Application of cotton enoyl coenzyme A reductase gene GhECRA10 in improvement of cotton fiber quality

Abstract

The invention provides an application of a cotton enoyl coenzyme A reductase gene GhECRA10 in improvement of cotton fiber quality. The cotton enoyl coenzyme A reductase gene GhECRA10 is predominantly expressed in the early stage of cotton fiber elongation, but the expression quantity is gradually reduced along with fiber development in the later stage. The gene is knocked out by using a gene editing system to create a mutant for functional verification, and the result shows that the mutated GhECRA10 gene can significantly increase the fiber length and strength, significantly reduce the micronaire value (A-grade high-quality fiber), enlarge seeds and increase the hundred-grain weight of the seeds, thereby providing a new way and means for improving the quality and yield of cotton and having broad application prospects. And the method has extremely high application value and potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant gene breeding, and in particular relates to the application of cotton enoyl-CoA reductase gene GhECR_A10 in improving cotton fiber quality. Background Art

[0002] Cotton is an important cash crop in my country, and cotton fiber is a crucial raw material for the textile industry. Improving cotton fiber quality has long been a breeding goal for cotton researchers. Studies have shown that the content of very-long-chain fatty acids (VLCFAs) affects cotton fiber elongation. VLCFAs are fatty acids with a carbon content of 20 or more carbon atoms and serve as precursors for the synthesis of sphingolipids, triacylglycerols, cuticular waxes, and suberin. C16:0, C18:0, and C18:1 fatty acids synthesized in plastids are exported to the cytoplasm as acyl-CoA and then further elongated to VLCFAs in the endoplasmic reticulum (ER). The entire process is initiated by fatty acid elongase (FAE), which catalyzes four sequential enzymatic reactions (Von Wettstein-Knowles, 1987): condensation (3-ketoacyl-coa synthase, KCS), reduction (β-ketoacyl reducer, KCR), dehydration (β-hydroxyacyl-coa dehydratase, HCD), and a second reduction (enoyl-coa reducer, ECR). As the key enzyme catalyzing the final step of fatty acid elongation, ECR plays a crucial role. The impact of ECR ​​on cotton fiber development has not been reported, with more research focusing on the effects of KCS and KCR on cotton fiber development. Summary of the Invention

[0003] This study isolated and cloned the GhECR_A10 gene, encoding enoyl-CoA reductase, located on chromosome ChrA10 from upland cotton. The full-length cDNA nucleotide sequence of this gene in the standard upland cotton line TM-1 and the amino acid sequence of the encoded protein are provided. Using this gene as the target gene, the CRISPR / Cas9 editing system was used to mutate the GhECR_A10 gene. Functional analysis of the gene revealed that the GhECR_A10 gene affects cotton fiber quality, significantly increasing fiber length and strength while significantly reducing micronaire value (Grade A high-quality fiber). This gene can be used to cultivate new cotton germplasm with excellent fiber quality for production applications.

[0004] The technical route taken by the present invention is as follows: In a first aspect of the present invention, a gene for regulating cotton fiber length is provided. The gene is the GhECR_A10 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO. 2.

[0005] In a second aspect of the present invention, a vector for knocking out the gene is provided, wherein two sgRNA sequences are connected in series with tRNA and then connected to the pRGEB32-Cas9 gene editing vector, and the nucleotide sequences of the sgRNAs are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0006] The third aspect of the present invention provides a host bacterium carrying the recombinant vector, wherein the host bacterium is Agrobacterium GV3101.

[0007] The fourth aspect of the present invention provides the application of the gene in regulating the elongation and development of cotton fibers. Through the Agrobacterium-mediated cotton genetic transformation method, the GhECR_A10 gene is mutated in the transgenic recipient material Jin668, thereby causing the cotton fiber length and strength to increase significantly and the micronaire value to decrease significantly (Grade A high-quality fiber).

[0008] Furthermore, the significantly increased length and strength of the cotton fiber and the significantly reduced micronaire value (grade A high-quality fiber) are achieved by inserting or deleting bases in the GhECR_A10 gene in the cotton, resulting in amino acid changes.

[0009] Specifically, the present invention provides the use of the cotton enoyl-CoA reductase gene GhECR_A10 in any of the following applications, wherein the amino acid sequence encoded by the cotton enoyl-CoA reductase gene GhECR_A10 is shown in SEQ ID NO: 2, A1) Improve cotton fiber quality; A2) Preparation of products with improved cotton fiber quality; A3) Cultivate high-quality cotton germplasm; A4) Prepare and cultivate cotton germplasm products with excellent fiber quality.

