Application of cellulose synthase-like gene GhCSLC4 in improvement of cotton yield traits

Abstract

The invention discloses application of a cellulose synthase-like gene GhCSLC4 in improvement of cotton yield traits, and belongs to the field of biotechnology application. According to the invention, a biotechnology is utilized to determine that the GhCSLC4 gene has a promotion effect on fiber cell elongation. Researches show that the silent cotton cellulose synthase gene GhCSLC4 inhibits fiber initiation and elongation, and the root length and the number of stem hairs of an arabidopsis plant with heterologous expression of GhCSLC4 are both higher than those of a wild control. The GhCSLC4 gene has important effects and application prospects in improvement of cotton fiber yield traits and cultivation of new high-yield cotton varieties.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology application, and relates to the application of a cellulose synthase-like gene GhCSLC4 in improving cotton yield traits. The present invention screens out a cellulose synthase-like gene derived from upland cotton (Gossypium hirsutum) and names it GhCSLC4. The gene ORF has a full length of 1989 bp and encodes 662 amino acids. A secondary separation population is constructed by hybridizing a low-lint-percentage marine-land introgression material L283 with a high-lint-percentage receptor parent TM-1. Combined with fine positioning, displacement mapping, gene structure and expression analysis, a cellulose synthase-like gene GhCSLC4 is identified in the chromosome introgression fragment region. The expression level of the gene in different fiber development stages of TM-1 is higher than that of L283, and the expression level in 10-day fiber is the highest. Inhibiting the expression of GhCSLC4 in cotton results in fewer fiber cell protrusions, shorter fiber length, and fewer mature fiber spirals. Heterologous expression of GhCSLC4 in Arabidopsis thaliana resulted in longer root length and root cells, more vigorous growth of rosette leaves, more stem hairs, and taller main stems, promoting plant growth. The present invention uses biotechnology to clarify that GhCSLC4 has a promoting effect on plant cell elongation, and proposes that overexpression of GhCSLC4 can promote fiber cell elongation and increase cotton fiber yield breeding value. Background Art

[0002] Upland cotton (Gossypium hirsutum) and Sea island cotton (Gossypium barbadense) are two allotetraploid cultivated cotton species. Upland cotton accounts for more than 90% of the global cotton planting area and has the characteristics of high fiber yield and wide adaptability. Sea island cotton 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 upland cotton and the negative correlation between fiber quality and yield. By constructing cotton chromosome segment introgression lines (CSILs), we can further fine-tune the excellent loci related to yield or quality, break the negative correlation between quality and yield, and obtain new high-yield and high-quality cotton varieties.

[0003] Chromosome segment introgression lines are also called single segment introduction lines (SSIL), single segment substitution lines (SSSL), near isogenic introduction lines (NIL), chromosome replacement lines (CSSL), etc. They are mainly constructed through backcrossing and selfing, combined with molecular marker-assisted selection (MAS), and are a set of families with only one or a few specific donor chromosome segments, and the rest of the genetic background is exactly the same as the recipient parent. The main characteristics of this type of family are: the genome of each introgression line individual contains only one or a few chromosome segments from the donor parent, and the rest are the same as the recurrent parent; the introgression line population is a permanent separation population, which can be used for multi-point phenotypic identification for many years; the target segment can be divided into shorter segments after backcrossing and recombination. In addition, the use of chromosome segment introgression lines can minimize the influence of genetic background between strains when investigating target traits and studying gene effects, so this population is of great significance for the location of QTL.

[0004] At the same time, the use of chromosome fragment introgression lines is also an ideal material for QTL fine positioning. There are generally two methods: the first is to use the overlapping groups in the introgression line to finely locate the QTL through the permutation mapping method; the second method is to construct a secondary segregation population of QTL for QTL fine positioning. Xiao et al. (2023) used a single segment substitution line (CSSL-106) and its recurrent parent CCRI45 to backcross and construct a secondary segregation population of 2852 strains. The fiber length-related QTLqFL-A12-5 was finely located in the 18.8Kb region on chromosome A12, and GH_A12G2192 (GhTPR), which encodes a tetratripeptide repeat superfamily protein, was identified as a candidate gene for regulating cotton fiber development. Feng et al. (2022) selected the genetically stable introgression line IL197 of Upland cotton × Asian cotton with small bolls and small cotton seeds, constructed F2 and F3 secondary segregation populations, and further narrowed the QTL q(BW+SI)-A12-1 on chromosome A12 that simultaneously controls boll weight (BW) and seed index (SI) to 60.09 kb through substitution mapping. RT-qPCR analysis preliminarily identified TIP41-like family protein (TIP41L) as a candidate gene.

