Method for improving fiber length by changing extraplasm acidification degree

Abstract

The invention belongs to the technical field of plant expression vectors and application thereof, and discloses a method for improving fiber length by changing extracellular body acidification degree, which comprises the following steps: fusing a specific promoter with a PM < H + >-ATP enzyme gene to construct a plant expression vector for specifically expressing PM < H + >-ATP enzyme, and integrating the plant expression vector into a cotton genome to obtain the fiber length. According to the method, the pH value of fiber cytoplasm exosome is moderately reduced in the fiber elongation process, the length of the cotton fiber can be remarkably increased, and high-quality fiber is provided for the cotton industry and the textile industry. According to the method for improving the length of the cotton fiber, AHA related genes are specifically expressed in fiber cells of cotton, then the acidification degree of cell walls in the fiber elongation process is regulated and controlled, the cell walls are moderately acidified and relaxed, development of the cotton fiber is controlled, and the purpose of increasing the length of the cotton fiber is achieved. Test results prove that the length of the cotton fibers improved by the method for improving the characters of the cotton fibers is obviously increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant expression vectors and their applications, and in particular relates to a method and system for improving fiber length by changing the acidification degree of apoplast. Background Art

[0002] Cotton, known as "white gold," is an important crop that provides natural fibers for the textile industry (Serna and Martin, 2006) and is a vital component of the global economy and ecological environment. Globally, cotton forms the foundation of the textile industry chain, supporting the development of a series of related industries including spinning, weaving, printing and dyeing. Cotton fiber is widely used in various fields due to its excellent properties such as softness and comfort, moisture absorption and breathability, and easy dyeing and processing. It plays an indispensable role. It is not only the preferred material for clothing manufacturing, but also widely used in many fields such as medical supplies and furniture decoration, with huge market demand. As a source of natural fiber, cotton fiber is one of the main raw materials of the textile industry. Every 1.76 kg of seed cotton can produce 1 kg of cotton fiber (Gao et al., 2020).

[0003] Fiber length is one of the most important indicators for evaluating cotton fiber quality and a crucial factor in determining yarn value (Naoumkina et al., 2019; Hafeez et al., 2021). Cotton can be divided into three categories based on fiber length and linear density: long-staple cotton, coarse-staple cotton, and fine-staple cotton. Long-staple cotton fibers are slender and have strong individual fibers, making textiles durable and comfortable. However, they are prone to producing numerous neps and entanglements during carding and thus have a low yield. Coarse-staple cotton fibers are coarse and elastic, but of lower quality. Fine-staple cotton is predominant in terms of quantity, accounting for 85% of the world's total cotton fiber production and is currently the main cultivated cotton variety. Cotton fiber quality directly affects the quality of textiles, so improving cotton fiber yield and quality has always been a primary goal of cotton germplasm improvement.

[0004] Sea Island cotton fibers are characterized by their long, strong, and fine fibers, but their low yields result in a high price per kilogram. While Upland cotton strains offer high yields, their quality is inferior to that of Sea Island cotton. Breeders have long been concerned with developing new cotton varieties that combine high yield with superior fiber quality. This is difficult to achieve through traditional breeding alone, so we have employed genetic engineering to create new cotton germplasm.

[0005] Cotton fibers develop from single-cell protrusions of the ovule epidermis, with aspect ratios reaching up to 3000. Fiber differentiation and development can be divided into four main phases: initiation, elongation, secondary cell wall (SCW) biosynthesis, and dehydration and maturation. These four phases occur sequentially but overlap, making it an ideal model system for studying single-cell differentiation, elongation, and secondary wall thickening (Lee et al., 2007). The acid growth theory may explain the enormous elongation potential of fiber cells. This theory posits that auxin activates the plasma membrane H+-ATPase, leading to H+ production. + Trans-plasma membrane outflow and the decrease of apoplast pH affect cell wall extensibility by regulating the activity of cell wall modification related proteins, including expansins (EXP), xyloglucan endotransglycosylase / hydrolase (XTH) and pectin methylesterase, thereby activating cell wall modification related enzymes such as expansins, pectin methylesterase and xyloglucan endotransglycosidase / hydrolase, thereby relaxing the cell wall. + The absorption of iodine causes an increase in turgor pressure, which promotes the expansion of the cell wall and causes cell elongation (Du et al., 2020; Ak et al., 2014), but the application of acid growth theory to cotton fibers is still unclear.

