A gene for down-regulating leaf fuzz trait of upland cotton and application thereof

Abstract

The application belongs to the field of cotton molecular biology, and particularly relates to a negative regulation upland cotton leaf pubescence trait gene and application thereof. The gene is named as GhGIR1, has only one exon without intron, and has a length of 315 bp. The nucleotide sequence of the gene is shown in the sequence table SEQ ID No. 1. The application regulates the development of upland cotton leaf pubescence by participating in the plant hormone signal transduction pathway.

Description

Technical Field

[0001] This invention belongs to the field of cotton molecular biology, specifically relating to a gene that negatively regulates the leaf pubescence trait of upland cotton and its application. Background Technology

[0002] In plant taxonomy, cotton belongs to the Malvaceae family (Malva family). Malvaceae ), Cotton ethnic group ( Gossypieae ), genus Gossypium ( Gossypium The genus *Gossypium* is a model crop for studying the origin, evolution, and domestication of polyploids. Cultivated cotton is one of the world's most important economic crops, and cotton fiber is a natural source of textile raw materials. Allotetraploid upland cotton (… Gossypium hirsutum Cotton (L.) is the world's largest source of renewable textile fibers, accounting for over 90% of global production. The trichomes of cotton originate from single epidermal cells, primarily covering the surface of leaves and stems, and play a crucial role in various biological processes, including regulating transpiration and photosynthesis, regulating leaf surface temperature, and self-defense against pests and diseases. The trichomes covering the surface of terrestrial plants are an adaptive mechanism evolved over long periods to resist abiotic stress. Therefore, trichome traits can serve as an important indicator for screening new cotton varieties.

[0003] The type of pubescence varies considerably among different cotton varieties. Studies have shown that cotton varieties from tropical regions have sparse pubescence, while those from subtropical regions are densely covered with pubescence. Furthermore, the type of pubescence varies with latitude; the lower the latitude, the less pubescent the plant, and the higher the latitude, the more pubescent the plant. The pubescence on cotton leaves and stems plays a role in resisting aphid damage. The density and length of the pubescence on cotton leaves and stems are closely related to aphid resistance, as the pubescence can prevent aphids from laying eggs and feeding. In addition, cotton pubescence, fibers distributed in seed epidermal cells, and epidermal trichomes in Arabidopsis thaliana share certain similarities in structure and development, and exhibit similar regulatory mechanisms during development. Cotton pubescence can be observed and counted during the seedling stage, facilitating research. Therefore, studying the developmental mechanism of cotton pubescence can provide a good model for the development of cotton fibers and plant epidermal trichomes.

[0004] Plant hormones such as gibberellin (GA), cytokinin (CK), salicylic acid (SA), ethylene, and jasmonate acid (JA) play important regulatory roles in the differentiation and development of plant epidermal hairs. Cotton pubescence and cotton fibers are both single-celled epidermal projections, exhibiting similar regulatory mechanisms. Cotton pubescence and fibers are regulated by the activation of the triadic complex R2R3MYB protein (GLABRA1, GL1), the bHLH transcription factor (GLABRA3, GL3), and the WD40 protein (TRANSPARENT TESTA GLABRA1, TTG1). The MYB / bHLH / WD40 trimer formed by these proteins binds to the promoter of the downstream gene homology protein GLABROUS2 (GL2) and activates its expression, promoting the initiation of epidermal hair cells. The homologous genes of GL2 in cotton are also discussed. GhHD1 Silencing inhibits the development of leaf pubescence and delays the initiation of cotton fiber cells; while GhHD1 The number of fibers starting to grow in the overexpression lines was significantly increased. When Asian cotton was used... GaHOX1 Arabidopsis epidermal hair mutant gl2 Heterologous expression restored the hairless phenotype of the mutant. Additionally, some MYB-class transcription factor family genes and MADS protein family genes are also involved in the development of cotton fiber cells and epidermal hairs. Therefore, understanding the developmental mechanism and regulatory mechanisms of cotton epidermal hairs, and identifying the factors regulating epidermal hair development, has significant application value for improving cotton fiber yield and screening new cotton varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a gene that negatively regulates the leaf pubescence trait of upland cotton and its application.

[0006] A gene that negatively regulates the leaf pubescence trait in upland cotton, named GhGIR1,

[0007] The gene has only one exon and no introns, and is 315 bp in length. The nucleotide sequence of the gene is shown in SEQ ID No. 1 of the sequence listing.

[0008] A protein encoded by a gene that negatively regulates the leaf pubescence trait of upland cotton, wherein the gene encoding the protein is a diploid cotton gene, consisting of 105 amino acids, as shown in SEQ ID No. 2.

