Cotton rhythm regulation-related secretory peptide GhRALF1 and transcription factor GhTCP14a and their expression genes and applications

Abstract

The present invention relates to a cotton rhythm regulation-related secretory peptide GhRALF1, whose amino acid sequence is shown in SEQ ID NO.5, or a sequence with 0, 1, or multiple amino acid mutations but unchanged biological activity. The present invention relates to the expression gene of the secretory peptide GhRALF1, and its related applications. The present invention also relates to the transcription factor GhTCP14a and the application of its expression gene. The inventors obtained the method of ovule outer integument cells by enzymatic hydrolysis, and separated the fiber cell group by single cell sequencing. It was subsequently found that the growth of fiber is a rhythmic process. In addition to the regulation of the core rhythmic gene on this process, it is also regulated by the small molecule peptide GhRALF1 and the transcription factor GhTCP14a. Changing its rhythmicity can accelerate fiber growth. By regulating the expression of rhythmic genes, secretory peptides and transcription factors, the length of cotton fibers can be changed. It can be extended to the field of cotton breeding technology to effectively improve the quality of fibers.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to cotton rhythm regulation-related secretory peptide GhRALF1 and transcription factor GhTCP14a, and expression genes and applications thereof. Background Art

[0002] Cotton (Gossypium spp.) is a major cash crop in the world. It is not only the world's main natural fiber source, but also an important oil and protein crop second only to soybean. The development of cotton fiber has attracted extensive attention from cotton researchers around the world. So far, many genes related to cotton fiber development have been reported, but the complex molecular regulatory network of cotton fiber development remains to be explored.

[0003] The circadian clock acts as a metronome to control physiological processes such as stomatal opening and closing, hypocotyl elongation, and flowering time. In tomatoes and soybeans, changes in the biological clock can promote their growth, which is consistent with the view that regulating the circadian clock of plants can improve yield and other agronomic traits. In cotton, both fatty acid metabolism and ethylene synthesis pathways related to fiber development are rhythmic, but whether there is a direct connection between circadian rhythm and fiber development remains unknown. Summary of the invention

[0004] One of the purposes of the present invention is to provide cotton rhythm regulation-related secretory peptide GhRALF1 and its expression gene and application.

[0005] The present invention confirms that the elongation of cotton fiber cells is a rhythmic process, which is regulated by rhythmic genes and multiple factors, mainly including secretory peptide GhRALF1 and transcription factor GhTCP14a. These two factors are also regulated by rhythmic genes. Changing their rhythmicity can improve the quality of fiber.

[0006] The technical solutions for achieving the above-mentioned purpose include the following.

[0007] The first aspect of the present invention is to provide a cotton rhythm regulation-related secretory peptide GhRALF1, whose amino acid sequence is shown in SEQ ID NO.5, or a sequence having 0, 1, or multiple amino acid mutations but the biological activity of cotton rhythm regulation remains unchanged.

[0008] In some embodiments, the cotton is Gossypium hirsutum or Gossypium barbadense.

[0009] The second aspect of the present invention is to provide a cotton rhythm regulation-related gene GhRALF1 expressing the secretory peptide GhRALF1 as described above.

[0010] The third aspect of the present invention is to provide the application of the cotton rhythmic regulation-related secretory peptide GhRALF1 and / or the gene GhRALF1.

[0011] Application of the above cotton rhythmic regulation-related secretory peptide GhRALF1 and / or the above gene GhRALF1 in regulating the rhythmic growth of cotton.

[0012] Application of the cotton rhythmic regulation-related secretory peptide GhRALF1 and / or the gene GhRALF1 in regulating cotton fiber growth.

[0013] In some embodiments, the application includes inhibiting the expression of cotton rhythmic regulation-related secretory peptide GhRALF1 to promote cotton fiber elongation.

[0014] The application of the cotton rhythmic regulation-related secretory peptide GhRALF1 and / or the gene GhRALF1 in regulating cotton fiber length in cotton breeding.

[0015] The fourth aspect of the present invention is to provide a method for regulating cotton fiber growth, comprising inhibiting or promoting the expression of the gene GhRALF1 as described above in cotton plants or cotton cells; or

[0016] Reduce or increase the expression level of the secretory peptide GhRALF1.

[0017] In some embodiments, the expression of the above-mentioned gene GhRALF1 in cotton plants or cotton cells is inhibited to promote cotton fiber elongation;

[0018] Or reducing the expression level of the secretory peptide GhRALF1 to promote cotton fiber elongation.

[0019] In some of the embodiments, the method comprises inhibiting the expression of the cotton GhRALF1 gene; the inhibiting the expression of the GhRALF1 gene is achieved by gene editing the cotton GhRALF1 gene.

[0020] The fifth aspect of the present invention is to provide a biological agent for regulating cotton fiber growth, including substances that inhibit or promote the expression of the gene GhRALF1 in cotton plants or cotton cells, or reduce or increase the expression level of the secretory peptide GhRALF1.

[0021] In some embodiments, the substance is an enhancer or an inhibitor.

[0022] In some of the embodiments, the regulator is an inhibitor, and the regulation refers to promoting the elongation of plant cells and / or cotton fibers.

