Application of keratin related protein gene GbKAP related to cotton fiber quality character

Abstract

The invention discloses application of a keratin related protein gene GbKAP related to cotton fiber quality characters, and belongs to the field of biotechnology application. The growth vigor and plant height of root cells, main roots, hypocotyls and rosette leaves of an arabidopsis thaliana plant for heterologous expression of the cotton keratin related protein gene GbKAP are superior to those of a control group. The GbKAP plays an important role and has an application prospect in promoting cell elongation, improving the quality character of cotton fibers and cultivating new varieties of high-quality cotton fibers.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology and relates to application of a keratin-associated protein gene GbKAP related to cotton fiber quality traits. The full-length ORF of the gene is 567 bp and encodes 187 amino acids. A secondary segregating population is constructed by crossing an excellent fiber quality Hai-Lu introgression line material L240 and a receptor parent TM-1, and a gene GbKAP derived from the Hai7124 introgression and significantly related to fiber quality traits is identified by combining fine mapping, substitution mapping, structure and expression analysis. The expression level of the gene in the 5-10 day fiber development period of L240 is higher than that of TM-1, and the expression amount in the 10-day fiber is the highest. GbKAP is heterologously expressed in Arabidopsis, and the root cells, main roots and hypocotyls are longer, the rosette leaves are more vigorous, and the plants are higher, promoting plant growth. The application uses biotechnology to determine that GbKAP has a promoting effect on plant cell elongation, and proposes a breeding utilization value of promoting fiber cell elongation by overexpression of GbKAP. BACKGROUND

[0002] Two allohexaploid cultivated cotton species are Gossypium hirsutum and Gossypium barbadense. The planting area of Gossypium hirsutum is more than 90% of the global cotton planting area, and it has the characteristics of high fiber yield and wide adaptability. Gossypium barbadense has excellent fiber quality and disease resistance. At present, the important bottleneck restricting the further improvement of cotton varieties is the narrow genetic background of Gossypium hirsutum and the negative correlation between fiber quality and yield. By constructing cotton chromosome segment introgression lines (CSILs), introducing excellent fiber quality genes of Gossypium barbadense into Gossypium hirsutum is an effective way to broaden the genetic variation of Gossypium hirsutum, break the negative correlation between quality and yield, and obtain new cotton varieties with high yield and excellent quality.

[0003] Chromosome segment introgression lines are also known as single segment introgression lines (SSIL), single segment substitution lines (SSSL), near-isogenic lines (NIL), chromosome substitution lines (CSSL), etc. They are constructed by backcrossing and selfing, combined with marker-assisted selection (MAS), and are a set of families that have only one or a few specific donor chromosome segments, while the rest of the genetic background is completely the same as the recipient parent. The main characteristics of this type of family are: each individual of the introgression line contains only one or a few chromosome segments from the donor parent, and the rest is the same as the recurrent parent; the introgression line population is a permanent segregating population and can be phenotyped for many years and at multiple sites; the target segment can be divided into shorter segments through re-backcrossing and recombination. In addition, the use of chromosome segment introgression lines for the study of target traits and gene effects can minimize the influence of genetic background between lines, so this population is of great significance for QTL mapping.

[0004] Chromosome segment introgression lines are also ideal materials for fine mapping of QTLs. There are generally two methods: the first is to use overlapping populations in introgression lines to map QTLs by substitution mapping; the second method is to construct secondary segregating populations of QTLs and use them for fine mapping of QTLs. Xiao et al. (2023) used single segment substitution line (CSSL-106) and its recurrent parent CCRI45 to construct a secondary segregating population of 2852 plants, fine-mapped the QTL qFL-A12-5 related to fiber length to an 18.8 Kb region on chromosome A12, and identified GH_A12G2192 (GhTPR), a protein encoding a tetatricopeptide repeat superfamily, as a candidate gene for regulating cotton fiber development. Feng et al. (2022) selected a stable introgression line IL197 of Gossypium hirsutum × Gossypium arboreum with small bolls and seeds, constructed F2 and F3 secondary segregating populations, and further narrowed down the QTL q(BW + SI)-A12-1 on chromosome A12 that controls both boll weight (BW) and seed index (SI) to 60.09 kb through substitution mapping, and identified TIP41-like family protein (TIP41L) as a candidate gene through RT-qPCR analysis.

