Application of silencing GhPEX4 gene in synergistically regulating fiber fineness and length of cotton

Abstract

This invention discloses silence. GhPEX4 The application of genes in the synergistic regulation of cotton fiber fineness and length belongs to the field of plant genetic engineering technology. This invention is the first to identify and verify the peroxisome biogenetic protein gene in cotton. GhPEX4 Its function in the coordinated regulation of fiber fineness and length. Through QTL mapping and near-isogenic transcriptome sequencing, a stable QTL region for fiber fineness was precisely identified within 5.1 Mb. GhPEX4 This is the only key candidate gene that shows significant differential expression across multiple fiber development stages. Functional validation demonstrates that silencing this gene using VIGS technology... GhPEX4 Subsequently, the diameter of cotton single fibers decreased significantly by 14.3%, the fiber length increased by approximately 3 mm, the cell wall thickness decreased by 5.3%, and the lumen diameter decreased by 49.5%, while the fiber strength remained largely unchanged. Heterologous overexpression of this gene in Arabidopsis thaliana resulted in its conserved function of promoting cell wall thickening, growth, and material accumulation.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to silencing. GhPEX4 Application of genes in the coordinated regulation of cotton fiber fineness and length. Background Technology

[0002] Cotton is the world's most important natural fiber crop, and its economic value is mainly determined by two core traits: fiber yield and quality. Among them, fiber fineness (reflecting the thickness of the fiber) directly affects yarn strength and the hand feel and luster of textiles, and is a key quality indicator for measuring textile processing performance.

[0003] PEX4 (Peroxin-4) is a class of E2 ubiquitin-binding enzymes located on the peroxisome membrane. In yeast and Arabidopsis thaliana, it is responsible for monoubiquitination of the PEX5 receptor, mediating the introduction of peroxisome matrix proteins and receptor cycling. In recent years, significant progress has been made in the identification and functional studies of cotton E2 family members, particularly in the functions of GhUBC family genes in fiber development. GhUBC2L mediates histone H2A / H2B monoubiquitination in conjunction with E3 ligases (GhUbox8, etc.), regulating the expression of cell cycle and organ development-related genes, thereby positively regulating fiber elongation and organ size (DOI:10.7666 / d.Y3396905). However, there are currently no direct reports on the function of PEX4 (Peroxin-4) genes in cotton fiber development. Summary of the Invention

[0004] The purpose of this invention is to provide silence. GhPEX4 The application of genes in the synergistic regulation of cotton fiber fineness and length aims to address the problems existing in the aforementioned technologies.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is silence. GhPEX4 Application of genes in the coordinated regulation of cotton fiber fineness and length.

[0006] The second technical solution of this invention is silence. GhPEX4 Application of gene recombinant vectors in the synergistic regulation of cotton fiber fineness and length.

[0007] The third technical solution of this invention is a method for synergistically regulating the fineness and length of cotton fibers, which is silent. GhPEX4 Gene expression reduces the fineness of cotton fibers and increases their length.

[0008] The fourth technical solution of this invention is a method for cultivating new cotton varieties with finer and longer cotton fibers, which involves the cotton plant... GhPEX4Gene silencing is achieved; The GhPEX4 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0009] Based on the above technical solution, the present invention has the following technical effects: This invention is the first to identify and verify the peroxisome biogenic protein gene in cotton. GhPEX4 Key functions in the coordinated regulation of fiber fineness and length. By integrating QTL mapping and near-isogenic transcriptome sequencing, a precise 5.1 Mb stable QTL region for fiber fineness was identified. GhPEX4 This is the only key candidate gene that shows significant differential expression across multiple fiber development stages. Functional validation demonstrates that silencing this gene using VIGS technology... GhPEX4 Subsequently, cotton single fiber diameter decreased significantly by 14.3%, fiber length increased by approximately 3 mm, cell wall thickness decreased by 5.3%, and lumen diameter decreased by 49.5%, while fiber strength remained largely unchanged. More importantly, heterologous overexpression of this gene in Arabidopsis resulted in a 52.2% increase in taproot diameter, a 52.9% increase in root hair cell wall thickness, and a significant increase in biomass accumulation in stems, leaves, and siliques, indicating that… GhPEX4 It has a conserved function in plants to promote cell wall thickening, growth, and material accumulation. Attached Figure Description

[0010] Figure 1 To analyze differentially expressed genes within the stable QTL interval (5.1 Mb) of fiber fineness (micron value).

[0011] Figure 2 Comparison of gene expression patterns between two nearly isogenic lines within the QTL interval of 5.1 Mb malclon value.

[0012] Figure 3 for GhPEX4 Gene structure diagram.