[0005] Plant cell walls contain three types of polysaccharides: cellulose, hemicellulose, and pectin. The enzymes that synthesize polysaccharides play an important role in plant growth, development, and stress response. Cellulose synthase-like (CSL) has a similar protein structure to cellulose synthase and participates in hemicellulose biosynthesis. It is a key enzyme in the biosynthesis of hemicellulose and affects the quality of plant cellulose to a certain extent. Based on the different sequence and structural characteristics, CSL proteins are divided into 9 subfamilies, namely CSLA-CSLH and CSLJ. Among them, CSLA is mainly responsible for the synthesis of mannan, which is very important for pollen tube growth and embryonic development in Arabidopsis; CSLC is involved in the synthesis of xyloglucan (XyG) in root hairs, and the length of mutant root hairs is significantly shortened. In addition, it also plays an important role in restricting cell wall extension and cell elongation during the root ethylene response; CSLD is involved in the biosynthesis of β-glucan-containing polysaccharides required for root hair elongation, plays a role in plant root hair growth and regulates plant tolerance to osmotic stress; CSLF genes have been shown to be involved in the synthesis of new (1,4)-β-linked xylan, playing a key role in root development (Goubet et al. 2003; Gajek et al. 2021; Favery et al. 2001; Zhu et al. 2010; Lou et al. 2022). Therefore, studying the mechanism of action of cellulose synthase-like genes in the fiber development process is beneficial to improving fiber yield and quality at the molecular level. Summary of the invention

[0006] The purpose of the present invention is to provide application of cellulose synthase-like gene GhCSLC4 in improving cotton yield traits.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] In the first aspect, the present invention claims the use of the GhCSLC4 gene as shown in SEQ ID NO.1 or a biological material related to the GhCSLC4 gene in the following (a1) or (a2) or (a3) ​​or (a4):

[0009] (a1) Improving cotton yield traits, or preparing products for improving cotton yield traits;

[0010] (a2) increasing cotton yield or preparing products for increasing cotton yield;

[0011] (a3) cultivating new high-yield cotton germplasm, or preparing products for cultivating new high-yield cotton germplasm;

[0012] (a4) Promote plant growth.

[0013] Furthermore, the biological material related to the GhCSLC4 gene is the biological material described in (b1) or (b2) or (b3) below:

[0014] (b1) protein GhCSLC4 encoded by the GhCSLC4 gene;

[0015] (b2) primers for amplifying the full-length coding sequence of the GhCSLC4 gene;

[0016] (b3) Biological material containing the GhCSLC4 gene.

[0017] Furthermore, the protein encoded by the GhCSLC4 gene in (b1) has an amino acid sequence as shown in SEQ ID NO.2.

[0018] Furthermore, the primers used to amplify the full-length coding sequence of the GhCSLC4 gene in (b2) are shown as SEQ ID NO.5 and SEQ ID NO.6.

[0019] Furthermore, the biological material containing the GhCSLC4 gene described in (b3) is as follows (c1) to (c9):

[0020] At least one of:

[0021] (c1) an expression cassette containing the GhCSLC4 gene;

[0022] (c2) a recombinant vector containing the GhCSLC4 gene, or a recombinant vector containing the expression cassette of (c1);

[0023] (c3) a recombinant microorganism containing the GhCSLC4 gene, or a recombinant microorganism containing the expression cassette described in (c1), or a recombinant microorganism containing the recombinant vector described in (c2);

[0024] (c4) a transgenic plant cell line containing the GhCSLC4 gene, or a transgenic plant cell line containing the expression cassette described in (c1), or a transgenic plant cell line containing the recombinant vector described in (c2);

[0025] (c5) transgenic plant tissue containing the GhCSLC4 gene, or transgenic plant tissue containing the expression cassette described in (c1), or transgenic plant tissue containing the recombinant vector described in (c2);

[0026] (c6) a transgenic plant organ containing the GhCSLC4 gene, or a transgenic plant organ containing the expression cassette described in (c1), or a transgenic plant organ containing the recombinant vector described in (c2);

[0027] (c7) a transgenic plant containing the GhCSLC4 gene, or a transgenic plant containing the expression cassette described in (c1), or a transgenic plant containing the recombinant vector described in (c2);

[0028] (c8) tissue cultures produced from regenerable cells of the transgenic plant described in (c7);

[0029] (c9) Protoplasts produced from the tissue culture described in (c8).

[0030] Furthermore, the above application is to use the GhCSLC4 gene as a target gene, over-express the GhCSLC4 gene through genetic engineering methods, improve cotton yield traits, increase cotton yield, cultivate new high-yield cotton germplasm or promote the growth of target plants.

[0031] Furthermore, the plant is cotton or Arabidopsis thaliana.

[0032] In a second aspect, the present invention claims a method for improving cotton yield traits or increasing cotton yield, which improves cotton yield traits or increases cotton yield by overexpressing the GhCSLC4 gene with a nucleotide sequence such as that shown in SEQ ID NO.1 in cotton.

[0033] In a third aspect, the present invention claims protection for a GhCSLC4 gene related to cotton yield traits, wherein the gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0034] In a fourth aspect, the present invention claims protection for a biological material related to the above-mentioned GhCSLC4 gene, wherein the biological material is the biological material described in (d1) or (d2) or (d3) below:

[0035] (d1) a protein GhCSLC4 encoded by the GhCSLC4 gene having an amino acid sequence as shown in SEQ ID NO.2;

[0036] (d2) primers as shown in SEQ ID NO.5 and SEQ ID NO.6 for amplifying the full-length coding sequence of the GhCSLC4 gene;

[0037] (d3) An expression cassette, recombinant vector, transgenic cell line or recombinant bacteria containing the GhCSLC4 gene.

