Use of ghprt lp68 protein in regulating cotton fiber development

Abstract

The application belongs to the technical field of plant genetic engineering, and specifically provides application of GhPRTLP68 protein in regulation of cotton fiber development. The amino acid sequence of the GhPRTLP68 protein is shown as SEQ ID No. 1, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 2. Research finds that the GhPRTLP68 gene is significantly up-regulated in the secondary wall thickening stage of cotton fiber, overexpression of the gene can increase the length and strength of cotton fiber, and after inhibition of the gene expression, the length of cotton fiber is shortened and the strength is reduced. The application determines the key role of the GhPRTLP68 protein and the coding gene in the development of cotton fiber, provides important gene resources and a new method for breeding transgenic cotton varieties with excellent fiber quality, and has important application value for solving the problem of dependence on imported high-quality cotton and promoting the development of cotton breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of GhPRTLP68 protein in regulating cotton fiber development. Background Technology

[0002] Cotton is the world's most important natural fiber crop, and its fiber quality directly determines the grade and value of textiles. As the world's largest producer and exporter of textiles, my country's cotton industry's stability and development are of great significance to the national economy. However, the fiber quality of my country's main cotton varieties, especially high-quality cotton suitable for high-end textiles, still relies to some extent on imports, posing supply chain risks under the complex international situation. Therefore, identifying key genes regulating cotton fiber development and quality formation, and using molecular breeding technology to target and improve existing varieties to cultivate new cotton varieties with superior fiber quality has become an urgent need to ensure the security of my country's cotton industry and enhance its competitiveness.

[0003] The quality of cotton fibers is mainly determined by their length, strength, and fineness. Among these, the fiber elongation development stage determines its final length, while the secondary wall thickening stage primarily contributes to fiber strength and fineness. Therefore, elucidating the molecular regulatory network during the secondary wall thickening stage of cotton fibers is a key scientific issue for improving fiber strength. Thaumatin-like proteins (TLPs) are a class of proteins associated with plant disease resistance; some members are known to exert resistance by hydrolyzing β-1,3-glucan in the cell walls of pathogenic fungi. Studies have shown that the strength of cotton fibers is closely related to the content and metabolism of β-1,3-glucan during their development. For example, applying potassium fertilizer can alleviate the negative impact of drought on fiber strength by enhancing the activity of β-1,3-glucanase in the fibers. This suggests that members of the TLP family may play a less fully understood role in cotton fiber development, especially in strength formation, by participating in the metabolism of polysaccharides in fiber cell walls.

[0004] Based on the above background, the inventors have developed a TLP family gene that is specifically upregulated during the secondary wall thickening stage of cotton fibers— GhPRTLP68 —Functional studies were conducted. Experiments revealed that overexpression GhPRTLP68 This gene can significantly increase the length and strength of cotton fibers, while inhibiting its expression using interference or gene editing techniques leads to shorter cotton fibers and decreased strength. This result reveals for the first time... GhPRTLP68 It plays a crucial role in the positive regulation of cotton fiber development. Therefore, this invention is proposed to provide new gene targets and effective strategies for molecular breeding of cotton fiber quality. Summary of the Invention

[0005] In response to the current situation in my country's cotton industry, where the fiber quality of major cotton varieties, particularly length and strength, needs improvement, and high-quality cotton germplasm resources are relatively scarce, this invention aims to provide a new gene resource and its application method that can effectively regulate cotton fiber development and improve fiber quality (especially length and strength). Specifically, this invention discloses for the first time the novel function of the GhPRTLP68 protein and its encoding gene in positively regulating cotton fiber development, solving the technical problem in existing technologies of lacking the ability to improve cotton fiber quality through this specific target.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The first aspect of this invention provides a novel use for the GhPRTLP68 protein and its gene: It reveals for the first time the application of the GhPRTLP68 protein (amino acid sequence as shown in SEQ ID No. 1) or its encoding gene (nucleotide sequence as shown in SEQ ID No. 2) in increasing cotton fiber length and / or enhancing cotton fiber strength. Based on this discovery, the protein or gene can be used to prepare transgenic cotton with longer fibers and / or greater fiber strength.

