Cotton fiber strength major gene ntl9bs41 and application thereof

Abstract

The application relates to a cotton fiber strength major gene NTL9 BS41 and application thereof. Through cotton transgenic technology, NTL9 BS41 is successfully introduced into Gossypium hirsutum and high expression of the gene in cotton fibers is realized, the fiber strength of cotton is significantly enhanced without negative influence on other fiber qualities, overexpression of NTL9 BS41 can effectively enhance the density and uniformity of cellulose microfibril accumulation, and the structure and performance of cotton fibers are significantly optimized. In addition, the gene is derived from Gossypium barbadense, compared with Gossypium hirsutum, there is an insertion type variation of 24 bp nucleotides in the CDS of the gene, and a molecular marker capable of effectively distinguishing Gossypium hirsutum and Gossypium barbadense haplotypes is successfully developed. The application provides new gene resources, technical means and germplasm resources for cotton fiber quality improvement, and has far-reaching application value and potential for promoting the progress of cotton breeding work.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the cotton fiber strength major gene NTL9. BS41 And its applications. Background Technology

[0002] Cotton is one of the most important natural fiber crops. Cotton fiber is widely used in textiles, clothing, and home furnishings. Fiber strength is a key indicator of cotton fiber quality and plays a decisive role in the quality and performance of textile products. High-strength cotton fibers can withstand greater tensile forces, reducing breakage and defects, and improving the efficiency and stability of textile production. High-strength cotton fibers also possess abrasion resistance, tensile strength, and wrinkle resistance, extending the service life of textiles. Improving cotton fiber strength has long been an important goal of cotton breeding. However, traditional breeding methods relying on phenotypic selection are time-consuming and inefficient.

[0003] Upland cotton and Sea Island cotton are the two most important cultivated varieties in the world. Upland cotton has high yields and wide adaptability, but its fiber quality is generally average. Sea Island cotton has low yields, but its fiber quality is excellent. Fully exploring the genetic resources of the superior fiber quality in Sea Island cotton and introducing them into upland cotton through genetic engineering is an effective way to achieve genetic improvement of upland cotton fiber quality.

[0004] Fiber strength is a typical quantitative trait, easily influenced by the environment. In recent years, although some studies have been reported on genes related to cotton fiber development, the identification and functional research of major genes affecting cotton fiber strength remain relatively limited. Existing research mostly focuses on the preliminary mapping of fiber strength QTLs; the identification of strength genes, their specific mechanisms of action in cotton fiber strength formation, and their potential in practical applications have not yet been fully elucidated. Summary of the Invention

[0005] This invention aims to identify and utilize the major fiber strength gene in upland cotton and develop efficient application technologies based on this gene to achieve rapid and precise improvement of upland cotton fiber quality. This invention provides a cotton fiber strength gene NTL9. BS41 The NTL9 genotype of sea island cotton is specifically highly expressed in upland cotton fibers. BS41 Genes can significantly improve the strength of cotton fibers, increase the compactness of cellulose microfibrils, and improve the quality of cotton fibers, thereby providing new genetic resources for cotton breeding and creating new germplasm.

[0006] This invention provides a major gene for cotton fiber strength, NTL9. BS41 The protein sequence it encodes is shown in SEQ ID NO:3.

[0007] Furthermore, its CDS sequence is shown in SEQ ID NO:2, and its genomic nucleotide sequence is shown in SEQ ID NO:1.

[0008] The present invention also provides a product containing any one of the following a1)-a3):

[0009] a1) An expression cassette encoding any of the genes described above;

[0010] a2) A recombinant vector containing the expression cassette described in a1);

[0011] a3) Recombinant microorganisms containing the expression cassette described in a1 or the recombinant vector described in a2).

[0012] This invention also provides the cotton fiber strength major gene NTL9. BS41 The molecular marker was amplified using primer pairs as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0013] This invention also provides a method for amplifying the aforementioned major gene NTL9 for cotton fiber strength. BS41 The primer pairs for molecular markers, the sequences of which are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0014] This invention also provides the application of the above-mentioned genes, products, molecular markers or primer pairs in regulating plant fiber strength, preparing products that regulate plant fiber strength, identifying or assisting in the identification of plant fiber strength, preparing products for identifying or assisting in the identification of plant fiber strength or in plant breeding.

[0015] Furthermore, the plant includes cotton.

[0016] The present invention also provides a method for enhancing cotton fiber strength or cultivating cotton varieties with high fiber strength, comprising introducing any of the above-mentioned genes or products into cotton for overexpression.

[0017] Furthermore, NTL9 BS41 A fiber-specific expression vector was constructed using the CDS sequence, and NTL9 was expressed via Agrobacterium-mediated genetic transformation. BS41 Genes were introduced into cotton receptors to achieve high expression of NTL9 in the fibers. BS41 Genetically modified cotton.

[0018] This invention also provides a method for distinguishing between Sea Island cotton and Upland cotton, and for identifying or assisting in the identification of cotton variety fiber strength. The method includes extracting sample DNA, performing PCR amplification using the primer pair shown in SEQ ID NO:4 and SEQ ID NO:5, and identifying cotton varieties with molecular markers as Sea Island cotton varieties by gel electrophoresis, whose fiber strength is significantly higher than that of cotton varieties without bands.

