Application of GhCOL2 gene in regulation and control of cotton flower primordium differentiation

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly discloses application of a GhCOL2 gene in regulation and control of cotton flower primordium differentiation. The coding sequence of the GhCOL2 gene is as shown in SEQ ID No.1 (sequence identifier number 1). According to the invention, the GhCOL2 gene is knocked out by using sgRNA target sequences as shown in SEQ ID No.2 and SEQ ID No.3 through a CRISPR / Cas9 gene editing technology, so that the quantity of the cotton flower primordia can be effectively increased. The invention provides new gene resources and technical means for cotton flower organ development regulation and breeding, and has important agricultural application value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more particularly to... GhCOL2 Application of genes in regulating cotton flower primordia differentiation. Background Technology

[0002] Cotton is an important economic crop, and floral primordium differentiation directly affects the formation of floral organs and yield. Breeding cotton varieties with well-developed floral primordium differentiation is crucial for increasing cotton yield. To achieve this goal, it is necessary to elucidate the regulatory mechanisms of floral primordium differentiation at the molecular level. Transcription factors play a key regulatory role in plant growth and development; therefore, identifying the key genes regulating cotton floral primordium differentiation is of great value for cotton genetic improvement. Currently, research on the key genes regulating floral primordium differentiation in cotton is not yet in-depth, and there is a lack of effective gene resources and technical means to directionally improve the characteristics of cotton floral primordium differentiation. Summary of the Invention

[0003] To address the pressing technical problems that need to be solved in this field, the present invention provides a GhCOL2 Application of genes in regulating cotton flower primordia differentiation.

[0004] The specific technical solution is as follows: First aspect of protection of the present invention GhCOL2 The use of genes or related biological materials in at least one of the following: A1) Regulating cotton flower primordium differentiation or preparing products that regulate cotton flower primordium differentiation; A2) Cultivate cotton that promotes the differentiation of flower primordia or prepare products that promote the differentiation of cotton flower primordia; A3) Preparation of genetically modified cotton; The GhCOL2 The gene's Gene ID in the CottonFGD database is: Ghir_D12G005870.1 Its nucleotide sequence contains the sequence shown in SED ID No. 1.

[0005] Furthermore, the biomaterial includes one or more of the following: B1) contains the above GhCOL2 Recombinant vectors of genes, recombinant microorganisms, or transgenic cotton cell lines; B2) contains negative regulation GhCOL2 Nucleic acid molecules expressing genes, recombinant vectors, recombinant microorganisms, or transgenic cotton cell lines.

[0006] The containing GhCOL2 The original vectors for gene recombination vectors include the PRGEB32 vector.

[0007] The containing GhCOL2 Recombinant microorganisms include Agrobacterium tumefaciens EHA105.

[0008] The negative regulation GhCOL2 The nucleic acid molecules expressing the gene include a single guide RNA (hereinafter referred to as sgRNA), the nucleotide sequence of which is shown in SEQ ID No. 2 and SEQ ID No. 3; The inhibition GhCOL2 The original vectors for recombinant gene expression vectors include the PRGEB32 vector.

[0009] The inhibition GhCOL2 Recombinant microorganisms that express genes include Agrobacterium tumefaciens EHA105.

[0010] Furthermore, the regulation of cotton flower primordia differentiation includes: regulating the number of cotton flower primordia.

[0011] Furthermore, the regulation of the number of cotton flower primordia includes: overexpression GhCOL2 Genes that reduce the number of cotton flower primordia; Or, by inhibiting GhCOL2 Gene expression increases the number of cotton flower primordia.

[0012] The second aspect of protection of the present invention inhibits GhCOL2 The application of gene-expressing biological materials in the cultivation of cotton that promotes floral primordium differentiation. GhCOL2 The nucleotide sequence of the gene contains the sequence shown in SED ID No. 1.

[0013] Furthermore, the biomaterial includes: inhibition GhCOL2 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic cotton cell lines.