[0005] Keratin-associated proteins (KAPs) are mainly found in mammals and are structural components of mammalian hair fibers. Keratin is an important component of the cytoskeleton and plays a key role in cell morphology maintenance and cell mechanical strength. Keratin-associated proteins can interact with keratin to enhance the stability and function of keratin fibers. Chen et al. (2024) identified the gene OsRGL2 encoding keratin-associated protein (KAP) by map-based cloning, overexpression of OsRGL2 gene increased grain size by affecting cell proliferation in rice spike husk, and significantly improved yield per plant. So far, the role of KAP in plants has been rarely reported. SUMMARY

[0006] The purpose of the present application is to provide an application of a keratin-associated protein gene GbKAP related to cotton fiber quality traits.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008] In the first aspect, the present application claims the application of GbKAP gene or biological material related to the GbKAP gene in (a1) or (a2) or (a3) as follows:

[0009] (a1) improving cotton fiber quality traits, or preparing products for improving cotton fiber quality traits;

[0010] (a2) breeding of new cotton germplasm with improved fiber quality, or preparing products for breeding of new cotton germplasm with improved fiber quality;

[0011] (a3) or promoting the growth of target plants;

[0012] The GbKAP gene is as follows (b1) or (b2) or (b3):

[0013] (b1) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 1;

[0014] (b2) a DNA molecule derived from cotton and having more than 95% identity with (b1) and encoding the same protein;

[0015] (b3) a DNA molecule hybridizing or complementary to the DNA sequence defined in (b1) under stringent conditions and encoding the same protein.

[0016] Further, in the above application, the biological material related to the GbKAP gene is at least one of (c1)-(c11) as follows:

[0017] (c1) the protein GbKAP encoded by the GbKAP gene;

[0018] (c2) a primer pair for cloning the GbKAP gene;

[0019] (c3) an expression cassette containing the GbKAP gene;

[0020] (c4) a recombinant vector containing the GbKAP gene, or a recombinant vector containing the expression cassette of (c3);

[0021] (c5) a recombinant microorganism containing the GbKAP gene, or a recombinant microorganism containing the expression cassette of (c3), or a recombinant microorganism containing the recombinant vector of (c4);

[0022] (c6) a transgenic plant cell line containing the GbKAP gene, or a transgenic plant cell line containing the expression cassette of (c3), or a transgenic plant cell line containing the recombinant vector of (c4);

[0023] (c7) a transgenic plant tissue containing the GbKAP gene, or a transgenic plant tissue containing the expression cassette of (c3), or a transgenic plant tissue containing the recombinant vector of (c4);

[0024] (c8) a transgenic plant organ containing the GbKAP gene, or a transgenic plant organ containing the expression cassette of (c3), or a transgenic plant organ containing the recombinant vector of (c4);

[0025] (c9) a transgenic plant containing the GbKAP gene, or a transgenic plant containing the expression cassette of (c3), or a transgenic plant containing the recombinant vector of (c4);

[0026] (c10) a tissue culture produced from regenerable cells of the transgenic plant of (c9);

[0027] (c11) a protoplast produced from the tissue culture of (c10).

[0028] Further, the protein GbKAP in (c1) is as follows (d1) or (d2) or (d3) or (d4):

[0029] (d1) a protein with an amino acid sequence as shown in SEQ ID NO. 2;

[0030] (d2) a fusion protein obtained by linking a protein tag to the N terminus or / and C terminus of the protein of (d1);

[0031] (d3) a protein related to the cotton fiber quality trait or / and the plant growth, which is obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid residue sequence shown in SEQ ID NO. 2;

[0032] (d4) a protein derived from cotton and having 98% or more identity with (d1) and related to the cotton fiber quality trait or / and the plant growth.

[0033] Further, the nucleotide sequences of the upstream and downstream primers in (c2) are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0034] Further, the above-mentioned application is to use the GbKAP gene as a target gene, to overexpress the GbKAP gene by genetic engineering method, to improve the cotton fiber quality trait or to cultivate new cotton germplasm with improved fiber quality, or to promote the growth of target plants.

