Cotton fiber strength major gene NTL9BS41 and application thereof

Abstract

The invention relates to a cotton fiber strength major gene NTL9BS41 and application thereof. Through a cotton transgenic technology, the NTL9BS41 is successfully introduced into upland cotton and high-quantity expression of the NTL9BS41 in cotton fibers is realized, the fiber strength of the cotton is remarkably enhanced without negative influence on the quality of other fibers, the accumulation density and uniformity of cellulose microfibrils can be effectively enhanced by over-expression of the NTL9BS41, and the structure and performance of the cotton fibers are remarkably optimized. Besides, the gene is derived from sea island cotton, and compared with upland cotton, the gene CDS has insertion type variation of 24bp nucleotide, so that the molecular marker capable of effectively distinguishing haplotypes of the upland cotton and the sea island cotton is successfully developed. The invention provides new gene resources, technical means and germplasm resources for cotton fiber quality improvement, and has profound application value and potential for promoting the cotton breeding process.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a cotton fiber strength major gene NTL9 BS41 and its applications. Background Art

[0002] Cotton is one of the important natural fiber crops. Cotton fiber is widely used in textiles, clothing, home furnishings and other fields. Fiber strength is a key indicator to measure the quality of cotton fiber, and plays a decisive role in the quality and performance of textile products. Cotton fibers with high strength can withstand greater tensile force, reduce broken ends and defects, and improve the efficiency and stability of textile production; cotton fibers with high strength are wear-resistant, tensile-resistant and wrinkle-resistant, and can extend the service life of textiles. For a long time, improving cotton fiber strength has been an important goal of cotton breeding. However, traditional breeding methods that rely on phenotypic selection have long cycles and low efficiency.

[0003] Upland cotton and Sea island cotton are the two most important cultivated varieties in the world. Upland cotton has high yield and wide adaptability, but its fiber quality is average. Sea island cotton has low yield, but its fiber quality is very excellent. Fully tapping the genetic resources of excellent 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 that is easily affected by the environment. In recent years, although there have been some reports on cotton fiber development-related genes, the identification and functional studies of cotton fiber strength major genes are still relatively rare. Most of the existing studies have focused on the preliminary positioning of fiber strength QTLs, and the identification of strength genes and their specific mechanisms of action in the formation of cotton fiber strength and their potential in practical applications have not been fully revealed. Summary of the invention

[0005] The present invention aims to identify and utilize the fiber strength major gene in Sea Island cotton, and develop efficient application technology based on the major gene to achieve rapid and accurate improvement of upland cotton fiber quality. The present invention provides a cotton fiber strength gene NTL9 BS41 NTL9 of the Sea Island cotton genotype is specifically expressed in the fibers of Upland cotton. BS41 The gene can significantly improve the strength of cotton fibers, increase the compactness of cellulose microfibril stacking, and improve the quality of cotton fibers, thereby providing new genetic resources for cotton breeding and creating new germplasm.

[0006] The present invention provides a cotton fiber strength major gene NTL9 BS41 The protein sequence encoded by it 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) A recombinant microorganism containing the expression cassette described in a1) or the recombinant vector described in a2).

[0012] The present invention also provides a cotton fiber strength major gene NTL9 BS41 The molecular marker, and the primer pair sequence for amplifying the molecular marker are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0013] The present invention also provides a method for amplifying the cotton fiber strength major gene NTL9. BS41 A primer pair for molecular labeling, wherein the sequences of the primer pair are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0014] The present invention also provides the use of the above-mentioned genes, products, molecular markers or primer pairs in regulating plant fiber strength, preparing products for regulating plant fiber strength, identifying or assisting in identifying plant fiber strength, preparing products for identifying or assisting in identifying 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 high fiber strength cotton varieties, comprising introducing any of the above genes or products into cotton for overexpression.

[0017] Furthermore, NTL9 BS41 The CDS sequence of NTL9 was used to construct a fiber-specific expression vector and NTL9 was transformed into NTL9 by Agrobacterium-mediated genetic transformation. BS41 Gene introduction into cotton receptors resulted in high expression of NTL9 in fibers BS41 Genetically modified cotton.

[0018] The present invention also provides a method for distinguishing between sea island cotton and upland cotton, and identifying or assisting in identifying the fiber strength of cotton varieties, comprising extracting sample DNA, performing PCR amplification using the primer pair shown in SEQ ID NO:4 and SEQ ID NO:5, and wherein the sample having a band under gel electrophoresis is a cotton variety with a molecular marker, namely, a sea island cotton variety, and its fiber strength is significantly higher than that of a cotton variety without a band.