[0023] In some of the embodiments, the regulator is a promoter, and the regulation refers to inhibiting the elongation of plant cells and / or cotton fibers.

[0024] In some embodiments, the substance is an RNAi interference sequence that inhibits the expression of the above-mentioned gene GhRALF1.

[0025] The second purpose of the present invention is to provide cotton rhythm regulation-related transcription factor GhTCP14a and its expression gene and application.

[0026] The fifth aspect of the present invention is to provide a cotton rhythm regulation-related transcription factor GhTCP14a, whose amino acid sequence is shown in SEQ ID NO.8, or a sequence with 0, 1, or multiple amino acid mutations but unchanged biological activity.

[0027] The sixth aspect of the present invention is to provide a cotton rhythmic regulation-related gene GhTCP14a of the expression transcription factor GhTCP14a, whose sequence is shown in SEQ ID NO.9, or a sequence having 0, 1, or multiple nucleotide mutations but unchanged biological activity.

[0028] The seventh aspect of the present invention is to provide the use of the cotton rhythmic regulation-related transcription factor GhTCP14a and / or the gene GhTCP14a in regulating the rhythmic growth of cotton.

[0029] The seventh aspect of the present invention is to provide the use of the cotton rhythm regulation-related transcription factor GhTCP14a and / or the gene GhTCP14a in regulating cotton fiber growth.

[0030] In some of the embodiments, the expression of cotton rhythm regulation-related gene GhTCP14a is promoted to promote cotton fiber elongation.

[0031] The eighth aspect of the present invention is to provide the use of the cotton rhythm regulation related transcription factor GhTCP14 and / or the gene GhTCP14a mentioned above for regulating cotton fiber length in cotton breeding.

[0032] The ninth aspect of the present invention is to provide a method for regulating cotton fiber growth, comprising inhibiting or promoting the expression of the gene GhTCP14a as described above in cotton plants or cotton cells; or

[0033] Reduce or increase the expression level of the above transcription factor GhTCP14a.

[0034] In some embodiments, the expression of the gene GhTCP14 as described above in cotton plants or cotton cells is promoted to promote cotton fiber elongation;

[0035] Or promoting the expression of the above-mentioned transcription factor GhTCP14a to promote cotton fiber elongation.

[0036] The tenth aspect of the present invention is to provide a method for increasing fiber length, comprising extending the illumination time for cotton plants.

[0037] In the present invention, the inventors obtained the outer integument cells of the ovule by enzymatic hydrolysis, and separated the fiber cell populations by single-cell sequencing, and then found that the growth of the fiber is a rhythmic process. In addition to the regulation of the core rhythmic genes on this process, it is also regulated by the small molecule peptide GhRALF1 and the transcription factor GhTCP14a. Changing its rhythmicity can accelerate fiber growth. By regulating the expression of rhythmic genes, secretory peptides and transcription factors, the length of cotton fibers can be changed. This can be extended to the field of cotton breeding technology to effectively improve the quality of fibers. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A single-cell sequencing solution.

[0039] Figure 2 A large number of protoplasts from the outer integuments of the ovule were produced for enzymatic hydrolysis.

[0040] Figure 3 The ovule sections are before and after enzymatic hydrolysis.

[0041] Figure 4 Identification of module-enriched genes.

[0042] Figure 5 Clustering of 251 module-enriched genes.

[0043] Figure 6 Single cell clustering.

[0044] Figure 7 Single cell clustering for WT and FL.

[0045] Figure 8 The reproducibility of single-cell sequencing.

[0046] Fig. 9 Batch effect for WT and FL single cell sequencing.

[0047] Fig.10 is the number of marker genes.

[0048] Fig.11 GO annotation for C3 marker genes.

[0049] Fig.12 In situ hybridization for XTH gene.

[0050] Fig.13 This is a diagram of the rootless development trajectory.

[0051] Fig.14 It is a relative expression of time.

[0052] Fig.15 The developmental trajectory of fiber cells.

[0053] Fig.16 Validation of developmental trajectories for fluorescence quantification.

[0054] Fig.17 Single-cell expression of RALF.

[0055] Fig.18 This is the alignment of RALF small peptide sequences.

[0056] Fig.19 The ovules cultured in vitro were treated with RALF peptide.

[0057] Fig. 20 The effect of RALF on the activity of ovule proton pump.

[0058] Fig.21 Transcriptome sequencing before and after GhRALF1 treatment.

[0059] Fig. 22 Expression of GhRALF1 and changes in pH and fiber length.

[0060] Fig.23 Rhythmic gene expression.

[0061] Fig.24 qRT-PCR verification of rhythmically expressed genes.

[0062] Fig.25 Single-cell clustering based on single-cell chromatin accessibility sequencing.

[0063] Fig.26 The difference in chromatin accessibility between WT and FL.

[0064] Fig. 27 A method for predicting fiber cell-specific K-mer motifs.

[0065] Fig.28 are k-mers with the potential to regulate specific genes.

[0066] Fig.29 is the G-box motif contained in M1.

[0067] Fig.30 It is a TCP / TCP-like motif.

[0068] Fig.31 is the proportion of C3 highly expressed genes targeted by TCP / TCP-like motifs.

[0069] Fig.32 GO annotation for TCP / TCP-like target genes.