[0009] A primer pair for amplifying the gene that negatively regulates the leaf pubescence trait of upland cotton, wherein the forward primer is F: 5'-ATGAGTGGTGGAAATGGTCCC-3'; and the reverse primer is R: 5'-CTAATTTTTAATGGCGGGGAGA-3'.

[0010] A plant silencing vector for the gene that negatively regulates the leaf pubescence trait of upland cotton.

[0011] A method for regulating the development of epidermal pubescence in cotton using the aforementioned gene for negative regulation of upland cotton leaf pubescence traits, characterized in that the gene GhGIR1 regulates the development of upland cotton leaf pubescence by participating in plant hormone signal transduction pathways.

[0012] This invention provides a gene that negatively regulates the leaf trichome trait in upland cotton. This gene is highly expressed in the leaves and stems of trichome-free materials and controls epidermal trichome development. Silencing the GhGIR1 gene in cotton significantly increases the number of leaf trichomes. When the GhGIR1 gene of this invention interacts with GhHD1, it inhibits the transcriptional activity of the GhHD1 gene. The GhGIR1 gene regulates the development of cotton epidermal trichomes by participating in plant hormone signal transduction pathways. This results in excellent control of aphids and other pests during cotton growth. This invention is the first to elucidate the biological function of this gene at the gene and protein levels, which is of great significance for exploring the developmental mechanism of epidermal trichomes and breeding cotton varieties with resistance to abiotic stress. Attached Figure Description

[0013] Figure 1 This is the sequence listing for SEQ ID No. 1;

[0014] Figure 2 This is the sequence listing for SEQ ID No. 2;

[0015] Figure 3 Analysis of GhGIR1 gene expression in cotton leaves and stems;

[0016] Figure 3 A is a transcriptional component of the GhGIR1 gene;

[0017] Figure 3 B represents the quantitative fluorescence result of the GhGIR1 gene;

[0018] Figure 4 Subcellular localization analysis of the GhGIR1 gene in tobacco leaf epidermal cells;

[0019] Figure 5 Gene structure and evolutionary analysis of GhGIR1 homologs from Arabidopsis thaliana and different cotton species;

[0020] Figure 6Phenotypic analysis of TRV:00 and TVR:GhGIR1 gene-silenced lines;

[0021] Figure 6 A: Distribution of epidermal hairs on the leaves and stems of the Cotton 3080 plant;

[0022] Figure 6 B: Observation of epidermal hairs in plants treated with TRV:00 empty vector;

[0023] Figure 6 C: Analysis of changes in epidermal hair in GhGIR1 gene-silenced lines;

[0024] Figure 6 D: qRT-PCR detection of GhGIR1 gene silencing efficiency;

[0025] Figure 6 E: Statistics on the number of leaf trichomes before and after GhGIR1 gene silencing;

[0026] Figure 6 F: TRV:CLA cotton silencing line albino phenotype;

[0027] Figure 7 Interaction analysis between GhHD1 and GhGIR1;

[0028] Figure 7 A: Yeast two-hybrid experiments verified the interaction between GhHD1 and GhGIR1. GhHD1 was linked to the bait vector pGBKT7, and GhGIR1 was linked to the prey vector pGADT7.

[0029] Figure 7 B: The firefly enzyme complementation experiment verified the interaction between GhHD1 (fused to the N-terminus of the firefly enzyme LUC) and GhGIR1 (fused to the C-terminus of the firefly enzyme) in tobacco epidermal cells. Two Agrobacterium strains containing the corresponding recombinant plasmids were simultaneously injected into the same tobacco cell.

[0030] Figure 8 Analysis of the transcriptional activity of GhGIR1 in inhibiting GhHD1;

[0031] Figure 8 A: Schematic diagram of the vector structure used in the figure. Report vector: 35S-REN-Gal4-luc vector, GalSKGhHD1: CDS of Gal4BD fused with GhHD1, GalSKGhGIR1: CDS of Gal4BD fused with GhGIR1, SKGhGIR1: CDS of GhGIR1 constructed on 62SK vector;

[0032] Figure 8B: The luciferase activity analysis experiment showed that GhGIR1 could significantly inhibit the transcriptional activation activity of GhHD1. LUC / REN represents the relative luciferase activity. Each experiment was performed in 10 biological replicates. Lowercase letters indicate that the difference was significant under the condition of P<0.05.

[0033] Figure 8 C is a schematic diagram of fluorescence imaging corresponding to D;

[0034] Figure 8 D: Qualitative results of luciferase activity.

