Application of GhATG18a gene in accelerating cotton callus induction

Abstract

This invention discloses the application of the GhATG18a gene in accelerating callus induction in cotton. The invention constructs a pCAMBIA2300-GhATG18a overexpression vector from the full-length CDS fragment of this gene, then introduces it into Agrobacterium, and transforms cotton using Agrobacterium-mediated transformation to obtain transgenic lines. qRT-PCR results show that, compared with the CCRI24 control material, the expression level of the GhATG18a gene in the overexpression lines OE-GhATG18a-1 and OE-GhATG18a-2 is significantly upregulated. Phenotypic observation and histological sections both show that the OE-GhATG18a line overexpressing this gene accelerates the process of somatic cell dedifferentiation and callus induction in cotton, thereby improving the genetic transformation efficiency of cotton. Therefore, overexpressing this gene and applying it to commercial cotton varieties that are difficult to genetically transform can help accelerate the callus induction process, thereby improving the genetic transformation efficiency and providing technical support for breeding high-yield and stress-resistant high-quality cotton germplasm.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to the application of the GhATG18a gene in accelerating cotton callus induction. Background Technology

[0002] Cotton, as one of the world's most important economic crops, is a core raw material for the textile industry and an important source of oil, occupying a strategic position in my country's national economy. However, current major varieties face severe challenges in terms of fiber quality, yield, and stress resistance, and traditional breeding techniques are insufficient to meet the needs of industrial upgrading. Although genetic engineering technology has provided a new path for crop improvement, cotton genetic transformation systems have long been limited by bottlenecks such as genotype dependence, high somatic embryo deformity rate, and long regeneration cycle. Despite recent breakthroughs in cotton transgenic technology in my country (such as the "stem cell-based" transformation system developed by the Chinese Academy of Agricultural Sciences), mainstream commercial varieties such as upland cotton still face the common problem of low transformation efficiency, severely restricting the application effectiveness of transgenic technology in breeding.

[0003] Somatic embryogenesis, as a core pathway for the expression of plant cell totipotency, reprograms cell fate through three stages: callus initiation (dedifferentiation), callus proliferation, and embryogenic transformation (redifferentiation). Its efficiency is synergistically regulated by hormonal signaling, mechanical damage, and stress response networks. Systematic analysis of the molecular regulatory mechanisms of somatic embryogenesis, screening of key callus initiation (dedifferentiation) regulators, and development of broadly applicable genetic transformation tools will be a crucial breakthrough in overcoming genotype limitations and establishing an efficient cotton genetic improvement system, providing theoretical support and technological reserves for ensuring national cotton security. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of the GhATG18a gene in accelerating cotton callus induction.

[0005] To achieve the above objectives, one aspect of the technical solution of the present invention is to provide an application of the GhATG18a gene in accelerating the induction of cotton callus.

[0006] Furthermore, the nucleotide sequence of the GhATG18a gene is shown in SEQ ID NO.3.

[0007] Furthermore, the GhATG18a gene was amplified using the CCRI24 genome as a template and with primers shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] Furthermore, the amino acid sequence encoded by the GhATG18a gene is shown in SEQ ID NO.4.

[0009] Furthermore, the GhATG18a gene was constructed into the pCAMBIA2300 vector and transformed into cotton to enhance the expression of the GhATG18a gene in cotton materials and accelerate the process of cotton somatic cell dedifferentiation to induce callus.

[0010] Furthermore, the pCAMBIA2300 vector sequence is shown in SEQ ID NO.5.

[0011] Furthermore, the cotton in question is upland cotton.

[0012] On the other hand, the technical solution of the present invention is to provide a recombinant expression vector containing the GhATG18a gene and genetically engineered bacteria.

[0013] On the other hand, the technical solution of the present invention is to provide the application of the recombinant expression vector and genetically engineered bacteria in accelerating the process of cotton somatic cell dedifferentiation to induce callus.

[0014] On the other hand, the technical solution of the present invention is to provide the protein encoded by the GhATG18a gene.