[0019] Beneficial effects:

[0020] (1) The present invention performed map-based cloning of the major QTL loci for cotton fiber strength and identified 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 fiber, but also provides important clues for a deeper understanding of the molecular mechanism of fiber development.

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

[0022] (3) The present invention is to synthesize the sea island cotton genotype NTL9 BS41 The introduction of genes into upland cotton has achieved the optimization and improvement of upland cotton fiber quality. This innovative improvement can significantly improve fiber strength without changing other fiber quality traits and plant structure, providing new germplasm resources for the field of cotton breeding.

[0023] (d) The present invention uses scanning electron microscopy to detect that NTL9 BS41 The high expression of can improve the uniformity and compactness of cellulose microfibril arrangement, thereby enhancing the structural stability and physical properties of cotton fibers. This paper, for the first time, deeply analyzes the cytological mechanism that affects the strength of cotton fibers from the perspective of cell wall structure, providing key insights into the scientific reasons for the formation of cotton fiber strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1The map-based cloning of the major gene 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 scanning detected the major locus for fiber strength, qFS-A11-1. D: Fine positioning of qFS-A11-1. E: Four candidate genes within the positioning interval. F: Expression levels of the four candidate genes during the three fiber secondary wall thickening periods. G: Gene coding region variation of the candidate gene NTL9. H: Molecular marker amplification effect of the 24bp InDel variation on the second exon in E22, BS41, and 3-79.

[0026] Figure 2 This is the variation analysis of 24bp InDel in the CDS of NTL9 gene in cotton species. A: The variation of 24bp InDel in various cotton species, the red box represents the 24bp deletion type, and the orange box represents the 24bp insertion type. B: The variation type and frequency distribution of 24bp InDel 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 ​​of materials with different variation types. Blue represents the 24bp deletion genotype, and orange represents the 24bp insertion genotype.

[0027] Figure 3 To express NTL9 specifically and at high levels in fibers BS41 Effects on fiber quality. A: Detection of the expression of NTL9 in 20DPA fibers, Jin668 as the control, OE1, OE2 and OE3 as NTL9 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 from overexpressing materials. Scale bar = 1 cm. E: Jin668 and NTL 9 BS41 Plant morphology of overexpressing materials at the boll-opening stage and after removing leaves. Scale bar = 10 cm.

[0028] Figure 4 To express NTL9 specifically and at high levels in fibers BS41 Effects on fiber cell wall structure. A: Helical structure of mature cotton fibers of E22, BS41 and 3-79. Scale bar = 50 μm. B: Cell wall surface structure of mature cotton fibers of E22, BS41 and 3-79. Scale bar = 5 μm. C: Jin668 and NTL9 BS41 Cell wall surface structure of mature cotton fibers from overexpressing materials. Scale bar = 5 μm. DETAILED DESCRIPTION

[0029] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

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

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

[0032] Source and phenotype of materials: In the early stage of this laboratory, sea island cotton (Pima3-79, hereinafter referred to as 3-79) was used as the donor parent, and upland cotton 'Emian 22' (hereinafter referred to as E22) was used as the recipient parent. E22 and 3-79 were hybridized to obtain the F1 generation. Subsequently, a BC1 population was constructed with E22 as the male parent. After multiple generations of self-pollination, a backcross inbred line (BIL) population of sea island cotton and upland cotton containing 54 strains was finally obtained (Nie et al. 2015. A BIL population derived from G. hirsutum and G. barbadense provides aresource for cotton genetics and breeding. PLoS One, 10, 10: e0141064). Among them, a backcross inbred line material named BS41 was selected, which is characterized by excellent fiber quality ( Figure 1 A). In terms of fiber strength, the fiber strength of BS41 (33.93±0.87 cN / tex) was significantly increased 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 positioning population and genetic analysis of fiber strength: The present invention used E22 as the female parent and BS41 as the male parent for hybridization to obtain the F1 generation and self-pollinate the F1 generation. Subsequently, an F2 generation segregation population (1230 individual plants) was constructed in 2018. For each F2 generation plant, 10 cotton bolls were collected from the middle of the plant during the boll opening period and then the cotton fibers were harvested by ginning. The quality of mature fibers was measured using an HVI (High Volume Instrument) (HFT9000, Premier, India) instrument, and the weight of each fiber sample was not less than 8.5 g. The frequency distribution statistics of fiber strength found that the fiber strength showed an approximately normal continuous distribution in the F2 population ( Figure 1 B), indicating that fiber strength is a typical quantitative trait.