[0019] Beneficial effects:

[0020] (1) This invention performs map-based cloning of the major QTL locus for cotton fiber strength and identifies the major gene NTL9 for cotton fiber strength for the first time. BS41 This discovery not only provides new genetic resources for the improvement of cotton fibers, but also offers important clues for a deeper understanding of the molecular mechanisms of fiber development.

[0021] (2) This invention identified a 24bp mutation site in NTL9. The average fiber strength of the sea island cotton genotype (24bp insertion) material was about 1.72 cN / tex higher than that of the upland cotton genotype (24bp deletion) material. Molecular markers were developed for this mutation site. These markers can effectively distinguish between upland cotton and sea island cotton genotypes, which is beneficial for marker-assisted selection breeding of cotton.

[0022] (3) This invention utilizes the NTL9 genotype of sea island cotton. BS41 Gene introduction into upland cotton has optimized and improved the fiber quality. This innovative improvement significantly enhances fiber strength without altering other fiber quality traits or plant structure, providing new germplasm resources for cotton breeding.

[0023] (d) This invention demonstrates, through scanning electron microscopy, that NTL9 BS41 High expression of this compound can improve the uniformity and density of cellulose microfibrils, thereby enhancing the structural stability and physical properties of cotton fibers. This invention, for the first time, delves into the cellular mechanisms affecting cotton fiber strength from the perspective of cell wall structure, providing crucial insights into revealing the scientific reasons for cotton fiber strength formation. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1Map-based cloning of major genes for fiber strength. A: Phenotypic comparison of hand-combed fibers from E22, BS41, and 3-79. Scale bar = 1 cm. B: Frequency distribution of fiber strength in the F2 population. C: QTL scan detected the major fiber strength locus qFS-A11-1. D: Fine mapping of qFS-A11-1. E: Four candidate genes within the mapping interval. F: Expression levels of the four candidate genes at three stages of secondary fiber wall thickening. G: Variations in the coding region of candidate gene NTL9. H: Molecular marker amplification of the 24 bp InDel variation on the second exon in E22, BS41, and 3-79.

[0026] Figure 2 This section presents a variation analysis of the 24bp InDel genotype in the NTL9 gene CDS across cotton varieties. A: Variation of the 24bp InDel genotype in various cotton varieties; red boxes represent the 24bp deletion type, and orange boxes represent the 24bp insertion type. B: Variation types and frequency distribution of the 24bp InDel genotype in cotton germplasm resources; blue represents the 24bp deletion genotype, and orange represents the 24bp insertion genotype. C: Violin plot of fiber strength phenotypic values ​​for materials with different variation types; blue represents the 24bp deletion genotype, and orange represents the 24bp insertion genotype.

[0027] Figure 3 To specifically and highly express NTL9 in fibers BS41 The impact on fiber quality was studied. Specifically, the expression level of A:NT L9 in 20DPA fibers was detected, with Jin668 as the control and OE1, OE2, and OE3 representing NTL 9. BS41 Overexpression materials. B: Phenotypic value of fiber strength. C: Phenotypic value of fiber length. D: Jin668 and NTL9 BS41 Phenotypic comparison of hand-combed fibers of overexpression materials. Scale bar = 1 cm. E: Jin668 and NTL 9 BS41 Plant morphology during the boll opening stage of the overexpression material and plant morphology after leaf removal. Scale bar = 10cm.

[0028] Figure 4 To specifically and highly express NTL9 in fibers BS41 Effects on fiber cell wall structure. A: Helical structure of mature cotton fibers E22, BS41, and 3-79. Scale bar = 50 μm. B: Cell wall surface structure of mature cotton fibers E22, BS41, and 3-79. Scale bar = 5 μm. C: Jin668 and NTL9. BS41 Cell wall surface structure of mature cotton fibers used as overexpression material. Scale bar = 5 μm. Detailed Implementation

[0029] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0030] Example 1: QTL mapping and candidate gene analysis for cotton fiber strength

[0031] (1) QTL positioning of cotton fiber strength

[0032] Material source and phenotype: Our laboratory previously used Pima 3-79 (hereinafter referred to as 3-79) as the donor parent and E22 (hereinafter referred to as E22) as the recipient parent. E22 was crossed with 3-79 to obtain the F1 generation. Subsequently, a BC1 population was constructed using E22 as the male parent. After several generations of self-pollination, a backcross inbred line (BIL) population of 54 lines from both Pima 3-79 and E22 was finally obtained (Nie et al. 2015. A BIL population derived from G. hirsutum and G. barbadense provides source for cotton genetics and breeding. PLoS One, 10, 10:e0141064). Among them, one backcross inbred line, named BS41, was selected, characterized by its excellent fiber quality (…). Figure 1 A). In terms of fiber strength properties, the fiber strength of BS41 (33.93±0.87 cN / tex) is significantly improved by nearly 30% compared with E22 (26.20±0.14 cN / tex) (Table 1).