[0006] TRANSMEMBRANE KINASE (TMK) is one of the leucine-rich repeat receptor-like kinases (RLKs) first identified in Arabidopsis thaliana three decades ago and is considered a key player in cell surface auxin signaling (Chang et al., 1992; Xu et al., 2014; Cao et al., 2019; Yu et al., 2023). Previous studies have determined that the kinase domain of TMK can phosphorylate substrates such as plasma membrane H + TMK proteins are known to express auxin through the regulation of mitogen-activated protein kinase kinases 4 / 5 (MKK4 / 5), tryptophan aminotransferase 1 (TAA1), and abscisic acid insensitive 1 and 2 (ABI1 / 2), leading to a rapid auxin response and affecting various developmental stages of plants (Huang et al., 2019; Wang et al., 2020; Li et al., 2021; Lin et al., 2021; Yang et al., 2021; Zhang et al., 2023). However, the molecular mechanism by which TMK proteins perceive auxin has long remained unclear. Recent studies have demonstrated that apoplast-localized ABP1-like proteins (ABL1 and ABL2) act as co-receptors with TMKs to regulate auxin responses and various plant developmental processes (Yu et al., 2023).

[0007] It is impossible to artificially regulate the pH of the cell wall during fiber cell elongation, but it can be achieved by adjusting the PM H+ -ATPase (AHA) or TMK activity, thereby regulating the internal pH of the cell, thereby achieving the purpose of moderately acidifying the cell wall to allow fiber cells to elongate.

[0008] Through the above analysis, the problems and defects of the existing technology are as follows:

[0009] K + The absorption of acid causes an increase in turgor pressure, which promotes the expansion of cell walls and causes cell elongation. However, the application of acid growth theory to cotton fibers is still unclear. Summary of the Invention

[0010] In response to the problems existing in the prior art, the present invention provides a method for improving fiber length by changing the acidification degree of the apoplast.

[0011] The present invention is implemented as follows: a method for improving fiber length by changing the acidification degree of the apoplast comprises:

[0012] Step 1, obtaining a fiber-specific expression promoter;

[0013] Step 2, obtaining genes related to changing the acidification degree of the apoplast of fiber cells;

[0014] Step 3, fusing the specific promoter isolated and cloned in step 1 with the related gene isolated and cloned in step 2 to construct a plant expression vector that specifically expresses AHA and TMK;

[0015] Step 4, genetically transforming cotton using the specific plant expression vector obtained in step 3;

[0016] Step 5: further culture and cultivate the cotton obtained in step 4 to obtain transgenic cotton plants.

[0017] Furthermore, the specific expression promoter in step 1 may be a natural promoter isolated and cloned from an animal, plant or microorganism, or may be an artificially modified or designed synthetic promoter.

[0018] Furthermore, the cotton AHA / TMK-related gene described in step 2 may be a natural gene isolated and cloned from an animal, plant or microorganism, or an artificially modified or designed gene.

[0019] Furthermore, the method of fusing the specific promoter described in step (3) with the auxin synthesis-related gene to construct an expression vector that specifically expresses the auxin synthesis-related gene is a conventional method in the art, and the vector used can be a conventional vector used in the field of plant transgenics;

[0020] The specific promoters are fiber-specific promoters GhHOS3 and GhEXPA2, which are highly expressed mainly during the fiber elongation period.