[0036] Initial positioning of fiber strength QTL: In the screening of polymorphic molecular markers, 5152 pairs of molecular markers on the high-density molecular marker genetic linkage map constructed in the laboratory in the early stage 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), and polymorphic molecular markers were screened for parents E22 and BS41, and 359 polymorphic markers were finally identified. Subsequently, the present invention uses the BSA (Bulked Segregation Analysis) method to perform preliminary detection of fiber strength gene loci. Specifically, 20 plants with extreme phenotypes (high fiber strength and low fiber strength) were selected from the F2 population, and DNA from the leaves of the plants was extracted. The DNA was mixed in equal amounts to construct extreme pools of fiber length and fiber strength. The polymorphic molecular markers between parents screened out on the genetic map were used to further screen the polymorphic markers between the pools, and 16 polymorphic markers between the pools were screened out on the A11 chromosome. Based on the reference genomes of upland cotton TM-1 and sea island cotton 3-79 (Wang et al., 2019), the present invention further developed InDel molecular markers (insertion and deletion fragments of not less than 4bp) in this section of the A11 chromosome. After screening for polymorphism between parents, 37 pairs of markers were finally used to perform genotyping and fiber strength QTL scanning on the F2 population. The QTL Icimapping 4.0 software was used to scan the F2 population for QTL. The results of QTL detection showed that there was a major QTL for fiber strength between markers FSID-19 (FSID-19_F: 5'GGCA CTATTCTTGATCTGAGTA 3'; FSID-19_R: 5'ACAAAACCCTAAATTCTACAT3') and markers HAU3356 (HAU3356_F: 5'AGCGCCAATTCCCAGACCTT 3'; HAU3356_R: 5'AGGGTTTTGTTGCTGGGGCA 3'), which was named qFS-A11-1 ( Figure 1 C), corresponding to the approximately 51 kb interval from 1.474 Mb to 1.526 Mb of the upland cotton reference genome, the LOD value was as high as 118.18, the explanation rate of phenotypic variation was 13.19%, and the allele type of BS41 provided a positive phenotypic effect value.

[0037] Fine mapping of fiber strength QTL: Recombinant plants within the major QTL interval of fiber strength were selected from the 2018 F2 population, and the F2 derived from these recombinant plants were further mapped. 2:3 Family (including 180 plants, Hainan in 2019) and F 2:4 The family (including 254 plants, Wuhan in 2020) was genotyped and phenotypic identified for fiber strength. After genotypic and phenotypic analysis of the recombinant plants, qFS-A11-1 was finally located 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'TTCACACGGATGGTGGAGTT3') ( Figure 1 D) The marker sequence was aligned to the reference genome of upland cotton TM-1, and the physical position corresponded to the range of 1508822 bp to 1523454 bp on chromosome A11 of cotton, with an interval size of 14.6 kb.

[0038] (2) Candidate gene analysis

[0039] The localization interval contains 4 candidate genes ( Figure 1 E), respectively: thymidylate 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 inhibitor Agrn (Ghir_A11G001710). In order to determine the causal gene of fiber strength, the present invention designed 4 pairs of primers for these 4 genes respectively:

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

[0045] RNA was extracted using the Tiangen kit (Tiangen Biochemical Technology, DP411). 3 μg of RNA was extracted from each sample and reverse transcribed into cDNA.

[0046] The expression level was detected by qRT-PCR. The qRT-PCR reaction system was 15 μL: 7.5 μL cDNA template, 7 μL SybrGreen-mix (BIO-RAD), 0.25 μL forward and reverse primers. After mixing, it was placed in a real-time fluorescence quantitative PCR instrument (ABIPrism 7500 system). The program was set as follows: Stage 1, 95°C 30s, 1 cycle; Stage 2, 95°C 5s + 60°C 35s, 40 cycles. The fluorescence intensity value was read at the end of each cycle, and then 2 -ΔΔCT Methods The relative expression levels of genes were calculated, and GhUBQ7 was used as the internal reference gene (GenBank accession number No. DQ116411).