[0035] Figure 9 Transcriptome analysis of TRV: GhGIR1 and TVR:00;

[0036] Figure 9 A: Volcano plots show the distribution between changes in all gene expression levels and p-values;

[0037] Figure 9 B: Venn diagrams represent the number of transcription factors contained in downregulated genes;

[0038] Figure 9 C: Venn diagram shows the number of transcription factors included in downregulated genes;

[0039] Figure 9 D: Analysis of differentially regulated gene KEGG enrichment pathways;

[0040] Figure 9 E: Analysis of upregulated differentially regulated KEGG enrichment pathways;

[0041] Figure 10 Cloning of the GhGIR1 gene cDNA sequence. In the figure, M represents DNA Marker, and the red arrow indicates the location of the target gene. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Example

[0043] A gene that negatively regulates the leaf pubescence trait in upland cotton, named GhGIR1,

[0044] The gene has only one exon and no introns, and is 315 bp in length. The nucleotide sequence of the gene is shown in SEQ ID No. 1 of the sequence listing.

[0045] A protein encoded by a gene that negatively regulates the leaf pubescence trait of upland cotton, wherein the gene encoding the protein is a diploid cotton gene, consisting of 105 amino acids, as shown in SEQ ID No. 2.

[0046] A primer pair for amplifying the gene that negatively regulates the leaf pubescence trait of upland cotton, wherein the forward primer is F: 5'-ATGAGTGGTGGAAATGGTCCC-3'; and the reverse primer is R: 5'-CTAATTTTTAATGGCGGGGAGA-3'.

[0047] A plant silencing vector for the gene that negatively regulates the leaf pubescence trait of upland cotton.

[0048] A method for regulating the development of epidermal pubescence in cotton using the aforementioned gene for negative regulation of upland cotton leaf pubescence traits, characterized in that the gene GhGIR1 regulates the development of upland cotton leaf pubescence by participating in plant hormone signal transduction pathways.

[0049] Sources of genes negatively regulating leaf pubescence traits in upland cotton:

[0050] This invention utilizes genome-wide association analysis (GWAS) to analyze the leaf pubescence trait of 723 upland cotton natural populations, identifying a candidate gene, Gh_A11G366200, that regulates the leaf pubescence trait. This gene was named GhGIR1. Transcriptome data analysis and quantitative real-time PCR revealed that the expression level of this gene in the leaves and stems of the non-pubescent material was significantly higher than that in the pubescent material. Figure 3 This indicates that the gene may be a candidate gene controlling leaf trichome development. Using upland cotton leaf genomic DNA and cDNA as templates, the sequence of this target gene was obtained. Sequence alignment revealed that this gene is a 315 bp gene with no introns and an unknown function, as shown in SEQ ID No. 1. The protein encoded by this gene consists of 105 amino acids (aa), as shown in SEQ ID No. 2. Figure 3 In this context, ZM19 and TY02-6 represent the hairy cotton materials Zhongmian Institute 19 and Taiyuan 02-6, respectively; SY04-92 and Aizimian3080 represent the non-hairy cotton materials Suyuan 04-92 and Aizimian 3080, respectively; L: leaf, S: stem. The error bars represent the mean (±SD) of three biological replicates for each sample. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0051] Interaction between GhGIR1 and GhHD1 at the protein level

[0052] Subcellular localization results showed that GhGIR1 was expressed in both the cell membrane and the nucleus. Figure 4 Phylogenetic analysis revealed that GhGIR1 (GhA11G366200) is related to Gossypium. tomentosum GotomA11G383100, Gossypium mustelinum The closest relative is GomusA11G358100 from Arabidopsis thaliana; the next closest relative is AtGIR1 (AT3G11600) from Arabidopsis thaliana. Gene function annotation shows that GhGIR1 and its homologs are of unknown function, indicating that GhGIR1 is a novel protein. Figure 5 ).