[0035] In the technical solution of the present application, the plant is cotton or Arabidopsis.

[0036] In the technical solution of the present application, the cotton fiber quality trait is at least one of fiber length, fiber strength and fiber uniformity.

[0037] In a second aspect, the present application claims to protect a method for improving the cotton fiber quality, which overexpresses the GbKAP gene with the nucleotide sequence shown in SEQ ID NO. 1 in cotton.

[0038] In a third aspect, the present application claims to protect a GbKAP gene related to the cotton fiber quality trait, which has the nucleotide sequence shown in SEQ ID NO. 1.

[0039] In a fourth aspect, the present application claims to protect biological materials related to the aforementioned GbKAP gene, which are at least one of the following (e1)-(e5):

[0040] (e1) a protein GbKAP with the amino acid sequence shown in SEQ ID NO. 2, which is encoded by the GbKAP gene;

[0041] (e2) an expression cassette containing the GbKAP gene;

[0042] (e3) a recombinant vector containing the GbKAP gene, or a recombinant vector containing the expression cassette of (e2);

[0043] (e4) a recombinant microorganism containing the GbKAP gene, or a recombinant microorganism containing the expression cassette of (e2), or a recombinant microorganism containing the recombinant vector of (e3);

[0044] (e5) a transgenic plant cell line containing the GbKAP gene, or a transgenic plant cell line containing the expression cassette of (e2), or a transgenic plant cell line containing the recombinant vector of (e3).

[0045] The present application shows that overexpression of the gene GbKAP with the nucleotide sequence as shown in SEQ ID NO. 1 in Arabidopsis thaliana can make the root cells of the overexpression Arabidopsis thaliana strain longer, the main root longer, the hypocotyl longer, the rosette leaf more vigorous, and the plant taller, thus promoting plant growth.

[0046] The present application utilizes a high-fiber quality land-sea introgression line material L240 to cross with the receptor parent TM-1 to construct a secondary segregation population, combines the resequencing data of L240 to obtain a precise introgression segment, develops InDel markers for the insertion / deletion sites on the introgression segment, performs linkage analysis on the fiber quality phenotype values and genotypes of the secondary segregation population, and identifies a FU QTL qFU-D04. Further substitution mapping is performed on the recombinants in the candidate segment to obtain a candidate interval of the QTL. Combined with the differential expression characteristics of the genes in the candidate interval in L240 and TM-1 at different fiber development periods, a gene GbKAP with ID number GB_D04G0512 is found. The expression of the gene in the 5-15 day fiber development period of L240 is higher than that in TM-1, and the expression amount in the 10 day fiber is the highest. The parent L240 has advantages in fiber length, fiber strength and fiber uniformity compared with TM-1. Overexpression of the gene GbKAP with the nucleotide sequence as shown in SEQ ID NO. 1 in Arabidopsis thaliana can make the root cells of the overexpression Arabidopsis thaliana strain longer, the main root longer, the hypocotyl longer, the rosette leaf more vigorous, and the plant taller, thus promoting plant growth. The above results show that GbKAP plays a role in promoting plant growth and development.

[0047] The development process of the technical scheme of the present application specifically includes the following steps:

[0048] 1. Cross a high-quality fiber land-sea introgression line material L240 with TM-1 to construct (L240 x TM-1) F2 / F 2:3 a secondary segregation population. Investigate the fiber quality traits (fiber length, fiber strength, micronaire value, fiber uniformity and fiber elongation rate) of the secondary segregation population to obtain phenotype data.

[0049] 2. Combine the whole genome resequencing information of L240 to develop InDel markers for the insertion / deletion sites on the introgression segment, and screen polymorphic InDel primers. Use the polymorphic InDel primers to identify the genotype of each single plant of the secondary segregation population to obtain genotyping data.

[0050] 3. Utilizing F2 single plants and F 2:3 The fiber quality detection data of the strain and the InDel marker genotype data of the F2 single plants are subjected to QTL mapping to detect a stable fiber uniformity related QTL (qFU-D04), and combining substitution mapping, the candidate interval of the QTL is determined.