[0010] A5) Controlling cotton seed size and / or 100-seed weight; A6) Preparing products for regulating cotton seed size and / or cotton seed 100-grain weight.

[0011] Furthermore, the nucleotide sequence of the cotton enoyl-CoA reductase gene GhECR_A10 is shown in SEQ ID NO: 1.

[0012] Furthermore, fiber quality includes fiber length, fiber strength and / or micronaire value.

[0013] Furthermore, the expression of cotton enoyl-CoA reductase gene GhECR_A10 or the function or activity of its protein is inhibited or reduced, thereby increasing cotton fiber length and fiber strength, reducing micronaire value, increasing seed size, and increasing 100-grain seed weight.

[0014] The present invention also provides a composition comprising at least one of the following: B1) sgRNA, B2) an expression vector containing the sgRNA, B3) an engineered bacterium containing the sgRNA, Wherein, the sgRNA includes sgRNA1 and / or sgRNA2, the sequence of sgRNA1 is shown as SEQ ID NO: 3, and the sequence of sgRNA2 is shown as SEQ ID NO: 4.

[0015] sgRNA1: 5'-GCAGTCCTGATGGTAGT-GGAGGG-3' sgRNA2: 5'-CCCCGAAGATGGGTTATTCTTCC-3'.

[0016] The present invention also provides the use of the above composition in any of the following: C1) Increase cotton fiber length and fiber strength, and reduce micronaire value; C2) Preparation of products that increase cotton fiber length and fiber strength and reduce micronaire value; C3) Increase cotton seed size and improve 100-grain weight; C4) Prepare products for increasing cotton seed size and improving 100-grain weight of cotton seeds.

[0017] The present invention also provides a method for improving cotton fiber quality and / or cotton seed size and 100-grain weight, comprising inhibiting or reducing the expression of the cotton enoyl-CoA reductase gene GhECR_A10 or the function or activity of its protein, thereby improving cotton fiber length and fiber strength, reducing micronaire value, increasing seed size, and improving 100-grain weight of seeds, wherein the amino acid sequence encoded by the cotton enoyl-CoA reductase gene GhECR_A10 is shown in SEQ ID NO: 2.

[0018] Furthermore, the expression of the cotton enoyl-CoA reductase gene GhECR_A10 or the function or activity of its protein is inhibited or reduced by one or more of the following methods: gene knockout, gene silencing, gene mutation and introduction of GhECR_A10 gene encoding protein expression and / or activity inhibitors.

[0019] Furthermore, gene knockout includes one or more selected from the following groups: zinc finger nuclease (ZFN) technology, transcription activating effector nuclease (TALEN) technology and CRISPR / Cas system-mediated gene editing technology; gene silencing includes one or more selected from the following group: RNA interference (RNAi), virus-induced gene silencing (VIGS), transcription factor deficiency and CRISPR-Cas9 system-mediated gene silencing.

[0020] Furthermore, the sgRNA used in the CRISPR / Cas9 system includes sgRNA1 and / or sgRNA2, wherein the sequence of sgRNA1 is shown in SEQ ID NO: 3, and the sequence of sgRNA2 is shown in SEQ ID NO: 4.

[0021] Beneficial Effects: The present invention isolated and cloned the GhECR_A10 gene encoding enoyl-CoA reductase, located on chromosome ChrA10, from upland cotton. The full-length cDNA nucleotide sequence of this gene in the standard upland cotton line TM-1 and the amino acid sequence of the encoded protein were provided. Furthermore, it was found that the cotton enoyl-CoA reductase gene GhECR_A10 is dominantly expressed during the early stages of cotton fiber elongation, but its expression level gradually decreases with fiber development in the later stages. Functional verification of mutants created by knocking out this gene using a gene editing system revealed that mutating the GhECR_A10 gene significantly increased fiber length and strength, significantly reduced micronaire value (grade A high-quality fiber), increased seed size, and increased 100-grain weight. This provides a new approach and means for improving cotton quality and yield, and has extremely high application value and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1Figure 2 shows the expression profiles of GhECR genes in different tissues and organs of the upland cotton genetic standard line TM-1. Figure A shows the expression profiles of four GhECR genes in different cotton tissues, and the FPKM data have been normalized. Figure B shows the expression profiles of GhECR_A10 and GhECR_D10 genes in different cotton tissues, and the data are FPKM values. Root: root; stem: stem; left: leaf; petal: petal; pistil: pollen; torus: receptacle; stamen: stamen; calycle: sepal; 5, 10, 20, 25 DPA_Fiber: fiber at 5 days, 10 days, 20 days, and 25 days after anthesis; -3, -1, 0, 1, 3, 5, 10, 20, 25, 35 DPA_Ovule: ovule at -3 days, -1 day, 0 day, 1 day, 3 days, 5 days, 10 days, 20 days, 25 days, and 35 days after anthesis. Data from ADDINEN.CITE ADDIN EN.CITE.DATA (Zhang et al., 2015).