[0033] Table 1. Fiber quality analysis of E22, BS41 and 3-79

[0034]

[0035] Construction of the target population and genetic analysis of fiber strength: This invention uses E22 as the female parent and BS41 as the male parent for hybridization to obtain the F1 generation, which is then self-crossed. Subsequently, in 2018, an F2 segregating population (1230 individual plants) was constructed. For each F2 plant, cotton fibers were harvested from the middle 10 bolls at the boll opening stage, and the quality of mature fibers was determined using an HVI (High Volume Instrument) (HFT9000, Premier, India) instrument. Each fiber sample weighed no less than 8.5g. Statistical analysis of the frequency distribution of fiber strength revealed that fiber strength exhibited an approximately normal continuous distribution in the F2 population. Figure 1 B) indicates that fiber strength is a typical quantitative trait.

[0036] Preliminary QTL mapping for fiber strength: In the screening of polymorphic molecular markers, 5152 pairs of molecular markers from the high-density genetic linkage map previously constructed in our laboratory were first selected (Li et al. 2016a. Structure, evolution, and comparative genomics of tetraploid cotton based on a high-density genetic linkage map. DNA Research, 23, 3: 283-293) to screen for polymorphic molecular markers in the parents E22 and BS41, ultimately identifying 359 polymorphic markers. Subsequently, this invention uses the BSA (Bulked Segregation Analysis) method for preliminary detection of fiber strength gene loci. Specifically, 20 plants each with extreme phenotypes (high fiber strength and low fiber strength) were selected from the F2 population. DNA was extracted from the leaves of each plant, and the DNA was mixed in equal amounts to construct extreme pools for fiber length and fiber strength, respectively. Polymorphic molecular markers between parents selected from the genetic map were then used to further screen for polymorphic markers between pools, identifying 16 polymorphic markers on chromosome A11. Based on the reference genomes of upland cotton TM-1 and sea island cotton 3-79 (Wang et al., 2019), this invention further developed the InDel molecular marker (insertion / deletion fragment not less than 4 bp) in this region of chromosome A11. After screening for polymorphism between parents, 37 pairs of markers were finally used for genotyping and QTL scanning of fiber strength in the F2 population. QTL scanning of the F2 population was performed using QTL Icimapping 4.0 software. The QTL results showed a principal QTL for fiber strength between marker FSID-19 (FSID-19_F: 5'GGCA CTATTCTTGATCTGAGTA 3'; FSID-19_R: 5'ACAAAACCCTAAATTCTACAT3') and marker HAU3356 (HAU3356_F: 5'AGCGCCAATTCCCAGACCTT 3'; HAU3356_R: 5'AGGGTTTTGTTGCTGGGGCA 3'), named qFS-A11-1. Figure 1 C), corresponding to a 51kb interval of 1.474Mb to 1.526Mb in the upland cotton reference genome, has a high LOD value of 118.18, explaining 13.19% of the phenotypic variation. The BS41 allele provides a positive phenotypic effect value.

[0037] Fine mapping of fiber strength QTLs: Recombinant plants within the major QTL intervals for fiber strength were selected from the 2018 F2 population, and further fine mapping was performed on F2 plants derived from these recombinant plants. 2:3 The family (containing 180 individual plants, Hainan, 2019) and F 2:4 Genotypic identification and fiber strength phenotypic identification were performed on the family (containing 254 individual plants, Wuhan, 2020). After genotypic and phenotypic analysis of the recombinant individual plants, qFS-A11-1 was finally mapped to the interval between markers FSID-45 (FSID-45_F: 5'TCATAGGCGAAAGTCGTCAAT 3'; FSID-45_R: 5'ACACATGACCCATATATACCTCA 3') and FSID-33 (FSID-33_F: 5'GCCACTTTGATTAGAGCGTC 3'; FSID-33_R: 5'TTCACACGGATGGTGGAGTT 3'). Figure 1 D). The marker sequence was aligned to the upland cotton TM-1 reference genome, and its physical location corresponds to chromosome A11 of cotton from 1508822bp to 1523454bp, with a range size of 14.6kb.

[0038] (2) Candidate gene analysis

[0039] The localization interval contains 4 candidate genes ( Figure 1 E), namely: thymidine synthase gene Ts (Ghir_A11G001680), unannotated gene Ghir_A11G001690, NAC transcription factor family gene NT L9 (Ghir_A11G001700), and a gene encoding a putative serine protease repressor Agrn (Ghir_A11G001710). To determine the causal genes for fiber strength, this invention designed four pairs of primers for each of these four genes:

[0040] qRT-Gh_A11G0145(qRT-Gh_A11G0145_F:5'AGGGAACGACAATGGCG GAAAA 3'; qRT-Gh_A11G0145_R:5'TGACGGCGGAAAGCTTAGCAAT 3')

[0041] qRT-Gh_A11G0146(qRT-Gh_A11G0146_F:5'CTTCTGTCTTGGTGTTCGG ATCATAC 3'; qRT-Gh_A11G0146_R:5'ACCTGCCTCGGAAAGACCACTA 3')

[0042] qRT-Gh_A11G0147(qRT-Gh_A11G0147_F:5'AGTTCGAGCATGGCACAA ACGA 3'; qRT-Gh_A11G0147_R:5'CGACTGCTTGAGTCGTCACTGT 3')

[0043] qRT-Gh_A11G0148(qRT-Gh_A11G0148_F:5'GCCCCGTCCTTATTCATGT CCC 3'; qRT-Gh_A11G0148_R:5'AGCCTGACCAGGGAAAGATCCA 3')

[0044] We examined the expression of these four genes at three stages (20 DPA, 25 DPA, and 30 DPA) of secondary wall thickening in cotton fibers of E22 and BS41.