[0051] 4. Combining the expression of the genes in the candidate interval at different fiber development periods of the parents, the gene GbKAP is determined as the candidate gene of the fiber uniformity QTL, the expression of the gene at the fiber development period of 5-15 days of L240 is higher than that of TM-1, and the expression amount in the fiber of 10 days is the highest. The CDS sequence of the gene is SEQ ID NO. 1, and the encoded amino acid sequence is like SEQ ID NO. 2. Sequence analysis between the parents shows that there is one non-synonymous SNP mutation in the gene in L240 and TM-1.

[0052] 5. The application also constructs an overexpression Arabidopsis thaliana transgenic strain of GbKAP. Compared with the wild type WT, the Arabidopsis thaliana strain heterogeneously expressing the cotton GbKAP gene as shown in SEQ ID NO. 1 has longer root cells, longer main roots, longer hypocotyls, more vigorous rosette leaves, and higher plants. It is shown that overexpression of GbKAP significantly promotes cell elongation and plant growth of Arabidopsis thaliana plants.

[0053] The application has the following advantages:

[0054] 1. The application utilizes linkage analysis and substitution mapping to identify a QTL (qFU-D04) related to uniformity in a sea-land introgression line material with excellent fiber quality, combines the expression of the genes in the candidate interval at different fiber development periods of the two parents L240 and TM-1, clones the gene GbKAP, and takes it as the candidate gene of the QTL.

[0055] 2. According to the analysis of different haplotypes by substitution mapping, the recombinant population can be divided into two types, i.e. type 1 (G1-G6) and type 2 (G7-G8). It is found through statistical analysis that there are significant differences in fiber uniformity and fiber length between the single plants corresponding to different haplotypes.

[0056] 3. The expression levels of GbKAP and its homologous genes at different fiber development periods of the two parents L240 and TM-1 are obtained through transcriptome data and verified by quantitative data. GbKAP and its homologous genes are expressed higher in the fiber development period of 5-15 days of L240 than in TM-1, and the expression amount in the fiber of 10 days is the highest, indicating that the gene plays a role in the fiber initiation and elongation period.

[0057] 4. L240 has advantages in fiber length, fiber strength and fiber uniformity compared with TM-1; in F 2:4 Among the recombinant population, different recombinant types have significant differences in fiber uniformity and fiber length. It is shown that the gene GbKAP has significant advantages in improving fiber quality traits, which can be used to improve cotton fiber quality traits and cultivate new cotton varieties with high quality.

[0058] 5. Compared with wild type (WT), the Arabidopsis line with overexpression of cotton GbKAP gene as shown in SEQ ID NO. 1 has longer root cells, longer main roots, longer hypocotyls, more vigorous rosette leaves and higher plants, indicating that overexpression of GbKAP gene promotes plant growth and development. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 . Genetic mapping of fiber uniformity QTL site qFU-D04.

[0060] Wherein, a is the initial mapping of fiber uniformity QTL site qFU-D04 in (L240 × TM-1) F2 and F 2:3 population. Blue line and green line are the LOD curves of the QTL in F2 and F 2:3 population mapping, LOD>2.5. b is the genetic linkage map of D04 chromosome introgression fragment. c is the replacement mapping of qFU-D04 and the comparison of fiber uniformity (FU) and fiber length (FL) of different introgression fragments with TM-1. ** represents P<0.01, and * represents P<0.05.

[0061] Figure 2 . Expression patterns and sequence comparison of GbKAP and its homologous genes in L240 and TM-1 at different fiber development stages.

[0062] Wherein, a is the expression pattern analysis of GbKAP and its homologous genes in L240 and TM-1 at different fiber development stages. The left Y axis and the column chart represent the RT-qPCR of GbKAP and its homologous genes; the right Y axis and the line chart represent the RNA-seq; the X axis represents the fiber tissues at 5-25 DPA. b is the amino acid sequence alignment of GbKAP and its homologous genes in Gossypium hirsutum. There is 1 non-synonymous SNP.

[0063] Figure 3 . Subcellular localization map of GbKAP in tobacco cells.

[0064] Wherein, a is the subcellular localization of 35S: GbKAP-eGFP. AtPIP2 is a plasma membrane marker. b is the subcellular localization of GbKAP after plasmolysis of 35S: GbKAP-eGFP tobacco leaves.

