AsTN6 protein and its encoding gene that can regulate oat tiller number

Abstract

This invention relates to the field of plant genetic engineering technology, and more particularly to the AsTN6 protein and its encoding gene, which can regulate the number of oat tillers. This invention provides the first cloning of... AsTN6 Genes were found to be overexpressed in oat plants after being transformed with an overexpression vector. AsTN6 Genes can promote oat tillering traits. This invention provides a new molecular tool for regulating oat tillering traits, achieving targeted improvement of oat tillering traits through precise gene regulation. It breaks through the bottleneck of oat tillering trait breeding, provides effective technical support for promoting the high-quality development of the oat industry, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the AsTN6 protein and its encoding gene that can regulate the number of oat tillers. Background Technology

[0002] Feed oats ( Oatmeal Oat (L.) is an annual allohexaploid plant belonging to the genus *Oat* of the Poaceae family. With its excellent drought resistance, cold tolerance, salt and alkali tolerance, and tolerance to poor soil conditions, it has become a core cultivated forage species in arid, semi-arid, saline-alkali, and barren regions. In its green fodder state, oat provides fresh nutrition for livestock; after silage, it can be supplied year-round and out of season; and as hay, it is easy to store and transport. It plays an irreplaceable role in improving the economic benefits of regional animal husbandry and ensuring the supply of livestock products.

[0003] Tillering is a key agronomic trait in the construction of plant architecture for forage oats, directly affecting plant population structure, photosynthetic area allocation, and resource utilization efficiency, thus significantly impacting biomass production and forage quality. Developing forage oat varieties with suitable tillering capacity is a core direction for optimizing existing breeding systems and improving the overall performance of varieties.

[0004] However, current technologies lack sufficient key gene discovery for regulating tillering in forage oats. There is a lack of clearly defined gene resources that can efficiently improve tillering traits in forage oats, making it difficult to achieve targeted improvement of tillering ability through precise gene regulation. This hinders effective technical support for overcoming breeding bottlenecks in forage oats and promoting high-quality industrial development. Therefore, there is an urgent need to discover key genes regulating tillering in forage oats. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention provides a protein capable of effectively regulating oat tillering, namely the AsTN6 protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0006] SEQ ID NO.2:

[0007] MKKAKEITVLCDAQVAIIMFSSTGKYHEFISPGYDVKGIFDRYQQAQGTSLWTEQYENMQRTLSHLKDINRSLRTEIRQRMGEDLDVLEFDELRGLEQNVDAALKEVRQRKYHVITTQTETYKKKVKHSQEAYKNLQQELGMREDPAYGFVDNPAASGWDGVAAVAMSGASAADMYAFRVVPSQPNLHGMAYGGSHDLRLG

[0008] Furthermore, the present invention provides the gene encoding the AsTN6 protein.

[0009] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.1. SEQ ID NO.1 is... AsTN6 CDS sequence of the gene.

[0010] SEQ ID NO.1:

[0011] ATGAAGAAGGCCAAGGAGATCACCGTGCTCTGCGACGCACAGGTCGCCATCATCATGTTCTCCTCCACCGGCAAGTACCACGAGTTCATCAGCCCAGGCTACGATGTCAAGGGGATCTTTGACCGCTACCAGCAGGCCCAAGGGACCAGCC TGTGGACCGAGCAGTATGAGAATATGCAGCGCACGCTGAGCCATCTCAAGGACATCAACCGGAGCCTGCGCACCGAGATCAGGCAAAGGATGGGTGAAGATCTGGATGTGCTGGAGTTCGACGAGCTGCGTGGTCTTGAGCAAAATGTCGAT GCAGCTCTCAAGGAGGTTCGCCAGAGGAAGTATCATGTGATCACCACGCAAACTGAAACCTACAAGAAGAAGGTGAAGCACTCCCAGGAGGCATACAAGAACCTGCAGCAGGAGCTGGGCATGCGCGAGGACCCGGCGTATGGGTTCGTGG ACAACCCGGCGGCGAGTGGGTGGGACGGCGTCGCGGCGGTGGCGATGAGCGGCGCCTCCGCGGCAGACATGTACGCCTTCCGCGTGGTGCCCAGCCAGCCCAACCTGCACGGCATGGCCTACGGCGGCTCACACGACCTGCGTCTCGGCTAA

[0012] Furthermore, the present invention provides biological materials containing the aforementioned gene.