[0014] Furthermore, promoting the differentiation of flower primordia includes increasing the number of cotton flower primordia.

[0015] A third aspect of the present invention protects a method for promoting cotton flower primordia differentiation, comprising: inhibiting... GhCOL2 Gene-expressing biological materials are transferred into cotton.

[0016] A fourth aspect of this invention protects a method for cultivating cotton that promotes floral primordia differentiation, comprising: inhibiting... GhCOL2 Gene-expressing biological materials are transferred into cotton.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is aimed at GhCOL2 Genes were designed to work with sgRNA and a targeted gene was constructed. GhCOL2The CRISPR-Cas9 vector, compared with wild-type Jin668, yielded Ghcol2 The mutant has a significantly increased number of flower primordia, providing a new technical means for improving cotton flower primordia differentiation.

[0018] (2) This invention discloses GhCOL2 The expression of the gene in cotton leaves and stems exhibits a clear diurnal rhythm and participates in the cotton's light signal response process.

[0019] (3) The present invention also clarifies that GhCOL2 The specific expression pattern of the gene in the shoot apical meristem of the flower primordium differentiation reveals its regulatory role. Attached Figure Description

[0020] Figure 1 In Example 1 GhCOL2 A schematic diagram of the gene structure and CRISPR-Cas9 target sites; among which... Figure 1 A is GhCOL2 Gene structure; Figure 1 B is a schematic diagram of the CRISPR-Cas9 target.

[0021] Figure 2 In Example 2 GhCOL2 Sequence analysis of gene-edited mutants.

[0022] Figure 3 In Example 3 GhCOL2 Statistics on the number of floral primordia in gene-edited mutants.

[0023] Figure 4 In Example 4 GhCOL2 The diurnal rhythm expression pattern of genes in cotton leaves and stems.

[0024] Figure 5 In Example 5, the cotton shoot tip meristem GhCOL2 In situ hybridization analysis of genes. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0026] Unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the reagents and materials used are commercially available.

[0027] In this invention, the GhCOL2 The coding sequence of the gene is shown in SED ID No. 1.

[0028] SED ID No. 1:

[0029] For details on the specific components of the various culture media used in the transgenic process of this invention, please refer to the reference: Xiao Li # Yuanlong Wu, Zhenping Liu, Hengling Wei, Hantao Wang, and Shuxun Yu*. Apicalmeristem transcriptome analysis identifies a role for the blue light receptor gene. GhFKF1 In cotton architecture development. The crop journal, 2024, 12(4):1126-1136, specifically: MS basal medium (based on the preparation of 1L stock solution): Potassium nitrate (KNO3) 38.0g, magnesium sulfate (MgSO47H2O) 7.4g, potassium dihydrogen phosphate (KH2PO4) 3.4g, and calcium chloride (CaCl22H2O) 8.8g were dissolved in an appropriate amount of deionized water and brought to a final volume of 1L to prepare MS macro-element stock solutions, which were stored at 4℃ for later use. MS micro-element stock solutions, iron salt stock solutions, and organic component stock solutions were prepared according to conventional methods.

[0030] Sterile seedling culture medium (per 1L): MS macro-element stock solution 25mL, glucose 15g, plant gel (Phytagel) 2.6g; adjust pH to 6.1-6.2, sterilize and use for cotton seed surface disinfection and aseptic seedling culture.

[0031] Dedifferentiation medium (per 1 L): 50 mL MS macro-element stock solution, 10 mL MS micro-element stock solution, 10 mL iron salt (Molysite) stock solution, 10 mL inositol (Inose) stock solution, 10 mL ammonium nitrate (NH4NO3) stock solution, 1 mL L-glycine (L-Gly) stock solution (1 mg / mL), 1 mL B5 organic complex stock solution, 1 mL 2,4-dichlorophenoxyacetic acid (2,4-D) stock solution (1 mg / mL), 0.2 mL kinetin (KT) stock solution (1 mg / mL), 0.9-1.0 g magnesium chloride hexahydrate (MgCl26H2O), 30 g glucose, and 2.6 g plant gel; adjust the pH to 5.85-5.95, sterilize, and use for callus induction in explants.