[0065] Figure 4 Phenotype analysis of Arabidopsis thaliana overexpressing GbKAP gene.

[0066] Wherein, a is the relative expression level of GbKAP in overexpressing Arabidopsis thaliana lines and wild type (WT). b is the identification of transgenic GbKAP positive clones. c is the phenotype observation of root cell of GbKAP transgenic lines and WT. The scale is 50 μm. d is the comparative analysis of root cell length of transgenic Arabidopsis thaliana and WT. e is the phenotype observation of root length of GbKAP transgenic lines and WT. The scale is 1 cm. f is the comparative analysis of root length of transgenic Arabidopsis thaliana and WT. g is the phenotype observation of hypocotyl of GbKAP transgenic lines and WT. The scale is 1 cm. h is the comparative analysis of hypocotyl length of transgenic Arabidopsis thaliana and WT. i is the phenotype observation of rosette leaf growth of GbKAP transgenic lines and WT. The scale is 4 cm. j is the phenotype observation of stem height of GbKAP transgenic lines and WT. The scale is 10 cm. k is the comparative analysis of stem height of GbKAP transgenic lines and WT. ** indicates P < 0.01, and * indicates P < 0.05. DETAILED DESCRIPTION

[0067] In order to make the technical problems solved by the present application, technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below in combination with embodiments.

[0068] The experimental methods used in the following examples are conventional methods unless otherwise specified

[0069] Example 1 Obtaining fiber uniformity QTL qFU-D04 by fine mapping and substitution mapping and excavating candidate genes thereof

[0070] A set of Gossypium hirsutum TM-1 background Gossypium barbadense Hai7124 introgression line population was constructed, and multi-year fiber quality identification was carried out, and it was found that a Gossypium barbadense land introgression line material L240 had excellent fiber quality, and the fiber length, fiber strength and uniformity were significantly higher than those of the receptor TM-1 (Table 1). A (L240 x TM-1) F2 / F 2:3 Secondary segregation population, fine mapping and candidate gene excavation work of high-quality loci contained in L240 introgression fragment were carried out. Combined with whole genome resequencing information of L240, InDel markers were developed for insertion / deletion sites on the introgression fragment, and polymorphic InDel primers were screened. Using the polymorphic InDel primers, the genotypes of F2 single plants were identified, and genotyping data were obtained. F 2:3 Phenotype investigation was carried out on the five fiber quality traits of the F2 single plants and F 2:3The fiber quality data of the lines and the InDel marker genotyping data of the F2 single plants were subjected to QTL mapping, and a stable fiber uniformity (FU) QTL (qFU-D04) was detected (a in Figure 1 ). 2:4 The 56 recombinants detected in F 2:4 were genotyped and investigated for fiber quality traits, and substitution mapping was carried out. The 56 recombinants were divided into 8 groups, and the FU (fiber uniformity) and FL (fiber length) of the G1-G6 groups were significantly higher than TM-1, but had no significant difference with L240, and the FU and FL of the G7-G8 groups were significantly lower than L240, but had no significant difference with TM-1. qFU-D04 was finely mapped between D04-7.0 and D04-7.8 (b-c in Figure 1 ).

[0071] Table 1 Comparison of phenotypic values of parents

[0072] TM-1 L240 FL 26.53±1.34 29.43±0.59** FS 26.70±0.78 31.04±0.90** MIC 5.36±0.15 4.62±0.80 FU 82.57±1.09 84.44±0.66* FE 6.23±0.72 6.50±0.41

[0073] Based on the transcriptome data of TM-1 and L240 parents at different fiber development periods, sequence information and functional annotation information of genes, a candidate gene GbKAP was found, which was higher than TM-1 at 5-15 days of fiber development period, and the expression amount was the highest at 10 days of fiber development period (a in Figure 2 ). Sequence analysis showed that there was one non-synonymous SNP mutation between the two parents (b in Figure 2 ). Subcellular localization results showed that GbKAP was a membrane-located protein (c in Figure 3 ). Based on the expression and gene sequence results, it was indicated that GbKAP was a candidate gene of fiber uniformity QTL (qFU-D04), which played an important role in cotton fiber development.