[0021] Furthermore, the plant expression vector specifically expressing GhAHA4:

[0022] Contains at least PM H + -nucleotide sequence for expressing GhAHA4 consisting of an ATPase gene and a cotton fiber-specific promoter;

[0023] The nucleotides contain at least PM H + -ATPase gene GhAHA4 and cotton fiber-specific promoter HOS3 gene promoter, and the GhHOS3 gene promoter is located 5' upstream of GhAHA4;

[0024] The nucleotide sequence has the sequence shown in SEQ ID No. 1.

[0025] The plant expression vector specifically expressing GhTMK3A:

[0026] The invention at least contains a nucleotide sequence for expressing GhTMK3A composed of a TMK gene and a cotton fiber-specific promoter;

[0027] The nucleotide sequence at least comprises the TMK gene GhTMK3A and the cotton fiber-specific promoter GhEXPA2 gene promoter, and the GhEXPA2 gene promoter is located 5' upstream of GhTMK3A;

[0028] The nucleotide sequence has the sequence shown in SEQ ID No. 2.

[0029] Another object of the present invention is to provide two transformants obtained by transforming a host with the plant expression vector.

[0030] Another object of the present invention is to provide the application of the plant expression vector in improving cotton fiber traits.

[0031] Another object of the present invention is to provide a method for preparing a transgenic plant containing the plant expression vector, comprising the following steps:

[0032] 1) PM H + -ATPase gene and TMK gene are operably linked to cotton fiber-specific promoter;

[0033] 2) Construct a PM H + -Plant expression vector of ATPase gene and TMK gene with cotton-specific promoter.

[0034] Another object of the present invention is to provide a method for encoding PM H +-ATPase and TMK related gene nucleotides, characterized in that they have the nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2.

[0035] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0036] First, the technical problem to be solved by the present invention is that cotton breeding usually adopts hybrid breeding or genetic engineering breeding. However, although traditional hybrid breeding can quickly gather multiple excellent genes, the genetic background of the offspring is complex, and a lot of selection and identification work is required. Gene editing using CRISPR-Cas9 technology can produce new traits in cotton, but because there are many cotton genes, most gene mutations or overexpressions will not cause a huge change in fiber quality due to functional redundancy. However, the present invention takes a different approach and uses related genes in the acid growth theory to construct an expression vector that can regulate the activity of AHA and thus regulate the pH in fiber cells. It is transferred into cotton to moderately acidify the cell wall of the fiber cells, thereby causing fiber elongation, which can be used to improve cotton fiber traits.

[0037] "Genetically modified cotton," as used in this article, refers to cotton grown by transferring genes from other organisms through molecular biology and biotechnology, thereby modifying the genetic material of the modified cotton. The genes used for modification can originate from plants, animals, microorganisms, or be artificially synthesized.

[0038] Through extensive preliminary research and analysis, the present invention believes that since moderately acidifying the cell wall during fiber elongation can cause fiber elongation, the idea of ​​regulating cell wall pH by regulating the activity of AHA should be feasible. To achieve precise regulation of apoplast acidification during the fiber elongation stage, this study selected a suitable promoter based on the following strategy: First, the dynamic changes in apoplast pH during fiber development and the expression patterns of the GhAHA4A gene and GhTMK3A were comprehensively considered; second, to avoid adverse effects on other agronomic traits, promoters with fiber-specific expression characteristics were specifically selected to effectively regulate the cell wall acidification process. Through multi-tissue transcriptome analysis of cotton, this study found that the GhHOS3 promoter and GhEXPA2 exhibit ideal expression characteristics: they are specifically highly expressed during fiber elongation and almost not expressed in other tissues. This tissue-specific expression pattern makes them ideal tools for regulating apoplast acidification during fiber elongation.

[0039] The present method for improving cotton fiber length specifically expresses AHA and TMK-related genes in cotton fiber cells, thereby regulating the degree of cell wall acidification during fiber elongation. This results in moderate cell wall acidification and relaxation, controlling cotton fiber development, and ultimately increasing cotton fiber length. Experimental results demonstrate that the cotton fibers improved by the present method significantly increase in length.