[0045] RNA extraction was performed using the Tiangen reagent kit (Tiangen Biotech, DP411). 3 μg of RNA was taken from each sample and reverse transcribed into cDNA.

[0046] Expression levels were detected using qRT-PCR. The qRT-PCR reaction mixture consisted of 15 μL: 7.5 μL cDNA template, 7 μL SybrGreen-mix (BIO-RAD), and 0.25 μL forward and reverse primers. After mixing, the mixture was placed in a real-time quantitative PCR instrument (ABIPrism 7500 system). The program was set as follows: Stage 1, 95℃ for 30 s, 1 cycle; Stage 2, 95℃ for 5 s + 60℃ for 35 s, 40 cycles. Fluorescence intensity was read at the end of each cycle, and then analyzed using 2... -ΔΔCT The relative expression levels of the gene were calculated using the method, with GhUBQ7 used as an internal reference gene (GenBank accession number DQ116411).

[0047] qRT-PCR results showed that NTL9 was the only candidate gene with the highest expression level, while the other three genes had lower expression levels. During the three stages of secondary cell wall thickening, the expression level of NTL9 in BS41 was significantly higher than that in E22. Figure 1 F). Therefore, the present invention selects the NTL9 gene as the causal gene for fiber strength.

[0048] In the third-generation cotton reference genome of our laboratory (Wang et al. 2019. Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense. Nature Genetics, 51, 2: 224-229), the gene corresponding to this gene in upland cotton TM-1 is Ghir_A11G001700 (named GhNTL9 in this invention), and the gene corresponding to the gene in sea island cotton 3-79 is Gbar_A11G001260 (named NTL9 in this invention). BS41 )

[0049] NTL9 BS41 The gene consists of 5 exons and 4 introns. Its genomic nucleotide sequence (as shown in SEQ ID NO.1, with underlines indicating exons) is as follows:

[0050] AAAATGGCCTATGGGGACATCTTCCTTTTCGCTTAAACTCTTTCAAATCCAAATACTAATTCGCCCTGAATCTTCTTCCTTTTAGGGTTTCAAACGATCCTTTATGCTTTGAATCTCCTCCTCCTTTTTGGGTTTCAAACGATCCTTTATTTGGGGGATTAACAGCAGTTTTACC ATGGCTGTTATCTCACTGAATTCACTGCCAATCGGAATCAGATTCC GACCAACGGACGAGGAGTTAATCGATTTTTACTTGCGAGCTAAAGTGAACGGGAAAAGGAAAGATGAGATCGGGGT TATACGGGAAGTTGATGTTTGTAAATGTGAACCGTGGGATTTGCCTCATTTGTCTGCTGTAAAGGCTCGTGACCCG GAATGGTTTTTCTTTTGCCCACTGGACCGGAAGTATCCGAATGGAAACCGGCTTAATAGGGCTACCGAGGCGGGTT [[ID=!4]]ATTGGAAGGCTACGGGTAAGGATAGAAAGATAAAGTCTGGGTCTTGCTTGATTGGCATGAAGAAAACTTTGGTGTT TTATACGGGTCGGGCTCCTAAAGGGAAGCGAACTAATTGGGTTATGCATGAGTACCGTACTACTCTTGATGAACTT GATGGAACCAAGCCTGGACAG GTTCGATTACTGGGTCTTTCTTAATTCTTTTGATTGTTATGTTTTTTTTTTTAATTCAGAATTTGATGGGTTAATTTGTTTTGTTGTGTAG AATCCTTTTGTAATTTGTCGTTTGTTCAAGAAACAAGATG AGACTATTGAAGATATTAATGGTGATGATGTTGATCCTGGGGCCTCTTCTCATACGGAAGAGGTGCAATCGGAGTT CGACGGGCCTTCGGTGGAAGGAGGAGCCGGAAAAGTTCCCGACGGAGGAGCCGGAAAAGTTCCCGACGAAACTCGG CCTGTGGGTTTATGTAATGAAGCCGTTGCACCTATATTTGACTGCAACAACGATGGGTTTAATGCTTGTGAACTGG AAGAAACGGCACCTGCACAG GTGAGAAAATGGAACTGGATTTGGATTTGGGTTTTGCAATCATTGCTGTAGTGCTTATGTGGTTTGATGTGGTTTTCAG GTGGATCTACTTGGAGAAGCTTTGTTTCAAATTTGTGATCCAATGATGGAGCC ATTGGATTGGAAGCTCTTTTCACCATTGCATTCGCAGATCGAAGCCGAGGGGGCACCTTGGATGTTTGACCATGTC GGGAACAGTTTTGGTGGGGTGAAGTTCGAGCATGGCACAAACGAAAATGATGCTGAATTCATGAACTCTATCCTGA It should be noted that in the provided text, there seems to be a potential error in line where the "!4" in the ID might be a mistake. I've translated it as is based on the instructions. If this is an incorrect ID format, it might need to be corrected before further processing or analysis. ATAATTCGGATGACTATTACAGTGACGACTCAAGCAGTCGAAGGAATTCAGTTATTGAAACCGAGACCCCAAAGTC AATGGCCTTTGGTACAAATGGTGGATTGTATTGCAAACCTGATGCTGAACCGGCTCGAGTATTGGTGAGTAGTAGAACCAAGCTTGTTACATATTTGTTTACTGTCTCAATATTCATTGAAATCATGAAATGCCTCATTGAACTCCTATTCAAAATTCATACAGCCTGGGACAGGAACTGCCAACGGAGGAGCATTCTAT AATGTGCTAAATAGCAATGGAGAACCAA GCAACCATGTGAACACAGCTTGTAACGTCGACACTGCACCTACCATCAGGATTCGATCTCGTGCTCATCGAATTCA GCCAGACACAGAGAACTTCGACACACAGGGTATTGCATCGAGAAGGTGTAAACTTGTGGTCCATTCACCTCGCTTC GAAGAGAAAGAACATGAATCGAAACCCATCCTTACGAAG GTGAGAAATTTGTAAACTATGACATAGTGTTAAGCCATTGCCAAATCCAGATATTTGGGCATTAATAGAGTTGTCTTTTACAGGGTGTAAAAGCCATGGAAGAGTATATCAGTGTTGGCAA CGATGCTGCTCGAAGGACCATGGACGAACCCCAGATTTTCGAGACAAGCAAAAGAGATGTTTTGAGGT CGAAGAGCAAGATTCCAGTCTCTGAAGCGGTTTCATATCGATGTTTGAAGAGGTTTTCAGCTCGTCGCCAGCATAA GCCGTTCAGTGTTATTATGTTTCGGGTCGTTGCTGTAATGTTAATCTTGTTTGTAGCTTTGGTTAGCACATTGAAT GTTTTGTAA TTTGATGCTGTTTTTGTGTTGATTTTTTGTTAGTAATTAAACTATTAATCATATTTACAATATTGATATATGCAAATTAATTAATTCGAGTTAAAACAAAATTAAGATTTACTTAACTGATTCCC。

[0051] NTL9 BS41 The CDS sequence of the gene (shown in SEQ ID NO.2) is:

[0052]

[0053] NTL9 BS41 The protein sequence encoded by the gene (as shown in SEQ ID No. 3) is as follows:

[0054] MAVISLNSLPIGIRFRPTDEELIDFYLRAKVNGKRKDEIGVIREVDVCKCEPWDLPHLSAVKARDPEWFFFCPLDRKYPNGNRLNRATEAGYWKATGKDRKIKSGSCLIGMKKTLVFYTGRAPKGKRTNWVMH EYRTTLDELDGTKPGQNPFVICRLFKKQDETIEDINGDDVDPGASSHTEEVQSEFDGPSVEGGAGKVPDGGAGKVPDETRPVGLCNEAVAPIFDCNNDGFNACELEETAPAQVDLLGEALFQICDPMMEPLDW KLFSPLHSQIEAEGAPWMFDHVGNSFGGVKFEHGTNENDAEFMNSILNNSDDYYSDDSSSRRNSVIETETPKSMAFGTNGGLYCKPDAEPARVLPGTGTANGGAFYNVLNSNGEPSNHVNTACNVDTAPTIRI RSRAHRIQPDTENFDTQGIASRRCKLVVHSPRFEEKEHESKPILTKGVKAMEEYISVGNDAARRTMDEPQIFETSKRDVLRSKSKIPVSEAVSYRCLKRFSARRQHKPFSVIMFRVVAVMLILFVALVSTLNVL

[0055] Further analysis was conducted on sequence variations of the candidate gene NTL9. Gene primers (Ghir_A11G001700_F: 5'ATGGCTGTTATCTCACTGAATTCAC 3'; Ghir_A11G001700_R: 5'TGCTTGTCGTCATCTTCTGTAC 3') were designed for NTL9 in this invention, and the CDS sequences (NTL9) of NTL9 in E22 and BS41 were cloned respectively. E22 and NTL9 BS41 Comparative sequencing revealed that the CDS sequence of NTL9 in BS41 was identical to that of Sea Island cotton, indicating that the gene was introduced from Sea Island cotton. BS41The genomic sequence corresponding to the gene is SEQ ID NO.1, the CDS sequence is SEQ ID NO.2, and the protein sequence is SEQ ID NO.3. Compared with the CDS sequence of E22, BS41 has a SNP at 139 bp after the start codon ATG (changing from T in E22 to G in BS41), resulting in an amino acid substitution (changing from phenylalanine in E22 to valine in BS41). There is a 24 bp inserted InDel at 583 bp (the second exon), and a 12 bp deleted InDel at 1356 bp (the fifth exon). Figure 1 G), but neither of the two InDels caused frameshift mutations.