[0008] A second aspect of this invention provides the application of related biomaterials: applying biomaterials related to the GhPRTLP68 protein to improve the quality of cotton fibers. These biomaterials include, but are not limited to: expression cassettes containing nucleic acid molecules encoding the protein, recombinant vectors (such as plant expression vectors), and recombinant microorganisms containing these nucleic acid elements (e.g., Agrobacterium for plant transformation). These biomaterials can be used to achieve overexpression of the GhPRTLP68 protein-encoding gene in cotton, thereby increasing fiber length and enhancing fiber strength.

[0009] The third aspect of this invention provides a method for breeding and improving cotton varieties, and provides two specific molecular breeding methods:

[0010] Method 1 is a method for cultivating cotton with increased fiber length: the method includes overexpressing the gene for the GhPRTLP68 protein in cotton to obtain cotton plants with increased fiber length. Preferably, the overexpression of this gene is achieved through transgenic technology, for example, by introducing a plant expression vector integrating the nucleotide sequence shown in SEQ ID No. 2 into cotton.

[0011] Method two is a method for cultivating cotton with enhanced fiber strength: the method includes overexpressing the gene for the GhPRTLP68 protein in cotton to obtain cotton plants with enhanced fiber strength. Similarly, it is preferably achieved through transgenic technology using an expression vector containing the sequence shown in SEQ ID No. 2.

[0012] The beneficial effects of this invention are:

[0013] (1) First time to reveal new functions: This invention has for the first time clarified the key role of GhPRTLP68 protein and its gene in cotton fiber development, especially in positively regulating fiber length and strength, which expands the functional understanding of the sweet protein family and provides a brand-new gene target with independent intellectual property rights for cotton fiber quality improvement.

[0014] (2) Clear application in breeding: This invention not only discovers gene function, but also provides specific technical solutions and approaches for applying this function to cotton molecular breeding (such as through overexpression technology), directly transforming basic research results into breeding technology, which is highly operable.

[0015] (3) Highly targeted and with high application value: This invention directly targets the key economic trait of cotton fiber quality (length and strength). Its application can effectively serve the major needs of my country in cultivating high-quality and high-yield new cotton varieties. It has important theoretical and practical significance for reducing dependence on foreign high-quality cotton varieties and enhancing the competitiveness of my country's cotton industry. Attached Figure Description

[0016] Figure 1 Expression pattern analysis of GhPRTLP68. qRT-PCR detection. GhPRTLP68 Expression levels at different developmental stages of cotton fibers. Standard error is calculated based on three biological replicates. UBQ7 As an internal reference gene.

[0017] Figure 2 for GhPRTLP68 It affects cotton fiber development. (A) Quantitative fluorescence detection of transgenic cotton. GhPRTLP68 Level of expression. (B) GhPRTLP68 Comparison of fiber length between overexpression and interference lines and wild-type cotton, scale bar = 1 cm. (C and D) Statistical results of fiber length and strength between overexpression and interference lines and wild-type cotton. Error bars represent the mean ± SD of three independent experiments. ** and * indicate statistical significance at the 0.01 and 0.05 probability levels, respectively.

[0018] Figure 3 For gene editing GhPRTLP68 Inhibits cotton fiber development. (A) Gene-edited strain GhPRTLP68 Sequencing results for edit type. (B) Wild type and GhPRTLP68 Comparison of cotton fiber lengths from gene-edited lines, scale bar = 1 cm. Detailed Implementation

[0019] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0020] Explanation of the sequence list:

[0021] SEQ ID No. 1:

[0022] MAISSGIYLLFLLNFFSFGTIFSSATSFTLENRCSFTVWPGSLTANGPPLGDGGFVLAPGSSSRLQPPPGWSGRFWGRTGCNFDNSGSGKCVTGDCGGALKCNGGGIPPVSLIEFTLNGHDN KDFYDISLVDGYNMAVAVKAVGGTGTCQYAGCVNDLNTNCPAELQMMDSGSVVACKSACAAFNMPEYCCTGAHGTPQTCSPTKYSQLFKNACPTAYSYAYDDATSTMTCTGADYLITFCPTS*

[0023] SEQ ID No. 2:

[0024] ATGGCGATTTCCTCTGGGATTTATCTTCTTTTTCTACTGAATTTCTTCTCATTTGGGACGATATTTTCTTCAGCAACAAGCTTTACACTCGAAAATCGTTGTAGTTTCACAGTATGGCCTGGTTCCCTGACCGCAAATGGCCCTCCCCTTGGTGATGGTGGTTTTGTATTGGCTCCTGGTTCATCATCCCGACTCCAGCCTCCACCTGGTTGGTCTGGTCGCTTTTGGGGTCGAACTGGCTGCAATTTCGATAACTCTGGCTCAGGCAAATGTGTAACCGGTGACTGTGGTGGCGCGTTAAAATGCAACGGCGGTGGCATCCCACCCGTTTCCCTGATCGAATTCACCCTTAATGGACATGACAATAAGGACTTTTATGACATTAGTCTCGTAGATGGTTACAACATGGCCGTAGCAGTCAAGGCAGTTGGTGGTACAGGGACTTGCCAATATGCAGGTTGCGTCAATGACCTCAACACAAATTGCCCCGCCGAGTTACAGATGATGGACTCAGGTTCTGTCGTCGCTTGTAAAAGCGCCTGTGCCGCCTTCAACATGCCGGAATATTGCTGCACTGGTGCACATGGTACGCCTCAGACTTGCTCACCTACGAAGTACTCACAATTGTTCAAAAATGCATGCCCTACGGCTTACAGTTATGCTTACGATGACGCCACTAGTACCATGACTTGCACCGGGGCGGATTATCTGATCACATTTTGTCCAACCAGCTGA

[0025] SEQ ID No.3:

[0026] GTTCATCATCCCGACTCCAGCC

[0027] SEQ ID No.4:

[0028] TACCACCAACTGCCTTGACTGC

[0029] SEQ ID No.5:

[0030] ATGGCGATTTCCTCTGGGAT

[0031] SEQ ID No. 6:

[0032] TCAGCTGGTTGGACAAAATGTGATC

[0033] SEQ ID No. 7:

[0034] GACGCACAATCCCACTATCC

[0035] SEQ ID No. 8:

[0036] AAAAGCGACCAGACCAACCA

[0037] SEQ ID No. 9:

[0038] GGAATATTGCTGCACTGGCG

[0039] Experimental materials:

[0040] The cotton varieties selected for this experiment were Zhongmian Institute 36, Zhongmian Institute 24, and Jin668, which were planted in the experimental field of the National Southern Breeding Research Institute of the Chinese Academy of Agricultural Sciences, and managed under normal field management conditions.

[0041] Experimental reagents and consumables:

[0042] Enzymes and kits: PrimeSTAR® GXL DNA Polymerase high-fidelity enzyme, real-time fluorescence kit, RNA reverse transcription kit, gel extraction kit, and PCR product purification kit were all purchased from Takara Bio Engineering (Dalian) Co., Ltd. ClonExpress ® The Ultra One Step Cloning Kit was purchased from Vazyme; the plasmid small-scale extraction kit was purchased from Magen; the restriction endonucleases were purchased from NEB; and the DNA Marker and plant total RNA extraction kit were purchased from TIANGEN.

[0043] Other medicines: Agarose was a Spanish original product; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. were domestically produced analytical grade; ampicillin, etc. were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; and Escherichia coli competent cells were purchased from Beijing Tiangen Biotech Co., Ltd.

[0044] Culture media: LB liquid medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L; LB solid medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L, Agar powder 15 g / L, bring to a final volume of 1 L;

[0045] LB selective medium: Before plating LB, add the appropriate concentration of antibiotics when the medium has been autoclaved and cooled to 55 degrees Celsius, shake well and then plating.