[0070] Fig.33 Gel retardation experiments confirmed that TCP protein binds to the TCP / TCP-like motif.

[0071] Fig.34 The expression changes of GhTCP14a under 16-h and 24-h daylight conditions.

[0072] Fig.35 Comparison of WT and FL regarding polysomes and energy production.

[0073] Fig.36 Comparison of WT and daytime polysomes and energy production.

[0074] Fig.37 The effect of 24 hours of continuous light on fiber length.

[0075] Fig.38 This is the expression pattern of GhRALF1 in Example 4.

[0076] Fig.39 This is the size detection result of the complete protein sequence of GhRALF1 in Example 4. DETAILED DESCRIPTION

[0077] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0078] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0079] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0080] The development of single-cell sequencing technology has made it possible to detect gene expression with single-cell accuracy. However, due to the presence of cell walls in plants, the difficulty of obtaining protoplasts by enzymatic hydrolysis of plant tissues has greatly limited the application of single-cell sequencing in plants. So far, it has only been successfully used in the roots and leaves of Arabidopsis, rice, corn, poplar stems and Physcomitrella patens, and has not been reported in cotton.

[0081] >GhTCP14a (CDS sequence)

[0082] ATGGAAGGAGGAGGAAGTGATGATCATCACCACCACCACCACAGCCACCACCATCACCACCAACAACCGCTCCATCATCA

[0083] TCATCGTCACCACCACCGCCCTACTTTCCCATTCCAATTACTAGAGAAGAAAGAAGAAGATAACCAACCTTGTTCCTCCT

[0084] CTTCTTCCCTTTCCTTTCCCTTCCCTAACCGGTTTCATCTTCCTCGGCCGATCAACACACCTCGACTCGTTCCATTTCAACC

[0085] CACCAGATCTCGCCGGAAACTTCCAAGACTCCACCGCCTAAACGTACTTCGACTAAAGACCGACACACCAAGGTAGATGG

[0086] GCGTGGTCGTAGGATCCGCATGCCGGCTCTTTGTGCAGCTAGGGTTTTTCAGCTTACTCGCGAGTTGGGTCATAAATACG

[0087] ATGGTGAAACGATCGAGTGGTTGCTACAACAAGCTGAGCCGGCGGTGATCGCCGCTACGGGGACTGGTACCATCCCTGCA

[0088] AACTTCACTTCCCTCAATATCTCACTCCGTAGCTCCGGCTCAAGCATGTCGGTTCCTTCTCAGCTTCGGTCCCTCGGGTTT

[0089] TAGCTCCAACTTTTCTATGCAACAACGTAGAAGCTTGTTCCCCGGAATCGGACTCGAAACGACGCCGACGCCGACGACGT

[0090] TCCTTAACTTCCAATCTAGTAGTACTTCGTTGGGAATATCGGAAGAAGCCGAAGAAAGTAGTTTAGGAAGGAAACGAAGA

[0091] CCCGAACAAGATTTATCTTCACAACATCATCAAATGGGGAGTTACTTGTTGCAATCAAGCACCGGTACAATTCCGGCGAG

[0092] TCACCACGGTCAAGTTCCGGCTAACTTTTGGATGGTAACTAATTCTAATAATCAAGTTATGAGTGGTGGTGACCCTATAT

[0093] GGACATTTCCTTCAGTTAATAACAGTGGTTTGTATAGGGGAACTATGTCTAGTGGGTTGCATTTCATGAACTTTCCAACA

[0094] CCAATGGCCTTGTTACCAGGCCAACAATTGGGTTCAAGCAGTAGCGGTGCCGATGGAGGCGGCGGCGGAGGTGGCAGTGG

[0095] TGGAAGTAGTGGCATAAGTGAAGGACATTTAAATATGTTAGCTGGATTGAATCCTTATAGACAAGACTCCGGTGGCACCGGCGTTTCGGAATCTCAAGTGAGTGGCTCACATTCACACCACGGTGGTGGAGGTGGCAGCGATGAATGA(SEQ ID NO.9)>GhTCP14 a(Amino acid sequence)

[0096] MEGGGSDDHHHHHHSHHHHHQQPLHHHHRHHHRPTFPFQLLEKKEEDNQPCSSSSSLPFPSLPVSSSSADQHTSTRSIST

[0097] HQISPETSKTPPPKRTSTKDRHTKVDGRGRRIRMPALCAARVFQLTRELGHKYDGETIEWLLQQAEPAVIAATGTGTIPA

[0098] NFTSLNISLRSSGSSMSVPSQLRSSGFSSNFSMQQRRSLFPGIGLETTPTPTTFLNFQSSSTSLGISEEAEESSLGRKRR

[0099] PEQDLSSQHHQMGSYLLQSSTGTIPASHHGQVPANFWMVTNSNNQVMSGGDPIWTFPSVNNSGLYRGTMSSGLHFMNFPTPMALLPGQQLGSSSSGADGGGGGGGSGGSSGISEGHLNMLAGLNPYRQDSGGTGVSESQVSGSHSHHGGGGGSDE*(SEQ ID NO.8)

[0100] >GhRALF1 (CDS sequence)