[0056] The origin of the 24bp InDel variant in NTL9 was analyzed using a genome database. The cotton species analyzed included diploids such as *Gossypium herbaceum* (A1), *Gossypium asiaticum* (A2), and *Gossypium raymondii* (D5), and allotetraploids such as *Gossypium uplandense* (AD1), *Gossypium seashore* (AD2), *Gossypium pubescens* (AD3), *Gossypium chrysogenum* (AD4), *Gossypium darwinii* (AD5), *Gossypium ekkermannii* (AD6), and *Gossypium steffen* (AD7). The results showed that in *Gossypium herbaceum* and *Gossypium asiaticum* of the A genome, and in *Gossypium seashoreense* and *Gossypium darwinii* of the AD genome, this variant was a 24bp insertion type (red box); in *Gossypium raymondii* of the D genome, and in *Gossypium uplandense*, *Gossypium pubescens*, *Gossypium ekkermannii*, and *Gossypium steffen* of the AD genome, this variant was a 24bp deletion type (orange box). Based on the time of cotton species differentiation, this variant was found to occur after the formation of allotetraploids, during the period when *Gossypium seashoreense* and *Gossypium darwinii* differentiated from other cotton species. Figure 2 A). Furthermore, Sea Island cotton retained the original variant type (24bp insertion type), while upland cotton lost this 24bp fragment as cotton varieties differentiated.

[0057] Subsequently, the variation of 24bp InDel was analyzed in 4180 cotton materials, and it was found that 99.6% of upland cotton varieties carried NTL9. E22 Allelic type (24bp deletion), while 89.7% of Sea Island cotton varieties carry NTL9. BS41 Equivalent (24bp insertion) ( Figure 2 B). Among them, the average fiber strength of the 1233 materials carrying the upland cotton genotype (24bp deletion) was 28.90 cN / tex, and the average fiber strength of the 5 materials carrying the sea island cotton genotype (24bp insertion) was 30.62 cN / tex. The sea island cotton genotype showed an increase of approximately 1.72 cN / tex compared to the upland cotton genotype. Figure 2 C).

[0058] Molecular markers (SEQ ID NO.4-5) were developed for this 24-bp InDel. The detailed sequence information of the molecular markers is as follows: NTL9-24_F: 5'TCTTCTCATACGGAAGAGGTG 3' (SEQ ID NO.4); NTL9-24_R: 5'GTTGTTGCAGTCAAATATAGGT 3' (SEQ ID NO.5). This site was amplified in 503 upland cotton resource materials in our laboratory, verifying that the majority of upland cotton (99.4%) indeed belonged to NTL9. E22 Alleles ( Figure 2 (D) This molecular marker can effectively distinguish between upland cotton and sea island cotton genotypes and can serve as a gene marker for fiber strength genes. This marker can be used to directly select the genotype of NTL9 and can be applied to marker-assisted selection breeding.

[0059] Example 2: NTL9 BS41 Overexpression in cotton fibers

[0060] (1) Overexpression of NTL9 by the fiber-specific promoter Gb_expa2 BS41 Carrier construction

[0061] NTL9 BS41 The CDS sequence was constructed into the fiber-specific promoter expression vector Gb_expa2. The Gb_expa2 vector was provided by Dr. Yang Li, a graduate of our laboratory. Using cDNA from the 20DPA fiber of BS41 as a template, NTL9 was amplified using primers with added BP reaction adapters. BS41 Gene. The specific primer sequences after adding the adapter are as follows:

[0062] BP-NTL9_F:5'GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGC TGTTATCTCACTGAATTCAC3'

[0063] BP-NTL9_R:5'GGGGACCACTTTGTACAAGAAAGCTGGGTGTTACAA AACATTCAATGTGCTAACC3'

[0064] PCR amplification reaction system 20μL: 10μL cDNA template, 7μL ddH2O, 2μL 10×LongTaq Buffer, 0.4μL dNTP, 0.2μL Forward / Reverse Primer, 0.2μL LongTaq.

[0065] PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 sec, 56℃ for 90 sec, 72℃ for 30 sec, 32 cycles; 72℃ for 5 min. Correct NTL9 containing the BP adapter was obtained by PCR amplification. BS41 Excerpt.

[0066] PCR amplification products were purified using a kit. The purified products were then used for BP enzyme ligation (BP enzyme purchased from Invitrogen, USA). The reaction mixture consisted of: 1.5 μL ddH2O, 1 μL pDONER221 (Kan+), 2 μL purified PCR product, and 0.5 μL Gateway BP Clonase. The reaction was carried out at 25°C for 4 hours. The mixture was then transformed into E. coli strain TOP10 and plated with Kan+. + LB dish.