[0015] The beneficial effects of this invention are:

[0016] This invention integrates CCRI24 callus initiation transcriptome data from upland cotton to screen for the autophagy gene GhATG18a, which is significantly upregulated in the early dedifferentiation stage (0-3 days). This invention provides the application of the GhATG18a gene in somatic embryogenesis of upland cotton. The full-length CDS fragment DNA sequence of this gene was used to construct the pCAMBIA2300-GhATG18a overexpression vector, which was then introduced into Agrobacterium and transformed into cotton using Agrobacterium-mediated transformation to obtain transgenic lines. The expression level of GhATG18a in the overexpression lines was detected by qRT-PCR. The results showed that compared with the CCRI24 control material, the expression level of the GhATG18a gene in the overexpression lines OE-GhATG18a-1 and OE-GhATG18a-2 was significantly upregulated. After seed treatment, the OE-GhATG18a and CCRI24 lines were cultured as sterile seedlings under dark conditions. Hypocotyl segments from these sterile cotton seedlings were used as explants on callus induction medium for induction regeneration. Phenotypic changes at various stages of somatic embryogenesis were observed. Results showed that the OE-GhATG18a line, overexpressing this gene, exhibited hypocotyl swelling two days after callus induction, initiating dedifferentiation earlier than CCRI24. Histological section analysis further confirmed that the OE-GhATG18a line formed secondary meristem cells approximately one day earlier than the control, with an onset time shortened by about 30%. This indicates that the GhATG18a gene plays a crucial role in cotton somatic embryogenesis. Therefore, overexpressing this gene and applying it to commercially viable cotton varieties with difficult genetic transformation can accelerate callus induction, providing technical support for cultivating high-yield and stress-resistant high-quality cotton germplasm. Attached Figure Description

[0017] Figure 1 Transcriptome data analysis of CCRI24 callus initiation period in upland cotton.

[0018] Figure 2 Analysis of the GhATG18a protein domain.

[0019] Figure 3 This is a schematic diagram of the pCAMBIA2300-GhATG18a overexpression vector.

[0020] Figure 4 The image shows the PCR detection results of the GhATG18a gene in transgenic cotton plants. In the figure, OE-GhATG18a-1 and OE-GhATG18a-2 are two transgenic lines; CCRI24 is the non-transgenic control; V is the plasmid positive control; and M is the marker III molecular weight standard.

[0021] Figure 5Analysis of GhATG18a gene expression levels in the OE-GhATG18a strain.

[0022] Figure 6 The study investigated the induction of somatic embryogenesis from explants of GhATG18a gene-transformed materials and CCRI24 control materials from day 0 to 30. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] The cotton variety used in the following examples is upland cotton (Gossypium hirsutum L.) variety CCRI24, sourced from the germplasm bank of the Cotton Research Institute, Chinese Academy of Agricultural Sciences, hereinafter referred to as CCRI24. The hormones used in the following examples are from Sigma-Aldrich; the culture media and other products are from reagent companies.

[0026] Aseptic seedling culture medium: solutes and their concentrations are: NH4NO3 1650mg / L, KNO3 1900mg / L, KH2PO4 170mg / L, MgSO4·7H2O 370mg / L, CaCl2·2H2O 440mg / L, sucrose 25g / L, plant gel 2g / L; solvent is distilled water; pH 5.8.

[0027] MSB medium: Solutes and their concentrations are as follows: NH4NO3 1650 mg / L, KNO3 1900 mg / L, KH2PO4 170 mg / L, MgSO4·7H2O 370 mg / L, CaCl2·2H2O 440 mg / L, FeSO4·7H2O 27.85 mg / L, Na2EDTA 37.25 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MOO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, COCl2·6H2O 0.025 mg / L, inositol 100 mg / L, VB1 10 mg / L, VB6 1.0 mg / L, nicotinic acid 1.0 mg / L, sucrose 28 g / L, agar 5.6 g / L; solvent: distilled water; pH 5.8.

