摘　要

 

棉花被认为是世界上所有可以直接产生天然纤维的最重要经济作物之一,其所产生的棉纤维也是我们进行纺织业的一种重要资源。我国一直以来都是世界上主要的产棉国,棉花的生产对于我国的国民经济具有重要意义。棉纤维主要成分为纤维素，棉纤维发育分为起始、伸长、过渡（转变）、次生壁加厚、脱水成熟5个相互重叠的时期。通过对参与棉纤维发育的基因进行筛选和功能验证，有助于解析棉纤维发育的分子机制，进而为棉花产量的提高和纤维品质的改善提供重要的理论基础。

 

WRKY基因作为植物中重要的转录因子，在植物的生长发育中发挥着十分重要的作用。本实验室挖掘到一批可能参与棉纤维发育相关的基因。在本研究中我们挑选了其中在纤维起始期高表达的GhWRKY53基因进行克隆和功能鉴定，并对GhWRKY53所属的陆地棉Ⅲ亚组中的其他WRKY基因进行家族分析。主要内容如下：

 

1、之前的研究人员通过对陆地棉TM-1中的WRKY家族成员进行鉴定和系统发育分析，得到239个GhWRKY基因，其中属于III亚组的WRKY基因有21个。本研究重点对这21个基因进行深入分析。系统发育分析表明，陆地棉III亚组WRKY基因有三个分支，在拟南芥中也有同样的分支模式；多序列比对、保守motif分析和基因结构分析表明陆地棉III亚组WRKY基因序列高度相似，都具有保守的WRKY结构域和C2HC 锌指结构域，内含子和外显子数量基本一致，在系统发育树中聚在一起的成员具有相似的基序组成和基因结构特征，表明陆地棉III亚组WRKY基因高度保守且基因的结构不同，其功能可能也存在差异；利用公开的RNA-seq数据分析了陆地棉III亚组WRKY基因在植物组织中的表达模式，发现其主要在根、茎和花托中高表达；对陆地棉III亚组WRKY基因在陆地棉纤维发育时期的表达模式进行分析，发现其主要在-3DPA-3DPA和30DPA特异表达，参与棉花纤维起始和成熟的发育过程。

 

2、棉纤维起始相关基因GhWRKY53的克隆和功能鉴定。GhWRKY53基因全长 3321 bp，含有 3个外显子，2个内含子，CDS 长度为 1008 bp，编码 335个氨基酸，含有典型的 WRKY保守序列。对GhWRKY53在纤维发育阶段的表达模式进行分析，发现该基因在纤维起始和成熟过程中特异表达；GhWRKY53的亚细胞定位实验表明，其定位在细胞核中；GhWRKY53在拟南芥中的异源表达可以显著增加表皮毛的数量；通过酵母双杂交实验，从棉花纤维cDNA文库中筛选出42个与GhWRKY53有相互作用倾向的蛋白，并对其进行去冗余和重复频率统计，最终得到14个与GhWRKY53有相互作用倾向的蛋白；之后，通过生物信息学和酵母双杂交点对点验证实验重点对GhWKY53与GhRAX3的相互作用进行验证，并对GhRAX3的功能进行初步验证；为了在棉花中验证上述结果，利用Crispr-Cas9技术构建了GhWRKY53基因敲除载体，进行棉花遗传转化。

 

本研究对陆地棉III亚组WRKY基因成员进行了详细的解析，主要在拟南芥中对GhWRKY53基因的功能进行了初步探索，为进一步研究GhWRKY53基因在棉纤维发育中的作用奠定了基础,为棉纤维发育研究提供了新的思路和方法。

 

关键词：陆地棉，基因家族，纤维起始，Ⅲ亚组WRKY基因，表达模式

 

Abstract

 

Cotton is considered one of the most important economiccrops that can directly produce natural fiber globally. The cotton fiber it makes is also an essential resource for our textile industry.China has always been a significant cotton producer globally,and cotton production is incredibly powerful to our national economy. The main component of cotton fiber is cellulose.The development of cotton fiber can be divided into five overlapping stages: initiation,elongation, transition (transformation), secondary wall thickening, and dehydration maturation. The screening and functional verification of genes involved in cotton fiber development help analyze cotton fiber development's molecular mechanism. It then provides an essential theoretical basis for improving cotton yield and fiber quality.As a critical transcription factor in plants,the WRKY gene plays a crucial role in plant growth and development. Our laboratory found several genes that may be involved in cotton fiber development.In this study, we selected the GhWRKY53 gene,which was highly expressed in the fiber initiation stage for cloning and functional identification. We carried out a family analysis of other WRKY genes in the third subgroup of upland cotton to which GhWRKY53 belongs. The main contents are as follows: 1.Through identification and phylogenetic analysis of WRKY family members in upland cottonTM-1, 239 GhWRKY genes were obtained, of which 21 WRKY genes belong to the III subgroup. This study focuses on the in-depth analysis of these 21 genes.Phylogenetic analysis showed three branches of the WRKY gene in the upland cotton III subgroup, and the same branching pattern was found in Arabidopsis thaliana. Multiple sequence alignment, conservative motif analysis,and gene structure analysis showed that the WRKY gene sequence of upland cotton III subgroup was highly similar,with conserved WRKY domain and C2HC zinc finger domain,the number of introns and exons were the same, and the members clustered together in the phylogenetic tree had similar motif composition and gene structure characteristics, indicating that the WRKY gene of upland cotton III subgroup was highly conserved and the gene structure was different. Its functions may also be other.The upland cotton III subgroup WRKY gene expressionin plant tissue was analyzed using open RNA-seq data. It was found that it was mainly highly expressed in root, stem, and receptacle.The expression pattern of the upland cotton III subgroup WRKY gene during upland cotton fiber development was analyzed. It was found that it was mainly expressed in-3DPA-3DPA and 30DPA, which was involved in cotton fiber initiation and maturation. 2.Cloning and functional identification of cotton fiber initiation-related gene GhWRKY53.The full length of the GhWRKY53 gene is 3321bp, which contains three exons and two introns.The CDS length is 1008bp and encodes 335 amino acids,which includes a typical conserved sequence of WRKY. The expression pattern of GhWRKY53 during fiber development was analyzed,and it was found that the gene was explicitly expressed during fiber initiation and maturation. The subcellular localization of GhWRKY53 showed that it was located in the nucleus, and the heterologous expression of GhWRKY53 in Arabidopsis could significantly increase the number of epidermal hairs. 42 proteins that tend to interact with GhWRKY53 were screened from cotton fiber cDNA library by yeast two-hybrid experiment,and 14 proteins with the tendency to interact with GhWRKY53 were obtained by de-redundancy and repetition rate statistics. After that, the interaction between GhWKY53 and GhRAX3 was verified by bioinformatics and yeast two-hybrid point-to-point verification experiment, and the function of GhRAX3 was preliminarily verified. To verify the above results in cotton,the GhWRKY53 gene knockout vector was constructed by CRISPR-Cas9 technology, and cotton genetic transformation was carried out. In this study, the members of the WRKY gene of the III subgroup in upland cotton were analyzed in detail. The function of the GhWRKY53 gene was explored in Arabidopsis thaliana,which laid a foundation for further study on the role of the GhWRKY53 gene in cotton fiber development. It provided new ideas and methods for the study of cotton fiber development.

 

Keywords: Gossypium hirsutum, gene family,fiber initiation, subgroup Ⅲ WRKY gene, expression pattern