[0040] The method of the invention is simple and easy to implement, has remarkable effect, can provide high-quality fiber raw materials for the textile industry, and generates huge economic benefits.

[0041] Second, the expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: the embodiment of the present invention can increase the length of cotton fibers to about 32 mm, greatly improving the quality and yield of cotton fibers, and helping to improve the economic benefits of cotton planting.

[0042] In the past, efforts to improve cotton fiber quality often focused on the star genes that directly affect fiber elongation. This invention creatively applies the acid growth theory to cotton, providing a new perspective for studying fiber elongation.

[0043] For a long time, one of the core challenges facing the field of cotton breeding has been how to significantly improve fiber quality, especially fiber length, while ensuring high yield. Although traditional hybrid breeding can integrate excellent traits of multiple genes, it is limited by the complex genetic background and long screening cycle, making it difficult to accurately regulate the key physiological processes of fiber development. Although gene editing technologies (such as CRISPR-Cas9) can target and modify specific genes, due to the high redundancy of the cotton genome, mutations or overexpression of a single gene often cannot significantly change the fiber phenotype, resulting in limited practical application effects. In addition, although the acid growth theory provides an important theoretical basis for the cell elongation mechanism, its specific application path in cotton fiber elongation has always been unclear, especially how to accurately regulate the acidification degree of the apoplast through artificial means to promote fiber development. This problem has long been unresolved.

[0044] This invention innovatively combines acid growth theory with genetic engineering methods to achieve precise regulation of the acidification degree of cotton fiber cell walls for the first time. Specifically, by screening the fiber-specific promoter GhHOS3 / GhEXP2, the expression of GhAHA4 and GhTMK3A is driven in a targeted manner during the fiber elongation stage, effectively reducing the pH of the apoplast and activating the activity of cell wall relaxases, thereby significantly promoting fiber elongation. Experimental data showed that the fiber length of the transgenic strain increased from an average of 28.88 mm in the control group to 32.0–32.75 mm ( Figure 4 ), and the pH of the apoplast was significantly lower than that of the wild type ( Figure 4), fully verifying the feasibility and effectiveness of the technical solution. This breakthrough not only overcomes the limitations of traditional breeding and gene editing techniques, but also fills the practical gap in the application of acid growth theory to cotton fiber. It successfully solves the industry's difficult problem of achieving both high yield and high quality, and provides a new molecular regulation strategy for cotton breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a flow chart of a method for improving fiber length by changing the acidification degree of the apoplast, provided in an embodiment of the present invention.

[0046] Figure 2 This is a diagram of the ProGhHOS3::GhAHA4 expression vector provided in an embodiment of the present invention.

[0047] Figure 3 This is a diagram of the ProGhEXPA2::GhTMK3A expression vector provided in an embodiment of the present invention.

[0048] Figure 4 It is a q-PCR analysis diagram of GhAHA4 in ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic lines and a pH diagram of the fiber apoplast fluid of ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic cotton at 10 DPA, provided by the embodiments of the present invention.

[0049] Figure 5 Figure 1 shows the fiber length and statistics of ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic cotton plants provided in this embodiment. A wild-type TM-1 plant grown under identical conditions was used as a control. B Statistical analysis of mature fiber length of ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A plants. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0051] like Figure 1 As shown, the method for improving fiber length by changing the acidification degree of the apoplast provided by the embodiment of the present invention includes the following steps:

[0052] S101, obtain fiber-specific expression promoter;

[0053] S102, obtaining AHA- and TMK-related genes;

[0054] S103, the specific promoter isolated and cloned in S101 is fused with the AHA / TMK-related gene isolated and cloned in S102 to construct a plant expression vector specifically expressing AHA / TMK;

[0055] S104, delivering the specific plant expression vector obtained in S103 to Weimi Company for genetic transformation;

[0056] S105, further culturing and cultivating the cotton obtained in S104 to obtain transgenic cotton plants.

