AsTN7 protein and its application in regulating oat tillering

Abstract

This invention relates to the field of plant genetic engineering technology, and more particularly to the AsTN7 protein and its application in regulating oat tillering. This invention provides the first cloning of... As TN7 Genes were found to be overexpressed in oat plants after being transformed with an overexpression vector. As TN7 Genes can promote tillering traits in oats. This invention successfully identified and validated key genes regulating oat tillering, providing novel targets and molecular tools for oat molecular breeding. Through precise gene regulation, targeted and efficient improvement of oat tillering ability was achieved, effectively overcoming the bottleneck of precise control over tillering traits in traditional oat breeding. This breakthrough significantly enhances the population building ability and biomass potential of oat varieties, laying a solid technical foundation for breeding high-yield, high-quality oat varieties and promoting the development of the oat industry towards precision and efficiency.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the AsTN7 protein and its application in regulating oat tillering. Background Technology

[0002] Feed oats ( Avena sativa L. Oats (Oxalis corniculata) are annual allohexaploid plants belonging to the genus Oxalis in the Poaceae family. With their outstanding drought resistance, cold tolerance, salt and alkali tolerance, and tolerance to poor soil conditions, they have become a core cultivated forage species in arid and semi-arid regions, saline-alkali lands, and infertile soil areas. Forage oats have significant multi-faceted utilization value: green fodder can directly provide high-quality fresh forage; silage allows for year-round off-season supply; and processing into hay facilitates long-term storage and long-distance transportation. Forage oats are crucial for ensuring stable and high yields in animal husbandry, promoting the large-scale and sustainable development of the livestock industry, and playing an irreplaceable role in improving the economic benefits of animal husbandry and stabilizing the supply of livestock products in the market.

[0003] Tillering, as a core agronomic trait in the construction of forage oat plant architecture, directly determines the rationality of plant population structure, photosynthetic area allocation efficiency, and resource utilization efficiency, thereby significantly regulating biomass accumulation and forage quality. Therefore, breeding forage oat varieties with suitable tillering ability has become a key direction for optimizing breeding technology systems and improving the comprehensive agronomic performance of varieties.

[0004] Currently, there are still significant shortcomings in the discovery of key genes regulating tillering in forage oats. There is a lack of clearly defined gene resources that can be used to efficiently improve tillering traits, making it difficult to achieve targeted improvement of tillering ability through precise gene editing or regulation technologies. This restricts the breakthrough of bottlenecks in forage oat breeding and the high-quality development of related industries.

[0005] Against this backdrop, identifying key genes that regulate tillering traits in feed oats has become a pressing technical challenge in this field. Summary of the Invention

[0006] To address the aforementioned technical challenges, this invention, for the first time, cloned a gene capable of positively regulating oat tillering traits, named... AsTN7 The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] ATGTCGGCCGGGCAGATCACGCCGGCAGAACAAGTGTGCTACGTGCACTGCAACTTCTGCAACACAGTACTTGCGGTGAGCGTTCCTGGGAACAGCATGCTGAGCATTGTGACTGTTCGGTGTGGGCACTGCACAAATCTACTATCAGTGAACCTGAGAGGGCTGTTGCCCTCATCAGTCCCTCCTGCTACTACACAAGATCATCATATCCAGGAGAATGCGAGCAAGGTCGTCCATGGCGTCAACGGCTTCCATCAAGCTGGCGGGGTCGTCCATTATTCAGATCAGTTCGGTGGCTCTTCTTCTTCTTCTTCGTCGTCGTCGACCAAGTTCCGGCTGCCCATGATGATGTTCTCGCAGCAGAACGAGCTGCTGCAGGAGCAGACGCTGCACACACGCCCAGCCCCGGAGAAGAGGCAGCGCGTTCCATCTGCGTATAACAGATTCATCAAGGAAGAGATACGAAGGATCAAGGCGAACAACCCCGACATTAGCCACAGGGAAGCCTTCAGCACAGCGGCAAAGAACTGGGCACATTACCCCAACATCCATTTCGGGCTACACCCGGGCGGCCGCGGCGACGGTGGCAGCAAGAAGCTCGCCGTTGACGTCGACGCGCCGCCGGCTCCCAAGAAGATCCATGCTAGTTTCTGCTCATAG

[0009] Based on this, the following technical solutions are proposed.

[0010] In a first aspect, the present invention provides the AsTN7 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0011] SEQ ID NO.2:

[0012] MSAGQITPAEQVCYVHCNFCNTVLAVSVPGNSMLSIVTVRCGHCTNLLSVNLRGLLPSSVPPATTQDHHIQENASKVVHGVNGFHQAGGVVHYSDQFGGSSSSSSSSSST KFRLPMMMFSQQNELLQEQTLHTRPAPEKRQRVPSAYNRFIKEEIRRIKANNPDISHREAFSTAAKNWAHYPNIHFGLHPGGRGDGGSKKLAVDVDAPPAPKKIHASFCS

[0013] Secondly, the present invention provides AsTN7 The gene is the gene encoding the AsTN7 protein.

