Gene ghabh for regulating fiber quality of cotton and application thereof

Abstract

The application discloses a gene for regulating cotton fiber quality GhABH and application thereof, and relates to the field of genetic engineering GhABH The nucleotide sequence of the gene is shown as SEQ ID NO:1, the amino acid sequence of the encoded protein of the gene is shown as SEQ ID NO:2, and the cotton fiber quality includes fiber length, strength and fineness. GhABH After the expression of the gene is down-regulated through VIGS, the fiber becomes longer, thinner but the strength is increased, microscopic observation shows that GhABH the fiber diameter is reduced after silencing, but the cell wall is significantly thickened and the spiral density on the surface is increased, which indicates that GhABH the gene has the potential of synchronously improving fiber length, strength and fineness. GhABH Overexpression of the gene in Arabidopsis results in shorter root hairs and thinner stem cell walls. GhABH These results collectively show that GhABH the gene can cooperatively improve the length, fineness and strength of cotton, and is a promising candidate gene in the molecular breeding strategy for targeting cotton fiber quality improvement.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to an α / β-hydrolase gene that regulates the length, strength, and fineness of cotton fibers. GABH And its applications. Background Technology

[0002] Upland cotton ( Gossypium hirsutum Cotton is the most important renewable natural fiber source for the global textile industry. Originating from the outermost epidermal cells of the ovule, cotton development involves four distinct but partially overlapping stages: initiation, elongation (primary cell wall synthesis), secondary cell wall (SCW) deposition, and maturity. Fiber quality, primarily determined by fiber length (FL), strength (FS), and fineness / micronaire value (FM), is closely related to these developmental stages. During the elongation stage, rapid primary cell wall synthesis drives fiber elongation, while secondary cell wall deposition subsequently increases fiber thickness and affects FL and FM. Notably, the initiation time and duration of secondary cell wall synthesis critically influence fiber elongation, cell wall thickness, and ultimately determine core quality traits such as cotton length, fineness, and strength. These characteristics directly determine the commercial value of cotton.

[0003] Modern cotton breeding focuses on improving cotton length, strength, and fineness by identifying functional genes. Significant progress has been made in the identification of key genes, including those regulating fiber initiation. GhMYB25- like (Zhao G, Le Y, Sun M, et al. A dominant negative mutation of GhMYB25 -like alters cotton fiber initiation, reducing lint and fuzz. The Plant Cell 36(8): 2759-2777.); regulates fiber elongation. BLH1 , KNOX6 (Jia T, Wang H, Cui S, et al. Cotton BLH1 and KNOX6 antagonistically modulate fiber elongation via regulation of linolenic acidbiosynthesis. Plant Communications 5(7): 100887.), GhATL68b (Li X, Huang G, Zhou Y, et al. GhATL68bregulates cotton fiber cell development byubiquitinating the enzyme required for β-oxidation of polyunsaturated fattyacids. Plant Communications 5(10): 101003.)、 SMXL7 (Sun Y, Tian Z, Zuo D, etal. Strigolactone-induced degradation of SMXL7 and SMXL8 contributes togibberellin- and auxin-mediated fiber cell elongation in cotton. The PlantCell 36(9): 3875-3893.)、 GhMYB4 (Duan Y, Shang X, Wu R, et al. Thetranscription factor GhMYB4 represses lipid transfer and sucrose transportergenes and inhibits fiber cell elongation in cotton. Plant Physiology 197(1):637.)、 GhLCBK1 (Zhang J, Meng Q, Wang Q, et al. Cotton sphingosine kinase GhLCBK1 participates in fiber cell elongation by affecting sphingosine-1-phophate and auxin synthesis. International Journal of BiologicalMacromolecules, 267:131323.)、 GhHDZ76 (Wu C, Xiao S, Zhang X, et al. GhHDZ76, a cotton HD-Zip transcription factor, involved in regulating the initiation and early elongation of cotton fiber development in G. hirsutum. Plant Science 345: 112132.) and GhMYB201 (Suo Q, Fang N, Zeng J, et al. R2R3 MYB transcription factor GhMYB201 promotes cotton fiber elongation via cell wall loosening and very-long-chain fatty acid synthesis. International Journal of Molecular Sciences 25(17): 9559.); for regulating fiber strength GbCML7 (Zhao N, Wang W, Jiang K, et al. A calmodulin-like gene ( GbCML7 ) for fiber strength and yield improvement identified by resequencing core accessions of a pedigree in Gossypium barbadense. Frontiers in Plant Science 12: 815648.) and GhbHLH18 (Gao Z, Sun W, Wang J, et al. GhbHLH18 negatively regulates fiber strength and length by enhancing lignin biosynthesis in cotton fibers. Plant Science 286: 7-16.); for regulating fiber fineness Gh_D04G0865 (Naoumkina M, Florane CB, Kim HJ, et al. Overexpression of an actin Gh_D04G0865gene in cotton reduced fineness offiber. Crop Science 63:740-749. ) and GhROD1 (Ding B, Liu B, Zhu X, et al.Downregulation of the GhROD1 Genes improve cotton fiber fineness by decreasing Acyl pool saturation, stimulating small heat shock proteins (sHSPs), and reducing H2O2 production. (International Journal of Molecular Sciences 25(20): 11242.). It is noteworthy that multiple pleiotropic genes that synergistically regulate multiple fiber quality traits have become highly promising targets for genetic improvement. Traditional locus / gene assessments typically focus on single fiber traits, which may overlook potential trait trade-offs—some alleles beneficial to one fiber trait may adversely affect other traits. Therefore, discovering new stable multi-trait QTLs and genes that can synergistically optimize fiber length, fineness, and strength is crucial for precision cotton breeding.