[0053] To investigate the function of GhGIR1 in regulating epidermal trichome development, a VIGS vector for GhGIR1 was constructed and injected into the leafless trichome cotton material, Cotton 3080. Two weeks after injection, the albino phenotype began to appear in cotton seedlings. Figure 6 F) indicates that the VIGS experiment was successful. To detect the silencing efficiency of GhGIR1 in the material 'Aizi Cotton 3080', after albinism, leaf RNA was extracted from control plants (CK), negative control plants (TRV:00), and gene-silenced plants (TRV: GhGIR1), and gene expression levels were detected using qRT-PCR. It was found that the gene expression level of the TRV: GhGIR1 silenced line was significantly lower than that of the TRV:00 and CK lines in the leaves, while there was no significant difference in gene expression levels between CK and TRV:00. Figure 6 D). Phenotypic observation was conducted on lines with high gene silencing efficiency 21 days after injection into cotton seedlings. It was found that the number of leaf pubescence and stem pubescence in the silencing lines was significantly higher than that in TRV:00 plants. Figure 6 (C and E). The above results further demonstrate that GhGIR1 negatively regulates pubescence development by inhibiting pubescence initiation. Figure 6A: Distribution of epidermal hairs on leaves and stems of *Cotton aubergine* 3080 plants; B: Observation of epidermal hairs in plants treated with the TRV:00 empty vector; C: Analysis of changes in epidermal hairs in GhGIR1 gene-silenced lines; D: qRT-PCR detection of GhGIR1 gene silencing efficiency; E: Statistical analysis of leaf hair number before and after GhGIR1 gene silencing; F: Albinism phenotype of TRV:CLA cotton silencing lines. Studies have shown that GhHD1 promotes the initiation of leaf hairs and cotton fibers. The expression level of this gene in materials with abundant leaf hairs is significantly higher than that in materials with few leaf hairs. GhGIR1 negatively regulates leaf hair development. Therefore, is there a protein-level interaction between the two? This invention utilizes yeast two-hybrid technology to verify the mechanism by which GhHD1 and GhGIR1 regulate leaf hair development. GhHD1 was constructed into the pGBKT7 vector and fused with a GAL4 protein encoding a DNA-binding domain. Simultaneously, the self-activation activity of GhHD1 on the pGBKT7 vector was detected. GhGIR1 was constructed into the pGADT7 vector and co-transformed with GhHD1-pGBKT7 into Y2H yeast competent cells. The results were then evaluated on SD-TL and SD-TLHA plates containing 50 mM 3-AT. Figure 7 ,in Figure 7 A: Yeast two-hybrid assays verified the interaction between GhHD1 and GhGIR1. GhHD1 was ligated to the bait vector pGBKT7, and GhGIR1 was ligated to the prey vector pGADT7. B: Firefly enzyme complementation assays verified the interaction between GhHD1 (fused to the N-terminus of the firefly enzyme LUC) and GhGIR1 (fused to the C-terminus of the firefly enzyme) in tobacco epidermal cells. Two Agrobacterium strains containing the corresponding recombinant plasmids were simultaneously injected into the same tobacco cell. Figure 7 As shown in Figure A, all plaques could grow on the SD-TL amino acid-deficient culture plate, while only the positive control and plaques co-transformed with GhGIR1-pGADT7 and GhHD1-pGBKT7 grew on the SD-TLHA / 50 mM 3-AT culture plate; the others could not grow normally. These results indicate that GhGIR1 and GhHD1 interact at the protein level.

[0054] To ensure the reliability of the interaction results, we employed a luciferase complementation assay to further verify the interaction effect through transient expression in tobacco leaves. GhGIR1 was ligated into the JW772 35S LUC-C vector to form the GhGIR1-LUC-C recombinant vector; GhHD1 was ligated into the JW771 35S LUC-N vector to form the GhHD1-LUC-N recombinant vector. These vectors were then transformed into tobacco epidermal cells, and the fluorescence signal was observed. The results are as follows: Figure 7As shown in Figure B, only tobacco leaves transformed with GhGIR1-LUC-C+GhHD1-LUC-N emitted fluorescence, while other combinations and the empty vector control did not. These results further confirm that GhGIR1 and GhHD1 can interact at the protein level.

[0055] Interactions between GhGIR1 and GhHD1 at gene expression levels:

[0056] This invention performed dual-luciferase activity analysis in tobacco epidermal cells. The p35S-REN-5XGAL4-TATA-LUC reporter vector was used, containing REN luciferase driven by the 35S promoter and LUC luciferase driven by 5XGAL4-TATA. The CDS sequences of GhGIR1 and GhHD1 fused with Gal4DBD, as well as the single GhGIR1, were cloned into the pGreenⅡ62-SK vector to form SKGhGIR1, GalSKGhHD1, and GalSKGhGIR1 recombinant vectors as effector plasmids. The constructed vector structures are shown below. Figure 8 As shown in Figure A, the LUC / REN ratio and fluorescence signal intensity were used to determine whether the activity was activated or inhibited. The results (8B, D) showed that compared to the control, the luciferase activity of GalSKGhHD1 was significantly increased, while the luciferase activity was significantly decreased upon the addition of SKGhGIR1. The luciferase activity of GalSKGhGIR1 was relatively low and did not differ from the control. These results indicate that GhGIR1 can inhibit the transcriptional activity of GhHD1. Figure 8 A: Schematic diagram of the vector structure used in the figure. Reporting vector: 35S-REN-Gal4-luc vector. GalSKGhHD1: Gal4BD fused with the CDS of GhHD1. GalSKGhGIR1: Gal4BD fused with the CDS of GhGIR1. SKGhGIR1: The CDS of GhGIR1 constructed into the 62SK vector. B: The luciferase activity analysis experiment showed that GhGIR1 can significantly inhibit the transcriptional activation activity of GhHD1. LUC / REN represents the relative luciferase activity. Each experiment was performed in 10 biological replicates. Lowercase letters indicate that the difference is significant under the condition of P<0.05. C is the fluorescence imaging diagram corresponding to D. D: Qualitative results of luciferase activity.

