Functional analysis of 4-coumarate: CoA ligase gene Gh4CL30 in upland cotton

doi:10.6048/j.issn.1001-4330.2024.06.001

Abstract

Abstract:

【Objective】 To preliminarily study the biological function of 4-coumarate-CoA ligase (4CL) family gene gh4cl30, which might provide theoretical basis and genetic resources for cotton plant breeding. 【Methods】 Virus-induced gene silencing technology and gene editing technology was used to obtain gh4cl30-silenced and edited plants and determine the flavone and lignin contents of the gene suppressed and knockout plants to investigate field phenotype, seed size and fiber quality. 【Results】 Both gene silencing and gene editing of the gene resulted in dwarfing of the plants and a significant reduction of lignin content in the plant stalks.Field phenotypic measurements showed that seed size and fiber length were significantly reduced in the gene edited plants. 【Conclusion】 Gh4CL30 affects cotton growth and development mainly through regulating lignin biosynthesis.

Key words: Gossypium hirsutum L.; 4CL; lignin; gene editing; plant height

摘要：

【目的】研究4-香豆酸辅酶A连接酶(4CL)家族基因Gh4CL30的生物学功能,为棉花株型育种提供理论依据和基因种质资源。【方法】利用病毒诱导的基因沉默技术和基因编辑技术获得Gh4CL30沉默和编辑植株,测定该基因抑制及敲除植株的黄酮和木质素含量,调查棉花田间表型性状、种子大小和纤维品质。【结果】Gh4CL30基因沉默和敲除植株中木质素合成相关基因表达量显著下降,4-香豆酸辅酶A连接酶含量显著减少,茎秆中木质素含量显著降低,其株高、种子大小和纤维长度显著降低。【结论】Gh4CL30通过调控木质素生物合成功能影响棉花的生长发育。

关键词: 陆地棉, 4-香豆酸辅酶A连接酶, 木质素, 基因编辑, 株高

CLC Number:

S188

GONG Junming, XIONG Xianpeng, ZHANG Caixia, SHAO Dongnan, CHENG Shuaishuai, SUN Jie. Functional analysis of 4-coumarate: CoA ligase gene Gh4CL30 in upland cotton[J]. Xinjiang Agricultural Sciences, 2024, 61(6): 1301-1309.

巩隽铭, 熊显鹏, 张彩霞, 邵东南, 程帅帅, 孙杰. 陆地棉4-香豆酸辅酶A连接酶基因Gh4CL30的功能分析[J]. 新疆农业科学, 2024, 61(6): 1301-1309.

Figures/Tables 8

Tab.1 Type I Gh4CL genes in G.hirsutum

基因名称 Genes Name	基因ID Gene ID	染色体 Chromosome	蛋白长度 Protein Length (aa)	等电点 Isoelectric Point	亚细胞定位 Subcellular localization
Gh4CL6	GH_A05G0051	A05	543	5.835	Plasma membrane
Gh4CL7	GH_A05G1439	A05	543	6.09	cytosol
Gh4CL12	GH_A10G0499	A09	129	10.388	chloroplast
Gh4CL24	GH_D05G0056	D05	540	6.25	chloroplast
Gh4CL25	GH_D05G1456	D05	543	5.872	cytosol
Gh4CL30	GH_D10G0525	D10	543	5.284	cytosol

Fig.1 Expression patterns of six type I Gh4CL in different tissues of cotton Note: A:Expression patterns of six type I Gh4CLs in different tissues of cotton;B:Relative expression of six type I Gh4CL genes in cotton roots;C:Relative expression of six type I Gh4CL genes in cotton stems

Fig.2 Phenotype and determination of relevant indicators in Gh4CL30 gene silenced plants Note: A:Phenotypic differences between TRV:Gh4CL30 and TRV:00 plants;B:Relative expression of Gh4CL30 in TRV:00 and TRV:Gh4CL30 plants;C:Differences in plant height and internode length between TRV:00 and TRV:Gh4CL30 plants.From bottom to top, the first to fourth internode lengths of cotton plants are shown;D:Lignin content of TRV:00 and TRV:Gh4CL30 plants;E:Flavonoid metabolite content of TRV:00 and TRV:Gh4CL30 plants

Fig.3 Gh4CL30 gene structure and selection of editing targets and types of gene editing Note:A:Two editing targets were selected in the first exon region of the Gh4CL30 gene;B:Type of gene editing of the mutant

Fig.4 Phenotype, 4CL enzyme activity and relative expression of Gh4CL30 in WT and 4CL30 plants Note: A:Phenotypic comparison of 4CL30 and WT plant heights;B:Plant height statistics of 4CL30 edited plants and WT; C:The 4CL enzyme activity of WT and 4CL30 plants was different;D:Relative expression of Gh4CL30 gene in stems of WT and 4CL30 edited plants

Fig.5 Lignin content of 4CL30 and WT plants and expression of genes related to lignin synthesis Note:A:Staining of stem sections of 4CL30 and WT plants at the same node using hydrochloric acid-phloroglucinol;B:Determination of lignin content in 4CL30 and WT plants;C:Expression of genes related to lignin synthesis in stem and root tissues of 4CL30 and WT plants

Fig.6 Cytological observations of 4CL30 and WT plants Note:A:Transverse and longitudinal paraffin sections of same-node stems of 4CL30 and WT plants;B:Cell counts in longitudinal sections;C:Cell counting in transverse sections (Cells in red boxes of the same size are counted at the same scale; a small number of cells indicates a longer cell length and a larger cell size.)

Fig.7 Phenotypic comparison of boll size and fiber length of WT and 4CL30 plants Note:A:From left to right are plant boll size, fiber length and seed size;B:Cotton boll weight (Cotton bolls on the first fruit branch of the same period) of WT and 4CL30 plants;C:Fiber length of WT and 4CL30 plants;D:Lint percent of WT and 4CL30 plants;E:Seed index of WT and 4CL30 plants

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