[0004] Although the alpha / beta hydrolase (ABH) superfamily exhibits diverse biochemical functions in plants through active site modifications (Mindrebo JT, Nartey CM, Seto Y, et al. Unveiling the functional diversity of the alpha / beta hydrolase superfamily in the plant kingdom. Current Opinion in Structural Biology 41: 233-246.), its specific role in cotton fiber quality formation remains unclear. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a gene for regulating cotton fiber quality. GABH This invention, through integrating QTL mapping and transcriptome analysis, and combining virus-induced gene silencing (VIGS) technology in cotton loss-of-function experiments and transgenic function verification in Arabidopsis thaliana, discovered an α / β-hydrolase gene. GABH It has the potential to simultaneously improve the length, strength, and fineness of cotton fibers.

[0006] This invention provides a gene for regulating cotton fiber quality. GABH The GABH The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0007] Furthermore, the aforementioned GABH The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0008] The present invention also provides the above-mentioned gene. GABH The application in regulating cotton fiber quality, the GABH The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.

[0009] Furthermore, the cotton fiber quality includes fiber length, strength, and fineness.

[0010] Furthermore, the application specifically involves: suppressing or reducing... GABH Gene expression can simultaneously improve fiber length, fineness, and strength (resulting in longer fibers, higher strength, and lower micronaire value).

[0011] The GABH The gene was obtained using homologous cloning in the A generation of the cross between cotton ji 1518 and the offspring. dh It was cloned from the cDNA of the leaf. GABH The gene's ORF is 921 bp in length and encodes 306 amino acids (excluding the stop codon).

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses VIGS downregulation GABH After expression, the fibers become longer and thinner but stronger, as shown by microscopic observation. GABH After silencing, the fiber diameter decreased, but its cell wall thickened significantly and the surface helical density increased, indicating that... GABH It has the potential to simultaneously improve fiber length, strength, and fineness (manifested as a reduction in micronaire value from 5.3 to 4.6). Overexpression in Arabidopsis thaliana... GABH This results in shorter root hairs, thinner stem cell walls, and a reduced number of vascular bundles. These results collectively indicate... GABH It can synergistically improve the length, fineness and strength of cotton, and is a very promising candidate gene in molecular breeding strategies that target the improvement of cotton fiber quality. Attached Figure Description