[0028] MSBI medium: 2,4-D (2,4-dichlorophenoxyacetic acid) and KT (6-furfurylaminopurine) were added to MSB medium to make the concentration of 2,4-D and KT 0.5 mol / L, thus obtaining MSBI medium.

[0029] Embryogenic callus induction medium: KT and IAA (indoleacetic acid) were added to MSB medium until their concentrations were both 0.5 mol / L to obtain embryogenic callus induction medium.

[0030] Embryomorph induction medium: KT and 6-BA (6-benzyladenine) were added to MSB medium until their concentrations were both 0.01 mol / L to obtain embryomorph induction medium.

[0031] Rooting medium: Add 6-BA to MSB medium until its concentration is 0.1 mol / L to obtain the rooting medium.

[0032] Example 1: Identification of the GhATG18a gene

[0033] Autophagy, a core mechanism for plants to cope with abiotic stress, plays a crucial role in stress signal-mediated cell reprogramming. This invention integrates CCRI24 transcriptome data from upland cotton at callus initiation and uses the pheatmap package in R to create heatmaps of gene expression levels, visualizing the expression levels of autophagy-related genes. The results show that the autophagy marker gene GhATG8 exhibits high expression within 3 days before hypocotyl induction in cotton, suggesting high autophagy activity during callus initiation, indicating that the autophagy pathway is involved in regulating callus initiation in cotton. Furthermore, the expression of the cotton autophagy gene GhATG18a-3 (hereinafter referred to as GhATG18a) was found to be relatively active during callus initiation, and its expression pattern was highly synchronized with the autophagy marker gene GhATG8, suggesting that autophagy genes play a key role in cotton dedifferentiation. Figure 1 ).

[0034] To investigate the conservation of GhATG18a in cotton, this invention retrieved the amino acid sequence of GhATG18a from the Cotton Functional Genomics Database (https: / / cottonfgd.net / ), and used the Pfam database (http: / / pfam.xfam.org / ) to identify the conserved domains of the protein and draw a schematic diagram. Sequence analysis showed that GhATG18a belongs to the conserved ATG18 family, its WD-40 domain mediates autophagosome membrane extension, and its homologs have been shown to be induced by biotic / abiotic stress in various plants. Figure 2 ).

[0035] Example 2: Cloning of the GhATG18a gene

[0036] Primers were designed based on the sequence of the GhATG18a gene (Gh_D12G0092) in upland cotton:

[0037] GhATG18a-F: 5'-AGAACACGGGGGACTCTAGAATGGCCACCCTCTCAGCTTATC-3'

[0038] (SEQ ID NO.1)

[0039] GhATG18a-R: 5'-TAGTCAGGCGCGCCGGTACCGAAAGCTGCCTCTGGCTTCAG-3'

[0040] (SEQ ID NO.2)

[0041] RNA was then extracted from the cotton variety CCRI24 (a cotton variety that is a highly efficient transgenic recipient for transformation), reverse transcribed into cDNA, and used as a template for PCR amplification using the primers described above. A 1302 bp PCR product was obtained, representing the GhATG18a gene with restriction enzyme sites (the GhATG18a gene sequence is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4), which was then set aside. The pCAMBIA2300 vector (sequence shown in SEQ ID NO.5) was then digested with Xba1 and Kpn1 restriction endonucleases to obtain a linearized vector. The PCR product and the linearized vector were ligated, and the ligation product was transformed into competent E. coli cells. After antibiotic selection, single clones were selected for sequencing. The correctly sequenced vector was named pCAMBIA2300-GhATG18a (structure shown in SEQ ID NO.4). Figure 3 (As shown). pCAMBIA2300-GhATG18a was transformed into Agrobacterium competent cells EHA105, and positive clones were picked and cultured for later use.