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

[0048] In the third-generation reference genome of cotton in this 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 ID corresponding to this gene in upland cotton TM-1 is Ghir_A11G001700 (named GhNTL9 in the present invention), and the gene ID corresponding to this gene in sea island cotton 3-79 is Gbar_A11G001260 (named NTL9 in the present invention). BS41 )

[0049] NTL9 BS41 The gene consists of 5 exons and 4 introns. The genomic nucleotide sequence of the gene (as shown in SEQ ID NO.1, underline indicates exons) is:

[0050] AAAATGGCCTATGGGGACATCTTCCTTTTCGCTTAAACTCTTTCAAATCCAAATACTAATTCGCCCTGAATCTTCTTCCTTTTAGGGTTTCAAACGATCCTTTATGCTTTGAATCTCCTCCTCCTTTTTGGGTTTCAAACGATCCTTTATTTGGGGGATTAACAGCAGTTTTACC ATGGCTGTTATCTCACTGAATTCACTGCCAATCGGAATCAGATTCC GACCAACGGACGAGGAGTTAATCGATTTTTACTTGCGAGCTAAAGTGAACGGGAAAAGGAAAGATGAGATCGGGGT TATACGGGAAGTTGATGTTTGTAAATGTGAACCGTGGGATTTGCCTCATTTGTCTGCTGTAAAGGCTCGTGACCCG GAATGGTTTTTCTTTTGCCCACTGGACCGGAAGTATCCGAATGGAAACCGGCTTAATAGGGCTACCGAGGCGGGTT ATTGGAAGGCTACGGGTAAGGATAGAAAGATAAAGTCTGGGTCTTGCTTGATTGGCATGAAGAAAACTTTGGTGTT TTATACGGGTCGGGCTCCTAAAGGGAAGCGAACTAATTGGGTTATGCATGAGTACCGTACTACTCTTGATGAACTT GATGGAACCAAGCCTGGACAG GTTCGATTACTGGGTCTTTCTTAATTCTTTTGATTGTTATGTTTTTTTTTTTAATTCAGAATTTGATGGGTTAATTTGTTTTGTTGTGTAG AATCCTTTTGTAATTTGTCGTTTGTTCAAGAAACAAGATG AGACTATTGAAGATATTAATGGTGATGATGTTGATCCTGGGGCCTCTTCTCATACGGAAGAGGTGCAATCGGAGTT CGACGGGCCTTCGGTGGAAGGAGGAGCCGGAAAAGTTCCCGACGGAGGAGCCGGAAAAGTTCCCGACGAAACTCGG CCTGTGGGTTTATGTAATGAAGCCGTTGCACCTATATTTGACTGCAACAACGATGGGTTTAATGCTTGTGAACTGG AAGAAACGGCACCTGCACAG GTGAGAAAATGGAACTGGATTTGGATTTGGGTTTTGCAATCATTGCTGTAGTGCTTATGTGGTTTGATGTGGTTTTCAG GTGGATCTACTTGGAGAAGCTTTGTTTCAAATTTGTGATCCAATGATGGAGCC ATTGGATTGGAAGCTCTTTTCACCATTGCATTCGCAGATCGAAGCCGAGGGGGCACCTTGGATGTTTGACCATGTC GGGAACAGTTTTGGTGGGGTGAAGTTCGAGCATGGCACAAACGAAAATGATGCTGAATTCATGAACTCTATCCTGA 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:

[0054] MAVISLNSLPIGIRFRPTDEELIDFYLRAKVNGKRKDEIGVIREVDVCKCEPWDLPHLSAVKARDPEWFFFCPLDRKYPNGNRLNRATEAGYWKATGKDRKIKSGSCLIGMKKTLVFYTGRAPKGKRTNWVMH EYRTTLDELDGTKPGQNPFVICRLFKKQDETIEDINGDDVDPGASSHTEEVQSEFDGPSVEGGAGKVPDGGAGKVPDETRPVGLCNEAVAPIFDCNNDGFNACELEETAPAQVDLLGEALFQICDPMMEPLDW KLFSPLHSQIEAEGAPWMFDHVGNSFGGVKFEHGTNENDAEFMNSILNNSDDYYSDDSSSRRNSVIETETPKSMAFGTNGGLYCKPDAEPARVLPGTGTANGGAFYNVLNSNGEPSNHVNTACNVDTAPTIRI RSRAHRIQPDTENFDTQGIASRRCKLVVHSPRFEEKEHESKPILTKGVKAMEEYISVGNDAARRTMDEPQIFETSKRDVLRSKSKIPVSEAVSYRCLKRFSARRQHKPFSVIMFRVVAVMLILFVALVSTLNVL