[0046] Main instruments: PCR amplification instrument (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), real-time PCR instrument (ABI7500), electric thermostatic incubator (Shanghai Senxin), thermostatic incubator shaker (Shanghai Zhicheng), artificial climate chamber.

[0047] Example 1: Cotton Genes GhPRTLP68 Expression pattern analysis

[0048] Previous expression profiling analysis of CCRI36 fibers at different developmental stages in cotton revealed in our laboratory... GH_D05G2004 ( GhPRTLP68 The expression of [a specific gene] was upregulated during the thickening stage of the secondary cell wall of fibers. Tissue expression levels were measured from roots, stems, leaves at the two-leaf stage, and fibers at 0, 3, 5, 7, 10, 15, 20, 25, and 30 DPA. All collected samples were immediately and rapidly frozen in liquid nitrogen and stored at −80°C for RNA extraction, reverse transcription to cDNA, and qRT-PCR. GhUBQ7 was used as an internal control for data normalization. Relative expression levels were calculated using the 2-ΔΔCt method. Figure 1 ).

[0049] Cloning process: Roots from different treatment stages of the treatment and control groups were rapidly frozen in liquid nitrogen, ground in liquid nitrogen, and stored at -80℃ for later use. Total RNA extraction from plants: RNA was extracted using a TIANGEN RNA extraction kit. cDNA synthesis was performed according to the instructions of the Toyobo FSQ-201 reverse transcription kit. The reverse transcription product cDNA solution was diluted 6-fold as a template for PCR and quantitative PCR was performed. Primers for quantitative PCR were as follows:

[0050] qrtGhPRTLP68-F:GTTCATCATCCCGACTCCAGCC

[0051] qrtGhPRTLP68-R: TACCACCAACTGCCTTGACTGC

[0052] PCR reaction system:

[0053]

[0054] PCR reaction procedure:

[0055]

[0056] cotton GhPRTLP68 Gene cloning: Obtained from COTTONOMICS GhPRTLP68 Based on the gene sequence, primers were designed to amplify the cDNA from the roots of *Cotton spp.* 36 obtained in the above steps. GhPRTLP68 The CDS sequence has an open reading frame of 735 bp, encoding 244 amino acids.

[0057] According to TaKaRa PrimeSTAR ® The GXL DNA Polymerase high-fidelity enzyme instruction manual specifies the PCR reaction system as follows:

[0058]

[0059] The PCR amplification program was as follows: 98℃ for 3 min; 98℃ for 10 s; 56℃ for 15 s; 68℃ for 1 min, 35 cycles; 68℃ for 10 min. The primer sequences are as follows:

[0060] GhPRTLP68-F: ATGGCGATTTCCTCTGGGAT

[0061] GhPRTLP68-R:TCAGCTGGTTGGACAAAATGTGATC

[0062] The target fragment was then cut and recovered using a gel recovery kit; the recovered product was then... ClonExpress ® The Ultra One Step Cloning Kit was used to construct and transform T-vectors into E. coli; single clones were picked from ampicillin-resistant LB medium and cultured overnight at 37°C; bacterial culture was verified by PCR, positive clones were picked and sent to Sangon Biotech for sequencing, and 60% glycerol was added to the bacterial culture with correct sequencing and stored at -70°C.

[0063] Example 2: Overexpression or silencing GhPRTLP68 Affecting cotton fiber quality

[0064] The clone obtained from Example 1 GhPRTLP68 The CDS was used to construct overexpression and interference vectors.

[0065] The plasmid extraction procedure is as described in the full-length gold standard. EasyPure @ The Plasmid MiniPrep Kit has been slightly improved.

[0066] 1) Take 5 mL of the amplified E. coli, centrifuge at 10000×g for 1 min, and discard the supernatant.

[0067] 2) Add 250 μL of solution RB (add RNase A to RB before use and store at 2-8℃), suspend and shake to mix.