[0074] Example 2 Analysis of phenotypic differences of different haplotypes based on substitution mapping

[0075] Using the method of t-test in statistics, we calculated the phenotypic differences of fiber quality traits between the two groups of haplotypes. The fiber length, fiber strength and fiber uniformity of haplotype 1 (L240 type) were significantly better than those of haplotype 2 (TM-1 type) (Table 1). F 2:4 The fiber uniformity and fiber length of haplotype 1 (G1-G6) were significantly better than those of haplotype 2 (G7-G8) (c in Figure 1 ).

[0076] Example 3 Expression level analysis of GbKAP at different fiber development periods of cotton

[0077] The fiber tissues of L240 and TM-1 at the fiber development stage of 5-25 DPA were collected for transcriptome sequencing to quantitatively detect the expression of GbKAP homologous genes. The reads obtained by sequencing were aligned with the TM-1 genome, and the calculated expression level was expressed as the average expression level of monomer genes in one million transcripts (TPM). The gene-specific quantitative primers were designed by Beacon Designer 7, and the quantitative detection was completed by AB7500 Real-Time PCR. The CT values of the internal reference primers and quantitative primers were read, and the relative expression = 2 -△CT The relative expression data of the genes were calculated for significant difference analysis. The experimental results are shown in Table 1. Figure 2 The expression of the gene in the fiber development stage of 5-15 days of L240 was higher than that of TM-1, and the expression in the fiber of 10 days was the highest, indicating that the gene had more obvious effect on the fiber elongation period (a in Table 1). Figure 2

[0078] Example 4 Subcellular localization analysis

[0079] In order to verify the subcellular localization of GbKAP, we constructed the pBin-eGFP4 vector of the gene to be detected, which fused the target gene and eGFP protein under the control of the 35S promoter of CaMV (35S-target gene-eGFP-NOS) (Liu et al. 2014). The 35S-GbKAP-eGFP-NOS vector was injected into the tobacco leaves of 5 weeks old by Agrobacterium. Three days after injection, the fluorescence signal was detected and photographed under the Zeiss LSM780 confocal microscope. The subcellular localization results showed that GbKAP was located in the cell membrane (a in Table 1). In order to further verify the localization results, we treated the tobacco leaf cells with 5% (5g / 100ml) NaCl solution for 10 min, and observed that the green fluorescence of GbKAP still overlapped with the red signal of AtPIP2A protein. Figure 3

[0080] Example 5 Phenotype identification of transgenic Arabidopsis thaliana with ectopic expression of GbKAP gene

[0081] ​​To further elucidate the function of GbKAP gene in plant growth and development, we selected PBI121 vector driven by the 35S promoter of cauliflower mosaic virus (CaMV), and developed recombinant primers (forward and reverse primer sequences are SEQ ID NO. 3 and SEQ ID NO. 4, respectively) targeting BamH1 and Sac1 enzyme cleavage sites. After cutting the PBI121 vector with BamH1 and Sac1 endonucleases, the CDS sequence of the target gene GbKAP was fused using homologous recombination to construct the expression vector. Further phenotypic investigation was carried out on three high-expression transgenic lines (OE-4, OE-5, and OE-6) in a-b) of Figure 4 . Surface-sterilized seeds were sown in rows on 1 / 2MS medium. The test was placed vertically in the growth chamber under long-day and dark conditions (24 / 22°C, 16h light / 8h dark); after 7 days, the root cell length of wild type (WT) and transgenic lines was observed under a Zeiss LSM780 confocal microscope, and the root cell length was measured using ImageJ software. The root cells of overexpression transgenic lines were longer (c-d) of Figure 4 . The main root length of wild type (WT) and transgenic lines was measured, and overexpression transgenic lines showed longer main root length after 7 days of light culture (e-f) of Figure 4 . The hypocotyl length of wild type (WT) and transgenic lines was measured, and overexpression transgenic lines showed longer hypocotyl length after 7 days of dark culture (g-h) of Figure 4 . In addition, surface-sterilized seeds were randomly sown on 1 / 2MS medium and placed horizontally in the growth chamber under long-day conditions (24 / 22°C, 16h light / 8h dark); after 10 days, wild type (WT) and transgenic lines were transferred to plastic cups filled with vermiculite (three plants per cup, and two plates for each clone), and placed in short-day conditions (24 / 22°C, 8h light / 16h dark) for growth; the rosette leaf growth of wild type (WT) and transgenic lines was observed, and the rosette leaves of transgenic lines were more vigorous (i) of Figure 4 . When wild type (WT) and transgenic lines bolted, the main stem height of wild type (WT) and transgenic lines was measured, and the main stem of transgenic lines was higher (j-k) of Figure 4 . The experimental results showed that the root cells of transgenic Arabidopsis were longer, the roots were longer, the hypocotyls were longer, the rosette leaves were more vigorous, and the main stems were higher. This indicates that overexpression of GbKAP significantly promotes cell elongation and plant growth in Arabidopsis.