[0013] Figure 1 It involves the development of resequencing sequence differences and specific region markers, where a: cotton fiber near-isogeninized line A based on resequencing. dh With Adl Sequence difference analysis; b: Development of molecular markers for QTL regions to stabilize fiber quality; Figure 2 This study uses KASP markers and transcriptome analysis to finely map pleiotropic QTLs for fiber quality, where a: QTL location associated regions and their differentially expressed genes; b: QTL region gene expression pattern diagram. Figure 3 yes GABH Association analysis of gene structure and key SNP sites with fiber quality traits, where a: GABH Gene structure; b: SNP 855th Locus typing and 180 F 2:3 Correlation analysis of fiber length in strains; c: SNP 855th Locus typing and 180 F 2:3 Correlation analysis of fiber strength of strains; d: SNP 855th Locus typing and 180 F 2:3 Correlation analysis of micronaire values ​​of strains; e: SNP 120th Locus typing and 180 F 2:3 Correlation analysis of fiber length in strains; f: SNP 120th Locus typing and 180 F 2:3 Correlation analysis of fiber strength of strains; g: SNP 120th Locus typing and 180 F 2:3 Correlation analysis of micronaire values ​​of strains; in the figure, ** indicates a significant difference at the 0.01 level; Figure 4 yes GABH Subcellular localization map; Figure 5 yes GABH Analysis of relative expression levels in silent and control lines, where CK: control line, ** indicates a significant difference at the 0.01 level; Figure 6 This is based on three fiber testing instruments (HVI1000, FAVIMAT, and AFIS PRO 2) for... GABH Analysis of fiber density in silent plants and control plants, where a: HVI1000 was used for evaluation. GABH The effect of gene silencing on fiber length; b: assessment using HVI1000. GABH The effect of gene silencing on fiber strength; c: assessment using HVI1000. GABH The effect of gene silencing on horse clone value; d: assessed using AFIS PRO. GABH The effect of gene silencing on fiber length distribution; e: assessment using AFIS PRO. GABHThe effect of gene silencing on mean fiber length; f: assessment using FAVIMAT. GABH The effect of gene silencing on fiber strength; g: assessed using FAVIMAT. GABH The effect of gene silencing on malar clone value; in the figure, ** indicates a significant difference at the 0.01 level; Figure 7 yes GABH Microscopic images of silent and control materials, where a: GABH Statistical analysis of the effect of gene silencing on fiber helix length; b: GABH Microscopic observation of the effect of gene silencing on fiber helix length; c: GABH Statistical analysis of the effect of gene silencing on cell wall thickness; d: GABH Microscopic observation of the effect of gene silencing on cell wall thickness; in the figure, ** indicates a significant difference at the 0.01 level; Figure 8 It is Arabidopsis overexpression GABH Verify its effects on root hair elongation and cell wall development, where a: obtain GABH overexpression lines; b: GABH Comparison of root length between overexpression lines and wild-type lines after 8 days of growth; c: GABH Statistical analysis of root length of overexpression lines and wild-type lines after 8 days of growth; d: GABH Comparison of stem growth diagrams between overexpression lines and wild-type lines; e: GABH Comparison of stem sections between overexpression lines and wild-type lines; f: GABH Comparison of cell wall thickness in stem sections of overexpression lines and wild-type lines; g: GABH Statistical analysis of cell wall thickness in stem sections of overexpression lines and wild-type lines; in the figure, ** indicates a significant difference at the 0.01 level. Detailed Implementation

[0014] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0015] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0016] Example 1: Fine mapping of QTLs and candidate genes for fiber quality GABH Identification Research methods: F1 generation of the Ji 1518 hybrid combination2:9 Within the population, two near-isogenic lines, A and B, with significant differences in fiber strength (FS) and fiber length (FL), were selected. dh With A dl And based on this, a secondary F1 generation containing 229 strains was constructed. 2:3 The graphing group. Based on A dh With A dl Single nucleotide polymorphisms (SNPs) identified in the target region were converted into KASP markers using PolyMarker (http: / / polymarker.tgac.ac.uk). Phenotypic identification of the population and parents was performed under three environments: Weixian County (20WX) in 2020, Weixian County (21WX) in 2021, and Hengshui City (21HS) in 2021. Each F... 2:3 Collect 30 cotton bolls from the family line and parents, and analyze fiber quality traits (length [FL, mm], strength [FS, cN·dtex]). -1 The micronaire value (and micronaire value) were measured by the Cotton Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs (Anyang, Henan) using the HVI1000 system. Multi-environment phenotypic data were analyzed using Optimal Linear Unbiased Prediction (BLUP) with R software. Linkage map construction and fiber quality-related QTL identification were performed using IciMappingv4.0, with the LOD threshold uniformly set to ≥2.5.

[0017] Research Results: Previous studies identified a stable QTL regulating cotton fiber length (FL), strength (FS), and fineness (FM) near chromosome HAU2349 on chromosome A06. This QTL has a significant impact on near-isogenic lines A. dh and A dl Whole-genome resequencing (20×) was performed, and KASP and InDel markers were developed in the above QTL regions. Figure 1 Using near-isogenic line A dh and A dl Constructing a structure containing 229 Fs 2:3 Secondary populations of the strain, combined with multi-environment BLUP phenotypic correction and effective KASP markers, finely mapped the major QTL to an 11.41 Mb region (containing 244 genes) on A06. Figure 2 a) This region explains 6.02%–10.81% of the phenotypic variation (Table 1). Transcriptome analysis revealed 16 differentially expressed genes in this region, among which… GABH Significant differences in the expression of (α / β-hydrolase homologous genes) were observed between the two parents, A. dh and A dl Different expression patterns in Chinese ( Figure 2 b). Therefore, homologous cloning was used to clone the A offspring of the cotton ji 1518 hybrid. dh Cloning in leaf cDNA GABHPrimer sequences are shown in Table 2. The Tm value is 55 degrees Celsius. Amplification and Sanger sequencing were performed using a standard amplification system. GABH Single nucleotide polymorphisms (SNPs) in [the data] are represented by A [a specific type of nucleotide polymorphism dh The DNA from the leaves was amplified and then sequenced using Sanger sequencing to identify genotypes (sample size ≥ 180 F1 samples). 2:3 (Lineage). Based on the best linear unbiased prediction (BLUP) fiber quality phenotypic values ​​obtained from three replicate experiments, the association between these SNPs and fiber traits was analyzed using Tukey's HSD test (significance level set at 100%). p <0.01). Analysis based on sequencing results. GABH Gene structure and sequence differences revealed two SNPs in this gene that were significantly associated with fiber length and fiber strength. Figure 3 Subcellular localization findings GABH Located in the cytoplasm ( Figure 4 In summary, GABH Considered a key candidate gene for regulating fiber quality formation ( GABH The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2. GABH The full-length ORF of the gene is 921 bp, encoding 306 amino acids (excluding the stop codon).

