GhHDZ12、GhHDZ15、GhHDZ50、GhHDZ116、GhHDZ119、GhHDZ176、GhHDZ188正向响应干旱胁迫，GhHDZ76负向响应干旱胁迫，且发现上述基因对各种胁迫的响应时间各有差异。进一步对GhHDZ146的功能研究发现，该基因定位于细胞核，在拟南芥中异源过表达显著增强了对盐胁迫的耐受性。【结论】在全基因组范围内从陆地棉中系统鉴定出205个GhHDZ家族成员。不同基因对各种胁迫的响应不一，且表达模式存在差异。GhHDZ146对盐胁迫具有正向响应功能。

关键词: 陆地棉, GhHDZ基因家族, 系统进化, 非生物胁迫, 表达模式

Abstract:

【Objective】 HD-Zip（Homeodomain and Leucine zipper）family is one of the plant-specific transcription factor families, which plays an important role in plant growth and development and stress response. In this study, the members of GhHDZ gene family were identified from the whole genome of Gossypium hirsutum L. and the expression characteristics of related genes were analyzed to provide support for further research. 【Method】 The members of GhHDZ gene family were identified from TM-1 genome of G. hirsutum L. by bioinformatics, and their physicochemical properties, phylogenetic relationship, chromosome location, gene replication, gene structure and cis-acting elements in promoter region were analyzed. Transcriptome data and real-time quantitative polymerase chain reaction（RT-qPCR）were used to analyze the expression patterns of GhHDZ family genes under different abiotic stresses. The subcellular localization of the target protein was detected by tobacco transient transformation method, and the function of the target gene was verified by transgenic overexpression method. 【Result】 A total of 205 GhHDZ genes were identified in upland cotton genome, and the GhHDZ family was divided into 4 subgroups by phylogenetic analysis. Fragment replication was the main reason for the evolution of the GhHDZ gene family, and the gene family has undergone a strong purification selection. GhHDZ family members had potential diversity of biological functions. The analysis of abiotic stress transcriptome combined with RT-qPCR analysis of 10 GhHDZ genes showed that GhHDZ12, GhHDZ46 and GhHDZ119 positively responded to cold stress, GhHDZ15 and GhHDZ188 negatively responded to cold stress, GhHDZ15, GhHDZ46, GhHDZ50, GhHDZ76, GhHDZ116, GhHDZ146, GhHDZ176, GhHDZ188 positively responded to heat stress, and GhHDZ15, GhHDZ50, GhHDZ76, GhHDZ116, GhHDZ119, GhHDZ146, GhHDZ176, GhHDZ188 positively responded to salt stress. GhHDZ12, GhHDZ15, GhHDZ50, GhHDZ116, GhHDZ119, GhHDZ176, GhHDZ188 positively responded to drought stress, while GhHDZ76 negatively responded to drought stress, and it was found that the response times of these genes to various stresses were different. Further study on the function of GhHDZ146 showed that the gene was localized in the nucleus and heterologous overexpression in Arabidopsis, which significantly enhanced the tolerance to salt stress. 【Conclusion】 205 members of GhHDZ gene family are systematically identified from G. hirsutum L. within the whole genome. Different genes have different responses to various stresses, and there are differences in expression patterns. The positive response function of GhHDZ146 to salt stress is confirmed.

Key words: Gossypium hirsutum L., GhHDZ gene family, phylogeny, abiotic stress, expression pattern

吴翠翠, 肖水平. 陆地棉HD-Zip家族全基因组鉴定及响应非生物胁迫的表达分析[J]. 生物技术通报, 2024, 40(2): 130-145.

WU Cui-cui, XIAO Shui-ping. Genome-wide Identification of HD-Zip Gene Family in Gossypium hirsutum L. and Expression Analysis in Response to Abiotic Stress[J]. Biotechnology Bulletin, 2024, 40(2): 130-145.