[0101] ATGGCGGTACTCAACTATAAGCTCGTTGTGATCTGCGCGACGCTCATGGCAGCAGCTGCGGCGGCGGTAATGACTGTTGATGCGAGTGGGGACCACCACCTGCAACACCCGATCCTGGGGAGGATACCGACCAGATCGCCATGCAAGGGCTCCATAGCCGAATGCTTAGCCGGGGAAGAGTTCGAGCTGGATTCGGAGAGCAGC CGGCGCATTTTA GCCACCACCAGGTACATCAGCTACGGCGCGCTTCAGAGGAACACCGTTCCTTGTTCCCGGCGTGGCGCTTCCTATTATAATTGCAAGCCTGGTGCTCAGGCTAACCCTTACAACCGTGGGTGCAGCCGTATTACCAGGTGCAGGGGCTGA(SEQ IDNO.7)

[0102] >GhRALF1 (Amino acid sequence)

[0103] MAVLNYKLVVICATLMAAAAAAVMTVDASGDHHLQHPILGRIPTRSPCKGSIAECLAGEEFELDSESS RRILATTRYISYGALQRNTVPCSRRGASYYNCKPGAQANPYNRGCSRITRCRG*((SEQ ID NO.6)

[0104] The above is the complete sequence of the small peptide. The small peptide will be cleaved before the mature peptide can function. The underlined part is the cleavage site. ATTRYISYGALQRNTVPCSRRGASYYNCKPGAQANPYNRGCSRITRCRG (SEQ ID NO. 5) is the amino acid sequence of its mature peptide.

[0105] The present invention is further described in detail below with reference to specific embodiments.

[0106] Example Ⅰ Single-cell sequencing of cotton ovule outer integument cells and identification of fiber cell populations

[0107] (I) Enzymatic hydrolysis to obtain ovule outer integument cells

[0108] The buffer used for enzymatic hydrolysis contained 10mM KCl, 2mM MgCl2, 2mM CaCl2, 0.1% BSA, 2mM MES pH5.7 and 600mM mannitol. The enzyme combination used was: 1.5% cellulase R-10, 1.0% hemicellulose, 0.3% Macerozyme R-10. Ovules from -2, -1, 0, 1, 2DPA (0DPA is the day of flowering, -2DPA is 2 days before flowering. 2DPA is 2 days after flowering) were enzymatically hydrolyzed at 50rpm in a 28℃ shaker for 1.5h, and then filtered with a 40μm mesh to remove unenzymatically hydrolyzed tissues and larger fragments. The filtrate was centrifuged at 100g for 10min using a horizontal rotor, and the supernatant was discarded. The obtained protoplast pellet was washed three times with 8% mannitol and finally resuspended with 500μL. After determining the cell viability using trypan blue staining, the hemocytometer was used for counting.

[0109] (ii) Lysis of ovule outer integument cells to obtain nuclei

[0110] The lysis buffer used for lysis contained 10mM Tris-HCL, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Tween20, 1% BSA, 0.1% NP-40, and 0.5% Dodecyl Maltoside (DDM). After lysis, the cells were filtered using a 40μm mesh. The filtrate was centrifuged at 500g for 3min, the supernatant was discarded and the cells were resuspended. The cell nuclei suspension was purified using a flow cytometer, and the collected cell nuclei were counted using a hemacytometer.

[0111] (III) Single-cell sequencing and data analysis

[0112] Single-cell transcriptome sequencing: Single-cell droplets were formed using a single-cell microfluidic chip from 10×Genomics, and the constructed library was sequenced using the BGISEQ-500 platform for PE100 sequencing.

[0113] Single-cell ATAC sequencing: Purified nuclei were cut with Tn5 transposase, and the constructed library was sequenced with MGISEQ-2000 for PE50.

[0114] After the expression profiles of individual cells were separated using Cellranger (v3.1.0), the batch effect was removed using the correctExperiments function in the Batchelor package (v1.2.4). The corrected data were reduced in dimension using the UMAP algorithm, and then the cells were grouped using monocle3 (v0.2.1).

[0115] Marker genes were predicted using the findMarkers function in the scran package, and differentially expressed genes were obtained using the edgeR package (v3.28).

[0116] (IV) RNA in situ hybridization

[0117] In order to prove that cluster 3 (cell cluster 3, i.e., C3) is a fibroblast, the primer pair Forward Primer: 5'-CTGGGACTGTCACTGCCTTT-3' (SEQ ID NO.1) and the primer pair Reverse Primer: 5'-AATTAATACGACTCACTATAGGGTGGACCAGTCGGTCTTTTCG-3' (SEQ ID NO.2) were used for in vitro amplification and transcription as antisense probes for RNA in situ hybridization. The primer pair Forward Primer: 5'-ATTTAGGTGACACTATAGCTGGGACTGTCACTGCCTTT-3' (SEQ ID NO.3) and Reverse Primer: 5'-TGGACCAGTCGGTCTTTTCG-3' (SEQ ID NO.4) were used for in vitro amplification and transcription as sense probes for RNA in situ hybridization as a control. The tissue section was 14 μm, and the hybridization of the probe was performed in a hybridization oven, incubated overnight at 45°C in the hybridization oven. The hybridization signals were detected using anti-digoxigenin-AP antibody and NBT / BCIP.