[0055] The sequence variation of the candidate gene NTL9 was further analyzed. The present invention designed gene primers for NTL9 (Ghir_A11G001700_F: 5'ATGGCTGTTATCTCACTGAATTCAC 3'; Ghir_A11G001700_R: 5'TGCTTGTCGTCATCTTCTGTAC 3'), cloned the CDS sequence of NTL9 in E22 and BS41 respectively (NTL9 E22 and NTL9 BS41 ), comparative sequencing revealed that the CDS sequence of NTL9 in BS41 was consistent with that of G. barbadense, indicating that the gene was introduced from G. barbadense. 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, there is a SNP at 139 bp after the start codon ATG in BS41 (from T in E22 to G in BS41), which leads to an amino acid substitution (from phenylalanine in E22 to valine in BS41), a 24 bp insertion InDel at 583 bp (the second exon), and a 12 bp deletion InDel at 1356 bp (the fifth exon). Figure 1 G), but neither InDel caused a gene frameshift mutation.

[0056] The genomic database was used to analyze the source of the 24bp InDel variation in NTL9. The analyzed cotton species included diploid cotton (A1), Asian cotton (A2) and Raymond cotton (D5), as well as allotetraploid upland cotton (AD1), sea island cotton (AD2), woolly cotton (AD3), yellow-brown cotton (AD4), Darwin cotton (AD5), Aikman cotton (AD6) and Stephen cotton (AD7). The results showed that in the A genome of cotton and Asian cotton, as well as the AD genome of sea island cotton and Darwin cotton, the variation was a 24bp insertion type (red box); in the D genome of Raymond cotton, as well as the AD genome of upland cotton, woolly cotton, Aikman cotton and Stephen cotton, the variation was a 24bp deletion type (orange box). According to the differentiation time of cotton species, it was found that the variation occurred after the formation of allotetraploidy, when sea island cotton and Darwin cotton were differentiated from other cotton species ( Figure 2 A). The sea island cotton retained the original variant type (24 bp insertion type), while the upland cotton lost the 24 bp fragment as the cotton species 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 allele (24 bp deletion), while 89.7% of the sea island cotton varieties carry NTL9 BS41 Allele (24 bp insertion) ( Figure 2 B). Among them, the average fiber strength of 1233 materials carrying the upland cotton genotype (24bp deletion) was 28.90 cN / tex, and the average fiber strength of 5 materials carrying the sea island cotton genotype (24bp insertion) was 30.62 cN / tex. The sea island cotton genotype was about 1.72 cN / tex higher than the upland cotton genotype material ( 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 NTL9-24_F: 5'TCTTCTCATACGGAAGAGGTG 3' (SEQ ID NO. 4); NTL9-24_R: 5'GTTGTTGCAGTCAAATATAGGT 3' (SEQ ID NO. 5). This locus was amplified in 503 upland cotton resource materials in our laboratory, and it was verified that most upland cotton (99.4%) were indeed NTL9 E22 Allele type ( Figure 2 D). This molecular marker can effectively distinguish upland cotton genotypes from sea island cotton genotypes and can be used as a gene marker for fiber strength genes. This marker can be used to directly select the genotype of NTL9 and can be used for molecular marker-assisted selection breeding.

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

[0060] (1) Fiber-specific promoter Gb_expa2 overexpresses NTL9 BS41 Vector construction

[0061] NTL9 BS41 The CDS sequence of NTL9 was constructed into the fiber-specific promoter expression vector Gb_expa2. The Gb_expa2 vector was provided by Dr. Li Yang, who has graduated from our laboratory. The cDNA of 20DPA fiber of BS41 was used as a template to amplify the NTL9 gene with primers that added BP reaction adapters. BS41 Gene. The specific sequence of the primer after adding the adapter is 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] The PCR amplification conditions were 95°C for 5 min, 95°C for 30 sec, 56°C for 90 sec, 72°C for 30 sec, 32 cycles, and 72°C for 5 min. The correct NTL9 containing the BP linker was obtained by PCR amplification. BS41 Fragment.