[0038] The present invention shows that when the cellulose synthase-like gene GhCSLC4 with a nucleotide sequence as shown in SEQ ID NO.1 is silenced in the introgression line L283, compared with the control plants, the silenced plants show fewer protrusions in the 0-day ovule fiber cells, shorter fiber length, fewer mature fiber spirals, and inhibited fiber initiation and elongation; when the cellulose synthase-like gene GhCSLC4 with a nucleotide sequence as shown in SEQ ID NO.1 is overexpressed in Arabidopsis, compared with the wild type (WT), the overexpressing Arabidopsis strain has longer roots, longer root cells, more vigorous rosette leaf growth, more stem hairs, and a taller main stem, thereby promoting plant growth.

[0039] The present invention uses a low lint content marine-terrestrial introgression material L283 to cross with a high lint content recipient parent TM-1 to construct a secondary segregation population, combines the resequencing data of L283, obtains a precise introgression segment, develops InDel markers for the insertion / deletion sites on the introgression segment, conducts linkage analysis on the yield trait phenotypic values ​​and genotypes of the secondary segregation population, identifies a lint content QTL qLP-D09, and further uses the recombinants in the candidate segment for replacement mapping to obtain the candidate interval of the QTL. Combined with the differential expression characteristics of genes in the candidate interval at different fiber development stages of L283 and TM-1, a cellulose synthase-like gene GhCSLC4 was discovered, ID number GH_D09G0294. The expression level of this gene in TM-1 at different fiber development stages is higher than that of L283, and the expression level is highest in 10-day fiber. Compared with L283, the parent TM-1 has advantages in lint content and lint index. In the parent TM-1, the cellulose synthase-like gene GhCSLC4 with a nucleotide sequence as shown in SEQ ID NO.1 was silenced. Compared with the control plants, the silenced plants showed fewer protrusions of 0-day ovule fiber cells, shorter fiber length, and significantly inhibited fiber initiation and elongation; in Arabidopsis, the cellulose synthase-like gene GhCSLC4 with a nucleotide sequence as shown in SEQ ID NO.1 was overexpressed. Compared with the wild type (WT), the overexpressing Arabidopsis strains had longer roots, longer root cells, more vigorous rosette leaves, more stem hairs, and taller main stems, which promoted plant growth. The above results indicate that GhCSLC4 plays a role in promoting cotton fiber development and plant growth and development.

[0040] The development process of the technical solution of the present invention specifically includes the following steps:

[0041] 1. A low lint percentage marine-land introgression material L283 was hybridized with a high lint percentage upland cotton TM-1 to construct an F2 secondary segregation population (L283×TM-1). Individual plants of the F2 population were planted in rows to obtain F 2:3 The yield traits (boll weight, seed index, lint percentage, lint index) of the secondary segregating population were investigated to obtain phenotypic data.

[0042] 2. Combined with the whole genome resequencing information of L283, InDel markers were developed for the insertion / deletion sites on the introgressed fragments, and polymorphic InDel primers were screened. The polymorphic InDel primers were used to identify the genotypes of the secondary segregation population and obtain genotyping data.

[0043] 3. Use F 2：3 QTL mapping was performed using the yield-related data of the lines and the InDel marker genotype data of the F2 plants, and a QTL for lint percentage, qLP-D09, was detected. Combined with substitution mapping, the candidate interval of the QTL was identified.

[0044] 4. Combining the expression of genes in the candidate interval at different fiber development stages between parents, a cellulose synthase-like gene GhCSLC4 was obtained as a candidate gene for the lint content QTL. Its expression in different fiber development stages of TM-1 was higher than that of L283, and the expression level was highest in 10-day fiber. The CDS sequence of this gene is SEQ ID NO.1, and the encoded amino acid sequence is SEQ ID NO.2. Sequence analysis between parents showed that this gene had two non-synonymous SNP mutations in TM-1 and L283.

[0045] 5. The present invention also constructed a GhCSLC4 silenced strain. Compared with the control plant, the strain silencing the cotton GhCSLC4 gene as shown in SEQ ID NO.1 had fewer protrusions in the 0-day ovule fiber cells, shorter fiber length, and fewer mature fiber spirals, indicating that silencing GhCSLC4 significantly inhibited fiber initiation and elongation.

[0046] 6. The present invention also constructed a GhCSLC4 overexpressing Arabidopsis transgenic strain. Compared with the wild type WT, the Arabidopsis strain heterologously expressing the cotton GhCSLC4 gene as shown in SEQ ID NO.1 had significantly longer roots, longer root cells, more vigorous rosette leaves, more stem hairs, and a taller main stem. This indicates that overexpression of GhCSLC4 significantly promotes cell elongation in Arabidopsis plants.

[0047] The advantages of the present invention are:

[0048] 1. The present invention uses linkage analysis and displacement mapping to identify a QTL (qLP-D09) related to lint content in the introgression fragment interval in a secondary segregation population constructed from the high lint content TM-1 and the low lint content marine-terrestrial introgression material L283. Combined with the expression characteristics of genes in the candidate interval at different fiber development stages between the two parents L283 and TM-1, a cellulose synthase-like gene CSLC4 was preliminarily identified as the candidate gene of the QTL.

[0049] 2. According to the different haplotypes of cellulose synthase-like gene CSLC4, the recombinant population can be divided into two types, namely type 1 (G1 and G4) and type 2 (G2 and G3). Through statistical analysis, it was found that there were significant differences in the lint content of individual plants corresponding to different haplotypes.