[0032] Redifferentiation medium (per 1L): MS macro-element stock solution 50 mL, MS micro-element stock solution 10 mL, iron salt stock solution 10 mL, inositol stock solution 10 mL Potassium nitrate (KNO3) stock solution (100g / L) 50mL, L-glycine stock solution (1mg / mL) 1mL, B5 organic complex stock solution 1mL, indole-3-butyric acid (IBA) stock solution (1mg / mL) 1mL, kinetin (KT) stock solution (1mg / mL) 0.3mL, glutamine (Gln) 1.0g, asparagine (Asn) 0.5g, glucose 30g, plant gel 2.6g; adjust pH to 6.1-6.2, sterilize, and use for bud differentiation of embryoids or callus tissue.

[0033] Rooting medium (per 1L): MS macro-element stock solution 25mL, MS micro-element stock solution 5mL, iron salt stock solution 5mL, inositol stock solution 10mL, L-glycine stock solution (1mg / mL) 1mL, B5 organic complex stock solution 1mL, glucose 15g, plant gel 2.6g; adjust pH to 5.90-5.95, sterilize, and use for root induction of regenerated seedlings.

[0034] Example 1 GhCOL2 Construction of CRISPR-Cas9 gene editing vector For cotton GhCOL2 The B-box domain region of the gene (whose gene coding sequence is shown in SEQ ID No. 1) Figure 1 A), design sgRNA1 and sgRNA2 ( Figure 1 B) The synthesis was commissioned to a biotechnology company. The activity of the sgRNA was verified through in vitro cleavage experiments to ensure its efficient guidance of Cas9 protein recognition and cleavage. GhCOL2 Gene target sequences were selected to avoid off-target effects. The validated sgRNA coding sequence was inserted into a CRISPR-Cas9 vector to construct a gene editing vector.

[0035] sgRNA1 (SEQ ID No. 2): 5'-CCCTGTTGTGCAAGGCAGACGCT-3'; sgRNA2 (SEQ ID No. 3): 5'-CCTCTGTGCTGGATGTGATGCCC-3'.

[0036] Example 2 GhCOL2 Obtaining gene-edited cotton plants The experimental steps for vector construction and detection and analysis of genetically transformed materials are as follows: This study employed CRISPR-Cas9 technology to implement a gene editing strategy, with specific procedures following existing research methods (Pengcheng Wang).# Jun Zhang # , Lin Sun, Yizan Ma, Jiao Xu, Sijia Liang, JinwuDeng, Jiafu Tan, Qinghua Zhang, Lili Tu, Henry Daniell, Shuangxia Jin*, Xianlong Zhang*. High efficient multisites genome editing in allotetraploidcotton (Gossypium hirsutum) using CRISPR / Cas9 system. Plant Biotechnol J, 2018, 16(1): 137-150.; Detailed steps are as follows: Plump, mature seeds of the Jin668 variety, provided by the cotton research team of the State Key Laboratory of Huazhong Agricultural University, were selected and sterilized for 10 minutes using a mercuric chloride solution with a concentration of 1 g mercuric chloride / 1000 ml double-distilled water. The sterilized seeds were inoculated into a sterile seedling culture medium and cultured in a 28℃ dark incubator for 3-5 days. The hypocotyls of the seedlings were then cut into small segments using a sharp scalpel to serve as explants.