[0118] Example 2 Inhibitory effect of GhRALF1 small peptide on fiber elongation (I) In vitro culture of cotton ovules and treatment with small peptides After 1DPA cotton bolls were disinfected with 10% sodium hypochlorite, the ovules in the cotton bolls were carefully peeled off and placed on the in vitro culture liquid culture medium, and cultured in the dark in an incubator at 28°C. The sequence of the in vitro synthesized GhRALF1 small peptide was ATTRYISYGALQRNTVPCSRRGASYYNCKPGAQANPYNRGCSRITRCRG (SEQ ID NO.5) and the sequence of the GhRALF2 small peptide was FWKRMRYYISYGALWANRIPCPPRAGRSYYTHNCFKVHGPVHPYTRGCSRITRCRR (SEQ ID NO.10), i.e., the mature peptide portion after the RRXL cleavage site. During the in vitro culture, the final concentration of 1 μM was used for treatment.

[0119] (II) Cotton ovule transcriptome sequencing

[0120] According to the above in vitro culture and small peptide treatment methods, 30 minutes later, the in vitro cultured ovules treated with and without small peptide treatment were taken, total RNA was extracted, and the constructed library was sequenced by PE150 using DNBSEQ.

[0121] (III) Cytoplasmic membrane H + -ATPase activity assay

[0122] The H+-ATPase activity of the ovule plasma membrane was measured using a Seahorse XF24 bioenergetic meter. The calibration solution was added to the probe plate the day before the experiment, and the probe plate was hydrated overnight at 28°C. Three ovules were placed in each well of the test plate. After adding an equal amount of test solution, the plate was placed in a 28°C carbon dioxide-free incubator for 45 minutes before being tested on the machine. A total of 23 cycles were tested, each cycle including mixing for 3 minutes, standing for 2 minutes, and then testing for 3 minutes. GhRALF1 and GhRALF2 peptides were injected into the test wells approximately 20 minutes after the test.

[0123] (IV) Detection of pH in cotton ovule apoplast and measurement of fiber cell length

[0124] Using ddH2O with pH=7.0 as the detection solution, the ovules of 0-2DPA cotton bolls were peeled into 1ml of the detection solution, and the pH of the cotton ovules was detected by a microelectrode pH meter. The fiber cell length was measured using paraffin embedding technology, and the sections were stained with safranin fast green. The scanned images were used to measure the fiber cell length using Caseviewer (V2.4). In order to minimize the differences, the cotton bolls we picked were all from cotton plants with the same growth conditions and on the branches at the same position. In addition, for the ovule sections, the ovules we selected were all from the same position in the cotton bolls.

[0125] (V) Ovule integument qRT-PCR (fluorescence quantitative PCR) and RNA-seq

[0126] The ovule outer integument cells were obtained using the above enzymatic hydrolysis method, extracted using the Tiangen polysaccharide polyphenol RNA extraction kit, and RNA extraction lysis solution was directly added to the collected protoplasts for lysis. Reverse transcription used OligodT primers and a one-step reverse transcription kit. The constructed library was sequenced by PE150 using DNBSEQ.

[0127] Example 3 GhTCP14a rhythmically controls translation and mitochondrial energy production during fiber elongation

[0128] (I) Gel retardation experiment

[0129] Use primer pair 5'FAM-GCAGAATTCACTTGtgggctaaACCAGTCTAGACGT-3'

[0130] (SEQ ID NO.11), 5'-ACGTCTAGACTGGTttagcccaCAAGTGAATTCTGC-3'(SEQ ID NO.12) were annealed to form a probe with a FAM label at the 5' end. The cold probe used the same probe sequence but without the FAM fluorescent label. The mutation probe was formed by annealing the primer pair 5'-GCAGAATTCACTTGaaaaaaaaACCAGTCTAGACGT-3'(SEQ ID NO.13), 5'-ACGTCTAGACTGGTttttttttCAAGTGAATTCTGC-3'(SEQ ID NO.14). After incubation of the protein with the probe at 25°C for 60 min, the protein was electrophoresed in a 6% non-denaturing PAGE gel for 1 hour.

[0131] (II) ATP content determination

[0132] The concentration of ATP was detected using an ATP detection kit. The sample was lysed with 200 μl of lysis buffer and centrifuged at 12,000 g for 5 min at 4°C. The supernatant was taken to determine the protein concentration and then the ATP concentration was detected. The fluorescence signal was detected using an ELISA reader FlexStation 3, and the ATP concentration was calculated using the protein content.

[0133] (III) Polyribosome analysis

[0134] After extraction, the polyribosomes were resuspended in extraction buffer and then loaded onto a 10%-50% sucrose gradient medium. The mixture was centrifuged at 35,000 rpm at 4°C for 4 hours using an ultracentrifuge. The ribosome components of each layer were detected by UV260 absorption spectroscopy.

[0135] Experimental results:

[0136] The structure of cotton ovules consists of a central embryo sac and two layers of covering outside the embryo sac, namely the inner integument and the outer integument. Fiber cells develop from the outermost cells of the outer integument. In order to determine the cell types of the outer integument, we performed single-cell transcriptome sequencing and single-cell chromatin accessibility sequencing on the outer integument of wild-type upland cotton and its glabrous mutant ( Figure 1 ). This hairless mutant does not develop into fiber cells and is a valuable material for studying the initiation and elongation of fiber cells.