[0068] 3) Add 250 μL of LB solution and gently invert the container 5 times to completely lyse the bacteria (within 5 minutes).

[0069] 4) Add 350 μL of solution NB, gently invert 5 times, and after the precipitate has completely formed, let it stand at room temperature for 2 min.

[0070] 5) Centrifuge at 12000×g for 5 min, and only aspirate the supernatant onto the centrifuge column. Centrifuge rapidly at 12000×g for 1 min, and discard the waste liquid.

[0071] 6) Add 650 μL of solution WB (already mixed with 80 ml of anhydrous ethanol), centrifuge at 12000×g for 1 minute, and discard the waste liquid.

[0072] 7) Repeat step 6).

[0073] 8) Centrifuge at 12000×g for 2 min, then let stand at room temperature for 30 min.

[0074] 9) Place the centrifuge column into a clean EP tube, add 30 μL of preheated ddH2O (pH>7.0) to the center of the centrifuge column, and incubate at 65°C for 15 min.

[0075] 10) Centrifuge at 10000×g for 2 min to elute the plasmid, and store in a -20℃ refrigerator.

[0076] Construction of overexpression and interference vectors:

[0077] Construction of overexpression vector: The extracted pBI121 binary vector was used... Bam HI and Sac I was subjected to double enzyme digestion, and overexpression and interference were used (the interference vector was a full-length interference, that is, the CDS sequence of the target gene was inserted backward into the overexpression vector). GhPRTLP68The gene, with adapter primers, was cloned from a correctly sequenced T vector. The product of enzyme digestion and gene cloning was then used... EasyPure @ The Quick Gel Extraction Kit is used for gel recycling. The specific steps are as follows:

[0078] 1) Cut the target DNA from the agarose gel, transfer it to a centrifuge tube and weigh it, calculating that 100 mg of gel equals 100 μL.

[0079] 2) Add GSB (3 times the gel volume) to the EP tube and mix by inverting the tube every 2 minutes in a 55°C water bath.

[0080] 3) When the temperature of the completely melted gel solution drops to the same as the room temperature, transfer it to a centrifuge column and place it at room temperature for 1 min. Centrifuge at 10000×g for 1 min and discard the waste liquid.

[0081] 4) Add 650 μL of WB solution to the centrifuge column, centrifuge at 10000×g for 1 min, and discard the waste liquid.

[0082] 5) Centrifuge at 10000×g for 2 min.

[0083] 6) Transfer the centrifuge column to another centrifuge tube, open the centrifuge column cap and let it stand for 15 min. Add 30 μL of preheated ddH2O (pH>7.0) to the center of the centrifuge column and let it stand at room temperature for 15 min.

[0084] 7) Centrifuge at 10000×g for 2 min, and store the resulting gel recovery product in a -20℃ refrigerator.

[0085] Using ClonExpress ® The II One Step Cloning Kit enables seamless cloning and ligation of the recovered enzyme digestion and PCR products, as shown in the following system:

[0086]

[0087] After gently mixing with a pipette, the PCR instrument was used to react at 37°C for 30 min, and then transferred to ice after cooling to 4°C.

[0088] The recombinant product was then transformed into E. coli, with the following specific steps:

[0089] 1) Thaw DH5α clone competent cells on ice.

[0090] 2) Add all the obtained recombinant products to the semi-thawed competent cells taken from the refrigerator, gently tap the centrifuge tube wall with your finger, and place it on ice for 30 min.

[0091] 3) Let it stand in a 42°C water bath for 45 seconds, then place it on ice for 2 minutes.

[0092] 4) Add 500 μL of empty LB liquid medium (1 g peptone, 0.5 g yeast extract and 1 g NaCl to 100 mL ddH2O), and shake in a shaker at 200 rpm and 37°C for 45 min.

[0093] 5) Using a sterilized yellow pipette tip, gently spread all the bacterial solution evenly onto a preheated (37°C) solid LB agar plate containing kanamycin resistance (liquid LB with an additional 15 g / L Agar), and let it air dry.