[0057] The specific expression promoter described in S101 provided in the embodiment of the present invention can be a natural promoter isolated and cloned from an animal, plant or microorganism, or it can be an artificially modified or designed promoter. The cotton AHA / TMK related gene described in S102 provided in the embodiment of the present invention can be a natural gene isolated and cloned from an animal, plant or microorganism, or it can be an artificially modified or designed gene. The method of fusing the specific promoter described in S103 provided in the embodiment of the present invention with an auxin synthesis related gene to construct an expression vector that specifically expresses the auxin synthesis related gene is a conventional method in the art, and the vector used can be a conventional vector used in the field of plant transgenics; the specific promoter is the fiber-specific promoter GhHOS3 and GhEXPA2, which are highly expressed during the fiber elongation period. The plant expression vector for specific expression of AHA provided in the embodiment of the present invention: contains at least a promoter consisting of PM H + -nucleotide sequence for expressing AHA consisting of ATPase gene and cotton fiber-specific promoter;

[0058] The nucleotides contain at least PM H + -ATPase gene AHA and cotton fiber-specific promoter HOS3 gene promoter, and the GhHOS3 gene promoter is located 5' upstream of AHA;

[0059] The nucleotide sequence has the sequence shown in SEQ ID NO.1;

[0060] The plant expression vector has Figure 2 and Figure 3 The structure shown.

[0061] The plant expression vector specifically expressing TMK provided in the embodiment of the present invention is:

[0062] The invention at least contains a nucleotide sequence for expressing GhTMK3A composed of a TMK gene and a cotton fiber-specific promoter;

[0063] The nucleotide sequence at least comprises GhTMK3A and a cotton fiber-specific promoter, GhEXPA2 gene promoter, and the GhEXPA2 gene promoter is located 5' upstream of GhTMK3A;

[0064] The nucleotide sequence has the sequence shown in SEQ ID NO. 2;

[0065] The plant expression vector has Figure 3 The structure shown.

[0066] Another object of the present invention is to provide a transformant obtained by transforming a host with the plant expression vector.

[0067] Another object of the present invention is to provide the application of the plant expression vector in improving cotton fiber traits.

[0068] Another object of the present invention is to provide a method for preparing a transgenic plant containing the plant expression vector, comprising the following steps:

[0069] (1) PM H + -ATPase gene operably linked to a cotton fiber-specific promoter;

[0070] (2) Constructing a PM H + -ATPase gene and cotton-specific promoter plant expression vector.

[0071] Another object of the present invention is to provide a method for encoding PM H + -ATPase-related gene nucleotide, characterized in that it has a nucleotide sequence as shown in SEQ ID NO.1.

[0072] The present invention is specifically implemented:

[0073] like Figure 2 : The backbone vector of ProGhHOS3::GhAHA4 expression vector used to construct the plant expression vector is the V062 vector provided by Weimi Company.

[0074] like Figure 4 : q-PCR analysis of GhAHA4 in ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic lines and pH measurement of fiber apoplastic fluid of ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic cotton at 10 DPA

[0075] The expression level of GhAHA4 was detected in the fibers of ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic cotton at 10 DPA. It was found that the transgenic lines all had high expression levels of GhAHA4.

[0076] The pH of the apoplast of ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic cotton fibers at 10 DPA was measured. It was found that the pH of the apoplast of the transgenic lines was lower than that of the wild type at the same stage.

[0077] like Figure 5 :Fiber length and statistics of ProGhHOS3::GhAHA4 and ProGhEXPA2::GhTMK3A transgenic cotton

[0078] A. Using wild-type TM-1 as a control under identical growth conditions, transgenic plants in the experimental field were photographed to measure fiber length. The fiber length of the overexpressing strains was significantly longer than that of the wild-type. Bar = 1 cm. B. Statistical analysis of mature fiber length in transgenic plants revealed that the fiber length of the transgenic strains ranged from 32.0 to 33.75 mm, significantly longer than the average fiber length of 28.88 mm in the control strain TM-1.