[0024] Figure 2 Figure 1 shows the expression level of the GhECR_A10 gene and its correlation with fiber length. Figure A shows qRT-PCR analysis of the GhECR_A10 gene in different tissues and organs of upland cotton Jin668. Leaf: leaf; Stigma: stigma; Anther: anther; Petal: petal; 0, 10, 15, 20, 25, 30 DPA_Ovule: ovule at 0, 10, 15, 20, 25, and 30 days after anthesis; 5, 10, 15, 20, 25, and 30 DPA_Fiber: fiber at 5, 10, 15, 20, 25, and 30 days after anthesis. Figure B shows the expression analysis of GhECR_A10 during fiber development in upland cotton resource populations; Figure C shows the correlation analysis of GhECR_A10 expression during fiber development stages and fiber length.

[0025] Figure 3 Subcellular localization analysis of GhECR_A10 protein in tobacco leaves.

[0026] Figure 4 This is an analysis of the GhECR_A10 gene editing type. Figure A shows the target editing type; Figure B shows the protein sequence alignment information after gene editing.

[0027] Figure 5 Phenotypic identification of GhECR_A10 gene-edited plants (harvested in Hainan in April 2024). Figure A shows fiber length, bars = 1 cm; Figure B shows seed size, bars = 1 cm; and Figure C shows 100-grain weight.

[0028] Figure 6 Phenotypic identification of GhECR_A10 gene-edited plants (harvested in Wuhan in October 2024). Figure A shows fiber length, bars = 1 cm; Figure B shows seed size, bars = 1 cm; Figure C shows 100-grain weight.

[0029] Figure 7 Cross-sectional observation of mature fiber cells in GhECR_A10 gene-edited plants. Figure A shows the cross-sectional view of mature fibers, with bars = 50 µm. Figure B shows the thickness of the mature fiber cell wall. DETAILED DESCRIPTION

[0030] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention with this. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0031] Example 1. Planting of plant materials Upland cotton (G. hirsutum acc. Jin668) (transgenic recipient / WT) was cultivated in an experimental field at Huazhong Agricultural University to obtain cotton fiber and ovule samples from various tissues and organs and at different developmental stages. Cotton fibers and ovules were harvested at specific developmental stages, immediately frozen in liquid nitrogen, and stored at -80°C until further use. Nicotiana benthamiana was cultivated in a greenhouse (25°C / 16 h light, 20°C / 8 h dark). T0-generation mutants generated using the CRISPR / Cas9 gene editing system were cultivated in a greenhouse (28°C / 16 h light, 20°C / 8 h dark). Subsequent mutants and a control (WT) were planted in Hainan in early October 2023 and harvested in mid-April 2024, and in Wuhan in mid-April 2024 and harvested in early October 2024, respectively, to evaluate cotton fiber quality and 100-grain weight.

[0032] 2. Identification of the GhECR gene in cotton enoyl-CoA reductase Four enoyl-CoA reductase genes, GhECR genes, were identified in upland cotton TM-1. According to published expression profile data, GhECR_A10 and GhECR_D10 were dominantly expressed during cotton fiber development, while their expression abundance was low in other parts ( Figure 1A); The expression abundance of GhECR_A10 gene is higher than that of GhECR_D10 gene ( Figure 1 B). Therefore, GhECR_A10 gene was selected as a candidate gene for subsequent gene function verification.

[0033] Specific RT primers RT-ECR_A10-F / R were designed for the GhECR_A10 gene as shown in Table 1. The cotton endogenous gene GhUBQ7 (Ghir_A11G011460) was used as the internal reference gene, and RT primers (GhUB7-F / R) were designed. The expression levels of the GhECR_A10 gene in various tissues of upland cotton Jin668 were analyzed by qRT-PCR.