[0050] 3. The expression level of cellulose synthase-like gene CSLC4 in different fiber development stages between the two parents was obtained through transcriptome data and further verified by quantitative analysis. The results showed that the expression level of this gene in TM-1 was higher than that in L283 at different fiber development stages, and the expression level was highest in 10-day fiber, followed by 5-day fiber, and low expression in 15-20-day fiber. This indicates that this gene plays a role in fiber initiation and elongation.

[0051] 4. Compared with L283, TM-1 has advantages in lint percentage and lint index; 2：4 In the recombinant population, different recombinant types have advantages in lint percentage, indicating that this type of cellulose synthase gene GhCSLC4 has significant advantages in improving yield traits and can be used to improve cotton yield traits and cultivate new high-yield cotton varieties.

[0052] 5. Compared with the control plants, the lines in which the cotton GhCSLC4 gene shown in SEQ ID NO.1 was silenced had fewer protrusions in the 0-day ovule fiber cells, shorter fiber length, and fewer mature fiber spirals, indicating that silencing GhCSLC4 significantly inhibited fiber initiation and elongation.

[0053] 6. Compared with the wild type (WT), the Arabidopsis strain overexpressing the cotton GhCSLC4 gene as shown in SEQ ID NO.1 has significantly longer roots, longer root cells, more vigorous rosette leaves, more stem hairs, and taller main stems, indicating that overexpression of the GhCSLC4 gene promotes cell elongation and enhances plant growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 .Genetic positioning of the lint content QTL locus qLP-D09.

[0055] a Lint content QTL locus qLP-D09 in (L283×TM-1)F 2:3 The solid black line represents the initial location of the QTL in the F 2:3 LOD curve of the preliminary population positioning, LOD>2.5. b Genetic linkage map of the D09 introgression fragment. c qLP-D09 substitution mapping and comparison of lint percentage between different introgression fragment materials and TM-1. ** indicates P<0.01, * indicates <0.05. d Comparison of lint percentage phenotypes between L283 and TM-1 of the Sea Island cotton Hai7124 introgression line in the Upland cotton TM-1 background.

[0056] Figure 2 .Expression patterns of cellulose synthase-like genes at different fiber development stages in L283 and TM-1.

[0057] The ordinate represents the expression level of the gene (the left ordinate and histogram represent the TPM value of the cellulose synthase-like gene in the transcriptome data; the right ordinate and line graph represent the RT-qPCR analysis results of the cellulose synthase-like gene); the abscissa represents the fiber tissue of fiber development 5-20DPA.

[0058] Figure 3 Subcellular localization of GhCSLC4

[0059] AtPIP2 is a plasma membrane marker. GhCSLC4 co-localizes with AtPIP2 in tobacco cells.

[0060] Figure 4 . Fiber phenotype analysis of plants with silencing of the GhCSLC4 gene.

[0061] a Analysis of the relative expression level of GhCSLC4 in CLCrV-GhCSLC4 and CLCrV-A plants. b Comparison of fiber length at different developmental stages between CLCrV-GhCSLC4 and CLCrV-A plants. Scale bar is 1 cm. c Comparative analysis of fiber length at different developmental stages. d Comparison of bulges in ovule fiber cells at day 0 and comparison of mature fiber spirals between CLCrV-GhCSLC4 and CLCrV-A plants. Scale bars are 50 μm and 100 μm, respectively. ** indicates P < 0.01, * indicates < 0.05.

[0062] Figure 5 . Phenotypic analysis of transgenic Arabidopsis thaliana overexpressing the GhCSLC4 gene.

[0063] a Identification of positive clones of transgenic Arabidopsis overexpressing the GhCSLC4 gene and analysis of GhCSLC4 expression. b Phenotypic observation of rosette leaf growth of GhCSLC4 transgenic lines and WT. c Phenotypic observation of root cells of GhCSLC4 transgenic lines and WT. Scale bar is 50 μm. d Comparative analysis of root cell length between transgenic Arabidopsis and WT. e Phenotypic observation of root length of GhCSLC4 transgenic lines and WT. Scale bar is 1 cm. f Comparative analysis of root length between transgenic Arabidopsis and WT. g Observation of stem epidermal hairs of GhCSLC4 transgenic lines and WT. Scale bar is 2.5 mm. h Comparison of the number of stem epidermal hairs. i Phenotypic observation of main stem height of GhCSLC4 transgenic lines and WT. j Comparative analysis of main stem height of GhCSLC4 transgenic lines and WT. ** indicates P < 0.01, * indicates < 0.05. DETAILED DESCRIPTION

[0064] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0065] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0066] Example 1 Identification of lint QTL qLP-D09 by fine mapping and displacement mapping and discovery of its candidate genes