[0094] 6) Incubate overnight upside down in a 37℃ incubator.

[0095] 7) On the second day, use a white pipette tip to select single spots with good growth and add them to 300 μL of LB culture medium containing the corresponding resistance. Shake at 37°C for 5 h.

[0096] 8) Use enzymes from Kangwei Century Company to perform PCR identification of positive single clones. Use the 35S universal primer upstream and the downstream primer of the target gene downstream. Refer to the instruction manual for specific PCR procedures.

[0097]

[0098] 9) Single clones showing bands on gel electrophoresis were sent to Sangon Biotech Ltd. for sequencing. The OE- GhPRTLP68 and Anti- GhPRTLP68 The bacterial culture was stored in glycerol at -80°C.

[0099] Transformation of Agrobacterium: The extracted OE- GhPRTLP68 and Anti- GhPRTLP68 The vector plasmid was transformed into Agrobacterium tumefaciens LBA4404 competent cells using the freeze-thaw method. The specific transformation process is as follows:

[0100] 1) Add 500 ng of plasmid to GV3101 competent cells and gently tap the EP tube wall with your finger. Then incubate the tubes on ice, in liquid nitrogen, in a 37°C water bath, and on ice for 5 min each.

[0101] 2) Add 700 μL of empty LB culture medium and incubate at 28°C in a shaker for 2.5 h;

[0102] 3) Spread 100 μL of bacterial culture evenly onto an LB agar plate containing both kanamycin (50 mg / L) and rifampin (50 mg / L) using a yellow pipette tip, and incubate at 28°C with the plate upside down for 2 days.

[0103] 4) Transfer the positive clones obtained by colony PCR to 50 mL of LB culture medium containing the corresponding antibiotic and incubate at 28°C in a shaker.

[0104] The activated Agrobacterium was used for genetic transformation of cotton, following the procedures outlined in Dr. Zhang Chaojun's dissertation:

[0105] 1) Transform the positive clone into Agrobacterium competent strain LBA4404, expand Agrobacterium culture, centrifuge and discard the supernatant, add invasion dye (MGL and AS), shake to suspend the bacterial culture, and activate at 28°C and 200 rpm / min for at least 30 min on a shaker.

[0106] 2) The recipient cotton (CCRI24) seeds were sterilized with mercuric chloride, washed with sterile water, and then placed in sterile seedling culture medium and cultured at 30℃ for 6 days.

[0107] 3) Cut the hypocotyl of the recipient seedling into small stem segments, infect them with activated Agrobacterium, and then dry them;

[0108] 4) Lay the hypocotyl flat on a co-culture medium containing filter paper and incubate in the dark at 20°C for 1-2 days;

[0109] 5) Transfer the hypocotyl to 2,4-D medium and place it in a light-protected culture room. Subculture once every 20-30 days.

[0110] 6) The callus tissue grows into rice-grain-sized granules, which are then transferred to a differentiation culture medium to further differentiate into embryoids;

[0111] 7) Subculture the differentiated seedlings into the rooting medium until they grow into healthy seedlings with good roots;

[0112] 8) Transfer the seedlings to water to harden them off. After about a week, plant them in the greenhouse.

[0113] The obtained transgenic positive strains were tested using the following primers:

[0114] 35S: GACGCACAATCCCACTATCC

[0115] jGhPRTLP68oe-R:AAAAGCGACCAGACCAACCA

[0116] jGhPRTLP68anti-R: GGAATATTGCTGCACTGGCG

[0117] Transgenic plant detection reaction system:

[0118]

[0119] PCR reaction procedure:

[0120]

[0121] In the obtained transgenic positive plants GhPRTLP68 The expression level was detected by qRT-PCR, and the fluorescence quantitative procedure was performed as described in Example 1.