[0018] Table 1. QTL information of fiber quality identified under different environments

[0019] Table 2 Primer Sequences

[0020] Example 2: Using VIGS to reduce GABH The expression level can synergistically improve cotton fiber length, strength, and fineness (fiber diameter, cell wall thickness, fiber surface helices, etc. all change). Using pCLCrVA vector as the backbone, construct GABH The primers for the silencing vector are shown in Table 3. Table 3 Primer Sequences

[0021] Then, the vector was transformed into Agrobacterium strain GV3101 using the freeze-thaw method, with pCLCrVA: 00 and pCLCrVA: GhCLA1 As negative and positive controls, pCLCrVA: GABH The expression levels of the fiber at 5-15 DPA were compared with those of the control pCLCrVA: 00 Compared to significantly reduced ( Figure 5 Fiber quality assessment used 14-20 VIGS-treated and control plants. Bolls opened within 7 days from 4-6 plants were combined to form a biological replicate, with three replicates per line. Fiber length, strength, and micronaire value were determined using HVI1000 (Uster), and fiber quality was further analyzed using FAVIMAT (Textechno) (100 bolls) and AFIS PRO 2 (Uster) (3000 bolls). Fiber surface and cell wall thickness were observed using electron fiber microscopy.

[0022] Validation using VIGS-mediated gene silencing technology revealed that inhibiting... GABH It can increase fiber length and strength, and decrease fiber diameter and fineness (manifested as a decrease in micronaire value from 5.3 to 4.6 and a decrease in Lin. Den.). Figure 6 Microscopic observation revealed GABH Silencing increases the number of spirals per unit length of fiber and thickens the cell wall. Figure 7 This gene exhibits a unique function of simultaneously optimizing multiple traits of fiber quality, which aligns with the goal of breeding high-quality varieties and has significant research value.

[0023] Example 3 GABH Regulates cell elongation and secondary cell wall thickening in Arabidopsis stems and roots remove GABH In addition to the VIGS experiment, we also constructed an overexpression... GABH Transgenic Arabidopsis thaliana lines. Two transgenic lines were selected ( GABH -OE-3 and GABH -OE-4), and the characteristics of its roots and stems were analyzed. Compared with the wild-type control (WT), overexpression of -OE-4) was significantly improved. GABH Arabidopsis plants ( Figure 8 a) After 8 days of vertical growth, the primary roots showed a significant shortening ( Figure 8 bc), the thickness of the stem cell wall decreases ( Figure 8 dg). In addition, GABH The stem diameter and vascular bundle number of the overexpression lines were also significantly reduced. Figure 8 ef).

[0024] In summary, this embodiment identified a stable QTL on chromosome A06 that is associated with both fiber length and strength using molecular marker technology. This QTL comprises an 11.41 Mb region containing 244 genes, of which 16 genes are differentially expressed during fiber development. It is noteworthy that... GABH Single nucleotide polymorphisms (SNPs) in the fibrous material were significantly correlated with fiber length and strength. Silence GABHThis leads to a decrease in fiber diameter but promotes helical structure formation and cell wall thickening, ultimately increasing fiber length, strength, and fineness simultaneously (manifested as a decrease in micronaire value from 5.3 to 4.6, requiring materials with relatively low micronaire values ​​for production). Conversely, overexpression in Arabidopsis thaliana... GABH This leads to shorter root hairs, thinner stem cell walls, and a reduced number of vascular bundles. GABH Transcriptomic analysis of VIGS fibers revealed potential downstream genes in this gene regulatory pathway. Therefore, GABH It is a key regulatory factor that can simultaneously improve fiber length, strength and fineness, providing a valuable target for molecular breeding for improving cotton fiber quality.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gene that regulates cotton fiber quality GhABH Its features are: The GhABH The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A gene for regulating cotton fiber quality according to claim 1 GhABH Its features are: The GhABH The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. A gene as described in any one of claims 1-2 GhABH Its application in regulating cotton fiber quality is characterized by: The GhABH The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that: The cotton fiber quality includes fiber length, strength, and fineness.

5. The application according to claim 3, characterized in that: The specific application involves: suppressing or reducing... GhABH Gene expression can simultaneously improve fiber length, fineness, and strength.

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