[0067] Through the multi-year yield trait identification of a set of sea island cotton Hai7124 introgression lines with a background of upland cotton TM-1, we found a sea island introgression line material L283 with extremely low lint percentage, whose lint percentage and lint index were significantly lower than those of the receptor TM-1. Figure 1 d, Table 1). By combining with TM-1 (L283×TM-1), the F2 secondary segregation population was derived, and the F 2:3 The strain population was used to identify the yield-related loci and candidate gene mining contained in the introgression fragment of L283. Combined with the whole genome resequencing information of L283, InDel markers were developed for the insertion / deletion sites on the introgression fragment, and polymorphic InDel primers were screened. The polymorphic InDel primers were used to identify the genotypes of the F2 plants in the secondary segregation population and obtain genotyping data. 2:3 The four yield traits of the strain population were investigated by phenotypic investigation. 2:3 The yield phenotypic data of the strain population and the InDel marker genotype data of the F2 individual plants were used for QTL mapping, and a lint percentage (LP) QTL (qLP-D09) was detected ( Figure 1 a). Further, F 2:3 The 16 recombinants detected in the experiment were planted and F 2:4 The 16 recombinant strains of the population were identified and genotyped and yield traits investigated, and permutation mapping was performed. The 16 recombinants were divided into 4 groups. The lint percentage of the G2-G4 group was significantly lower than that of TM-1, but not significantly different from that of L283. The lint percentage of the G1 and G5 groups was significantly higher than that of L283, but not significantly different from that of TM-1. qLP-D09 was finely located between D09-7.36 and D09-7.77, with a size of 70 kb ( Figure 1 bc).

[0068] Table 1 Comparison of phenotypic values ​​of parents

[0069]

[0070] Based on the transcriptome data of TM-1 and L283 parents at different fiber development stages, combined with the sequence information of genes in the localization interval and functional annotation information, a cellulose synthase-like gene CSLC4 was discovered. The expression level of this gene in TM-1 at different fiber development stages was higher than that in L283, and the expression level was highest in 10-day fiber ( Figure 2 ). Sequence analysis between the two parents showed that the gene had two non-synonymous SNP mutations between the two parents. Subcellular localization results showed that it is a membrane-localized protein ( Figure 3 ). It was speculated that the cellulose synthase-like gene CSLC4 was the candidate gene for the lint percentage QTL (qLP-D09), which plays an important role in the formation of cotton fiber yield.

[0071] Example 2 Phenotypic Difference Analysis of Cellulose Synthase Gene CSLC4

[0072] The phenotypic differences in lint percentage between the parents were calculated using the t-test method in statistics. The lint percentage and lint index of parent L283 were significantly lower than those of TM-1 (Table 1). 2:4 The lint percentage of group type 2 (G2-G4) was significantly lower than that of group type 1 (G1 and G5) ( Figure 1 c).

[0073] Example 3 Analysis of the expression level of GhCSLC4 at different fiber development stages in cotton

[0074] Fiber tissues of the parent L283 and TM-1 during the fiber development period of 5-20 DPA were collected for transcriptome sequencing and quantitative detection of the expression of the cellulose synthase-like gene CSLC4. The reads obtained by sequencing were compared with the TM-1 genome, and the expression level was calculated as the average expression level of monomeric genes per million transcripts TPM. Gene-specific quantitative primers were designed by BeaconDesigner 7, and quantitative detection was completed by AB7500 Real-Time PCR. The CT values ​​of the internal reference primer and the quantitative primer were read, and the relative expression was calculated using the relative expression level = Calculate the relative expression data of genes and perform difference significance analysis. Figure 2 As shown, the expression level of this gene in TM-1 was higher than that in L283 at different fiber development stages, and the highest expression level was in 10-day fibers, followed by 5-day fibers, and low expression in 15-20-day fibers. This indicates that this gene plays a role in fiber initiation and elongation ( Figure 2 ).

[0075] Example 4 GhCSLC4 subcellular localization analysis

[0076] To verify the subcellular localization of GhCSLC4, we constructed the pBin-eGFP4 vector of the gene to be tested, which fused the target gene and eGFP protein (35S-targetgene-eGFP-NOS) under the control of the 35S promoter of cauliflower mosaic virus (CaMV) (Liu et al. 2014). The 35S-GhCSLC4-eGFP-NOS vector was injected into 5-week-old tobacco leaves by Agrobacterium. Three days after injection, the fluorescence signal was detected and photographed under a Zeiss LSM780 confocal microscope. The subcellular localization results showed that GhCSLC4 was localized on the cell membrane ( Figure 3 ).

[0077] Example 5 Phenotypic Identification of Plants with Silencing GhCSLC4 Gene

[0078] To further clarify the role of GhCSLC4 gene in cotton fiber development, we selected cotton leaf crinkle geminivirus vector CLCrV, selected SpeI and AscI restriction endonuclease sites, cloned 400bp of the GhCSLC4 coding sequence into pCLCrVA vector (pCLCrV-GhCSLC4), and the forward and reverse primer sequences were SEQ ID NO.3 and SEQID NO.4, respectively. pCLCrVB was used as an auxiliary vector, and pCLCrV-Su and pCLCrVA were mixed with pCLCrVB at a ratio of 1:1 and then infiltrated into the cotyledons of TM-1 plants, respectively as positive and negative controls; pCLCrV-GhCSLC4 was infiltrated in the same way as the experimental group. When yellow spots appeared on the pCLCrV-Su positive control, 20 cotton plants inoculated with each combination were moved to large plastic pots and placed in a greenhouse (24 / 22℃, 16h light / 8h dark) until flowering. A sign with the date was hung on each flower, and cotton bolls at different developmental stages were subsequently sampled and phenotypes observed according to the date.