[0042] The gene sequence of GhATG18a is as follows:

[0043]

[0044] The amino acid sequence encoded by GhATG18a is as follows:

[0045] MATLSAYPNTNTNTNTNTNFLSPTDQSNHDRESIPPMQNHPSAEPDSDANPNFHSPLLDPPDSVPPAVAPSSLLHLSFNQDHGCFAAGTDHGFRIYNCDPFREIFRRDFDCGGGIGVVEMLFRCNILALVGGGPDPQYPPNKVMIWDDHQSRCISELSFRSEVRSVRLRRDRIIVVLEQKIFVYNFVDLKLLHQIETILNPKGLCAVSQGAGSLVLVCPGLQKGQVRVEHYASKRTKFIMAHDSRIACFALSQDGQLLATASTKGTLVRIYNTIDGSLLQEVRRGADRAEIYSLAFSSNAQWLAVSSDKGTVHVFSLKISAGSPGTTQSRSTSEPVVSSHSSLSFIKGVLPKYFSSEWSVAQFRLVEGSQYLVAFGHQKNTVVILGIDGSFYRCQFDPVNGGEMTQLEYHNFLKPEAAF(SEQ ID NO.4)

[0046] The sequence of the pCAMBIA2300 vector is as follows:

[0047]

[0048] Example 3: Agrobacterium-mediated genetic transformation of cotton

[0049] For detailed steps and methods of transforming cotton using Agrobacterium-mediated transformation, please refer to J Yuan, X Liu, H Zhao, et al. GhRCD1 regulates cotton somatic embryogenesis by modulating the GhMYC3-GhMYB44-GhLBD18 transcriptional cascade. [J]. The New Phytologist, 2023. The specific details are as follows:

[0050] 1) The seeds of CCRI24 are delinted with sulfuric acid, disinfected by soaking in 0.1% mercuric chloride for 5 minutes, then rinsed with sterile water 3-5 times, and sown on sterile seedling culture medium to germinate and grow into sterile seedlings.

[0051] 2) Before infection, the Agrobacterium strain containing pCAMBIA2300-GhATG18a was inoculated into LB liquid medium containing 50 μg / mL kanamycin, 25 μg / mL rifampin, and 50 μg / mL streptomycin for activation. On the day of cotton transformation, the activated bacteria were inoculated into 50 mL of fresh LB liquid medium containing 50 μg / mL kanamycin, 25 μg / mL rifampin, and 50 μg / mL streptomycin at a ratio of 1:100 (v / v) and cultured at 28°C and 190 rpm in a shaker until OD. 600 The value is between 0.3 and 0.4.

[0052] 3) On a sterile workbench, use a scalpel blade sterilized by an alcohol lamp flame to cut the hypocotyl of a 7-day-old sterile seedling into 0.5cm segments. Pour in cultured Agrobacterium and inoculate for about 3 minutes, then pour out the bacterial solution. Use sterile filter paper to remove the residual bacterial solution on the hypocotyl segments, and then transfer the hypocotyl segments to MSB medium. Incubate in a dark incubator at 23°C for 48 hours.

[0053] 4) Two days later, the hypocotyl segments were transferred to callus induction medium (MSBI) containing kanamycin (50 mg / L) and cephalosporin (200 mg / L). The culture was carried out at 28±2℃, light intensity of 2000 Lx, and a photoperiod of 16 h light / 8 h dark for about 30 days to obtain callus tissue.

[0054] 5) The formed callus was transferred to an embryonic callus induction medium containing kanamycin (50 mg / L) and cephalosporin (200 mg / L), and cultured for about 60 days at 28±2℃, light intensity 2000 Lx, and a photoperiod of 16 h light / 8 h dark to obtain embryonic callus.

[0055] 6) Subculture the differentiated embryogenic callus onto embryoid induction medium. Under conditions of 28±2℃, light intensity of 2000Lx, and a photoperiod of 16h light / 8h darkness, subculture once every 15-20 days until shoots emerge.

[0056] 7) The young shoot tissue was subcultured onto the seedling culture medium. After the seedlings emerged, they were further transferred to the regenerated seedling rooting culture medium for rooting culture. Rooting occurred after two weeks of culture at 28±2℃, light intensity of 2000 Lx, and a photoperiod of 16h light / 8h darkness. The regenerated seedlings were then removed from the culture medium and soaked in tap water at 28±2℃, light intensity of 2000 Lx, and a photoperiod of 16h light / 8h darkness for hardening off. After one week, they were transplanted into flowerpots to obtain plants of 24 overexpressing 35S::GhATG18a from the Chinese Cotton Research Institute.