[0013] Figure 4 During the fiber development process of two nearly isogenetic lines GhPEX4 qRT-PCR expression analysis. Note: ** p <0.01.

[0014] Figure 5 VIGS-mediated GhPEX4 Comparison of the relative expression levels of this gene in silenced plants and the uninfected control (CK).

[0015] Figure 6 For AFIS Pro based GhPEX4 Fiber length statistics of silent plants. Note: ** p<0.01, data are mean ± SD of 3 technical replicates (n = 3,000 roots per replicate).

[0016] Figure 7 To determine using the FAVIMAT single fiber testing system GhPEX4 Fiber mechanical properties of silent plants and controls. Note: Fmax, single fiber breaking strength; Tenac, fiber strength; Lin.Den, fiber fineness (fiber diameter). ** indicates extremely significant difference (…). P <0.01, Student's *t*-test); data are the mean ± standard deviation of three technical replicates, with 100 single fibers measured in each replicate.

[0017] Figure 8 for GhPEX4 The effect of silence on the length of a single fiber helix. Where, a: GhPEX4 Comparison of fiber spiral morphology between silent plants and controls (SEM images). b: GhPEX4 Statistical comparison of fiber single helix length between silent plants and controls. Note: ** indicates extremely significant difference. P <0.01); data are mean ± SD.

[0018] Figure 9 To reduce GhPEX4 The effects of expression on fiber cell wall thickness and lumen diameter were investigated. Specifically: a) Comparison of fiber cross-sectional morphology (paraffin sections) between GhPEX4 silenced plants and controls; b) Statistical comparison of fiber cell wall thickness; c) Statistical comparison of fiber lumen diameter. Note: ** indicates highly significant differences. P <0.01); data are mean ± SD.

[0019] Figure 10 Heterologous overexpression in Arabidopsis thaliana GhPEX 4. Effects on root development. Among them, a, GhPEX4 Comparison of root morphology between T3 generation overexpression plants and wild-type (WT) plants; b, GhPEX4 Statistical comparison of root length between T3 generation overexpression plants and wild-type plants; c, GhPEX4 Statistical comparison of taproot diameter between T3 generation overexpression plants and wild-type plants. Note: ** indicates extremely significant difference. P <0.01); data are mean ± SD.

[0020] Figure 11 for GhPEX4 Effect of overexpression on Arabidopsis cell wall thickness. Wherein, a) root cell wall morphology (electron microscopy); b) root cell wall thickness statistics; c) stem cell wall morphology (electron microscopy); d) stem cell wall thickness statistics. Note: ** P<0.01; data are expressed as mean ± SD.

[0021] Figure 12 Heterologous overexpression GhPEX4 The promoting effect on the accumulation of whole plant biomass in Arabidopsis thaliana. Among them, a, GhPEX4 Comparison of rosette leaf biomass between overexpression T3 generation plants and wild-type (WT) plants; b, GhPEX4 Comparison of stem and silique biomass at flowering time between T3 generation overexpression plants and wild-type plants. Note: ** indicates extremely significant difference (…). P <0.01); data are mean ± SD. Detailed Implementation

[0022] This invention identifies a key gene regulating cotton fiber fineness—the peroxisome biogenogen gene. GhPEX4 (Protein PEROXIN-4 isoform X2). When this gene is normally expressed, it has a positive effect on maintaining / promoting fiber fineness (maintaining or thickening fibers); however, when this gene is silenced, cotton fibers become significantly thinner.

[0023] The inventors performed transcriptome sequencing analysis on near-isogenic lines derived from Ji Mian 906 with different fiber fineness at six fiber development stages. Within a stable fiber fineness (micronaire value) QTL region (5.1 Mb), seven differentially expressed genes were identified. Ghi_A06G05386 Significantly differential expression was observed in five developmental stages across the two near-isogenic lines. This gene was annotated as Protein PEROXIN-4, and its homology in Arabidopsis thaliana is [missing information]. AT5G25760 ( PEX4 Therefore, it was named GhPEX4 To further verify its function, researchers silenced the virus-induced gene silencing (VIGS) technique in the 906-1 derivative line with thinner fibers. GhPEX4 Subsequently, fiber fineness decreased significantly, while fiber length increased. Overexpression in Arabidopsis thaliana... GhPEX4 This leads to thicker root hairs and thicker root hair cell walls, while also significantly increasing the overall biomass accumulation of the plant. In summary, this study, combining QTL mapping and near-isogenic transcriptome sequencing of fiber fineness, successfully identified and validated... GhPEX4 Closely related to the development of upland cotton fibers, it can regulate plant biomass accumulation in Arabidopsis thaliana, providing valuable candidate genes and theoretical basis for future targeted improvement of cotton fiber quality traits and yield regulation.