[0079] We collected fibers at 10 and 15 days to test the silencing efficiency of GhCSLC4. The expression level of GhCSLC4 in pCLCrV-GhCSLC4 plants was significantly reduced, indicating that the silencing effect was good ( Figure 4 a). Fibers were collected at 15 days, 20 days and maturity to measure fiber length. Fibers at 15 days, 20 days and maturity of pCLCrV-GhCSLC4 plants were significantly shorter ( Figure 4 bc); we also used scanning electron microscopy to observe the protrusions of the 0-day ovule fiber cells and the spiral of the mature fiber. The results showed that the ovule protrusions on the 0-day ovule surface of the pCLCrV-GhCSLC4 plant were significantly reduced, and the spiral of the mature fiber was reduced ( Figure 4 d).

[0080] Example 6 Phenotypic Identification of Transgenic Arabidopsis thaliana Ectopically Overexpressing GhCSLC4 Gene

[0081] In order to further clarify the function of GhCSLC4 gene in plant growth and development, we selected PBI121 vector driven by 35S promoter of cauliflower mosaic virus (CaMV), developed recombination primers for BamHI and SacI restriction sites, and the forward and reverse primer sequences were SEQ ID NO.5 and SEQ ID NO.6, respectively. After cutting PBI121 vector with BamHI and SacI endonucleases, the CDS sequence of target gene GhCSLC4 was fused by homologous recombination to construct expression vector. Further, the three high expression transgenic lines (OE-1, OE-2, OE-3) ( Figure 5 a) Conduct phenotypic investigation. Surface sterilized seeds were sown in rows on 1 / 2MS medium. The experiment was placed vertically in a growth chamber under long-day conditions (24 / 22℃, 16h light / 8h dark); after 7 days of cultivation, the taproot cell length of the 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 results showed that the root cells of the overexpressing transgenic lines were longer ( Figure 5 c, d); The main root lengths of the wild type (WT) and transgenic lines were measured simultaneously, and it was found that the overexpression transgenic lines showed longer main root lengths after 7 days of light culture ( Figure 5 ef). In addition, surface-sterilized seeds were randomly sown on 1 / 2MS medium and placed horizontally in a growth chamber under long-day conditions (24 / 22°C, 16 h light / 8 h dark). After 10 days of cultivation, the wild type (WT) and transgenic lines were transferred to plastic cups filled with vermiculite (three plants in one cup, two trays for each clone) and placed in short-day conditions (24 / 22°C, 8 h light / 16 h dark). The growth of rosette leaves of the wild type (WT) and transgenic lines was observed, and the rosette leaves of the transgenic lines were more vigorous ( Figure 5 b); After the wild type (WT) and transgenic lines were bolted, the main stems of the wild type (WT) and transgenic lines with uniform growth were selected and the number of epidermal hairs was observed using an ultra-depth of field microscope (at least five biological replicates were used for each measurement). The transgenic lines showed more main stem epidermal hairs ( Figure 5 g, h); the main stem heights of the wild type (WT) and transgenic lines were measured simultaneously, and the main stems of the transgenic lines were taller ( Figure 5 i, j). The experimental results showed that transgenic Arabidopsis overexpressing GhCSLC4 had longer roots, longer root cells, more vigorous rosette leaves, more stem hairs, and taller main stems. This indicates that overexpression of GhCSLC4 significantly promotes cell elongation, root length, and growth potential in Arabidopsis plants.

[0082] Based on the above results, this study used linkage analysis and substitution mapping to identify a lint percentage QTL (qLP-D09) in the introgression interval of chromosome fragments of L283, a marine-terrestrial introgression line with low lint percentage. Combined with the differential expression of genes in the candidate interval at different fiber development stages, a cellulose synthase-like gene CSLC4 was found to be the candidate gene for this QTL. This gene was highly expressed in different fiber development stages of TM-1, and the expression level was highest in 10-day-old fibers. CDS sequence analysis of the cellulose synthase-like gene between the parents showed that there were two non-synonymous SNP mutations in this gene. Compared with L283, the parent TM-1 had advantages in lint percentage and lint finger. The fibers of TM-1 plants with silenced GhCSLC4 gene were shorter, the fiber cell protrusions on the surface of the ovules on the day of flowering were fewer, and the mature fiber spirals were also fewer. In addition, the root length and stem hair number of Arabidopsis plants heterologously expressing GhCSLC4 were better than those of the control plants. Studies have shown that GhCSLC4 plays an important role and has application prospects in improving cotton yield traits and breeding new high-yield cotton varieties.