[0057] 8) PCR detection: RNA was extracted from plants overexpressing 35S::GhATG18a, reverse transcribed into cDNA, and used as a template. Amplification was performed using the pCAMBIA2300-GhATG18a vector sequence-specific primers OE-GhATG18a-F / R, followed by agarose gel electrophoresis. Positive plants were specifically amplified and detected. The PCR target band was 509 bp, indicating a positive plant OE-GhATG18a (e.g., ...). Figure 4 (As shown). The above experiment detected two positive lines, OE-GhATG18a-1 and OE-GhATG18a-2, which were used for later transplanting.

[0058] OE-GhATG18a-F: 5'-AGCACTAAAGGGACGCTGGTTC-3' (SEQ ID NO.6)

[0059] OE-GhATG18a-R: 5'-CCGTCATGGTCTTTGTAGTC-3' (SEQ ID NO.7)

[0060] 9) RNA extraction: RNA was extracted from positive plants OE-GhATG18a-1, OE-GhATG18a-2 and CCRI24. RNA extraction reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd. For specific experimental procedures, please refer to the instruction manual.

[0061] 10) Reverse transcription to synthesize cDNA

[0062] (1) Removal of genomic DNA from the system. Add 4 μL of 4×g DNAwiper Mix and 1 μg of template RNA to each clean centrifuge tube as required, and make up to 16 μL with RNase-free ddH2O. Gently pipette the mixture to mix well and incubate at 42°C for 2 min.

[0063] (2) Mix 4 μL of 5×HiScript II qRT superMix II with the system from step 1 thoroughly by blowing.

[0064] (3) Reverse transcription reaction. The mixture from step 2 was reacted at 50℃ for 15 min and then at 85℃ for 5 s. The product after the reaction is cDNA, which can be used directly for quantitative real-time PCR or stored at -20℃ for a long time.

[0065] 11) Real-time quantitative PCR (qRT-PCR)

[0066] The cDNA obtained from reverse transcription was diluted, and the diluted cDNA was used as a template for the reaction using the highly sensitive dye-based quantitative PCR detection kit from Nanjing Novizan Biotechnology Co., Ltd. Each reaction system contained 5 μL of 2×Cham Q Universal SYBR qPCR Master Mix, 0.5 μL of forward primer q-GhATG18a-F (10 μM), 0.5 μL of reverse primer q-GhATG18a-R (10 μM), 2 μL of cDNA template (100 ng), and 2 μL of RNase-free ddH2O. The reaction program was: 95℃, 30 s; 95℃, 15 s, 40 cycles; 60℃, 30 s, 40 cycles; 95℃, 15 s; 60℃, 60 s; 95℃, 15 s; stored at 10℃. Each sample had three replicates. Differences between samples were tested using Student's t-test (unpaired) with GraphPad Prism (v.9.0.0). Significance was categorized into different levels: ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, no significant difference.

[0067] q-GhATG18a-F: 5'-AGCACTAAAGGGACGCTGGTTC-3' (SEQ ID NO.8)

[0068] q-GhATG18a-R: 5'-CGTGATTGGGTAGTGCCAGGAG-3' (SEQ ID NO.9)

[0069] The expression level of GhATG18a in the overexpression lines was detected by qRT-PCR. The results showed that compared with CCRI24 plants, the expression level of GhATG18a gene in the overexpression lines OE-GhATG18a-1 and OE-GhATG18a-2 was significantly upregulated. Figure 5 ).

[0070] Example 4: Phenotypic characteristics of GhATG18a transgenic cotton callus during tissue culture.