[0037] In the vector construction stage, the CRISPR-P online tool (Liu, H., Ding, Y., Zhou, Y., Jin, W., Xie, K. & Chen, LL (2017). CRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants. Molecular Plant, 10, 530-532.) was used to construct the vector in cotton. GhCOL2The exon regions of the gene were screened and target sites of 23 base pairs in length (sequence characteristic 5'-N20NGG-3') were identified. The specificity of each candidate target site was then evaluated using a potential off-target site prediction website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Two independent single guide RNAs (sgRNAs) targeting the gene were subcloned into the modified vector pRGEB32GhU6.7-NPTⅡ. (This application modifies the PRGEB32 vector by replacing the eukaryotic resistance marker hygromycin with kanamycin resistance, and adding the cotton endogenous U6 promoter (numbered U6-7 and U6-9) to guide gRNA transcription. However, the rice Ubiquitin2 promoter still drives Cas9 expression, and the prokaryotic resistance is kanamycin. The modified vector is named pRGEB32-GhU6.7-NPTⅡ; for details of the vector modification, please refer to the reference Pengcheng Wang.) # Jun Zhang # , Lin Sun, Yizan Ma, Jiao Xu, Sijia Liang, Jinwu Deng, Jiafu Tan, Qinghua Zhang, Lili Tu, Henry Daniell, Shuangxia Jin*, Xianlong Zhang*. High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR / Cas9 system. Plant Biotechnol J, 2018, 16(1): 137-150.

[0038] The recombinant vector was introduced into Agrobacterium tumefaciens using electroporation. Agrobacterium tumefaciens L.) EHA105 strain. Immerse the explants in EHA105 bacterial solution for 2-3 minutes, gently shaking occasionally; then transfer to co-culture medium plates and incubate at 20°C in the dark for 48 hours.

[0039] The treated explants were inoculated into callus dedifferentiation medium and placed in a 27°C culture environment. A photocycle culture mode of 12 hours light / 12 hours dark was adopted. The explants were placed flat on the surface of the medium and transferred to fresh medium for subculture every 25 days.

[0040] After the embryoids have formed, they are transferred to a redifferentiation medium. When the seedlings have grown at least one true leaf, they are transferred to a rooting medium to induce the initiation and differentiation of root and shoot tip meristems.

[0041] Genomic DNA was extracted from candidate gene-edited cotton plants using the CTAB method, targeting... GhCOL2 Specific primers for detecting sgRNA1 (SEQ ID No. 4, SEQ ID No. 5) and sgRNA2 (SEQ ID No. 6, SEQ ID No. 7) were designed based on the sequences flanking the gene editing target. The fragment was then amplified by PCR to verify the detection. GhCOL2 Whether the expected gene editing occurs.

[0042] sgRNA1 detection primers: Forward primer: 5'-GgagtgagtacggtgtgcAACCGGGTCGCCTCGCGC-3' (SEQ ID No. 4); Reverse primer: 5'-gagttggatgctggatggGCGGATTGGCGGAATGGAT-3' (SEQ ID No. 5); sgRNA2 detection primers: Forward primer: 5'-GgagtgagtacggtgtgcTCCGCAGCTTGCACAGTGTAC-3' (SEQ ID No. 6); Reverse primer: 5'-gagttggatgctggatggGCACGCTCACATACCTCGCA-3' (SEQ ID No. 7).

[0043] The PCR reaction procedure is as follows: Pre-denaturation: 95℃ for 4 minutes; Denaturation: 95℃ for 30 seconds; Annealing: 59℃ for 30 seconds; Extension: 72℃ for 20 seconds; The above denaturation, annealing, and extension steps are repeated 28 times. Final extension: 72℃ for 5 minutes.

[0044] Sequencing analysis of PCR products and statistical analysis were performed. GhCOL2 Gene editing efficiency (including the proportion of base deletions, insertions, or substitutions) was screened to obtain... Ghcol2 Homozygous mutant. For example... Figure 2 As shown, compared to wild type Ghcol2 mutant GhCOL2 Gene sequence, the results showed, GhCOL2 The gene has undergone various forms of insertion or deletion mutations ( Figure 2 ).