[0057] To further elucidate the molecular mechanisms and regulatory networks of epidermal hair development in upland cotton, this invention analyzed the differentially expressed genes in the leaves of TRV:GhGIR1 silenced lines and TRV:00 control lines. Figure 9A). A total of 153 downregulated genes and 366 upregulated genes were screened. Among the downregulated genes, 76 were transcription factors, and among the upregulated genes, 197 were transcription factors. Figure 9 (B and C). KEGG enrichment analysis of differentially regulated genes revealed that the downregulated genes were mainly enriched in the plant hormone signal transduction and plant MAPK signal transduction pathways; the upregulated genes were mainly enriched in plant pathogen interactions and protein processing in the endoplasmic reticulum, indicating that the GhGIR1 gene regulates leaf trichome development by participating in the above pathways.

[0058] Cloning, identification, and functional analysis of the full-length GhGIR1 gene sequence:

[0059] Extraction of total RNA from cotton

[0060] Young cotton leaves and stems at the three-leaf stage were collected, rapidly ground in liquid nitrogen, and placed in pre-cooled 2.0 ml EP tubes. 600 μl of lysis buffer was added, and the tubes were shaken thoroughly and incubated at room temperature for 5 min. Total RNA was extracted using the Novizan RNA extraction kit according to the manufacturer's instructions. RNA quality and concentration were determined by 1.2% agarose gel electrophoresis and spectrophotometry. The tubes were stored at -80℃.

[0061] Cloning of the cotton GhGIR1 gene

[0062] Using total RNA as a template, cDNA was synthesized using the reverse transcription kit (AG11728) from Acrel Biotech Ltd., following the manufacturer's instructions. Gene-specific primers were designed based on the GhGIR1 gene sequence: forward primer F: 5'-ATGAGTGGTGGAAATGGTCCC-3' and reverse primer R: 5'-CTAATTTTTAATGGCGGGGAGA-3'. The GhGIR1 gene was obtained by PCR amplification using total cDNA as a template, and the results are as follows. Figure 10 As shown, the target gene with the corresponding band size was obtained, the PCR product was recovered and ligated into the T vector, and sent to Qingke Biotechnology Co., Ltd. for first-generation sequencing.

[0063] Through the above steps, the full-length 315 bp coding sequence (SEQ ID NO:1) of the transcribed protein in cotton was obtained, and its protein coding sequence (SEQ ID NO:2) was deduced, wherein the start codon is ATG and the stop codon is TAA.

[0064] The PCR procedure is shown in Table 1:

[0065]

[0066] The PCR system is shown in Table 2.

[0067]

[0068] Cotton GhGIR1 gene expression analysis

[0069] Using stem and leaf cDNA from the hairy cotton plants Zhongmian 19 and Taiyuan 02-6, and the less hairy cotton plants Aizi cotton 3080 and Suyuan 04-92 as templates, the NovoStart® Fast SYBR qPCR SuperMix (Cat. No.: E301) fluorescence quantitative PCR kit from Nearshore Biotechnology Co., Ltd. was used. histone3 As an internal reference gene, the GhGIR1 gene was validated by real-time PCR using forward primer F: 5'-TGAAACTGAACCTATCACCGCC-3' and reverse primer R: 5'-ACCTTCATGCATCTGGTTCGTCT-3'. Three technical replicates and three biological replicates were designed for each gene. The relative expression levels of the gene were analyzed in multi-epidermal hair and non-epidermal hair materials, according to a 2:1 ratio. –ΔΔCT Methods for calculating relative expression levels, such as Figure 3 The GhGIR1 gene was highly expressed only in the stems and leaves of leafless pubescent cotton varieties A. coggygria 3080 and Suyuan 04-92.

