Chenopodium quinoa willd CqARF2 gene mutant as well as identification method and application thereof

Abstract

The invention discloses a quinoa CqARF2 gene mutant as well as an identification method and application thereof. Chenopodium quinoa seeds are mutated on a large scale by utilizing a chemical mutagenic agent EMS, a CqARF2 gene function deletion mutant arf2 is adopted as a quinoa mutant, G at the 1050th site of the CqARF2 gene of the mutant is mutated into A, and amino acid at the 350th site is mutated into a termination codon, so that the translation process of CqARF2 protein is terminated in advance. The chenopodium quinoa CqARF2 gene function deletion mutant arf2 shows remarkable agronomic character improvement, seeds are large and full, compared with a wild type, the hundred-grain weight of the mutant seeds is increased by about 40%, the seed diameter is increased by about 10%, the seed thickness is increased by about 30%, and a certain foundation is laid for breeding new high-yield and high-quality chenopodium quinoa varieties.

Description

Technical Field

[0001] The present invention belongs to the field of biological technology and plant molecular breeding, and specifically relates to a quinoa CqARF2 Gene mutants and their identification methods and applications. Background Art

[0002] Quinoa ( Chenopodium quinoa Quinoa ( quinoa ) is an annual plant in the Chenopodiaceae family, a gluten-free pseudocereal with a tetraploid genome. Due to its rich nutritional value and strong environmental adaptability, quinoa has seen a surge in market demand. Therefore, improving quinoa's yield and quality has become a key issue that needs to be addressed.

[0003] Seed size is a key factor in determining crop yield and also influences seed nutritional composition and vigor. In recent decades, important genes controlling seed size have been discovered in the model plant Arabidopsis thaliana and various crops such as rice, maize, and castor. Among these, growth regulatory factors (GRFs) are among the most important regulators of seed size. GRFs have been found to positively regulate seed size in rice, soybean, and castor, with overexpression of GRFs leading to increased seed size and yield. Plant growth and morphology depend on the precise expression of key regulatory genes. In Arabidopsis, numerous transcription factors have been identified that control leaf growth and development. Auxin response factor 2 (ARF2) influences organ development by regulating cell number and size. Studies have shown that ARF2 acts as a transcriptional repressor, directly binding to the promoter of GRF5 and inhibiting its expression, thereby regulating organ development.

[0004] However, the above research has not yet been carried out on quinoa, and how to develop and cultivate quinoa varieties with large grains and high yields remains an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defects of the current existing technology, provide a new quinoa mutant that is non-genetically modified and has significantly enlarged seeds, and improve the quality of quinoa.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a quinoa CqARF2 gene mutants arf2 , which converts the nucleotide sequence of SEQ ID NO: 1 CqARF2 The G at position 1050 of the gene mutated to A, causing the amino acid at position 350 to mutate to a stop codon, resulting in premature termination of the CqARF2 protein translation process.

[0007] Furthermore, the mutant arf2 The nucleotide sequence is shown in SEQ ID NO.3.

[0008] Furthermore, the mutant arf2 It was obtained by ethyl methanesulfonate (EMS) mutagenesis combined with multi-generation screening.

[0009] In a second aspect, the present invention provides the quinoa CqARF2 gene mutants arf2 Application in regulating seed size.

[0010] Furthermore, the regulation is: quinoa CqARF2 gene mutants arf2 Increase seed size and weight.

[0011] In a third aspect, the present invention provides a quinoa arf2 The molecular identification method of mutants is as follows: extract quinoa DNA, according to CqARF2 Specific PCR primers were designed based on the nucleotide sequence of the gene. The products were recovered after PCR and sequenced, and compared with the wild-type sequencing sequence to determine the mutation status of quinoa.