[0034] Table 1 The results showed that GhECR_A10 was dominantly expressed in the early stages of fiber development (5-10 DAP), but as the fibers continued to elongate (10-15 DPA) and thicken (20-25 DPA), the expression level of the gene gradually decreased. Figure 2 A), similar patterns were observed in each fiber development stage of upland cotton populations ( Figure 2 B). The above expression analysis results indicate that GhECR_A10 exhibits a stage-specific regulatory pattern during cotton fiber development. Its expression level is high in the early stages of fiber elongation and gradually decreases with fiber elongation and secondary wall thickening. This dynamic expression pattern is conserved in upland cotton Jin668 and resource populations, indicating that this gene has consistent biological functions across different genetic backgrounds.

[0035] To clarify the relationship between the expression level of this gene and the length of mature fibers, the correlation between the expression level of GhECR_A10 at the fiber development stages (4 DPA, 8 DPA, 12 DPA, 16 DPA and 20 DPA) and the length of mature fibers was analyzed ( Figure 2 C), the results showed that the expression level of this gene was significantly negatively correlated with the length of mature fibers, that is, this gene negatively regulates fiber elongation.

[0036] In summary, the dominant expression of GhECR_A10 in the early stages of fiber elongation provides necessary support for the initiation of fiber elongation. The rapid decrease in expression as the fiber develops, especially in the later stages, is to avoid hindering the elongation and thickening of the fiber.

[0037] 3. Subcellular localization analysis of GhECR_A10 protein The GhECR_A10 gene sequence information was obtained from the reference genome of upland cotton TM-1. The gene has a full-length cDNA nucleotide sequence as shown in SEQ ID NO: 1 and an amino acid sequence as shown in SEQ ID NO: 2. Using the full-length cDNA nucleotide sequence of the gene as a reference sequence, BP-GhECR_A10-F / R primers with bp linker primers as shown in Table 1 were designed to amplify the full-length cDNA sequence of the GhECR_A10 gene and ligated to the expression vector pGWB741 fused with a GFP tag via BP-LR homologous recombination for subcellular localization analysis. The results showed that the GhECR_A10 protein co-localized with the endoplasmic reticulum marker protein in tobacco leaves ( Figure 3 ), indicating that the protein is localized in the endoplasmic reticulum.

[0038] 4. Creation of GhECR_A10 gene mutants To further explore the function of the GhECR_A10 gene, the CRISPR / Cas9 gene editing system was used to mutate the gene, and two target sgRNAs 1 and sgRNA 2 were designed in the coding region of the gene. The vector construction vector-related primers are shown in Table 1: ERC_A10.1.as, ERC_A10.1.s, ERC_A10.2.as, inf.ERC_A10.2.as. The editing efficiency of the obtained mutant strains was analyzed using the Hi-TOM system, and the target amplification primers used are shown in Table 1: CrECR-T1-F / R, CrECR-T2-F / R. The editing types of the two mutant materials CrECR-1 and CrECR-22 obtained at these two target positions are as follows: Figure 4 As shown in A; the amino acid sequence alignment information of mutant materials CrECR-1 and CrECR-2 is shown in Figure 4 As shown in B. The results showed that the amino acid sequence of GhECR_A10 in the mutant materials CrECR-1 and CrECR-2 was destroyed and mutated into an amino acid sequence different from that in the control WT.

[0039] 5. Investigation of agronomic traits of GhECR_A10 gene mutants To explore the effect of GhECR_A10 gene on cotton fiber, cotton fiber quality-related traits, including fiber length, fiber strength and micronaire value, as well as 100-grain seed weight, were investigated in Hainan in mid-April 2024 and Wuhan in early October 2024, respectively.

[0040] Table 2 Fiber quality analysis (harvested in Hainan in mid-April 2024) Table 3 Fiber quality analysis (harvested in Wuhan in early October 2024) As shown in Tables 2 and 3, in the two planting environments of Hainan in 2024 and Wuhan in 2024, the fiber length and fiber strength of the mutant strains CrECR-1 and CrECR-2 were significantly increased compared with the control WT material, while the micronaire value was significantly decreased compared with the control WT material. Manual combing of cotton fibers also showed that the fiber length of the mutant strains CrECR-1 and CrECR-2 was longer ( Figure 5 A, Figure 6 A), seeds become larger ( Figure 5 B, Figure 6 B), the weight of 100 seeds increased ( Figure 5 C, Figure 6 C).