[0071] 1) Sterile seedling cultivation

[0072] In this invention, T0 generation seeds and CCRI24 seeds of different transgenic cotton lines OE-GhATG18a-1 and OE-GhATG18a-2 that overexpress GhATG18a were delinted with sulfuric acid, the outer shell of the cotton seeds was removed, the cotton kernels were disinfected by soaking in 0.1% mercuric chloride for 5 minutes, then rinsed 3-5 times with sterile water, and sown on sterile seedling culture medium to germinate and grow into sterile seedlings under dark conditions.

[0073] 2) Callus induction

[0074] On a sterile workbench, the hypocotyl of a 7-day-old sterile seedling was cut into 0.5cm segments with a scalpel blade sterilized by an alcohol lamp flame and placed in cotton callus induction medium (MSBI) for tissue culture.

[0075] 3) Phenotypic observation

[0076] Observation begins from the in vitro culture stage. Callus blocks are taken at various time points according to experimental needs, and their phenotypes are observed and photographed.

[0077] 4) Observation of tissue sections

[0078] The steps for observing callus initiation through paraffin sections are as follows:

[0079] Fixation: Select hypocotyl segments of each material cultured for 0, 2, 3, and 7 days and fix them in FAA fixative (50% ethanol) as soon as possible. The volume of fixative should be no less than 5 times the volume of the sample.

[0080] Dehydration: Gradual dehydration using 75% (v / v) ethanol solution for 4 h, 85% (v / v) ethanol solution for 2 h, 90% (v / v) ethanol solution for 1.5 h, 95% (v / v) ethanol solution for 1 h, and 100% ethanol for 0.5 h.

[0081] Transparency test: Anhydrous ethanol: Xylene = 1:1 (v / v) for 10 min; Xylene was used for transparency test twice, 10 min each time.

[0082] Embedding and sectioning: Immerse in paraffin three times at 60℃, 1 hour each time. After paraffin embedding, section the paraffin block to a thickness of 4-5 μm. Attach the sections to a glass slide, and then remove the paraffin from the slide.

[0083] Staining: Immerse tissue sections in toluidine blue staining solution for 2-5 minutes, then rinse lightly with tap water to remove excess stain. After rinsing, examine under a microscope and differentiate appropriately using 0.1% glacial acetic acid, depending on the degree of staining. If the staining is adequate and differentiation is not necessary, dry the sections in a 60°C oven. After drying, clear the sections with xylene for 10 minutes, then mount them with neutral resin.

[0084] The results showed that, compared with the receptor CCRI24, the OE-GhATG18a lines (OE-GhATG18a-1, OE-GhATG18a-2) overexpressing the GhATG18a gene exhibited hypocotyl swelling 2 days after callus induction, initiating dedifferentiation earlier than CCRI24. Figure 6 Histological section results showed that the OE-GhATG18a strain formed secondary meristems in the pericycle region approximately one day earlier than the control, with the onset time shortened by about 30%. Figure 6 The consistent phenotypic results indicate that the GhATG18a gene plays an important role in cotton somatic embryogenesis.

[0085] This demonstrates that Agrobacterium overexpressing the GhATG18a gene can significantly accelerate the process of cotton somatic cell dedifferentiation and callus induction. This can be used to address the genotypic limitation problem in some mainstream cotton varieties with low transformation efficiency, providing technical support for the cultivation of high-yield and stress-resistant high-quality cotton germplasm.

Claims

1. Use of the GhATG18a gene in accelerating induction of cotton callus, characterized in that, The GhATG18a gene is constructed into a pCAMBIA2300 vector, and is transformed into cotton to enhance the expression of the GhATG18a gene in the cotton material, and to accelerate the process of inducing callus by the somatic cell dedifferentiation of the cotton; the amino acid sequence coded by the GhATG18a gene is shown as SEQ ID NO. 4; and the cotton is Gossypium hirsutum.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the GhATG18a gene is shown as SEQ ID NO.

3.

3. The application of recombinant expression vector containing GhATG18a gene and genetically engineered bacteria in accelerating the process of cotton somatic cell dedifferentiation to induce callus, characterized in that, The nucleotide sequence of the GhATG18a gene is shown as SEQ ID NO. 3; and the cotton is Gossypium hirsutum.

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