[0014] In some embodiments, the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] SEQ ID NO.1 is AsTN7 CDS sequence of the gene.

[0016] Thirdly, the present invention provides a biological material, wherein the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, plant cell or tissue.

[0017] In some implementations, the plasmid vector is pUBI-FLAG.

[0018] Fourthly, the present invention provides the application of the AsTN7 protein, the gene, or the biological material in regulating oat tillering traits.

[0019] In some implementations, the total number of oat tillers or the number of effective tillers is increased by enhancing the activity or expression of the AsTN7 protein or its encoding gene.

[0020] In some implementations, the total number of oat tillers or the number of effective tillers is reduced by decreasing the activity or expression of the AsTN7 protein or its encoding gene.

[0021] Preferably, overexpression in oats AsTN7 Genes that increase the total number of tillers or the number of effective tillers in oats.

[0022] In some implementations, the overexpression method is selected from one or more combinations of (1)-(5):

[0023] (1) By introducing a plasmid containing the gene;

[0024] (2) By increasing the copy number of the genes described on the chromosome;

[0025] (3) By altering the promoter sequence of the genes described on the chromosome;

[0026] (4) By operatively linking a strong promoter to the gene;

[0027] (5) By introducing enhancers.

[0028] Fifthly, the present invention provides the application of the AsTN7 protein, the gene, or the biological material in oat breeding or oat germplasm resource improvement.

[0029] In some implementations, the purpose of oat breeding or oat germplasm resource improvement is to increase the number of oat tillers.

[0030] Preferably, the number of tillers is the total number of tillers or the number of effective tillers.

[0031] In a sixth aspect, the present invention provides a method for regulating the number of oat tillers, comprising: increasing the total number of oat tillers or the number of effective tillers by increasing the activity or expression level of the AsTN7 protein or its encoding gene in oats;

[0032] Alternatively, by reducing the activity or expression level of the AsTN7 protein or its encoding gene in oats, the total number of oat tillers or the number of effective tillers can be reduced.

[0033] In some implementations, genetic engineering or hybridization techniques are used to increase or decrease the activity or expression level of the AsTN7 protein or its encoding gene in oats.

[0034] In some implementations, the oats are feed oats.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] The invention first cloned and obtained AsTN7 Genes were found to be overexpressed in oat plants after being transformed with an overexpression vector. AsTN7 Genes can promote tillering traits in oats. This invention successfully identified and validated key genes regulating oat tillering, providing novel targets and molecular tools for oat molecular breeding. Through precise gene regulation, targeted and efficient improvement of oat tillering ability was achieved, effectively overcoming the bottleneck of precise control over tillering traits in traditional oat breeding. This breakthrough significantly enhances the population building ability and biomass potential of oat varieties, laying a solid technical foundation for breeding high-yield, high-quality oat varieties and promoting the development of the oat industry towards precision and efficiency. Attached Figure Description

[0037] Figure 1 It is in genetically modified feed oat plantsAsTN7 The relative expression level of genes.

[0038] Figure 2 yes AsTN7 Tillering phenotypes of overexpressed transgenic feed oat plants (OE) and wild-type plants (CK). Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art may conduct corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust them to specialized companies, such as gene sequencing.

[0041] Example 1: Feed Oats AsTN7 Gene mapping and cloning

[0042] (1) Genome-wide association analysis AsTN7 Gene

[0043] Based on the previous construction of a core population of forage oat germplasm resources, genotypic and tillering phenotypic data of this core germplasm resource population were collected. Genome-wide association analysis (GWAS) was used to screen for SNP loci significantly associated with tillering traits in forage oats. The results revealed a region on chromosome 7C significantly associated with tillering traits. Haploview analysis of this region using linkage disequilibrium block (LD block) revealed that the core SNP in this region was located at 541566626 bp on chromosome 7C. Further haplotype analysis showed that this SNP locus had both A and G allelic variations. Germplasm resources carrying the A allelic variation had a higher tillering number than those carrying the G allelic variation, and the difference was highly significant (P ≤ 0.01). Further analysis using genome annotation files revealed that this SNP locus was located in the promoter region of gene 7C072164. 7C072164 was preliminarily identified as the major gene in this region and named [generic name missing]. AsTN7 .