[0066] The PCR amplification product was purified using a kit and used for BP enzyme ligation reaction (BP enzyme purchased from Invitrogen, USA). The reaction system was: 1.5 μL ddH2O, 1 μL pDONER221 (Kan+), 2 μL PCR purified product, 0.5 μL Gateway BP Clonase. The reaction conditions were 25°C for 4 hours, transformed into Escherichia coli strain TOP10, and coated with Kan. + LB dish.

[0067] The monoclonal strains were picked and tested positive with M13 forward primer 5'CCCAGTCACGACGTTGTAAAACG 3' and adapter reverse primer. The positive strains were sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing, and the mutation-free plasmids were returned for LR (LR enzyme purchased from Invitrogen, USA) recombination reaction. The LR reaction system was: 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 h, transformed into E. coli strain TOP10, and coated with Spe + LB dish. Use EXP_F: 5'TGCTTGTCGTCATCTTCTGTAC 3' and adapter reverse primer to detect positive. Extract plasmid.

[0068] The positive plasmid (expression vector) was transformed into Agrobacterium GV3101 by electroporation and then coated with Spe + and rif + LB dish, detect and pick the positive strains.

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

[0070] A. Sterile seedling culture: Take the upland cotton variety Jin668 as the genetic transformation recipient, select seeds with full grains and normal development, peel off the cotton seed husks, soak in 2% sodium hypochlorite solution for 10 minutes, then rinse with sterile water for more than 3 times, inoculate on sterile seedling culture medium, and insert the roots into the culture medium for support after the seeds turn white. Seal the culture medium and culture it in a dark incubator at 28℃ for 5-6 days.

[0071] B. Cut the hypocotyl into small pieces and use activated NTL9 BS41Infect the overexpression vector with Agrobacterium, discard the bacterial solution, and blow dry; spread the hypocotyls flat on the co-culture medium (MS medium + 2,4-D 0.1mg / L + KT0.1mg / L + 30g / L glucose + 2.6g / L Phytagel, pH: 5.85-5.95) with filter paper, and culture in the dark in a constant temperature incubator at 20°C for 36-48h;

[0072] C. Obtaining regenerated materials: The 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). Cultured in a light culture room at 28°C (16 h of light + 8 h of darkness), subcultured with fresh callus induction medium every 20-30 days.

[0073] D. When the callus tissue grows into rice-like particles, it is transferred to differentiation medium (MS medium + IBA 0.5 mg / L + KT0.1 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) and further differentiated into embryoids.

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

[0075] (1)NTL9 BS41 Expression level identification and phenotype identification of the offspring of overexpressing transgenic plants

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

[0077] Subsequently, the field phenotypes of T1 and T2 plants of OE1, OE2 and OE3 were investigated. The cotton bolls with normal mature cracking in the middle of the transgenic positive plants were harvested, and the quality of mature fiber was determined by HVI (HFT9000, Premier, India) instrument after ginning. The weight of each fiber sample was not less than 8.5 g, and each material was measured for 3 or more biological replicates (Table 2). The results of the fiber quality test of the 2022 T1 generation showed that compared with the fiber strength of the wild-type Jin668 (24.73±1.07 cN / tex), NTL9 BS41 The fiber strength of the overexpression lines (OE1, OE2 and OE3) (27.77±1.76 cN / tex, 29.62±1.00 cN / tex, 27.17±1.29 cN / tex) increased significantly by 10.08% to 19.77% ( Figure 3 B) The fiber quality measurement results of the 2023 T2 generation showed that NTL9 BS41 The fiber strength of the overexpression lines (OE1, OE2 and OE3) was also significantly improved compared with Jin668, while the other five fiber quality traits including fiber length did not differ much ( Figure 3 C, D).

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

[0079]

[0080] In addition, NTL9 BS41 The morphological structure of overexpressing plants (T2 generation) showed that overexpressing NTL9 BS41 There is no significant difference in plant structure with Jin668 ( Figure 3 E) shows that NTL9 BS41 Overexpression in fibers did not affect the normal growth and development of the plant.

[0081] Example 3: Structural characteristics of fiber cell walls

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

[0083] The fiber strength of upland cotton E22 (26.20±0.14 cN / tex) was low, while that of BIL material BS41 (33.93±0.87 cN / tex) and sea island cotton 3-79 (43.83±1.19 cN / tex) was high. The cell surface structure of normal mature cotton fibers of the three materials was observed by scanning electron microscopy.