[0067] Single-clonal strains were selected and tested positive using the M13 forward primer 5'CCCAGTCACGACGTTGTAAAACG 3' and the adapter reverse primer. Positive strains were sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing, and the non-mutant plasmid was returned for use in LR (LR enzyme purchased from Invitrogen, USA) recombination reactions. The LR reaction system consisted of: 2.5 μL ddH2O, 1 μL Gb_expa2 vector (Spe... + ), 1 μL BP plasmid, 0.5 μL Gateway LR Clonase. The reaction conditions were 25°C for 4 hours, followed by transformation into E. coli strain TOP10, and plated on Spe. + LB dish. Positive results were detected using EXP_F:5'TGCTTGTCGTCATCTTCTGTAC 3' with the reverse primer for the adapter. Plasmid was extracted.

[0068] The positive plasmid (expression vector) was transformed into Agrobacterium GV3101 by electroporation and plated on Spe. + and rif + LB dishes were used to detect and pick out positive strains.

[0069] (2) Agrobacterium-mediated genetic transformation of cotton

[0070] A. Sterile seedling culture: Using the upland cotton variety Jin668 as the genetic transformation recipient, select plump and normally developed seeds, remove the seed coat, soak in a 2% sodium hypochlorite solution for 10 minutes, rinse with sterile water more than 3 times, inoculate on sterile seedling culture medium, and after the seeds show white sprouts, insert the roots into the culture medium to support the seedlings, seal the opening and place in a 28℃ dark constant temperature incubator for 5-6 days.

[0071] B. Cut the hypocotyl into small segments and use the activated NTL9-containing... BS41Infect the *Agrobacterium* strain with the overexpression vector, discard the bacterial culture, and dry it. Spread the hypocotyls evenly on a co-culture medium (MS medium + 2,4-D 0.1 mg / L + KT 0.1 mg / L + 30 g / L glucose + 2.6 g / L Phytagel, pH: 5.85–5.95) with filter paper, and incubate in the dark at 20°C for 36–48 h.

[0072] C. Obtaining regenerated material: Infected hypocotyls were inoculated into DK callus induction medium (MS medium + 2,4-D 0.1 mg / L + KT 0.1 mg / L + 30 g / L glucose + 2.6 g / L Phytagel + kanamycin 50 mg / L + cephalosporin 400 mg / L, pH: 5.85–5.95). The culture was carried out at 28°C in a light-controlled culture chamber (16 h light + 8 h dark), and subcultured every 20–30 days using fresh callus induction medium.

[0073] D. When the callus tissue grows into rice-grain-sized granules, it is transferred to differentiation medium (MS medium + IBA 0.5 mg / L + KT 0.1 mg / L + glutamine 1.0 g / L + asparagine 0.5 g / L + 30 g / L glucose + 2.6 g / L Phytagel, pH: 6.1~6.2) to further differentiate into embryoids.

[0074] E. The differentiated seedlings are subcultured on rooting medium (1 / 2 MS medium + 15 g / L glucose + 2.6 g / L Phytagel, pH: 6.1-6.2) until they grow into well-rooted seedlings.

[0075] (1)NTL9 BS41 Identification of expression levels and phenotypic characteristics in the progeny of overexpressed transgenic plants

[0076] Transgenic plants were generated using the upland cotton variety Jin668 as the genetic transformation recipient. DNA was extracted from leaves of T0 (2021 Wuhan), T1 (2022 Wuhan), and T2 (2023 Wuhan) transgenic plants. Positive detection of the transgenic plants was performed using primers (F: 5'TGCTTGTCGTCATCTTCTGTAC 3' and R: 5'TGCTTGTCGTCATCTTCTGTAC 3'). Subsequently, RNA was extracted from 20 DPA fiber samples of T1 positive plants for gene expression level detection, using the same method described in Example 1, with primers qRT-Gh_A11G0147. The expression level detection results showed that, compared with Jin668, the expression level of NTL9 was significantly increased in transgenic plants OE1, OE2, and OE3. Figure 3 A).

[0077] Subsequently, the field phenotypes of T1 and T2 generation plants of OE1, OE2, and OE3 were investigated. Normally matured and split cotton bolls from the middle of transgenic positive plants were harvested. After ginning, the quality of mature fibers was determined using an HVI (HFT9000, Premier, India) instrument. Each fiber sample weighed at least 8.5 g, and each material was measured at least three times with biological replicates (Table 2). The fiber quality results of the 2022 T1 generation showed that, relative to the fiber strength (24.73±1.07 cN / tex) of wild-type Jin668, NTL9... BS41 The fiber strength of the overexpression lines (OE1, OE2, and OE3) was significantly increased by 10.08%–19.77% (27.77±1.76 cN / tex, 29.62±1.00 cN / tex, and 27.17±1.29 cN / tex, respectively). Figure 3 B). Measurements of fiber quality in the 2023 T2 generation showed that NTL9... BS41 The fiber strength of the overexpression lines (OE1, OE2, and OE3) was also significantly improved compared to Jin668, while the other five fiber quality traits, including fiber length, showed no significant differences. Figure 3 C, D).