[0044] (2) AsTN7 Gene cloning and protein sequence analysis

[0045] Tissues from roots, stems, leaves, and ears of the forage oat material ZXY1 at different stages, including seedling, tillering, jointing, booting, and heading stages, were extracted. Total RNA was extracted using the FAstPure Plant Total RNA Isolation Kit (Nanjing Novizan Biotechnology Co., Ltd.). RNA integrity was assessed by agarose gel electrophoresis, and RNA purity and concentration were determined using Nanodrop. The RNA was then reverse transcribed into cDNA using the HiScripte III All-in-one RT SuperMix Perfect for qPCR kit (Nanjing Novizan Biotechnology Co., Ltd.). Equal volumes were then mixed to form... AsTN7 Gene cloning was performed using a mixed cDNA. Using the aforementioned mixed cDNA as a template, primer pairs CDS-F and CDS-R were used for cloning. AsTN7 Gene coding region amplification. CDS-F is shown in SEQ ID NO.3, and CDS-R is shown in SEQ ID NO.4.

[0046] CDS-F:ATGTCGGCCGGGCAGATCACG (SEQ ID NO.3)

[0047] CDS-R: CTATGAGCAGAAACTAGCATG (SEQ ID NO.4)

[0048] The amplification system is as follows: 50 μL reaction system, containing 20 ng DNA and Mg. 2+ 2 μL of PCR buffer, 5 μL of dNTPs, 1.5 μL of KOD enzyme, and 1.5 μL each of forward and reverse primers were used. The PCR amplification program was as follows: 34 PCR cycles were performed after a 3-minute pre-denaturation at 95°C. The cycle consisted of 35 seconds of denaturation at 95°C, 35 seconds of annealing at 58°C, and 2 minutes of extension at 72°C. After the final cycle, the product was extended at 72°C for 10 minutes. The amplified product was then subjected to 1.5% agarose gel electrophoresis, and Sanger sequencing was performed to obtain the final product. AsTN7 The nucleotide sequence of the gene (shown in SEQ ID NO.1).

[0049] Using the online analytics platform Expasy AsTN7The gene CDS sequence (shown in SEQ ID NO.1) was translated into a protein sequence (shown in SEQ ID NO.2). The AsTN7 protein consists of 219 amino acid residues, with a molecular weight of 23.79 KD and an isoelectric point of 9.26. The primary structure functional domains of the AsTN7 protein were analyzed using the online analysis platform SMART, and it was found that 7aa-187aa is a YABBY conserved domain, indicating that the protein belongs to the YABBY transcription factor.

[0050] Example 2 AsTN7 Creation of transgenic feed oats by overexpression

[0051] (1) AsTN7 Construction of gene overexpression vectors

[0052] The plant expression vector pUBI-FLAG (from Weimi Biotechnology Co., Ltd.) was obtained by double digestion with the restriction endonucleases HindIII and BamHI; simultaneously, pUBI-FLAG-F and pUBI-FLAG-R were used for amplification. AsTN7 The CDS region is used to obtain the carrier arm. AsTN7 The CDS region of a gene.

[0053] pUBI-FLAG-F: (SEQ ID NO.5)

[0054] TGTTACTTCTGCAGAAGCTTATGTCGGCCGGGCAGAT

[0055] pUBI-FLAG-R: (SEQ ID NO.6)

[0056] TCTTTGTAGTCCATGGATCCTGAGCAGAAACTAGCA

[0057] The vector and fragment were recombined using the LanGene™ Seamless Cloning & Assembly Kit (Nanjing Novizan Biotechnology Co., Ltd.). The recombinant product was transformed into DH5α competent E. coli cells, plated on LB solid medium containing 50 mg / L Kan, and incubated upside down at 37°C for 10 h. Positive single clones were picked and sequenced for verification. AsTN7 Gene overexpression vector pUBI-FLAG:: AsTN7 .

[0058] (2) AsTN7 Creation of feed oats through overexpression

[0059] The built AsTN7 Gene overexpression vector pUBI-FLAG:: AsTN7Transformed into EHA105 Agrobacterium competent cells (Beijing Huayueyang Biotechnology Co., Ltd.), the cells were plated on LB solid medium containing 50 mg / L Kan and 25 mg / L Rif. After incubation at 28°C with inverted incubation for 60 h, positive single clones were picked and inoculated into LB liquid medium (containing 50 mg / L Kan and 25 mg / L Rif) and cultured at 28°C with shaking until OD. 600 =0.6-0.8, prepare Agrobacterium infection solution. Using Agrobacterium-mediated genetic transformation of forage oat callus, transgenic forage oat plants were obtained after infection, dark culture, induction, differentiation and transplantation.