[0070] Subcellular localization analysis of the GhGIR1 gene

[0071] Using TOYOBO high-fidelity enzyme KODone, cDNA from upland cotton leaves was used as a template. Primers with corresponding adapters and restriction sites were used: upstream: 5'-acgggggactcttgaggatccATGAGTGGTGGAAATGGTCCC-3' (underlined BamHI restriction site); downstream: 5'-ccgggtaccgagctcgaattcATTTTTAATGGCGGGGAGAAA-3' (underlined EcoRI restriction site). The amplification was performed according to the instructions, removing the stop codon. GhGIR1 Full-length CDS of the gene. The empty vector pCAMBIA2300_35S_GFP-HA was double-digested with BamHI and EcoRI restriction enzymes to obtain a linearized vector; the target gene was ligated to the linearized vector to obtain the 35S:: GhGIR1 -GFP plant expression vector. A recombinant plasmid containing the target gene was used to transiently transform tobacco leaves into Agrobacterium competent cells GV3101 via liquid nitrogen freeze-thaw conversion. The injected tobacco plants were cultured overnight in darkness and then placed under a 16 / 8 h photoperiod for normal growth for 2 days. Subcellular localization results of the green fluorescence signal were then observed using a laser confocal microscope, such as... Figure 4As shown, green fluorescence signals are expressed in both the cell nucleus and cytoplasm. In the figure, a and d are observations under excitation light, while b and e are observations under visible light.

[0072] Phenotypic analysis of TRV: GhGIR1 silent strains

[0073] Using the TOYOBO high-fidelity enzyme KODone, cDNA from upland cotton leaves was used as a template. Primers with corresponding adapters and restriction sites were used: upstream 5'-gtgagtaaggttaccgaattcATGAGTGGTGGAAATGGTCCC -3' (underlined EcoRI restriction site); downstream 5'-cgtgagctcggtaccggatccCTAATTTTTAATGGCGGGGAGA -3' (underlined BamHI restriction site). Following the manufacturer's instructions, the silenced GhGIR1 gene fragment was amplified and ligated into the TRV2 vector digested with EcoRI and BamHI to form the TRV:GhGIR1 vector. After transformation with E. coli, KAN plate screening, colony PCR identification, and first-generation sequencing, the correctly sequenced recombinant plasmid was transformed into Agrobacterium LBA4404 via liquid nitrogen freeze-thaw method for subsequent silencing of the GhGIR1 gene in cotton. Agrobacterium LBA4404 containing TRV1 (auxiliary material), TRV2 (negative control), TRA2:CLA1 (positive control), and TRV2:GhGIR1 were respectively inoculated into a solution containing 50 μg / mL Kan + and 50 μg / mL Rif + In liquid LB medium, the culture was incubated overnight at 28°C and 200 r / min until the logarithmic growth phase. The Agrobacterium culture was then collected in 50 mL centrifuge tubes and centrifuged at 6500 rpm for 10 min to collect the Agrobacterium cells. The cells were thoroughly resuspended in an equal volume of resuspension solution (200 μmol / L AS + 10 mmol / L MgCl2 + 1 mmol / L MES) and adjusted to OD600 = 1.0, then incubated at room temperature for 3 h. For bacterial injection, TRV2:GhGIR1, empty vector TRV2, and positive control TRA2:CLA1 were mixed with the helper vector TRV1 at a 1:1 ratio and injected into two cotyledons of cotton using a 1 mL syringe. After injection, the cotton was placed in a culture chamber covered with plastic film to maintain humidity and incubated in the dark at 25°C for 24 h. Subsequently, the culture was allowed to grow normally in an artificial climate chamber with a photoperiod of (25°C / 14h light and 23°C / 10h dark).

[0074] To detect the silencing efficiency of the GhGIR1 gene in cotton: When the albino phenotype appears, RNA is extracted from cotton young leaves that are 2-3 weeks old and reverse transcribed into cDNA. The gene silencing efficiency is then detected by qRT-PCR.

[0075] Leaf hair count: Take 3 leaves (second from the bottom, third from the bottom, and fourth from the bottom functional leaves) from each plant, and count 3 fields of view for each leaf. Use an inverted microscope at 40x magnification to count the leaf hairs in each field of view.