[0137] The inventors have invented a new method for preparing protoplasts, namely partial tissue enzymatic digestion (PTED). By using cellulase, hemicellulase and pectinase to enzymatically hydrolyze the ovules of wild-type and glabrous mutants of upland cotton -2, -1, 0, 1, 2DPA, and controlling the enzymatic hydrolysis time to 1.5 hours, protoplasts only from the outer integument are obtained, and the viability of the protoplasts can reach more than 85% ( Figure 2 By paraffin embedding and sectioning, and staining with safranin fast green, we compared the ovules before and after enzymatic hydrolysis and found that the enzymatic hydrolysis method only hydrolyzed the cells from the outer integument of the ovule ( Figure 3 ).

[0138] Protoplasts from the outer integument of WT (wild type of cotton) and FL (natural hairless mutant of cotton) were subjected to single-cell transcriptome sequencing and single-cell chromatin accessibility sequencing, respectively. In the final two replicates of single-cell transcriptome sequencing, we successfully captured 18064 and 21389 cells in WT and FL, respectively, detected 98263514 and 109914075 sequences, and detected 58081 and 59869 genes, respectively.

[0139] We used UMAP (Uniform Manifold Approximation and Projection for Dimension Reduction) to reduce the dimensionality of single-cell sequencing data. In order to understand the distribution of all detected cells, we identified 251 module-enriched genes expressed at specific locations in UMAP ( Figure 4 ), and these cells can be divided into 5 groups, of which cells expressing module-enriched genes are divided into 4 groups, and cells not expressing module-enriched genes are divided into one group ( Figure 5 ). According to the specific distribution of module-enriched genes on UMAP, the 12 cell clusters obtained by unsupervised clustering can be merged into 5 clusters C1-C5 ( Figure 6). The cells captured in the wild type were divided into five groups, C1, C2, C3, C4, and C5, and the hairless mutant was divided into four groups, C1, C2, C4, and C5 ( Figure 7 The strong correlation between the two replicates of single-cell transcriptome sequencing indicates the reproducibility of our experiment ( Figure 8 ). We also evaluated the batch effect between WT and FL in single-cell transcriptome sequencing ( Fig. 9 ).

[0140] To define these five cell clusters, we identified 668 marker genes from these five clusters, including 89 transcription factors and 579 non-transcription factors ( Fig.10 ), among which C3 has the largest number of marker genes, including 49 transcription factors and 290 non-transcription factors. After comparing the cell populations of WT and FL, we found that WT has a specific cell population C3. Considering the phenotypic differences between WT and FL samples, that is, the outermost cells of WT outer integument can develop into fibroblasts, while the outermost cells of FL cannot develop into fibers, we speculate that C3 is a fiber cell. This speculation can also be supported by the following reasons: First, the transcription factors in the C3 marker genes were annotated and it was found that the transcription factors specifically expressed in C3 are related to the differentiation and initiation of seed coat hairs ( Fig.11 ). Second, the genes expressed by C3 include several cotton fiber-related transcription factors that have been reported, such as MYB25, GhHOX3, etc., as well as genes related to cotton fiber traits analyzed by GWAS: GhTUA9 and qFWPB_D02. Third, GhTCP14a, GhFLA1, GbPDF1, GhMYB109, etc. have been proven to be specifically expressed in fiber cells. And we have proved by in situ hybridization that the marker gene XTH of C3 is specifically expressed in fiber cells ( Fig.12 ).

[0141] To explore the connections between these five cell groups, we used monocle3 to establish single-cell developmental trajectories. We first obtained an unrooted trajectory map ( Fig.13 To define the origin of developmental trajectories, we used published transcriptome data from different developmental periods to define the relative expression times of all single cells ( Fig.14 ), we finally identified C1-C3 as the trajectory of fiber cell development ( Fig.15 We used fluorescent quantitative PCR to prove that the genes highly expressed at different developmental times were consistent with the predicted results ( Fig.16 ).

[0142] Small secretory peptides play an important role in plant cell communication. We analyzed the highly expressed genes in the C3 cell group and found that 7 genes encoding RALF small peptides were highly expressed in fiber cells ( Fig.17 ), the amino acid sequences of the mature peptides of the seven RALF genes were compared, and it was found that the seven small peptide encoding genes could be divided into two groups, GhRALF1 and GhRALF2, based on their similarity. Fig.18 ), when the in vitro synthesized small peptides were used to treat the cotton ovules cultured in vitro, the small peptide GhRALF1 inhibited the growth of cotton ovules, while GhRALF2 had no significant effect on the growth of ovules ( Fig.19 To explore the molecular mechanism of GhRALF1 inhibition, we detected the proton pump activity of ovules after treatment with GhRALF1 peptide and found that GhRALF1 would lead to a decrease in proton pump activity ( Fig. 20 ), and transcriptome sequencing of ovules before and after treatment revealed that GhRALF1 treatment caused the inhibitory auxin gene response ( Fig.21 ). To explain the seemingly contradictory phenomenon that this small peptide is highly expressed in fiber cells but inhibits fiber elongation, we selected the outer integuments of ovules at four-hour intervals for three consecutive days and performed fluorescence quantitative PCR. We found that the expression of GhRALF1 is a rhythmic process. We also sliced ​​the ovules to count the fiber cell lengths and measure the pH of the ovules. We found that when GhRALF1 expression increased, the pH of the ovules increased. When GhRALF1 expression was low, the pH of the ovules was also low, and the fiber elongation rate was faster ( Fig. 22 ), indicating that the GhRALF1 peptide plays a role in controlling the rhythmic growth of fibers during their elongation.