[0079] Example 1 Preparation of cotton genome

[0080] 1. DNA Extraction

[0081] Take about 3 g of young leaves of upland cotton, place them in a 2 mL centrifuge tube with steel balls added in advance (two steel balls are placed in each tube), fully pre-cool them in liquid nitrogen, and then quickly place them in a high-speed low-temperature freeze grinder pre-cooled at 4°C, set the frequency to 60 Hz, and grind them for 90 seconds until they are powdery.

[0082] To the 2 mL centrifuge tube, add 650 μL of preheated (65°C) CTAB lysis buffer supplemented with β-mercaptoethanol. Shake thoroughly and heat in a 65°C water bath for 30 minutes, ensuring thorough mixing. Add 650 μL of a chloroform:isoamyl alcohol lysis buffer (24:1 volume ratio), mix thoroughly, and centrifuge at 4°C for 20 minutes. Pipette the supernatant into a new 1.5 mL centrifuge tube. Add 0.6 volumes of icy isopropanol, invert to mix thoroughly, and centrifuge at 4°C for 10 minutes.

[0083] Discard the supernatant and wash with 70% ethanol and then anhydrous ethanol, then let it dry overnight or in an oven for 30 minutes. Add 200 μL of ddH2O to test the DNA concentration and purity. Take 1 μL and dilute it 10-fold before running on a gel. Store at -20°C.

[0084] 2. RNA Extraction

[0085] 3 g of cotton fiber cultured in the experimental field was selected and cotton RNA was extracted using the Polysaccharide and Polyphenol Crop Cotton RNA Extraction Kit (Tiangen Company) according to the experimental operation instructions. Reverse transcription and cDNA synthesis were performed using the HiScript II 1st Strand cDNA Synthesis Kit (Novozymes). Prepare the mixture in an RNase-free centrifuge tube. Incubate the mixture at 65°C for 5 minutes and place on ice for 2 minutes. Add 2 μL of HiScript II Enzyme Mix and 10 μL of 2× RT Mix and mix thoroughly. Incubate at 25°C for 5 minutes, 50°C for 45 minutes, and 85°C for 2 minutes. After the reaction, dilute with 80 μL of RNase-free H2O. After gel analysis, store at -20°C until ready for use.

[0086] 3. Cloning of GhHOS3 promoter and GhAHA4

[0087]

[0088] The primer sequences used are:

[0089] ProHOS3-F: 5'-ggccagtgccaagctaagcttTTAATTTTTAATTTTTTTTCTCTACTAAGAGG-3', ProHOS3-R: 5'-ttatcccccatCTTGCTGCTGCCTTTACTACTCTAGC-3', and the template was the cotton DNA extracted in Example 1. The amplification program was as follows: 95°C, 5 min; 95°C, 30 sec, 55°C, 10 sec; 68°C, 30 sec; 35 cycles; and extension at 68°C for 5 min.

[0090]

[0091] The primer sequences used were: GhAHA4-F: 5'-agcagcaagATGGGGGATAAAAATGAAGTCTTG-3', GhAHA4-R: 5'-gtccttgtagtccatgtcgacCACCGTGTACGCTTGCTGG-3', and the template was the cotton cDNA obtained in Example 1. The amplification procedure was: 95°C, 5 min; 95°C, 30 sec; 55°C, 10 sec; 68°C, 30 sec; 35 cycles; and extension at 68°C for 5 min.

[0092] Example 2 Construction of a genetic vector for ProHOS3-driven GhAHA4 expression

[0093] 1. Vector digestion

[0094] The empty plasmid was double-digested with NEB restriction endonucleases Hind III and Sal I.

[0095]

[0096] Plasmid DNA was from Weimi Genetic Transformation Company.