图/表 11

表1 所用相关引物

Table 1 Primers used in the study

基因名称Gene name	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
GhHis3	F:CGGTGGTGTGAAGAAGCCTCAT	R:AATTTCACGAACAAGCCTCTGGAA
GhHDZ12	F:CAGGCAAAGCAAAGTGGAGA	R:AGATCTGCAGCTGGTTCACT
GhHDZ15	CGGGTGGGCACAAAATAAGT	TTGGTTTCAGAAATGGGGCC
GhHDZ46	F:CCACCACAAAAGCCTGTTGA	R:GGTCGGAGTAATCATTGCCT
GhHDZ50	F:CCAGCAAGTTTTTAGGGCGA	R:GGAACCACCGGCAAAGAATT
GhHDZ76	F:ATGCTGAGCTCCAAGTCCTT	R:ATCTTGCACAACACAGCCAG
GhHDZ116	F:GGCCCCATTTCTGAAACCAA	R:TGGCGACTCAACTTCTGTCT
GhHDZ119	F:ACCAGCTGCAGATCTCAACT	R:CCCTTGTTGGTGTTGGTGTT
GhHDZ146	F:CCACCACAAAAGCCTGTTGA	R:CGGGTCGGAGTAATCATTGT
GhHDZ176	F:ATGCTGAGCTCCAAGTCCTT	R:ATCTTGCACAACACAGCCAG
GhHDZ188	F:ACGTCGCACTTTTACCTTCG	R:ACCGTTTCAACCGATTCGAC
GhHDZ146-sub	F:TTGATACATATGCCCGTCGACATG	R:GTCGTGGTCCTTATAGTCGGATCCAT
GhHDZ146-sub	GCGGGTGGGAGGGTCTTCTCTTGTAA	AAGCCGAAGACCAGAAAGCATGATCTTC

图1 陆地棉HDZ蛋白的系统发育分析不同颜色分别代表HD-ZipI、II、III、IV亚组

Fig. 1 Phylogenetic analysis of HDZ proteins from G. hirsutum L. Different colors represent HD-ZipI, II, III and IV, respectively

图2 GhHDZ基因家族的染色体定位分布

Fig. 2 Chromosomal locations of GhHDZ gene family

图3 GhHDZ基因家族的共线性分析 A01-A13分别表示陆地棉的A亚组染色体，D01-D13分别表示陆地棉的D亚组染色体

Fig. 3 Syntenic relationship of GhHDZ gene family A01-A13 represent the A subgroup chromosomes of upland cotton, and D01-D13 represent the D subgroup chromosomes of upland cotton

图4 GhHDZ家族的系统发育、基因结构和基序分析 a：以全长蛋白质序列为基础构建GhHDZ的系统发育树；b：GhHDZ家族基因保守基序分析；c：GhHDZ家族基因结构分析。用GSDS绘制基因结构图谱。绿色方框表示外显子；灰色线条表示内含子。比例尺显示在底部

Fig. 4 Phylogenetic, gene structure, and motif analyses of GhHDZ family a: Phylogenetic relationships between GhHDZ members are based on the full-length protein sequences. b: Gene motif analysis of GhHDZ family. c: Gene structure analysis of GhHDZ family. Gene structure maps were drawn with GSDS. Green boxes indicated exons; grey lines indicated introns. The scale bar is shown at the bottom

图5 GhHDZ基因家族顺式调控元件的功能类型和数量

Fig. 5 Function types and number of cis-regulatory elements of GhHDZ gene family

图6 GhHDZ家族基因非生物胁迫下的表达模式

Fig. 6 Expression patterns of GhHDZ family genes under abiotic stress

图7 10个GhHDZs在不同时间对非生物胁迫的相对表达量

Fig. 7 Relative expressions of 10 GhHDZs to abiotic stress at different time

图8 GhHDZ146-GFP融合蛋白在烟草叶片中的亚细胞定位（Bar= 50 μm）

Fig. 8 Subcellular localizations of GhHDZ146 proteins in tobacco leaves (Bar= 50 μm)

图9 GhHDZ146过表达拟南芥的盐胁迫鉴定 a和b：GhHDZ146-OE株系和WT之间主根长度的比较。bar=1 cm；c和d：GhHDZ146-OE株系和WT之间根毛长度的比较。bar=50 μm。通过双尾t检验分析差异显着性。误差线代表平均值±SD。其中，WT表示野生型；OE表示GhHDZ146 过表达株系。* P<0.05，**P<0.01

Fig. 9 Salt stress identification of GhHDZ146 overexpression in Arabidopsis thaliana a and b: Comparison of root length between GhHDZ146-OE and WT. Bar=1 cm. c and d: Comparison of root hair length between GhHDZ146-OE and WT. Bar= 50 μm. The difference was analyzed by double-tailed t-test. The error line represents the average ±SD. Among them, WT represents wild type and OE represents GhHDZ146 overexpression strain. * P < 0.05，** P < 0.01

图10 GhHDZ146与其他物种已知功能基因的聚类

Fig. 10 Clustering of GhHDZ146 and known functional genes in other species

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