[0076] Phylogenetic analysis of the GhGIR1 gene

[0077] Genome, CDS, and protein sequence data of different cotton species were downloaded from the cottongen website (https: / / www.cottongen.org / ), mainly including 5 allotetraploid cotton species, 2 diploid cultivated cotton species, and some wild cotton species. The GhGIR1 protein was clustered using MEGA software, and the gene structure of the GhGIR1 gene was plotted on the online website GSDS (http: / / gsds.gao-lab.org / ). Figure 4 As shown in the figure, AT: Arabidopsis thaliana, TURN: Gossypium Turnerii, Godar: Gossypium Darwinii, Gh: Upland cotton, Goari: Gossypium Raymondii, Gotom: Gossypium pubescens, Gomus: Gossypium chrysogenum, EVM: Gossypium Davidsonii, Golob: Gossypium lobatum, Gbar: Gossypium Sea Island, Kirkii: Gossypium Krowitzky, Golon: Gossypium longipes, Ga: Gossypium Asiatum, Ghe: Gossypium herbaceum, KAA: Gossypium australis, Goarm: Gossypium horseradish, Gohar: Gossypium harkenii. The results showed that the homologous genes of Gossypium Davidsonii and Gossypium longipes contain two exons and one intron, while the homologous genes of other cotton species contain only one exon. GhGIR1 is most closely related to Arabidopsis thaliana AT3G11660.

[0078] Yeast interaction analysis of GhGIR1 and GhHD1

[0079] GhHD1 was ligated into the empty vector pGBKT7 containing the GAL4 DNA binding domain. The correctly sequenced pGBKT7-GhHD1 plasmid was transformed into competent yeast cells Y2H and simultaneously plated on SD-Trp and / SD / -Trp / -His solid medium containing 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM 3-AT. The results showed that no yeast cells grew on the / SD / -Trp / -His plate containing 50 mM 3-AT, indicating that 50 mM 3-AT inhibited the self-activation activity of GhHD1. The full-length GhHD1 was cloned and ligated into the prey vector pGADT7 to form the pGADT7-GhGIR1 recombinant vector. pGBKT7-GhHD1+pGADT7, pGADT7-GhGIR1+pGBKT7, pGBKT7-GhHD1+pGADT7-GhGIR1, the positive control pGBKT7-p53+pGADT7-LargeT, and the negative control pGBKT7-LaminC+pGADT7-LargeT were co-transfected into Y2H yeast competent cells and plated on SD / -Trp / -Leu culture plates and incubated at 30℃ for 3-5 days. Clones were resuspended in 100 μl of sterile ddH2O and serially diluted 10-fold. 10 μl of the bacterial culture was then spotted onto SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade culture plates containing 50 mM 3-AT, and incubated at 30℃ for 3-5 days to further determine the interaction between the bait protein GhHD1 and the target protein GhGIR1. Results are as follows: Figure 7 As shown in Figure A: Only the positive control and pGBKT7-GhHD1+ pGADT7-GhGIR1 were able to grow on SD / -Trp / -Leu / -His / -Ade culture plates containing 50 mM 3-AT.

[0080] Analysis of luciferase complementation assays of GhGIR1 and GhHD1

[0081] Cloning the full-length CDS sequence of GhGIR1, and then cloning GhGIR1 The connection to via kpn I and Sal The GhGIR1-LUC-C recombinant vector was formed by digesting the JW772 35S LUC-C vector with enzyme I; the full-length CDS of GhHD1 was cloned, and GhHD1 was ligated into the vector. kpn I and Sal The GhHD1-LUC-N recombinant vector was formed on the JW771 35S LUC-N vector digested with enzyme I and transformed into E. coli. E. coliThe correctly sequenced recombinant plasmid and its corresponding empty vector were transformed into GV3101 Agrobacterium competent cells containing the helper vector psoup19, and plated on culture plates containing kanamycin and rifampin and incubated at 28 °C for 3 days. Single colonies were picked, propagated, collected, and resuspended to OD. 600 =Approximately 1.0, GhGIR1-LUC-C+JW771 35S LUC-N, GhHD1-LUC-N+JW772 35S LUC-C, JW771 35S LUC-N+JW772 35S LUC-C, and GhGIR1-LUC-C+GhHD1-LUC-N were injected into tobacco leaves that had grown for 3-4 weeks in equal volumes. The leaves were then incubated in the dark for 24 hours and under normal conditions for 48 hours. The results were observed using a fully functional luminescent and fluorescence bio-image analysis system. The results are as follows: Figure 7 As shown in B, only the combination of GhGIR1-LUC-C+ GhHD1-LUC-N can detect fluorescence signals.