[0143] In order to systematically study the rhythmic growth of the above fibers, we sequenced the transcriptome of the ovule outer integument at 10 a.m. and 10 p.m. on -2DPA, -1DPA, 0DPA, 1DPA, and 2DPA, and found that a total of 29,282 genes had circadian expression, and these genes could be divided into four expression forms: L1, L2, L3, and N1, where L1, L2, and L3 were highly expressed during the day, and N4 was highly expressed at night ( Fig.23), cluster L3 includes plant rhythmic genes PRR5, PRR7, PRR9, CCA1, LNK1, LNK2, etc. There is no difference in the expression of most rhythmic genes between WT and fl, but when we extracted ovule outer integument RNA at a higher density, i.e., 4-hour intervals, for fluorescence quantitative PCR verification, we found that there were differences in the expression of PRR5 and CCA1 between WT and FL. The expression of PRR5 in WT was higher than that in FL, while the expression of CCA1 in FL was higher than that in WT, and both showed rhythmicity. Under continuous light, their rhythmicity still exists. Genes related to fiber development, such as GhHD1, GhHOX3, GhPDF1, GhMYB25, etc., were significantly enriched in N1. We then used qRT-PCR to verify the expression of GhHOX3 and GhMYB25, and found that they showed rhythmic expression in WT, but almost no expression in FL, and the rhythmicity in WT disappeared under continuous light. This proves that the expression of these genes related to fiber development is regulated by light ( Fig.24 ).

[0144] In summary, we believe that fiber elongation is rhythmic and is regulated by rhythmic genes and light-responsive genes. The GhRALF1 peptide controlled by rhythmic genes acts as an inhibitory factor in this complex process to regulate fiber elongation.

[0145] In addition, we want to explore the important factors that regulate the rhythmic elongation of fiber cells at the chromatin level. We captured 8992 and 13615 cell nuclei from WT and FL single-cell chromatin accessibility sequencing, respectively, and found a total of 216160 highly reliable chromatin open regions, which are widely distributed on the genome. However, we used various methods and were unable to cluster these cells, which indicates that the cells from the ovule outer integument are very similar in chromatin openness, and their differences are not enough for cell clustering ( Fig.25 ).

[0146] Therefore, we used the data measured by single-cell ATAC-seq as a batch of data to analyze the differences between WT and fl, and found a total of 11,538 differentially accessible chromatin regions. At the gene level, chromatin accessibility was highly enriched in WT and 95 genes, respectively ( Fig.26 ), for most genes, chromatin openness is highly correlated with the expression of their genes. We further studied the transcription factors that bind to chromatin accessible regions in ATAC-seq. We integrated chromatin accessibility and scRNA-seq data and developed a method to identify fibroblast-specific K-mer motifs in ACRs in gene regions 1kb upstream of TSS or promoter regions ( Fig. 27 We identified 590 k-mers with strong potential to regulate specific genes ( Fig.28) and evaluated their relative distribution around the transcription start site (TSS). Based on this, we found 317 K-mers enriched near the TSS, of which 247 showed directional specificity. These 590 K-mers can be divided into two categories: M1 and M2, which play an inhibitory and activating role on Cluster3 highly expressed genes, respectively. The inhibitory M1 contains multiple G-box motifs ( Fig.29 ), this motif has been reported to be bound by rhythmic gene PRR to inhibit the transcription of genes in rhythmic regulation. These results once again prove our conclusion above that key rhythmic genes are involved in the regulation of fiber elongation.

[0147] In addition, we found two groups of cis-regulatory elements with activation effects: TCP motif (TGGGCC / T) and TCP-like motif (TAGGGC / T) ( Fig.30 ), about 1 / 3 of the highly expressed genes in cluster 3 have one or more TCP / TCP-like motifs ( Fig.31 ), and the activation effects of these two types of motifs were only found in cluster 3. Through GO annotation, it was found that the genes regulated by TCP / TCP-like motifs were mainly related to protein translation and mitochondrial energy ( Fig.32 ).

[0148] Next, we analyzed the TCP protein family and found that GhTCP7, GhTCP14a, and GhTCP21 were specifically expressed in cluster 3. We used gel retardation experiments to prove that GhTCP14a can bind not only the TCP motif but also the TCP-like motif ( Fig.33 ), quantitative PCR demonstrated that GhTCP14a was expressed in circadian rhythm in WT, but was expressed at a lower level in FL, and maintained rhythm under continuous light, indicating that it was also controlled by the rhythm gene ( Fig.34 ). Protein translation and mitochondrial energy targeted by TCP also showed similar characteristics, that is, protein translation and mitochondrial energy in WT were stronger than those in FL ( Fig.35 ), and in WT the night is higher than the day ( Fig.36 ).