[0082] In conclusion, we identified a stable QTL (qFU-D04) for fiber uniformity in L240, a superior fiber quality landrace introgression line, by linkage analysis and substitution mapping. We found a keratin-associated protein gene GbKAP as the candidate gene for this QTL. The expression of GbKAP was high in 5-10 day fiber and the highest in 10 day fiber in L240. The CDS sequence analysis of GbKAP and its homologous genes between parents showed that there was a non-synonymous SNP mutation in this gene. The Arabidopsis plants heterologously expressing GbKAP had longer root length and higher plant height than the control plants. GbKAP has important role and application prospect in improving cotton fiber quality traits and breeding new cotton varieties with superior fiber.

[0083] SEQ ID NO. 1 (CDS sequence of GbKAP (GB_D04G0512) 567

[0085] Gossypium barbadense

[0086] ATGGCTGTGACCATCAAGCGAGCGGCCCTTATCGTTGCCGGTCTTGGCGTCTTATCATTCATATTGGGAGTCATAGCCGAAAACAAAAAGCCAGAAGCTGGAACCCCAATTCCTGGTAAAGGTGTGGTTATATGCAAGTATCCAAGGGATCCCTCCCTGGCCCTGGGCTACCTATCGGTTGCATTTCTGATGCTATCGACTATCGCTGGCTATTGGTCTCTGTTTTACCCTTACAAAGGGAAATCTGTTCCACACTCTGTCCTCTTTCAAAGCGCCAGTTTCTTCGTCTTCTTCAACATTGCCTTGTTTACAAGTGGGGTAGCAGCCACACTGCTGTTGTGGCCAACGATCACGGAGCACCTTCACTTGATTCGCAATGTTCATCACAATCCCACCACCACATGCCCTACTGCTAAGACTGGACTCCTTGGTGGTGGTGCCTTTGTATCCCTGGATTCAGCCCTCTTCTGGTTAGTTGCCCTTATGTTGGCTGATAATGCCCGACATGATCACTTTTATGATGTTGAAAAACACAGCAAAGCTCAGGTTCTCCCAGATGGCTGCTGA

[0087] SEQ ID NO. 2 (amino acid sequence of GbKAP (GB_D04G0512) 188

[0089] Gossypium barbadense

[0090] MAVTIKRAALIVAGLGVLSFILGVIAENKKPEAGTPIPGKGVVICKYPRDPSLALGYLSVAFLMLSTIAGYWSLFYPYKGKSVPHSVLFQSASFFVFFNIALFTSGVAATLLLWPTITEHLHLIRNVHHNPTTTCPTAKTGLLGGGAFVSLDSALFWLVALMLADNARHDHFYDVEKHSKAQVLPDGC

[0091] SEQ ID NO. 3 (forward primer sequence of GbKAP PBI121 overexpression vector) 41

[0093] ACGGGGGACTCTAGAGGATCCATGGCTGTGACCATCAAGCG

[0094] SEQ ID NO. 4 (reverse primer sequence of GbKAP PBI121 overexpression vector) 41

[0096] CGATCGGGGAAATTCGAGCTCTCAGCAGCCATCTGGGAGAA.