[0084] Since mature cotton fibers are dead cells that have been dehydrated and matured, they do not contain water. For scanning electron microscopes, they can be directly sprayed with gold for observation. The specific operation process is: first paste the sample, stick a conductive double-sided tape on the sample stage, comb the mature cotton fibers with a comb, and carefully stick the dry fibers to the double-sided tape with tweezers. Then spray gold and observe. Put the sample stage into the ion sputtering instrument, spray gold, and observe and take pictures with a JSM-6390LV biological scanning electron microscope.

[0085] Under a magnification of 400 times, normal mature cotton fibers have random left-handed or right-handed twists. By observing the twisted parts, it was found that the E22 mature fibers were twisted in rods, while the BS41 and 3-79 mature fibers were twisted in thin sheets. The degree of twist (fiber helicity) of BS41 and 3-79 was also higher ( Figure 4 A). Under a magnification of 3500 times, the surface of E22 fibers is relatively rough, and the stacking and arrangement of cellulose microfibrils are scattered, while the surface of BS41 and 3-79 fibers is relatively smooth, and the stacking and arrangement of microfibrils are finer and more compact ( Figure 4 B) It shows that strengthening the tightness of cellulose microfibril connection and stacking can enhance the structure and mechanical properties of cell wall.

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

[0087] Select NTL9 BS41 The cell wall surface morphology of mature cotton fibers grown normally in the middle of the positive T2 generation plants (2023) of the overexpression lines (OE1, OE2 and OE3) was observed. The cellulose microfibrils on the cell wall of Jin668 fibers were arranged in a scattered manner, while NTL9 BS41 The cellulose microfibrils on the cell walls of the overexpressed materials had narrower gaps and were arranged more compactly and densely ( Figure 4 C). The results showed that fiber strength is closely related to the stacking and arrangement of cellulose microfibrils and the cell wall structure. BS41 It can change the structural characteristics of cotton fiber cell walls, improve the uniformity and compactness of cellulose microfibril arrangement, and thus increase fiber strength.

[0088] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Cotton fiber strength major gene NTL9 BS41 , characterized in that, The protein sequence encoded by the gene is shown in SEQ ID NO:

3.

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

1.

3. Products containing any of the following a1)-a3): a1) an expression cassette encoding the gene according to any one of claims 1 to 2; a2) a recombinant vector containing the expression cassette described in a1); a3) A recombinant microorganism containing the expression cassette described in a1) or the recombinant vector described in a2).

4. Cotton fiber strength major gene NTL9 BS41 A molecular marker characterized in that The sequences of the primer pairs for amplifying the molecular marker are shown in SEQ ID NO.4 and SEQ ID NO.

5.

5. Used to amplify the cotton fiber strength major gene NTL9 described in claim 4 BS41 A primer pair for molecular labeling, characterized in that: The primer pair sequences are shown in SEQ ID NO.4 and SEQ ID NO.

5.

6. Use of the gene according to any one of claims 1-2, the product according to claim 3, the molecular marker according to claim 4, or the primer pair according to claim 5 in regulating plant fiber strength, preparing products for regulating plant fiber strength, identifying or assisting in identifying plant fiber strength, preparing products for identifying or assisting in identifying plant fiber strength, or in plant breeding.

7. The use according to claim 6, characterized in that: The plants include cotton.

8. A method for increasing cotton fiber strength or cultivating high fiber strength cotton varieties, characterized in that: The gene according to any one of claims 1 to 2 or the product according to claim 3 is introduced into cotton for overexpression.

9. The method according to claim 8, characterized in that NTL9 BS41 The CDS sequence of NTL9 was used to construct a fiber-specific expression vector and NTL9 was transformed into NTL9 by Agrobacterium-mediated genetic transformation. BS41 Gene introduction into cotton receptors resulted in high expression of NTL9 in fibers BS41 Genetically modified cotton.

10. A method for distinguishing between sea island cotton and upland cotton, and identifying or assisting in identifying the fiber strength of cotton varieties, comprising extracting sample DNA, performing PCR amplification using the primer pair shown in SEQ ID NO:4 and SEQ ID NO:5, and samples with bands under gel electrophoresis are cotton varieties with molecular markers, which are sea island cotton varieties, and their fiber strength is significantly higher than that of cotton varieties without bands.

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