[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0025] This invention provides a silent solution. GhPEX4 Application of genes in the coordinated regulation of cotton fiber fineness and length.

[0026] In some specific implementation schemes, the GhPEX4 The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.3.

[0027] In some specific implementation plans, silence GhPEX4 Gene expression reduces the fineness of cotton fibers and increases their length.

[0028] In some specific implementation schemes, the cotton is Ji Mian 906.

[0029] Embodiments of the present invention also provide silence GhPEX4 Application of gene recombinant vectors in the synergistic regulation of cotton fiber fineness and length.

[0030] This invention also provides a method for synergistically controlling the fineness and length of cotton fibers, silencing... GhPEX4 Gene expression reduces the fineness of cotton fibers and increases their length.

[0031] In some specific implementation schemes, the GhPEX4 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0032] This invention also provides a method for cultivating new cotton varieties with finer and longer cotton fibers, which improves the cotton plant's... GhPEX4 Gene silencing is achieved; The GhPEX4 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0033] Example 1 1. Influences on fiber fineness formation GhPEX4 Gene identification Two individual plants with significantly different fiber quality but identical main agronomic traits, disease resistance, and yield were selected from Jimian 906 (Ji Shenmian 20250001) and named 906-1 and 906-2, respectively. After five generations of continuous self-pollination, the differential traits were stably inherited, thus establishing a near-isogenic line derived from Jimian 906, which was used for subsequent research related to fiber quality.

[0034] The two fibers have a significant and consistent difference in fiber fineness (measured by micronaire value): 906-1 fibers are finer, and have slightly better length and strength than 906-2 (Table 1).

[0035] Table 1. Two-year average of fiber quality measurement data from near-isogenic lines derived from Ji Mian 906 (Shijiazhuang HVI1000 test).

[0036] To elucidate the genetic basis for the aforementioned differences in fiber fineness, we performed whole-genome resequencing on both lines and conducted transcriptome sequencing at six time points during fiber development: 0, 5, 10, 15, 20, and 30 DPA (days after flowering). Simultaneously, using marker primers for existing micronaire value QTL mapping hotspots (DOI: 10.1186 / s12864-016-2560-2; DOI: 10.1016 / j.jare.2022.11.009), we performed whole-genome alignment analysis, preliminarily identifying the 34.44–39.51 Mb region on chromosome A6 as a potential candidate region for stable regulation of micronaire value. Figure 1 The genome version number is WHU 1.0, link: Gossypium hirsutum (AD1) 'TM-1' genome WHU_v1 | Cottongen.

[0037] Further integration of resequencing and transcriptome data revealed that only seven genes showed differential expression between 906-1 and 906-2 within the 34.44–39.51 Mb range (Table 2). Among these, one gene encodes a peroxisome biogenic protein. GhPEX4 Significant and consistent differential expression was observed in all five fiber development stages across the two near-isogenic lines. Cluster analysis of expression patterns of all genes within the candidate regions revealed... GhPEX4 In 906-1, it belongs to the Pattern IV expression pattern, while in 906-2, this pattern is completely absent. Figure 2 Based on the above multiple screening strategies, we will GhPEX4 The gene was identified as a major differentially expressed candidate gene, and subsequent functional verification experiments were conducted.

[0038] Table 2. Differentially expressed genes and annotation information in QTL regions (5.1 Mb) based on fiber fineness (micronaire value).

[0039] 2. GhPEX4 Functional validation of candidate genes in cotton and their application in high-quality cotton breeding First, we analyzed GhPEX4 Gene structure ( Figure 3 ), GhPEX4The full-length mRNA is 1998 bp (SEQ ID NO.1), containing 4 exon regions and 3 introns. The full-length mRNA is 474 bp (SEQ ID NO.2), encoding 157 amino acids (SEQ ID NO.3).

[0040] SEQ ID NO.1: >GhPEX4 sequence

[0041] SEQ ID NO.2: >GhPEX4 cds ATGCAGGCATCAAGAGCGAGGTTGTTCAAGGAATACAAAGAGGTTCAGCGAGAGAAGGCGGCTGATCCCGATATTCAACTAGTTTGTGATGATTCCAACATATTTAAGTGGACTGCTCTTATCAAGGGACCATCTGAGACACCTTATGAAGGCGGAGTCTTCCAGCTTGCTTTTGCTGTCCCTGAGCAGTACCCTTTGCAGCCTCCTCAAGTGCGGTTCTTGACAAAAATATTCAT CCCAATGTGCATTTCAAGACAGGAGAGATTTGCCTTGATATATTGAAGAATGCTTGGAGCCCTGCATGGACACTTCAGTCTGTTTGTAGGGCTATAATAGCTTTGATGGCTCATCCGGAACCTGATAGCCCGCTTAACTGTGATTCAGGCAATCTTTTAAGGTCTGGTGATCTCAGAGGATACCAGTCTATGGCAAGAATGTACACCAGACTTGCTGCCATGCCCAAAAAAGGGTGA.