Claims

1. The application of the GbKAP gene or biological materials related to the GbKAP gene in the following (a1) or (a2) or (a3): (a1) Improve the quality traits of cotton fibers, or prepare products for improving the quality traits of cotton fibers; (a2) Breeding new cotton germplasm for fiber quality improvement, or preparing products for breeding new cotton germplasm for fiber quality improvement; (a3) or promote the growth of the target plant; The GbKAP gene is as follows (b1) or (b2) or (b3): (b1) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO.1; (b2) DNA molecules derived from cotton that are more than 95% identical to (b1) and encode the same protein; (b3) A DNA molecule that hybridizes or complements the DNA sequence defined in (b1) under strict conditions and encodes the same protein.

2. The application according to claim 1, characterized in that, The biological material associated with the GbKAP gene is at least one of the following (c1)-(c11): (c1) The protein GbKAP encoded by the GbKAP gene; (c2) Primer pair used to clone the GbKAP gene; (c3) An expression cassette containing the GbKAP gene; (c4) A recombinant vector containing the GbKAP gene, or a recombinant vector containing the expression cassette (c3); (c5) A recombinant microorganism containing the GbKAP gene, or a recombinant microorganism containing the expression cassette of (c3), or a recombinant microorganism containing the recombinant vector of (c4); (c6) A transgenic plant cell line containing the GbKAP gene, or a transgenic plant cell line containing the expression cassette of (c3), or a transgenic plant cell line containing the recombinant vector of (c4); (c7) Transgenic plant tissue containing the GbKAP gene, or transgenic plant tissue containing the expression cassette of (c3), or transgenic plant tissue containing the recombinant vector of (c4); (c8) A transgenic plant organ containing the GbKAP gene, or a transgenic plant organ containing the expression cassette (c3), or a transgenic plant organ containing the recombinant vector (c4); (c9) A transgenic plant containing the GbKAP gene, or a transgenic plant containing the expression cassette (c3), or a transgenic plant containing the recombinant vector (c4); (c10) Tissue cultures produced from regenerative cells of the transgenic plant described in (c9); (c11) Protoplasts produced from the tissue culture described in (c10).

3. The application according to claim 2, characterized in that, The protein GbKAP described in (c1) is either (d1) or (d2) or (d3) or (d4): (d1) A protein with the amino acid sequence shown in SEQ ID NO.2; (d2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (d1); (d3) Proteins related to cotton fiber quality traits and / or plant growth obtained by substituting and / or deleting and / or adding one or more amino acid residues into the amino acid sequence shown in SEQ ID NO.

2. (d4) is a protein derived from cotton and has more than 98% identity with (d1) and is associated with cotton fiber quality traits and / or plant growth.

4. The application according to claim 2, characterized in that, The nucleotide sequences of the upstream and downstream primers of the primer pair described in (c2) are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

5. The application according to claim 1, characterized in that, Using the GbKAP gene as the target gene, the GbKAP gene is overexpressed through genetic engineering methods to improve cotton fiber quality traits or cultivate new cotton germplasm with improved fiber quality, or to promote the growth of target plants.

6. The application according to claim 1, 2, 3 or 5, characterized in that, The plant in question is cotton or Arabidopsis thaliana.

7. The application according to claim 1, 3, or 5, characterized in that, The cotton fiber quality characteristics mentioned are at least one of fiber length, fiber strength, and fiber uniformity.

8. A method for improving the quality of cotton fibers, characterized in that, The GbKAP gene, with a nucleotide sequence as shown in SEQ ID NO.1, is overexpressed in cotton.

9. A GbKAP gene associated with cotton fiber quality traits, having a nucleotide sequence as shown in SEQ ID NO.

1.

10. A biomaterial associated with the GbKAP gene of claim 1, characterized in that, The biomaterial is at least one of the following (e1)-(e5): (e1) The amino acid sequence encoded by the GbKAP gene is shown in SEQ ID NO.2 for the protein GbKAP; (e2) An expression cassette containing the GbKAP gene; (e3) A recombinant vector containing the GbKAP gene, or a recombinant vector containing the expression cassette (e2); (e4) A recombinant microorganism containing the GbKAP gene, or a recombinant microorganism containing the expression cassette (e2), or a recombinant microorganism containing the recombinant vector (e3); (e5) A transgenic plant cell line containing the GbKAP gene, or a transgenic plant cell line containing the expression cassette (e2), or a transgenic plant cell line containing the recombinant vector (e3).

Images