[0012] Furthermore, the sequences of the specific PCR primers are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0013] In a fourth aspect, the present invention provides a method for increasing the particle size and weight of quinoa seeds, specifically comprising: CqARF2 The G at position 1050 of the gene mutated to A, and the quinoa CqARF2 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention constructs quinoa CqARF2 Loss-of-function mutants have achieved breakthrough improvements in key agronomic traits of seeds while ensuring normal plant growth, specifically a significant increase in seed size: arf2 Compared to wild-type (WT) seeds, the mutant's average seed diameter increased by approximately 10% and its average seed thickness increased by approximately 30%. The mutant's 100-seed weight was significantly increased, reaching approximately 40% higher than that of the WT. Therefore, this mutant has excellent application value and can be directly used in hybrid breeding or as a target for molecular marker development to accelerate the selection of new high-yield, high-quality varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 for CqARF2 Gene structure diagram; Figure 2 For quinoa WT and arf2 Sequencing result diagram; Figure 3 For quinoa WT and arf2Plant phenotype of the mutant at the seedling stage; Figure 4 For quinoa WT and arf2 Seed diameter comparison results, where a is a schematic diagram and b is a statistical chart; Figure 5 For quinoa WT and arf2 Seed thickness comparison results, where a is a schematic diagram and b is a statistical chart; Figure 6 For quinoa WT and arf2 Statistical chart of 100-grain seed weight. DETAILED DESCRIPTION

[0016] The following mainly describes the specific embodiments of the present invention in detail so that researchers in related fields can understand the operation steps of the present invention in more detail and apply them to actual research work. The preferred embodiments described herein are only used to explain the present invention, but the embodiments are not intended to limit the present invention.

[0017] In the present invention, CqARF2 The nucleotide sequence of the gene is shown in SEQ ID NO.1: CqARF2 The amino acid sequence of the gene is shown as SEQ ID NO.2 below: MSSSVASMKATATNKAGKTHTAAGVGTGKWGAVDTAYKWHACAGVTVRNVYYGHVASTNAADKMNYSWKCRVDVKATDVYAVMSVKKGTSDHHVHSCKTTASDTSTHGGSVRRHADCDMTRTVAKDHGNDWRRHRGRRHSGWSVVSSKRVAGDARGNGRVGVRRAMRGVVSSVSSHSMHGVATAHASSGTMTVYYKRTSAVYDYMSKNYSGMRKMRGSRTGTVGMDAHHRWDSKWRCKVRWDTSTMRDRVSWTATTNVTRAKRRVNVVTSSSSVHTRGSKVAADTSVGGYSRVGHYTRNVDSNDSAKNVWNSDDKADNVKASRKYAYNWSSMRAYADSTGVHNTASGHSYTAATSMKKTDDGRSMGNSWSMMGSSNMSSNMKVHSSSNVSYAGNVSYGGSDMAMRGHRDNHDGNWMGYDNHATRAMSRNDHKKDGNYKGSSSTTDSASSKRHDSTTTHYNCSDRSSSKGSKMDNACSKRMSSSRDAVKGGHSSRSCTKVKGAGRSVDSKNNYDVDCDKGKNKNWVYTDGDMMVGDDWCGMVRKYTKVKMTRNSKDNMAAGGSDKAKSSSSNASH Mutant arf2 The nucleotide sequence of [mutant] is shown as SEQ ID NO.3 below: The present invention will be further described below.

[0018] Example 1: Construction of Quinoa arf2 mutant Materials: The quinoa used in this study is the L048 strain from the Hangzhou Normal University Seed Bank. This variety can be consumed as both a grain and an animal feed. Approximately 5,000 plump, uniformly sized, undamaged seeds were selected and placed in distilled water in a 4°C cold room for 14 hours. After soaking, the seeds were transferred to 1X PBS buffer and stored at room temperature until ready for use.

[0019] EMS mutagenesis treatment: Prepare a 1 mg / mL concentration of ethyl methanesulfonate (EMS) aqueous solution, soak the seeds that have been treated at low temperature in the EMS solution, and treat them at room temperature for 8 hours. After the treatment, rinse the seeds with running water for at least 60 minutes to completely remove residual EMS.