[0082] Experimental analysis of transcriptional repression of GhGIR1

[0083] This invention first modifies the vector. Using the p35S-GAL4-TATA-LUC vector as a template, and employing primers 5XTATA-LUCHindIII-F: gtcgacggtatcgataagcttAGATCCGCTCGGAGGACAGT and 5XTATA-LUCBamHI-R: cgctctagaactagtggatccAGCGTGTCCTCTCCAAATGAAA, the 5XGAL4-TATA-LUC sequence is amplified by PCR and ligated into the pGreenII 0800-LUC vector digested with HindIII and BamHI to form the p35S-REN-5XGAL4-TATA-LUC recombinant vector, which serves as a reporter vector. The results are as follows: Figure 8 As shown in Figure A, the CDS of GhGIR1 was cloned into the pGreenII 62-SK vector to form the SKGhGIR1 recombinant vector. Using the yeast pGBKT7 plasmid as a template, the Gal4DBD sequence was cloned, and the full-length CDS sequences of GhHD1 and GhGIR1 were also cloned. Gal4DBD was fused with GhHD1 and GhGIR1 respectively via homologous recombination, and then ligated into the pGreenII 62-SK vector to form the GalSKGhHD1 and GalSKGhGIR1 recombinant vectors, which served as effector plasmids. Figure 8As shown in Figure A. To verify the inhibitory effect of GhGIR1 on GhHD1, the following combinations were injected into tobacco epidermal cells: 62SK+p35S-REN-5XGAL4-TATA-LUC (CK), GalSKGhHD1+p35S-REN-5XGAL4-TATA-LUC, SKGhGIR1+GalSKGhHD1, and GalSKGhGIR1+p35S-REN-5XGAL4-TATA-LUC. The ratio of effector to reporter vector was 2:1. The dual-luciferase activity was measured using the Dual-Luciferase Reporter Assay System kit (Promega, Cat. No. E1910) according to the manufacturer's instructions. The relative luciferase activity was calculated as LUC / REN. Figure 8 As shown in B and C. Simultaneously, a full-function luminescence and fluorescence bio-imaging analysis system was used for qualitative detection of fluorescence, such as... Figure 8 As shown in D. The results show that GhGIR1 can inhibit the transcriptional activity of GhHD1.

[0084] TRV: Transcriptome sequencing of GhGIR1 silencing lines

[0085] Total RNA was extracted from leaf samples of TRV:00 and TRV:GhGIR1 using the RNA extraction kit from Novizan. The RNA was analyzed by agarose gel electrophoresis and spectrophotometry. The qualified RNA was sent to Wuhan Aijibaike Biotechnology Co., Ltd. for cDNA library construction. Paired-end sequencing was performed using the Illumina HiSeq 4000 platform to obtain raw sequencing data. The raw data was converted to FASTQ format, and adapters and low-quality data were removed using FastQC software to obtain high-quality clean reads. The clean reads were aligned to the third-generation genome of *Cotton Uplande* TM-1 using TopHat2 software, and the sequences aligned to the reference genome were annotated. Gene expression levels were determined using fragments per kilobase of transcript per million fragments mapped (FPKM) values. Differential gene expression analysis was performed on the count values ​​of TRV:00 and TRV:GhGIR1 lines using the "Deseq2" R software package. mRNAs with FPKM < 0.5 were filtered out, and selection was based on a false discovery rate (FDR) < 0.01 and |log2 foldchange| ≥ 1. PA -value <0.01 was used as the criterion for screening differentially expressed genes. To elucidate the function of these differentially expressed genes, KEGG (http: / / www.genome.jp / kegg / ) enrichment pathway analysis was performed. The results showed that GhGIR1 is primarily involved in plant hormone signal transduction pathways. Among them, Figure 9 A: Volcano plot showing the distribution of all gene expression changes and p-values; B: Venn plot showing the number of transcription factors contained in downregulated genes; C: Venn plot showing the number of transcription factors contained in downregulated genes; D: KEGG enrichment pathway analysis of differentially regulated genes.

[0086] All primers used in the embodiments of this invention were synthesized at Qingke Biotechnology Co., Ltd. The Agrobacterium involved in this invention is Agrobacterium tumefaciens (…). Agrobacterium tumefaciens The competent strain LBA4404 can be purchased commercially (Shanghai Weidi Biotechnology Co., Ltd.), Escherichia coli ( Escherichia coli The competent DH5α cells were purchased from Kangwei Century Company. The competent yeast strain Y2H involved in this invention was preserved in our laboratory. All primers involved in the embodiments of this invention were synthesized at Qingke Biotechnology Co., Ltd.

Claims

1. A method for negatively regulating the development of epidermal pubescence in upland cotton by regulating genes responsible for the leaf pubescence trait, characterized in that, The method involves the gene GhGIR1 regulating the development of leaf trichomes in upland cotton by participating in plant hormone signal transduction pathways. Silencing the GhGIR1 gene in cotton increases the number of leaf trichomes. This gene has only one exon and no introns, and its length is 315 bp. The nucleotide sequence of the gene is shown in SEQ ID No. 1 of the sequence listing.

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