[0097] 2. Recovery, ligation and transformation of target gene and enzyme-digested vector:

[0098] First, the PCR product and the enzyme-digested vector were subjected to agarose gel electrophoresis, the target band was cut out, and the target band was Quick Gel Extraction Kit gel recovery kit recovery. The Basic Seamless Cloning and Assembly Kit was used for enzyme ligation. After the reaction, 10 μL of the ligation product was transferred into a Trans 1-T1 competent medium. Positive clones were selected for PCR testing and sent to Qingke Biotech Co., Ltd. for sequencing. Plasmids were extracted from the clones that were sequenced correctly. The plasmids were then transformed into Agrobacterium competent cells (EHA105). Positive clones were selected for PCR testing and cultured, and the bacterial culture was sent to Weimi Biotech Co., Ltd. for genetic transformation.

[0099] Example 3 Determination of pH of the extracellular fluid

[0100] Cotton bolls of GhAHA4-OE material 10 days old were carefully peeled open with a sharp blade, taking care not to scratch the internal tissue. Fibers were removed from each chamber and carefully placed into a 5ml syringe without a plunger, connected to a 1.5ml centrifuge tube. The entire assembly was then placed into a 50ml centrifuge tube and centrifuged at 1000 rpm at 4°C for 10 minutes. In a 96-well plate, 160 μL of this apoplastic fluid was mixed with 40 μL of a 100 mg / mL HPTS solution (Sigma-Aldrich). Fluorescence intensity was measured at 510 and 530 nm using an excitation wavelength of 460 nm. A standard curve was generated using Britton-Robinson universal buffer (0.05 M H₃BO₃, 0.05 M H₃PO₄, 0.05 M CH₃COOH) to determine the solution pH.

[0101] Sequence SEQ ID NO.1

[0102]

[0103] SEQ ID NO.2

[0104]

[0105] The first step of the present invention is to isolate and clone a fiber-specific expression promoter and an AHA gene associated with apoplast acidification using molecular biology techniques. The fiber-specific expression promoter can regulate gene expression during cotton fiber development, while the AHA gene (a member of the H+-ATPase family) is directly related to apoplast acidification, affecting fiber elongation and development by regulating the acidic environment of the cell wall. This step, through precise screening and verification, ensures that the isolated promoter and gene can be efficiently expressed and function during fiber development.

[0106] Using molecular cloning techniques, a fiber-specific promoter was fused to the AHA and TMK genes to construct a plant expression vector specifically expressing AHA / TMK. This vector design ensures that the AHA / TMK gene expression is activated only in fiber-specific tissues, without affecting other tissues, thereby achieving precise regulation. The vector also contains the necessary selectable marker gene and replication elements for subsequent genetic transformation and screening. The key to this step is to achieve precise regulation of apoplast acidification during fiber development.

[0107] Using Weimi's genetic transformation techniques (such as Agrobacterium-mediated or gene gun techniques), the constructed plant expression vector is transferred into recipient cotton plants. Following transformation, cotton cell lines carrying the target gene are screened for resistance and further cultured into transgenic plants. Through transgenic manipulation, apoplast acidification is targeted at specific stages of fiber development, thereby improving fiber elongation and extending fiber length.

[0108] The transgenic cotton obtained was cultivated to observe its growth and fiber development characteristics, and the effect of apoplastic acidification regulation on fiber length was experimentally verified. Microscopic observation and physiological and biochemical analysis confirmed the changes in apoplastic acidification in the transgenic cotton fibers and measured the increase in fiber length. The results showed that this method significantly improved fiber length by altering apoplastic acidification, providing an innovative molecular breeding method for the selection of high-quality cotton.

[0109] The embodiment of the present invention can increase the fiber length of upland cotton from about 28 mm to about 32 mm (e.g. Figure 5 The promotion and application of long-fiber cotton will help promote the upgrading of the textile industry, increase the added value and market competitiveness of textiles, and reduce my country's dependence on imported American and Australian cotton.