[0045] Example 3 Ghcol2Phenotypic identification of floral primordia of mutants In the transgenic cotton experimental field of Huazhong Agricultural University, wild-type cotton (Jin 668) and... Ghcol2 The mutants were subjected to phenotypic observation and statistical analysis, and conventional field water and fertilizer management was adopted.

[0046] The results showed that, compared with the wild type, Ghcol2 The mutant has a significantly greater number of floral primordia than the wild type. Figure 3 ),illustrate GhCOL2 Gene editing significantly promoted the differentiation of cotton flower primordia.

[0047] Example 4 GhCOL2 Circadian rhythm expression analysis of genes The upland cotton material TM-1 was planted in a light incubator with a temperature of 28℃ and a day length of 16 hours of light / 8 hours of darkness. When the second true leaf unfolded, stems and leaves were collected at 0:00, 6:00, 9:00, 12:00, 15:00, 18:00, 21:00 and 24:00 of each day, and were flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80℃ for RNA extraction and expression level detection and analysis.

[0048] The specific experimental steps of real-time quantitative PCR technology are as follows: Total RNA was extracted using TRIzol reagent (Invitrogen). 3 micrograms of total RNA were taken and complementary DNA (cDNA) was synthesized using Oligo(dT)18 primers and TransScript® II one-step method to remove genomic DNA and synthesize cDNA using a super mixture (TransGen).

[0049] Real-time quantitative PCR (qRT-PCR) detection: Gene transcription level detection and analysis were performed using Premix Ex Taq reagent (Takara, Takara Bio) and a QuantStudio 6 Flex real-time quantitative PCR system. The total reaction volume was 20 μL. 2 μL of 10-fold diluted cDNA was used as a template, and TB Green® Premix Ex Taq reagent (Takara, Takara Bio) was added for amplification. The reaction program was: 95℃ pre-denaturation for 30 seconds; followed by 40 cycles, each cycle consisting of 95℃ denaturation for 5 seconds and 60℃ annealing extension for 30 seconds. Gene expression levels were measured using the cotton ubiquitin protein gene. GhUBQ7 (GenBank accession number: DQ116441) and histone genes GhHis3 As an internal reference gene, it is used to standardize and correct RNA content.

[0050] The results show GhCOL2Gene expression exhibits a distinct diurnal rhythm, and the expression patterns differ between leaves and stems. Figure 4 This indicates that its expression is regulated by photoperiod and participates in the light signal response process of cotton.

[0051] Example 5 GhCOL2 In situ hybridization analysis of genes in cotton shoot tip meristem Shoot apical meristems of the early-maturing cotton variety Zhongmian 58 and the late-maturing cotton variety Xinluzhong 47 were analyzed at different growth stages (stage 1: vegetative growth period - day 10 after emergence; stage 2: transition period from vegetative to reproductive growth - day 20 after emergence; stage 3: reproductive growth stage - day 30 after emergence). GhCOL2 In situ hybridization analysis of genes. Plasmids with correct base orientation were linearized, and then RNA probes were synthesized using a digoxigenin-labeled mixture (Roche Diagnostics Products, Ltd.). The specific experimental steps of in situ hybridization are as follows: First, sections of cotton stem tip meristem were vacuum-treated and then fixed in FAA fixative (Saiwell Biotech, catalog number: G1112) for 24 hours. After fixation, the tissue samples were subjected to gradient alcohol dehydration, paraffin embedding, and embedding.

[0052] Paraffin-embedded blocks were sectioned using a microtome (Thermo Fisher Scientific, model HM 340E), and the sections were collected using a spreader and baked in a 62°C oven for 2 hours.

[0053] The slices were processed sequentially as follows: soaked in xylene I for 15 minutes → soaked in xylene II for 15 minutes → soaked in anhydrous ethanol I for 5 minutes → soaked in anhydrous ethanol II for 5 minutes, and then air-dried before being immersed in diethyl pyrocarbonate (DEPC) water.