[0020] The cleaned seeds were sown in the seedling tray matrix, and when the seedlings grew to 6 true leaves, they were transplanted into the field as the M1 generation.

[0021] The plants of the M1 generation are self-pollinated, and after the seeds mature, they are harvested individually, threshed, and dried to obtain the seeds of the M2 generation.

[0022] The M2 generation seeds were sown and raised as seedlings in individual lines, transplanted to the field, and cultured until the seeds matured. The seeds were harvested from individual plants, threshed, and dried to create the M3 generation. M3 lines with significantly higher 100-grain weight than the wild-type L048 control plants were selected as candidate mutant lines.

[0023] Example 2: CqARF2 Identification of Quinoa Genotypes by Gene Mutation Planting and sampling: M3 seeds with a large seed phenotype were sown in seedling trays and transplanted to the field when the quinoa plants had grown to 6 true leaves. When the plants reached the vegetative growth stage, approximately 100 mg of young leaf tissue was collected and quickly frozen in liquid nitrogen for DNA extraction.

[0024] Extraction of Quinoa Genomic DNA: This method uses the CTAB extraction method to extract total plant DNA. CTAB extraction buffer was prepared as follows: 2% CTAB (cetyl trimethyl ammonium bromide), 1% β-mercaptoethanol, 1.4 mol / L NaCl, 20 mM EDTA, and 100 mM Tris-HCl (pH 8.0). Quinoa leaves were thoroughly ground into a fine powder using a tissue disruptor. 600 μL of CTAB extraction solution was added and mixed thoroughly by vortexing. The mixture was then placed in a 65°C oven for 30 minutes for complete extraction, inverting every 10 minutes. After complete reaction, the tube was centrifuged at 14,000 rpm for 10 minutes. 500 μL of the supernatant was carefully transferred to a new 1.5 mL EP tube. An equal volume (500 μL) of isopropanol was added and gently inverted to mix thoroughly. The tube was then placed in a -20°C freezer for 1 hour to fully precipitate the DNA. Centrifuge at 14,000 rpm for 10 minutes, discard the supernatant, and retain the white precipitate. Wash the precipitate twice with 70% ethanol, discard the supernatant, dry it in a 65°C oven, and add 40 μL of ddH2O. The dissolved DNA solution can be used as a template in subsequent PCR reactions. Before use, determine the DNA concentration and purity using a microspectrophotometer.

[0025] CqARF2 Gene PCR amplification and sequencing primer design. Design specific primer pairs CqARF2 Amplification was performed using the upstream PCR primer F1: 5′TGTCATGACTCTGCCCAATTAA3′ (SEQ ID NO. 4) and the downstream PCR primer R1: 5′CCAGCGAACCTGTAAAAAGTAC3′ (SEQ ID NO. 5). The components listed in Table 1 were mixed thoroughly and centrifuged briefly. The PCR reaction program was as follows: initial denaturation at 95°C for 3 minutes; denaturation at 95°C for 1 minute, annealing at 55°C for 30 seconds, and extension at 72°C for 40 seconds for 33 cycles; followed by a complete extension at 72°C for 5 minutes.

[0026] Table 1 PCR product detection, purification, sequencing, and mutation analysis. Take 5 μL of PCR product and analyze it by electrophoresis on a 1% agarose gel to confirm the amplification of a specific band of the expected size. Purify the remaining PCR product to remove impurities such as primers and dNTPs.

[0027] The purified PCR product was sequenced using primer R1: 5′CCAGCGAACCTGTAAAAAGTAC3′ (SEQ ID NO. 5), and the obtained sequencing peaks and sequences were analyzed using SnapGene (or other sequence analysis software). The sequencing results were compared with those of the wild-type quinoa L048 line. CqARF2 Accurately compare the gene reference sequence to identify and confirm mutation sites such as base insertions, deletions, and substitutions in the target gene sequence, and determine the mutation type. CqARF2 Gene structure diagram Figure 1 As shown; arf2 The gene mutation forms in mutants are as follows Figure 2 shown.