[0122] The obtained positive plants and empty vector control plants were then planted in a transgenic nursery, following the procedure described in Example 1. Mature cotton fiber samples were subsequently harvested for quality testing. The results showed that, compared to the control, the overexpressing plants had longer and stronger fibers, while the interference plants had shorter and weaker fibers. Figure 2 (Table 1). Figure 2 ZM24 is the offspring of transgenic overexpression and interference materials obtained by using ZM24 as the recipient material. OE90, OE89, OE88 and Sil03, Sil05 and Sil08 are transgenic overexpression and interference materials obtained by using ZM24 as the recipient material.

[0123] Table 1. Comparison of fiber quality between transgenic and control cotton.

[0124]

[0125] Example 3: Gene Editing GhPRTLP68 Affecting cotton fiber quality

[0126] Construction of gene-editing vectors: based on cloned... GhPRTLP68 CDS sequence information was used to design sgRNA target sequences using CRISPR-P2.0 and CRISPR-GE online software, and sgRNA and guideRNA were tandemly linked using the overlap PCR extension method.

[0127] The gene editing vector p7N was used Bsa Enzyme I was used for digestion, and the digestion products were recovered by gel extraction. The sgRNA-guideRNA fragment was constructed into the digested p7N vector using the infusion method. The plasmid with correct sequencing was transformed into Agrobacterium and colony PCR was performed. The bacterial culture with correct bands was stored in glycerol at -80℃.

[0128] The constructed gene-editing vector was used for genetic transformation of cotton, and the specific steps are described in Example 2.

[0129] The GhPRTLP68 gene sequence obtained from the transgenic edited plants was analyzed by Hi-Tom sequencing.

[0130] The homozygous gene-edited plants were then planted in a transgenic nursery, following the procedure outlined in Example 1. Mature cotton fiber samples were subsequently harvested for quality analysis. Results showed that, compared to the control, the gene-edited plants had shorter and weaker fibers. Figure 3 (Table 2). Figure 3 ZhongKO1 and KO2 are offspring of transgenic knockout materials obtained by using Jin668 as the recipient material.

[0131] Table 2 Comparison of fiber quality between gene-edited and control cotton.

[0132] .

Claims

1. The application of a gene overexpressing GhPRTLP68 protein in simultaneously increasing cotton fiber length and enhancing cotton fiber strength, characterized in that, The amino acid sequence of the GhPRTLP68 protein is shown in SEQ ID No.

1.

2. The application of biomaterials related to the GhPRTLP68 protein of claim 1, characterized in that, The application is to simultaneously increase the length and strength of cotton fibers; The biomaterial is any one of the following C1) to C3): C1) An expression cassette containing a nucleic acid molecule encoding the GhPRTLP68 protein; C2) A recombinant vector containing a nucleic acid molecule encoding the GhPRTLP68 protein; C3) A recombinant microorganism containing a nucleic acid molecule encoding the GhPRTLP68 protein, or a recombinant microorganism containing the expression cassette described in C1), or a recombinant microorganism containing the recombinant vector described in C2), wherein the microorganism is Agrobacterium; The amino acid sequence of the GhPRTLP68 protein is shown in SEQ ID No. 1, and the nucleotide sequence of the nucleic acid molecule encoding the GhPRTLP68 protein is shown in SEQ ID No.

2.

3. A method for cultivating cotton with increased fiber length and enhanced fiber strength, characterized in that, The method includes overexpressing the gene for the GhPRTLP68 protein in cotton to obtain cotton with increased fiber length and enhanced fiber strength, wherein the amino acid sequence of the GhPRTLP68 protein is shown in SEQ ID No.

1.

4. The method according to claim 3, characterized in that, The gene that overexpresses GhPRTLP68 protein in cotton is obtained by using transgenic technology to increase the expression level of the gene encoding GhPRTLP68 protein in cotton.

5. The method according to claim 4, characterized in that, The method of increasing the expression level of the gene encoding the GhPRTLP68 protein in cotton using transgenic technology is achieved by introducing a cotton expression vector that integrates the nucleic acid molecule shown in SEQ ID No. 2 into cotton.

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