陆地棉WRKY基因家族全基因组鉴定及WRKY44在纤维发育中的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0390

摘要/Abstract

摘要：

目的探讨陆地棉WRKY基因家族特征及WRKY转录因子在棉纤维生长发育中的功能，为阐明其分子机制提供依据。方法对GhWRKY基因家族进行全基因组鉴定，分析染色体定位、理化性质、亚细胞定位、基因结构和启动子元件，结合实时荧光定量分析GhWRKY基因家族在纤维发育过程中的表达模式，并利用病毒诱导基因沉默技术解析GhWRKY44的功能。结果在陆地棉全基因组中，共鉴定到225个WRKY基因家族成员，可进一步分为3个亚家族；该家族基因不均匀分布在26条染色体上。启动子区域含有大量植物激素、生长发育、胁迫响应和光响应相关顺式作用元件。8个GhWRKYs（GhWRKY11/14/31/48/133/149/ 160/224）在纤维不同发育时期存在优势表达；其中GhWRKY14和GhWRKY133这2个基因在纤维不同发育时期具有相似的表达模式和三维结构，为拟南芥WRKY44在陆地棉中的2个直系同源基因（拷贝），因此将其命名为GhWRKY44。而GhWRKY44基因的沉默能够显著抑制表皮毛的生长发育。结论系统分析了陆地棉WRKY基因家族，筛选出在纤维生长发育方面的关键基因GhWRKY44，为解析WRKY44在纤维生长发育方面的功能提供研究基础。

关键词: 陆地棉, WRKY基因家族, WRKY44, 全基因组鉴定, 纤维发育, 表达分析

Abstract:

Objective To investigate the characteristics of the WRKY gene family in upland cotton (Gossypium hirsutum L.) and elucidate the roles of WRKY transcription factors in fiber growth and development, thereby providing insights into their underlying molecular mechanisms. Method Genome-wide identification of the GhWRKY genefamily was performed, including analyses of chromosomal localization, physicochemical properties, subcellular localization, gene structure, and promoter cis-elements. Moreover, real-time quantitative PCR (qPCR) was employed to analyze the expression patterns of the GhWRKY gene family during fiber development, and a virus-induced gene silencing approach was to analyze the function of GhWRKY44. Result A total of 225 WRKY family members were identified in the G. hirsutum genome, and classified into three subfamilies. These genes were unevenly distributed across 26 chromosomes. The promoter cis-elements analysis showed that the promoters of GhWRKYs contained numerous regulatory elements related to plant hormones, growth and development, stress responses, and light response. Eight GhWRKYs (GhWRKY11/14/31/48/133/149/160/224) had preferential expressions at different stages of fiber development. Among them, the gene GhWRKY14 and GhWRKY133 shared similar temporal expression patterns and three-dimensional structures and were identified as orthologs (copies) of Arabidopsis thalianaWRKY44 in G. hirsutum. Accordingly, both were collectively designated as GhWRKY44. Silencing of GhWRKY44 significantly inhibited the growth and development of trichomes. Conclusion This study provides a wide analysis of the WRKY gene family in G. hirsutum and screens the key gene GhWRKY44 in fiber growth and development, thus laying the foundation for further exploring the function of WRKY44 genes in fiber growth and development.

Key words: Gossypium hirsutum, WRKY gene family, WRKY44, genome-wide identification, fiber development, expression analysis

徐嘉妤, 赵阁, 唐叶, 刘雯雯, 彭清忠, 吴家和. 陆地棉WRKY基因家族全基因组鉴定及WRKY44在纤维发育中的表达分析[J]. 生物技术通报, 2025, 41(12): 124-138.

XU Jia-yu, ZHAO Ge, TANG Ye, LIU Wen-wen, PENG Qing-zhong, WU Jia-he. Genome-wide Identification of WRKY Gene Family in Gossypium hirsutum and Expression Analysis of WRKY44 in Fiber Development[J]. Biotechnology Bulletin, 2025, 41(12): 124-138.

图/表 12

表1 引物名称和序列

Table 1 Primer names and sequences

引物名称 Primer name	序列 Sequence （5′‒3′）
qGhhis3-F	GACACCAACCTTTGCGCGAT
qGhhis3-R	AGCGACTGATCCACACTTCTG
qGhWRKY11-F	CCGAGCTCTTGGACTCTCCTGT
qGhWRKY11-R	TGTTGCGCAGTTTGATTTGCGT
qGhWRKY14-F	AGGCAGAGCAGTCCGGAATAGG
qGhWRKY14-R	TGATACAAGCTTTGCCTGCGGT
qGhWRKY31-F	GCTCGATTACCCCACGCTTCAA
qGhWRKY31-R	GCCCAGTAGGCGCAGTGAATAA
qGhWRKY48-F	GTCTCCTCCTGTAGCAAGCCAG
qGhWRKY48-R	ACACAGGTGTCTTCACAACGGG
qGhWRKY133-F	GGTTGTCTGTGCTTCCGGCTTA
qGhWRKY133-R	GCTCCCTCCTTGCTTGCTTCTT
qGhWRKY149-F	CTTCTGCTGGGTGTCCTGTACG
qGhWRKY149-R	GAGCAATTGGTTGGCCATGTCG
qGhWRKY160-F	TGGTCAGAAGCAAGTGAAGAGTCC
qGhWRKY160-R	TCTCAATTACATGGCCCGTCTGA
qWRKY224-F	GATGGGTATCGATGGCGCAAGT
qWRKY224-R	TGCTTTCGGATCATGAGACGCC