[0060] (3) In genetically modified feed oat plants AsTN7 Gene expression level detection

[0061] Following step (2) of Example 1, RNA was extracted from the leaves of transgenic feed oat plants and reverse transcribed into cDNA. Using primer pairs RT-F and RT-R, qRT-PCR was performed on an ABI Q7 real-time PCR instrument to amplify and detect RNA in the transgenic plants. AsTN7 Gene expression levels.

[0062] RT-F: (SEQ ID NO.7)

[0063] TACTTGCGGTGAGCGTTCCTG

[0064] RT-R: (SEQ ID NO.8)

[0065] CGACGACGACGAAGAAGAAGAAG

[0066] The qRT-PCR system is: 2 × HQ SYBR qPCR Mix 10.0 μL, RT-F 0.4 μL, RT-R 0.4 μL, cDNA 2.0 μL, ddH2O 7.2 μL.

[0067] The qRT-PCR program is as follows: pre-denaturation at 95℃ for 2 min followed by 40 cycles (denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s).

[0068] Exploit 2 -ΔΔCt Method calculation AsTN7 The relative expression levels of genes in transgenic plants were as follows: Figure 1 As shown: Compared with wild-type plants (CK), the five overexpression lines (OE-9, OE-10, OE-20, OE-21, OE-22) showed... AsTN7Gene expression levels were significantly increased in all strains, with the highest expression level in the OE-20 line (approximately 11.32-fold). The relative expression levels of the OE-22, OE-21, OE-10, and OE-9 lines were approximately 9.91-fold, 7.98-fold, 6.61-fold, and 6.43-fold, respectively, indicating that... AsTN7 The gene was successfully overexpressed in transgenic feed oats.

[0069] Example 3 AsTN7 Phenotypic analysis of transgenic feed oats overexpressing

[0070] The wild type (CK) and two from Example 2 were used. AsTN7 Transgenic feed oat lines (OE-20 and OE-22) expressing the gene were planted in an artificial climate chamber (temperature 20℃-25℃, 12h light / 12h dark, humidity 60%-70%). Phenotypic indicators of each line were investigated at the milk stage of the feed oats, including traits such as effective tiller number, total tiller number, plant height, flag leaf length and width, spike length, and spikelet number, and significance analysis was performed.

[0071] The results are shown in Table 1 and Figure 2 As shown, compared to the wild type (CK), AsTN7 The total number of tillers and the number of effective tillers in transgenic feed oats overexpressed significantly increased. The OE-20 line with high expression level showed the largest increase (total tiller number increased by 511.76% and effective tiller number increased by 300.0% compared to CK). The OE-22 line with slightly lower expression level also showed a significant tillering promotion effect (tiller number increased by 470.59% and effective tiller number increased by 281.82% compared to CK). However, there were no significant differences in traits such as plant height, flag leaf length and width, spike length, and spikelet number between transgenic plants and control plants.

[0072] Table 1 AsTN7 Phenotypic comparison between overexpression transgenic feed oat lines and wild-type lines

[0073]

[0074] The above results indicate that AsTN7 Overexpression of the gene significantly promoted tillering ability in feed oats without affecting other yield-related traits, confirming... AsTN7 Genes are key positive regulators of oat tillering traits and can be used for targeted improvement of tillering traits in feed oats.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. AsTN7 protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. AsTN7 Genes, characterized by, The gene is the encoding gene of the AsTN7 protein as described in claim 1.

3. The gene of claim 2, wherein, Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A biomaterial, characterized by, The biological material is recombinant DNA.

5. A biomaterial, characterized by, The biological materials are expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, plant cells, or tissues.

6. The use of the AsTN7 protein of claim 1, the gene of claim 2 or 3, or the biomaterial of claim 4 or 5 in regulating oat tillering traits.

7. Use according to claim 6, characterized in that, Increase the total number of tillers or the number of effective tillers in oats by increasing the activity or expression of the AsTN7 protein or its encoding gene.

8. Use according to claim 6, characterized in that, The total number of tillers or the number of effective tillers in oats can be reduced by decreasing the activity or expression of the AsTN7 protein or its encoding gene.

9. The application of the AsTN7 protein of claim 1, the gene of claim 2 or 3, or the biomaterial of claim 4 or 5 in oat breeding or oat germplasm resource improvement.

10. A method for regulating the number of oat tillers, characterized in that, include: Increase the total number of tillers or the number of effective tillers in oats by increasing the activity or expression level of the AsTN7 protein or its encoding gene in oats; Alternatively, by reducing the activity or expression level of the AsTN7 protein or its encoding gene in oats, the total number of oat tillers or the number of effective tillers can be reduced; The amino acid sequence of the AsTN7 protein is shown in SEQ ID NO.

2.

11. The method of claim 10, wherein, The activity or expression level of AsTN7 protein or its encoding gene in oats can be increased or decreased through genetic engineering or hybridization techniques.

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