[0028] Example 3: arf2 Statistics of mutant seed traits Plant cultivation and basic phenotype observation: The wild type (WT, L048 line) and arf2 Seeds of mutants were raised synchronously and transplanted into large pots. Normal water and fertilizer management and pest and disease control were carried out under the same environmental conditions. About 30 days after transplanting, the nutritional growth characteristics such as plant height and leaf size were systematically observed and recorded, and photographed. Figure 3 shown.

[0029] Seed harvesting and processing: After the seeds are fully mature, harvest WT and arf2 Seeds of mutant plants were harvested and dried in an oven at 37°C for 48 hours to ensure that the moisture content of the seeds dropped below the safe storage level.

[0030] Determination of seed diameter and thickness: from the WT and arf2 Randomly select 20 seeds with intact morphology and no damage from the mutant seeds. Lay the seeds flat in a straight line, place a ruler accurate to 1 mm underneath, take pictures with a camera, and use Image J software to calculate the seed diameter. Figure 4 Each seed was placed vertically in a straight line, a ruler accurate to 1 mm was placed underneath, a camera was used to take a picture, and the seed thickness was calculated using Image J software, as shown in the figure. Figure 5 shown.

[0031] 100-grain weight determination: from the WT and arf2 Among the mutant seeds, 100 seeds with intact morphology and no damage were randomly selected, WT and arf2 Three copies of each mutant were prepared. Accurately weigh the mutants using a precision analytical balance (accuracy 0.1 mg) and record the total weight to obtain the 100-grain weight data. Figure 6 shown.

[0032] Data analysis and conclusion: Independent sample T test was used to compare the differences in various traits between WT and mutants and evaluate the statistical significance (P<0.05 was set as significant). Figure 4-6 As shown, arf2 The mutant showed significant improvements in key seed traits. Compared with the wild type WT, the average seed diameter of the arf2 mutant increased by about 10% (P<0.001), the average thickness increased by about 30% (P<0.001), and the average 100-grain weight increased significantly by about 40% (P<0.001). These quantitative data fully confirmed that CqARF2 Gene mutations can effectively promote the development of quinoa seeds, significantly increase seed volume and increase the weight of single seeds, providing important genetic materials and breeding methods for increasing quinoa yield.

[0033] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of quinoa CqARF2 gene mutants arf2 , characterized in that, The nucleotide sequence is SEQ ID NO: 1 CqARF2 The G at position 1050 of the gene mutated to A.

2. Quinoa according to claim 1 CqARF2 gene mutants arf2 , characterized in that, The mutant arf2 The nucleotide sequence is shown in SEQ ID NO.

3.

3. Quinoa according to claim 1 CqARF2 gene mutants arf2 , characterized in that, The mutant arf2 It was obtained by ethyl methanesulfonate (EMS) mutagenesis combined with multi-generation screening.

4. Quinoa according to any one of claims 1 to 3 CqARF2 gene mutants arf2 Application in regulating seed size.

5. The use according to claim 4, characterized in that The regulation is: quinoa CqARF2 gene mutants arf2 Increase seed size and weight.

6. A type of quinoa arf2 A method for molecular identification of mutants, characterized in that The method comprises the following steps: extracting quinoa DNA, CqARF2 Specific PCR primers were designed based on the nucleotide sequence of the gene. The products were recovered after PCR and sequenced, and compared with the wild-type sequencing sequence to determine the mutation status of quinoa.

7. The molecular identification method according to claim 6, characterized in that The sequences of the specific PCR primers are shown in SEQ ID NO: 4 and SEQ ID NO:

5.

8. A method for increasing the particle size and weight of quinoa seeds, characterized in that: The method comprises the following steps: CqARF2 The G at position 1050 of the gene mutated to A, and the quinoa CqARF2 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

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