[0042] SEQ ID NO.3: >GhPEX4 amino acid sequence MQASRARLFKEYKEVQREKAADPDIQLVCDDSNIFKWTALIKGPSETPYEGGVFQLAFAVPEQYPLQPPQVRFLTKIFHPNVHFKTGEICLDILKNAWSPAWTLQSVCRAIIALMAHPEPDSPLNCDSGNLLRSGDLRGYQSMARMYTRLAAMPKKG*.

[0043] To explore GhPEX4 This study compared the expression patterns of fibril RNA in near-isogenic lines 906-1 and 906-2, focusing on its function in fiber development. Based on fibril RNA from seven developmental stages, qRT-PCR validation results were highly consistent with transcriptomic data. GhPEX4 It maintained high expression throughout most of the development of 906-1 fibers, showing a significant difference from 906-2. Figure 4 Suppressing VIGS in 906-1 using VIGS technology. GhPEX4 back( Figure 5The length of mature fibers increased significantly by 3 mm. Figure 6 The fineness (single fiber diameter) decreased by 14.3%, and the length of a single helix shortened by 29.7% (i.e., the number of helices per unit length increased). Figure 7 , Figure 8 Meanwhile, the cell wall thickness decreased by 5.3% (); Figure 9 (a) The diameter of the cavity is reduced by 49.5% ( Figure 9 (b), while fiber strength (Tenac) showed no significant change ( Figure 7 In summary, further suppression in the context of slender fibers... GhPEX4 This allows for the modification of fibers to be "longer and thinner" without compromising strength. Notably, this study not only successfully identified and validated [the technology] through combined transcriptome and QTL analysis. GhPEX4 This key regulatory node further reveals its potential as a "two-way regulatory switch." This discovery provides highly operational genetic resources and precise improvement strategies for molecular design breeding of high-quality and high-yield cotton.

[0044] 3. GhPEX4 Functional validation and application prospects of candidate genes in Arabidopsis thaliana To verify GhPEX4 This study investigated heterologous overexpression of a conserved function in plant growth and development in Arabidopsis thaliana. Phenotypic observation of the T3 generation homozygous lines showed that overexpression... GhPEX4 It significantly promotes root development, as evidenced by a 52.2% increase in the diameter of the main root. Figure 10 The thickness of root hair cell walls increased significantly by 52.9%. Figure 11 Meanwhile, the biomass accumulation of the above-ground parts of the plant, including stems, leaves, and siliques, also significantly increased. Figure 12 The above results indicate that... GhPEX4 This gene possesses a conserved function in Arabidopsis thaliana, positively regulating vegetative growth and nutrient accumulation. This discovery provides multiple pathways for its breeding applications: in economic crops such as cotton, precise upregulation at different sites and stages... GhPEX4 The expression of this gene, or its homologous genes, is expected to simultaneously promote root development to improve nutrient absorption efficiency and synergistically enhance biomass accumulation, laying a material foundation for the combined improvement of fiber quality and yield. Furthermore, the gene's regulatory effect on root hair cell wall structure also provides a potential molecular tool for improving crop root architecture and enhancing stress resistance (such as drought and poor soil tolerance). Therefore, GhPEX4 It is not only a target for breeding high-quality fiber, but also a multi-effect gene resource with the potential for global regulation of the "root-sprout".

[0045] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Silence GhPEX4 Application of genes in the coordinated regulation of cotton fiber fineness and length.

2. The application according to claim 1, characterized in that, The GhPEX4 The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

3.

3. The application according to claim 2, characterized in that, silence GhPEX4 Gene expression reduces the fineness of cotton fibers and increases their length.

4. The application according to any one of claims 1-3, characterized in that, The cotton in question is Ji Mian 906.

5. Silence GhPEX4 Application of gene recombinant vectors in the synergistic regulation of cotton fiber fineness and length.

6. A method for synergistically controlling the fineness and length of cotton fibers, characterized in that, silence GhPEX4 Gene expression reduces the fineness of cotton fibers and increases their length.

7. The method according to claim 6, characterized in that, The GhPEX4 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

8. A method for breeding new cotton varieties with finer and longer cotton fibers, characterized in that, For cotton plants GhPEX4 Gene silencing is achieved; The GhPEX4 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

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