[0041] 6. Observation of the thickness of mature fiber cell wall in GhECR_A10 gene mutant In the fiber quality analysis, it was found that the micronaire value of the GhECR_A10 gene mutant strain was significantly reduced, indicating that the fiber fineness was reduced. Further, the mature fibers of the GhECR_A10 mutant and WT materials planted in Wuhan in 2024 were analyzed by semi-thin section. Figure 7 As shown in A, the thickness of the fiber cross section of the GhECR_A10 mutant is thinner than that of the WT. Statistical analysis of the fiber thickness showed that the fiber thickness of the GhECR_A10 mutant was significantly lower than that of the WT ( Figure 7 B), indicating that the mutation of GhECR_A10 gene can significantly reduce the thickness of cotton fiber cell wall and reduce fiber fineness.

[0042] In summary, the present invention has found through research that the expression level of the GhECR_A10 gene encoding enoyl-CoA reductase located on the ChrA10 chromosome shows a significant negative correlation with the mature fiber length, that is, it negatively regulates fiber elongation. Further use of the gene editing system to knock out the gene to create mutants for verification found that the mutation of the GhECR_A10 gene can significantly increase fiber length and strength, significantly reduce the micronaire value (A-grade high-quality fiber), enlarge seeds and increase the 100-grain weight of seeds, providing a new way and means to improve cotton quality and yield, and has extremely high application value and potential.

[0043] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. The use of cotton enoyl-CoA reductase gene GhECR_A10 in any of the following applications, characterized in that: The amino acid sequence encoded by the cotton enoyl-CoA reductase gene GhECR_A10 is shown in SEQ ID NO:

2. A1) Improve cotton fiber quality; A2) Preparation of products with improved cotton fiber quality; A3) Cultivate high-quality cotton germplasm; A4) Preparation and cultivation of high-quality cotton fiber germplasm products; A5) Controlling cotton seed size and / or 100-seed weight; A6) Preparing products for regulating cotton seed size and / or cotton seed 100-grain weight.

2. The use according to claim 1, characterized in that The nucleotide sequence of the cotton enoyl-CoA reductase gene GhECR_A10 is shown in SEQ ID NO:

1.

3. The use according to any one of claims 1-2, characterized in that Fiber quality includes fiber length, fiber strength and / or micronaire.

4. The use according to claim 3, characterized in that Inhibit or reduce the expression of cotton enoyl-CoA reductase gene GhECR_A10 or the function or activity of its protein, thereby increasing cotton fiber length and fiber strength, reducing micronaire value, increasing seed size, and increasing seed 100-grain weight.

5. A composition, characterized in that The composition comprises at least one of the following: B1) sgRNA, B2) an expression vector containing the sgRNA, B3) an engineered bacterium containing the sgRNA, Wherein, the sgRNA includes sgRNA1 and / or sgRNA2, the sequence of sgRNA1 is shown as SEQ ID NO: 3, and the sequence of sgRNA2 is shown as SEQ ID NO:

4.

6. Use of the composition according to claim 5 in any of the following: C1) Increase cotton fiber length and fiber strength, and reduce micronaire value; C2) Preparation of products that increase cotton fiber length and fiber strength and reduce micronaire value; C3) Increase cotton seed size and improve 100-grain weight; C4) Prepare products for increasing cotton seed size and improving 100-grain weight of cotton seeds.

7. A method for improving cotton fiber quality and / or cotton seed size and 100-grain weight, characterized in that: Inhibiting or reducing the expression of the cotton enoyl-CoA reductase gene GhECR_A10 or the function or activity of its protein, thereby increasing cotton fiber length and fiber strength, reducing micronaire value, increasing seed size, and increasing 100-grain seed weight, wherein the amino acid sequence encoded by the cotton enoyl-CoA reductase gene GhECR_A10 is shown in SEQ ID NO:

2.

8. The method according to claim 7, characterized in that The inhibition or reduction of the expression of the cotton enoyl-CoA reductase gene GhECR_A10 or the function or activity of its protein is achieved by one or more of the following methods: gene knockout, gene silencing, gene mutation and introduction of GhECR_A10 gene encoding protein expression and / or activity inhibitors.

9. The method according to claim 8, characterized in that Gene knockout includes one or more selected from the following groups: zinc finger nuclease technology, transcription activator effector nuclease technology and CRISPR / Cas system-mediated gene editing technology; gene silencing includes one or more selected from the following group: RNA interference, virus-induced gene silencing, transcription factor deficiency and CRISPR / Cas9 system-mediated gene silencing.

10. The method according to claim 9, characterized in that The sgRNA used in the CRISPR / Cas9 system includes sgRNA1 and / or sgRNA2, wherein the sequence of sgRNA1 is shown in SEQ ID NO: 3, and the sequence of sgRNA2 is shown in SEQ ID NO: 4.

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