[0149] In addition, we increased the daily light duration from 16 hours to 20 hours after the vegetative growth of cotton plants ended, and compared the obtained cotton fiber lengths (the experimental method was to adjust the light duration in the greenhouse to 20 hours, collect the cotton fibers after the cotton bolls opened, comb them along the middle seam of the cotton seeds, measure the length with a steel ruler, and compare them with the cotton fibers with a light duration of 16 hours). The increase in light duration can significantly increase the fiber length ( Fig.37 ).

[0150] Example 4 Inhibitory effect of GhRALF1 peptide on fiber elongation

[0151] (A) Whole ovule RNA-seq

[0152] We divided the 24 hours of a day into 6 time periods, and took the complete ovules at 10am, 2pm, 6pm, 10pm, 2am, and 6am for freezing, and extracted RNA using the Tiangen Polysaccharide Polyphenol RNA Extraction Kit. Take 100mg of fresh sample or sample stored at -80℃, quickly and thoroughly grind it into powder in liquid nitrogen, and add the powder to 600μL lysis solution (30μL β-mercaptoethanol was added in advance) for lysis. Reverse transcription used Oligo dT primer and one-step reverse transcription kit. The constructed library was sequenced by PE150 using DNBSEQ.

[0153] The original files of sequencing data were obtained and uploaded to the server. Fastp (v0.20.1) software was used to detect and remove the adapter sequences of double-end sequencing data for Clean Data. The data after removing the adapter sequences were aligned with the HAU_v1 genome of upland cotton using STAR (v2.7.6a) software. The results were quantified at the gene level using featureCounts to obtain the original expression count data of each gene in the corresponding sample.

[0154] (II) Preparation of GhRALF1 Antibody

[0155] A specific polypeptide was designed according to the sequence of the GhRALF1 small peptide, and the specific polypeptide CKBGAQANPYNRG (SEQ ID NO.15) was synthesized in vitro. New Zealand white rabbits were immunized, and after multiple titer tests, final bleeding was performed, and the antibodies were purified.

[0156] (VII) WB detection of GhRALF1 small peptide

[0157] Denature the protein or synthetic specific peptide to be loaded, adjust the loading amount, and run electrophoresis in the protein gel until the marker reaches the bottom of the gel, then stop the electrophoresis, transfer the membrane, block, incubate with the above-prepared antibody, incubate with the corresponding secondary antibody, and develop and observe the bands.

[0158] We performed transcriptome sequencing on intact ovules at different time points (4 hours apart) at 0DPA, 1DPA, and 2DPA. After normalizing the data, we extracted the expression of GhRALF1 and found that GhRALF1 still maintained a pattern of low expression during the day and high expression at night in intact ovules ( Fig.38 ).

[0159] In order to further explore the mechanism of action of GhRALF1, we designed a specific peptide sequence for GhRALF1, immunized rabbits, and finally obtained specific antibodies. We used the obtained antibodies to perform protein immunoblotting experiments on the synthesized mature peptide of GhRALF1 (ATTRYISYGALQRNTVPCSRRGASYYNCKPGAQANPYNRGCSRITRCRG SEQ ID NO.5), and found that the size calculated based on the mature peptide sequence of GhRALF1 was about 5.7KD, while the detected size was about 11KD, which was about twice the size. It is speculated that the mature peptide of GhRALF1 assembles into dimers to exert its effect. Western blotting experiments were performed on the extracted ovule membrane protein and the whole protein, respectively, and the detected size was about 26KD, while the complete protein sequence of GhRALF1 (MAVLNYKLVVICATLMAAAAAAVMTVDASGDHHLQHPILGRIPTRSPCKGSIAECLAGEEFELDSESSRRILATTRYI SYGALQRNTVPCSRRGASYYNCKPGAQANPYNRGCSRITRCRG SEQ ID NO.6) calculated that the size was 13KD ( Fig.39 ), which is also a two-fold relationship. Based on this result, we speculate that GhRALF1 aggregates into dimers when performing its function.

[0160] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A cotton rhythmic regulation-related secretory peptide GhRALF1, whose amino acid sequence is shown in SEQ ID NO.

5.

2. A cotton rhythmic regulation-related gene GhRALF1 expressing the secretory peptide GhRALF1 as claimed in claim 1.

3. Use of the cotton rhythm regulation-related secretory peptide GhRALF1 according to claim 1 and / or the gene GhRALF1 according to claim 2 in regulating cotton fiber growth, characterized in that: Inhibiting the expression of cotton rhythmic regulation-related secretory peptide GhRALF1 is used to promote cotton fiber elongation.

4. Use of the cotton rhythmic regulation-related secretory peptide GhRALF1 according to claim 1 and / or the gene GhRALF1 according to claim 2 for regulating cotton fiber length in cotton breeding, characterized in that: Inhibiting the expression of cotton rhythmic regulation-related secretory peptide GhRALF1 is used to promote cotton fiber elongation.

5. A method for regulating cotton fiber growth, characterized in that: Comprising inhibiting the expression of the gene GhRALF1 as claimed in claim 2 in cotton plants or cotton cells; or Reducing the expression level of the secretory peptide GhRALF1 described in claim 1.

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