[0083] SEQ ID NO.1 (CDS sequence of GhCSLC4 gene (GH_D09G0294)) 1989

[0085] Gossypium hirsutum

[0086] ATGGCTCTAAGTTCAGTTGTGGTCACCATGGAGAAGCCTAATAAGTTCTCTTTAGTGG

[0087] AAATCAATGGCTCAGATTCAACTCTGCTACTCGAGAAACAAAAAGCAGTGAGTCCCA

[0088] AACAGTTCACATGGTTCCTTTTCCTCAAAGCTAACAGAGTTTTTGCTTGTCTTTCATG

[0089] GCTGCCAATGGCTTTGAAAACCACGTTTTTATCGGTCAAGAAACGCATTGCATTATCT

[0090] GATGTGAGTGACGAAGAAGCCAAAAGCAGAAGATTGTATAGATTCATCAAAGTCTTT

[0091] CTTTTTATCTCTATATTTGCTTTGGTTTTAGAAATCTTTGCTCATTTGAAGAAATGGAAT

[0092] TTGAACATGATTCAACCATGGGAGGTTCAAGGTCTCTTCCAATGGTGTTATATGACAT

[0093] GGTTGTCATTTAGAGTTGATTATGTTGCTCCATTGGTGCTAACCATGTCCAAATTCTGC

[0094] ATTGTTCTCTTCTTGATTCAGTCTCTTGATCGCCTTATTCTTTGTTTGGGTTGTTTCTGG

[0095] ATTAAATATAAGAACTTGAAACCAAGAATCAATGGAGATGCTTATGATATTGAAGATG

[0096] GCTCAAGTTTTCCTATGGTTCTAGTGCAGATCCCAATGTGCAATGAAAGAGAGGTATT

[0097] TGCACAATCAATTGCAGCTGCTTCTCAGCTTGATTGGCCTAAAGACCGGATTCTAATT

[0098] CAAGTTCTAGATGATTCAGATGATGGGAATTTACAGCTTTTGATCAAAGATGAAGTTT

[0099] CATTATGGCGTGAGAAAGGGGTGAACATAATCTACCGGCATCGGTTAATCCGAACCGG

[0100] ATACAAAGCCGGCAACCTTAAATCAGCAATGGCATGTGATTATGTTAAGGACTATGAA

[0101] TTTGTTGCTATATTTGATGCAGATTTCCAGCCTAACCCTGATTTCCTCAAGCAAACCGT

[0102] GCCTCACTTCAAGGGTGACCCTGAGCTTGGTTTAGTTCAAGCTCGTTGGTCATTTGTG

[0103] AATAAAGATGAGAATTTGCTTACAAGGCTTCAAAACATCAATCTTTGCTTCCATTTCG

[0104] AAGTTGAGCAGCAAGTGAATGGTGCTTTCCTCAACTTCTTCGGATTCAACGGGACGG

[0105] CTGGCGTGTGGAGGATCAAGGCCTTGGAAGAATCCGGTGGTTGGCTCGAGAGAACA

[0106] ACTGTTGAGGACATGGATATCGCGGTTCGAGCACACTTAAATGGATGGAAATTCATCT

[0107] TCCTCAATGATGTTAAAGTGCTTTGTGAGCTGCCTGAGTCTTATGAAGCTTACAAGAA

[0108] ACAGCAGCACCGATGGCATTCAGGTCCGATGCAGTTGTTTCGGTTATGTCTCCCTGCA

[0109] ATTGTAACATCCAAGATATCGGTATCGAAGAAGGCCAATTTGATATTCCTGTTTTTCCT

[0110] ACTAAGGAAGCTTATACTTCCATTTTACTCCTTCACATTGTTCTGCATCATACTTCCATT

[0111] AACAATGTTCATTCCTGAAGCTGAACTTCCCCTTTGGGTTATCTGTTATATCCCAATTT

[0112] TCATGTCATTCCTAAACATCATCCCGGCCCTGAAATCCTTCCCTTTCTTAGTCCCGTAC

[0113] CTTCTGTTCGAAAACACAATGTCGGTAACCAAATTTAATGCCATGATCTCGGGACTAT

[0114] TCCAGCTCGGGAGCGCCTACGAATGGGTAGTAACGAAGAAAACAGGTCGATCATCAG

[0115] AATCCGACTTAGTGGCCTTAGCTGAAAGGGAATCGAAATCATTGAACGAAGAAAAGA

[0116] TCACGAGGAGACACTCCGAATCCGGGTTGGAGACATTGAATAAACTCAAAAAACAA

[0117] GAAGAAATCACCCCTTTGAAGAAGAGAAATCGACTCTATAGGAAAGAGCTGGTACTG

[0118] GCTTTTCTTCTACTCACTGCTGCTGCAAGAAGCTTACTGTCTGCACATGGAGTTCATTT

[0119] CTATTTCTTGTTGTTTCAAGGCTTGACTTTTCTAGTAGTTGGCTTGGATTTGATTGGTG

[0120] AACAAATAAGTTAASEQ ID NO.2(Amino acid sequence of GhCSLC4 protein (GH_D09G0294)) 662

[0122] Gossypium hirsutum

[0123] MALSSVVVTMEKPNKFSLVEINGSDSTLLLEKQKAVSPKQFTWFLFLKANRVFACLSWL

[0124] PMALKTTFLSVKKRIALSDVSDEEAKSRRLYRFIKVFLFISIFALVLEIFAHLKKWNLNMIQ

[0125] PWEVQGLFQWCYMTWLSFRVDYVAPLVLTMSKFCIVLFLIQSLDRLILCLGCFWIKYKN

[0126] LKPRINGDAYDIEDGSSFPMVLVQIPMCNEREVFAQSIAAASQLDWPKDRILIQVLDDSD

[0127] DGNLQLLIKDEVSLWREKGVNIIYRHRLIRTGYKAGNLKSAMACDYVKDYEFVAIFDAD

[0128] FQPNPDFLKQTVPHFKGDPELGLVQARWSFVNKDENLLTRLQNINLCFHFEVEQQVNGA

[0129] FLNFFGFNGTAGVWRIKALEESGGWLERTTVEDMDIAVRAHLNGWKFIFLNDVKVLCEL

[0130] PESYEAYKKQQHRWHSGPMQLFRLCLPAIVTSKISVSKKANLIFLFFLLRKLILPFYSFTLF

[0131] CIILPLTMFIPEAELPLWVICYIPIFMSFLNIIPALKSFPFLVPYLLFENTMSVTKFNAMISGLF

[0132] QLGSAYEWVVTKKTGRSSESDLVALAERESKSLNEEKITRRHSESGLETLNKLKKQEEIT

[0133] PLKKRNRLYRKELVLAFLLLTAAARSLLSAHGVHFYFLLFQGLTFLVVGLDLIGEQISSEQ ID NO.3 (forward primer sequence for constructing GhCSLC4 CLCrV silencing vector) 44