[0054] Add a proteinase K solution with a concentration of 20 μg / mL and incubate at 37°C for enzymatic digestion. After rinsing with pure water, wash the sample three times with phosphate-buffered saline (PBS) for 5 minutes each time.

[0055] Add prehybridization solution and incubate at 37°C for 1 hour; discard the prehybridization solution and add hybridization solution containing target gene probe (concentration of 400-1000 ng / mL), hybridize at 42°C for 16-20 hours.

[0056] Hybridization solution washing procedure: Wash with 2× standard citrate buffer (SSC) at 37°C for 10 minutes, wash twice with 1× SSC at 37°C (5 minutes each time), and wash with 0.5× SSC at room temperature for 10 minutes.

[0057] Add blocking serum and incubate at room temperature for 30 minutes; discard the blocking solution, add digoxigenin-labeled alkaline phosphatase secondary antibody (anti-DIG-AP), and incubate at 37°C for 50 minutes. Then wash four times with tris(hydroxymethyl)aminomethane buffered saline (TBS), 5 minutes each time.

[0058] Add 0.3-0.8 mL of 5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium (BCIP / NBT) colorimetric solution, incubate overnight in the dark, and then rinse the section with pure water.

[0059] The sheets were sealed with glycerin gelatin and dried at 37°C.

[0060] Finally, the hybridization sections were scanned using a Nikon ECLIPSE CI microscope, and the images were observed using Cas Viewer software. Positive expression of BCIP / NBT was detected as a bluish-purple color, with the intensity varying depending on the expression level.

[0061] generally, COL2 Genes perform their primary functions in leaves, while their rhythms in stems may be related to other processes (such as stem elongation) or provide local signals for the activation of floral meristems. Results showed that during floral primordium differentiation, GhCOL2 Genes have specific expression distributions in the shoot apical meristem ( Figure 5 This further demonstrates its regulatory role in cotton flower primordia differentiation.

Claims

1. GhCOL2 The use of genes or related biological materials in at least one of the following: A1) Regulating cotton flower primordium differentiation or preparing products that regulate cotton flower primordium differentiation; A2) Cultivate cotton that promotes the differentiation of flower primordia or prepare products that promote the differentiation of cotton flower primordia; A3) Preparation of genetically modified cotton; The GhCOL2 The gene's Gene ID in the Cotton FGD database is: Ghir_D12G005870.1 Its nucleotide sequence contains the sequence shown in SED ID No.

1.

2. The application as described in claim 1, characterized in that, The biomaterials include one or more of the following: B1) contains the above GhCOL2 Recombinant vectors of genes, recombinant microorganisms, or transgenic cotton cell lines; B2) contains negative regulation GhCOL2 Nucleic acid molecules expressing genes, recombinant vectors, recombinant microorganisms, or transgenic cotton cell lines.

3. The application as described in claim 1 or 2, characterized in that, The regulation of cotton flower primordia differentiation includes: regulating the number of cotton flower primordia.

4. The application as described in claim 3, characterized in that, The regulation of cotton flower primordia number includes: overexpression GhCOL2 Genes that reduce the number of cotton flower primordia; Or, by inhibiting GhCOL2 Gene expression increases the number of cotton flower primordia.

5. Inhibition GhCOL2 The application of gene-expressing biological materials in the cultivation of cotton that promotes floral primordium differentiation is characterized by, The GhCOL2 The nucleotide sequence of the gene contains the sequence shown in SED ID No.

1.

6. The application as described in claim 5, characterized in that, The biomaterial includes: inhibitory GhCOL2 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic cotton cell lines.

7. The application as described in claim 5, characterized in that, The promotion of flower primordia differentiation includes increasing the number of cotton flower primordia.

8. A method for promoting the differentiation of cotton flower primordia, characterized in that, include: Will suppress GhCOL2 Gene-expressing biological materials are transferred into cotton.

9. A method for cultivating cotton that promotes floral primordium differentiation, characterized in that, include: Will suppress GhCOL2 Gene-expressing biological materials are transferred into cotton.

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