[0110] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

[0111] Sequence Listing Information:

[0112] DTD version: V1_3

[0113] File name: A method for improving fiber length by changing the acidification degree of the apoplast.xml

[0114] Software Name: WIPOSequence

[0115] Software version: 2.3.0

[0116] Date of Generation: 2025-05-21

[0117] Basic Information:

[0118] Current application / IP office: CN

[0119] Current application / applicant file name:Henan University

[0120] Applicant's name: Henan University

[0121] Applicant's name / language:zh

[0122] Applicant's name or title / Latin name:HenanUniversity

[0123] Inventors: Zou Changsong, Li Cheng, Wen Qing, Liu Leidi, Cheng Xiangfei, Li Zhifang, Zheng Yuge, Wang Xinchun, Yu Chengde, Jiang Jing, Jia Mingzhu

[0124] Inventor Name / Language:zh

[0125] Inventor Name / Latin Title:Zou Changsong,Li Cheng,Wen Qing,Liu Leidi,Cheng Xiangfei,Li Zhifang,Zheng Yuge,Wang Xinchun,Yu Chengde,Jiang Jing,Jia Mingzhu

[0126] Invention Title: A method for improving fiber length by changing the degree of apoplast acidification (zh)

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[0183] >organism,syntheticconstruct

[0184] Residue:

[0185] gtccttgtag tccatgtcga ccaccgtgta cgcttgctgg 40END.

Claims

1. A method for improving fiber length by changing the acidification degree of apoplast, characterized in that: The following steps are involved: Step 1, obtaining a fiber-specific expression promoter; Step 2, obtaining AHA-related genes; Step 3, fusing the specific promoter isolated and cloned in step 1 with the AHA-related gene isolated and cloned in step 2 to construct a plant expression vector that specifically expresses AHA; Step 4: delivering the specific plant expression vector obtained in step 3 to Weimi Company for genetic transformation; Step 5: further culture and cultivate the cotton obtained in step 4 to obtain transgenic cotton plants.

2. The method for improving fiber length by changing the acidification degree of the apoplast as claimed in claim 1, characterized in that: The specific expression promoter in step 1 can be a natural promoter isolated and cloned from an animal, plant or microorganism, or an artificially modified or designed synthetic promoter.

3. The method for improving fiber length by changing the acidification degree of the apoplast as claimed in claim 1, characterized in that: The cotton AHA-related gene described in step 2 may be a natural gene isolated and cloned from an animal, plant or microorganism, or an artificially modified or designed gene.

4. The method for improving fiber length by changing the acidification degree of the apoplast as claimed in claim 1, characterized in that: The method of fusing the specific promoter described in step 3) with the auxin synthesis-related gene to construct an expression vector that specifically expresses the auxin synthesis-related gene is a conventional method in the art, and the vector used can be a conventional vector used in the field of plant transgenics; The specific promoter is the fiber-specific promoter HOS3, which is activated during the fiber elongation period.

5. The method for improving fiber length by changing the acidification degree of the apoplast as claimed in claim 1, characterized in that: The plant expression vector specifically expressing AHA: Contains at least PM H + -nucleotide sequence for expressing AHA consisting of ATPase gene and cotton fiber-specific promoter; The nucleotides contain at least PM H + -ATPase gene AHA and cotton fiber-specific promoter HOS3 gene promoter, and the HOS3 gene promoter is located 5' upstream of AHA; The nucleotide sequence has the sequences shown in SEQ ID No. 1 and SEQ ID No.

2.

6. A transformant obtained by transforming a host with the plant expression vector according to claim 5.

7. Use of the plant expression vector according to claim 5 in improving cotton fiber traits.

8. A method for preparing a transgenic plant containing the plant expression vector according to claim 5, comprising the following steps: 1) PM H + -ATPase gene or TMK gene is operably linked to cotton fiber-specific promoter; 2) Construct a PM H + -Plant expression vector of ATPase gene / or TMK gene and cotton-specific promoter.

9. A coded expression PM H + -nucleotides of ATPase-related genes, characterized in that It has a nucleotide sequence shown as SEQ ID NO.1.

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