[0078] Table 2. NTL9 BS41 Fiber quality analysis of overexpression materials

[0079]

[0080] In addition, for NTL9 BS41 Morphological observation of overexpressing plants (T2 generation) revealed that overexpression of NTL9... BS41 There was no significant difference in plant structure compared to Jin668. Figure 3 E). Indicates NTL9 BS41 Overexpression in fibers does not affect the normal growth and development of plants.

[0081] Example 3: Structural features of fibrous cell walls

[0082] (1) Fiber characteristics of parental materials E22, BS41 and 3-79

[0083] Upland cotton E22 (26.20±0.14 cN / tex) has low fiber strength, while BIL-based materials BS41 (33.93±0.87 cN / tex) and Sea Island cotton 3-79 (43.83±1.19 cN / tex) have high fiber strength. The cell surface structure of normal mature cotton fibers from these three materials was observed using scanning electron microscopy.

[0084] Since mature cotton fibers are dehydrated and matured dead cells, they do not contain water and can be directly sputtered with gold for observation under a scanning electron microscope. The specific procedure is as follows: First, attach the sample. Apply conductive double-sided adhesive tape to the sample stage, comb the mature cotton fibers, and carefully attach the dry fibers to the double-sided adhesive tape with tweezers. Then, sputter-coat the sample with gold and observe. After sputter-coating gold in the ion sputtering instrument, observe and photograph the sample using a JSM-6390LV biological scanning electron microscope.

[0085] At 400x magnification, normal mature cotton fibers exhibit random left- or right-handed twists. Observation of the twisted areas revealed that mature E22 fibers showed rod-like twists, while mature BS41 and 3-79 fibers were twisted into thin, elongated sheets. Furthermore, the degree of twisting (fiber helixity) of BS41 and 3-79 was also higher. Figure 4 A). At a magnification of 3500x, the surface of E22 fiber is relatively rough, and the arrangement of cellulose microfibrils is disordered, while the surfaces of BS41 and 3-79 fibers are relatively smooth, and the arrangement of microfibrils is finer and more compact. Figure 4 B). This indicates that strengthening the connection and compactness of cellulose microfibrils can enhance the structural and mechanical properties of the cell wall.

[0086] (2) Overexpression of NTL9 BS41 Fiber characteristics

[0087] Select NTL9 BS41 The cell wall surface morphology of mature cotton fibers from normally growing plants in the middle of positive T2 generation plants (2023) of overexpression lines (OE1, OE2, and OE3) was observed. The cellulose microfilaments on the cell walls of Jin668 fibers were dispersed, while those of NTL9 fibers were more dispersed. BS41 The cellulose microfibrils in the cell walls of the overexpressed material have narrower interstices and are arranged more compactly and densely. Figure 4 C). Observations showed that fiber strength is closely related to the stacking and arrangement of cellulose microfibrils and cell wall structure. Overexpression of NTL9 in the fibers... BS41 It can alter the structural characteristics of cotton fiber cell walls, improving the uniformity and density of cellulose microfibrils arrangement, thereby increasing fiber strength.

[0088] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in 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 protection scope of the present invention.

Claims

1. A major gene for cotton fiber strength, NTL9 BS41 or an expression cassette encoding the gene or a recombinant vector or a recombinant microorganism containing the expression cassette in any one of A1) modulating plant fiber strength; A2) preparing a product for modulating plant fiber strength; wherein The protein sequence encoded by the gene is shown as SEQ ID NO: 3, and the plant is cotton.

2. Use according to claim 1, characterized in that, The CDS sequence of the gene is shown as SEQ ID NO: 2, and the genomic nucleotide sequence is shown as SEQ ID NO:

1.

3. Use of a primer pair of a molecular marker of the amplified cotton fiber strength major gene NTL9 BS41 characterized in that, The sequences of the primer pair are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the plant is cotton.

4. A method of enhancing the strength of cotton fibers or breeding a high fiber strength cotton variety, characterized by, The cotton fiber strength major gene NTL9 BS41 An expression cassette encoding the cotton fiber strength major gene NTL9 BS4 or a recombinant vector or a recombinant microorganism containing the expression cassette is introduced into the cotton to overexpress, wherein the protein sequence encoded by the gene is shown as SEQ ID NO:

3.

5. The method of claim 4, wherein, The CDS sequence of NTL9 BS41 is constructed into a fiber-specific expression vector, and the NTL9 BS41 gene is introduced into a cotton recipient by using the agrobacterium-mediated genetic transformation method to obtain transgenic cotton with high expression of NTL9 BS41 gene in the fiber.

6. A method for distinguishing Gossypium barbadense and Gossypium hirsutum, identifying or assisting in identifying the fiber strength of a cotton variety, comprising extracting sample DNA, performing PCR amplification using the primer pair shown as SEQ ID NO: 4 and SEQ ID NO: 5, and under gel electrophoresis, the sample with two bands is a cotton variety with a molecular marker, which is a Gossypium barbadense variety, and the fiber strength is significantly higher than that of the cotton variety with only one band.

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