[0135] ATGGCATGCCTGCAGACTAGTTCAGCTTGATTGGCCTAAAGACC

[0136] SEQ ID NO.4 (reverse primer sequence for constructing GhCSLC4 CLCrV silencing vector) 45

[0138] TTCACTAGACCTAGGGGCGCGCCCACTTGCTGCTCAACTTCGAAA SEQ ID NO.5 (forward primer sequence for constructing GhCSLC4PBI121 overexpression vector) 43

[0140] ACGGGGGACTCTAGAGGATCCATGGCTCCAAGTTCAGTTGTGG

[0141] SEQ ID NO.6 (reverse primer sequence for constructing GhCSLC4 PBI121 overexpression vector) 49

[0143] CGATCGGGGAAATTCGAGCTCTTAACTTATTTGTTCACCAATCAAATCC。

Claims

1. Use of the GhCSLC4 gene as shown in SEQ ID NO.1 or a biological material related to the GhCSLC4 gene in the following (a1) or (a2) or (a3) ​​or (a4): (a1) Improving cotton yield traits, or preparing products for improving cotton yield traits; (a2) increasing cotton yield or preparing products for increasing cotton yield; (a3) cultivating new high-yield cotton germplasm, or preparing products for cultivating new high-yield cotton germplasm; (a4) Promote plant growth.

2. The use according to claim 1, characterized in that: The biological material related to the GhCSLC4 gene is the biological material described in (b1) or (b2) or (b3) below: (b1) protein GhCSLC4 encoded by the GhCSLC4 gene; (b2) primers for amplifying the full-length coding sequence of the GhCSLC4 gene; (b3) Biological material containing the GhCSLC4 gene.

3. The use according to claim 2, characterized in that: The protein GhCSLC4 encoded by the GhCSLC4 gene in (b1) has the amino acid sequence shown in SEQ ID NO.

2.

4. The use according to claim 2, characterized in that: The primers used to amplify the full-length coding sequence of the GhCSLC4 gene in (b2) are shown as SEQ ID NO.5 and SEQ ID NO.

6.

5. The use according to claim 2, characterized in that: The biological material containing the GhCSLC4 gene described in (b3) is at least one of the following (c1) to (c9): (c1) an expression cassette containing the GhCSLC4 gene; (c2) a recombinant vector containing the GhCSLC4 gene, or a recombinant vector containing the expression cassette of (c1); (c3) a recombinant microorganism containing the GhCSLC4 gene, or a recombinant microorganism containing the expression cassette described in (c1), or a recombinant microorganism containing the recombinant vector described in (c2); (c4) a transgenic plant cell line containing the GhCSLC4 gene, or a transgenic plant cell line containing the expression cassette described in (c1), or a transgenic plant cell line containing the recombinant vector described in (c2); (c5) transgenic plant tissue containing the GhCSLC4 gene, or transgenic plant tissue containing the expression cassette described in (c1), or transgenic plant tissue containing the recombinant vector described in (c2); (c6) a transgenic plant organ containing the GhCSLC4 gene, or a transgenic plant organ containing the expression cassette described in (c1), or a transgenic plant organ containing the recombinant vector described in (c2); (c7) a transgenic plant containing the GhCSLC4 gene, or a transgenic plant containing the expression cassette described in (c1), or a transgenic plant containing the recombinant vector described in (c2); (c8) tissue cultures produced from regenerable cells of the transgenic plant described in (c7); (c9) Protoplasts produced from the tissue culture described in (c8).

6. The use according to claim 1, characterized in that: The GhCSLC4 gene is used as a target gene, and the GhCSLC4 gene is overexpressed through genetic engineering methods to improve cotton yield traits, increase cotton yield, cultivate new high-yield cotton germplasm, or promote the growth of target plants.

7. The use according to claim 1 or 6, characterized in that: The plant is cotton or Arabidopsis thaliana.

8. A method for improving cotton yield traits or increasing cotton yield, characterized in that: Overexpression of the GhCSLC4 gene with a nucleotide sequence as shown in SEQ ID NO. 1 in cotton improves cotton yield traits or increases cotton yield.

9. A GhCSLC4 gene related to cotton yield traits, the gene having a nucleotide sequence as shown in SEQ ID NO.

1.

10. The biological material related to the GhCSLC4 gene according to claim 9, characterized in that: The biological material is the biological material described in (d1) or (d2) or (d3) below: (d1) a protein GhCSLC4 encoded by the GhCSLC4 gene having an amino acid sequence as shown in SEQ ID NO.2; (d2) primers as shown in SEQ ID NO.5 and SEQ ID NO.6 for amplifying the full-length coding sequence of the GhCSLC4 gene; (d3) An expression cassette, recombinant vector, transgenic cell line or recombinant bacteria containing the GhCSLC4 gene.

Images