图1 GhWRKY基因家族染色体定位W1为简写，全称为GhWRKY1。下同

Fig. 1 Chromosome mapping of GhWRKY gene familyW1 is abbreviated from GhWRKY1. The same below

图2 GhWRKY基因家族成员的系统进化树

Fig. 2 Phylogenetic tree of GhWRKY gene family members

图3 GhWRKYs的多序列比对

Fig. 3 Multi-sequence alignment of GhWRKYs

图4 GhWRKY基因家族成员保守基序和基因结构分析A：陆地棉GhWRKYs家族蛋白进化树；B：陆地棉GhWRKYs家族蛋白的保守基序；C：陆地棉GhWRKY基因家族的基因结构

Fig. 4 Conserved motifs and gene structure analysis of GhWRKY gene family membersA: Phylogenetic tree of the GhWRKY family protein in G. hirsutum. B: Conserved motif of GhWRKY proteins in G. hirsutum. C: Gene structure of the GhWRKY gene family in G. hirsutum

图5 GhWRKYs顺式作用元件

Fig. 5 GhWRKYs cis-acting elements

图6 GhWRKYs在纤维发育时期的表达量分析红框中标注的为纤维和胚珠不同发育时期优势表达基因。DPA：开花后天数。下同

Fig. 6 Expression analysis of GhWRKYs during fiber developmentThe genes highlighted in red boxes are predominantly expressed during different developmental stages of fiber and ovule. DPA: Day post anthesis. The same below

图7 GhWRKYs在棉纤维发育不同时期的表达量分析采用Tukey test进行统计分析，不同小写字母表示GhWRKYs在棉纤维发育各阶段表达量差异显著（P<0.05）

Fig. 7 Relative expressions of key GhWRKYs during different stages of cotton fiber developmentStatistical analyses were performed using the Tukey test. Different lower letters indicate significant difference of the expression of GhWRKYs at different stages of cotton fiber development (P<0.05)

图8 GhWRKY44同源分析A：GhWRKY14（Gh_A04G096000.1）、GhWRKY133（Gh_D04G134100.1）和AtWRKY44（AT2G37260）系统进化树；B：GhWRKY14、GhWRKY133和AtWRKY44氨基酸序列比对分析；C：GhWRKY14、GhWRKY133和AtWRKY44蛋白三维结构

Fig. 8 Homology analysis of GhWRKY44A: Phylogenetic tree of GhWRKY14 (Gh_A04G096000.1), GhWRKY133 (Gh_D04G134100.1) and AtWRKY44 (AT2G37260). B: Amino acid sequence comparison analysis of GhWRKY14, GhWRKY133 and AtWRKY44. C: 3D structures of GhWRKY14, GhWRKY133 and AtWRKY44 proteins

图9 亚细胞定位

Fig. 9 Subcellular localization

图10 TRV：GhWRKY44沉默植株的培育A：VIGS沉默载体示意图；B：农杆菌侵染棉花14 d后，TRV：GhPDS植株表现出明显的白化表型；C：RT-qPCR检测GhWRKY44沉默效率，n=35；以GhHis3为内参基因，误差线为3次生物学重复的标准误；采用独立方差t检验进行统计分析，*P<0.05，**P<0.01，***P<0.001，****P<0.0001。下同

Fig. 10 Cultivating of TRV:GhWRKY44-silenced plantsA: Schematic diagram of VIGS silencing vector. B: Fourteen days after Agrobacterium infestation of cotton, TRV:GhPDS plants showed a distinct albino phenotype. C: RT-qPCR detection of GhWRKY44, silencing efficiency, n=35. GhHis3 was used as the internal reference gene, and the error line was the standard error of three biological replicates. Statistical analysis was performed using an independent variance t-test, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. The same below

图11 GhWRKY44的沉默株系中表皮毛的表型鉴定A：GhWRKY44沉默植株的表皮毛形态；B：GhWRKY44沉默植株的表皮毛密度统计分析，n=15

Fig. 11 Phenotypic characterization of epidermal trichomes in GhWRKY44-silenced plantsA: Trichome morphology of GhWRKY44-silenced plants. B: Statistical analysis of trichome density of GhWRKY44-silenced plants, n=15

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