[0013] In some embodiments, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues.

[0014] Preferably, the plasmid vector is pUBI-FLAG.

[0015] Furthermore, the present invention provides the application of the AsTN6 protein, the gene, or the biological material in regulating oat tillering traits.

[0016] Preferably, the total number of oat tillers or the number of effective tillers is increased by increasing the activity or expression level of the AsTN6 protein or its encoding gene;

[0017] Alternatively, by reducing the activity or expression of the AsTN6 protein or its encoding gene, the total number of oat tillers or the number of effective tillers can be reduced.

[0018] Preferably, overexpression in oats AsTN6 Genes that increase the total number of tillers or the number of effective tillers in oats.

[0019] In some implementations, the overexpression method is selected from one or more combinations of (1)-(5):

[0020] (1) By introducing a plasmid containing the gene;

[0021] (2) By increasing the copy number of the genes described on the chromosome;

[0022] (3) By altering the promoter sequence of the genes described on the chromosome;

[0023] (4) By operatively linking a strong promoter to the gene;

[0024] (5) By introducing enhancers.

[0025] Furthermore, the present invention provides the application of the AsTN6 protein, the gene, or the biological material in oat breeding.

[0026] Preferably, the purpose of oat breeding is to increase the number of oat tillers.

[0027] Furthermore, the present invention provides the application of the AsTN6 protein, the gene, or the biological material in the improvement of oat germplasm resources.

[0028] Preferably, the purpose of the oat germplasm resource improvement is to increase the number of oat tillers.

[0029] Furthermore, 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 AsTN6 protein or its encoding gene in oats;

[0030] Alternatively, by reducing the activity or expression level of the AsTN6 protein or its encoding gene in oats, the total number of oat tillers or the number of effective tillers can be reduced.

[0031] In some implementation schemes, genetic engineering techniques or hybridization techniques are used to increase or decrease the activity or expression level of the AsTN6 protein or its encoding gene in oats.

[0032] Preferably, the oats are feed oats.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] The invention first cloned and obtained AsTN6 Genes were found to be overexpressed in oat plants after being transformed with an overexpression vector. AsTN6 Genes can promote oat tillering traits. This invention provides a new molecular tool for regulating oat tillering traits, achieving targeted improvement of oat tillering traits through precise gene regulation. It breaks through the bottleneck of oat tillering trait breeding, provides effective technical support for promoting the high-quality development of the oat industry, and has broad application prospects. Attached Figure Description

[0035] Figure 1 It is in genetically modified feed oat plants AsTN6 The relative expression level of genes.

[0036] Figure 2 yes AsTN6 Tillering phenotypes of transgenic feed oat plants and wild-type plants. Detailed Implementation

[0037] 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.

[0038] 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.

[0039] Example 1: Feed Oats AsTN6 Gene mapping and cloning

[0040] (1) Genome-wide association analysis AsTN6 Gene

[0041] 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 6C significantly associated with tillering traits. Haploview linkage disequilibrium block analysis of this region showed that the core SNP was located at 46434276 bp on chromosome 6C. Further haplotype analysis revealed two allelic variants, A and G, at which germplasm resources carrying the A allelic variant had a higher tillering number than those carrying the G allelic variant, 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 6C0199976. 6C0199976 was preliminarily identified as the major gene in this region and named [generic name missing]. AsTN6 .

[0042] (2) AsTN6 Gene cloning and protein sequence analysis

[0043] 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... AsTN6 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. AsTN6 Gene coding region amplification, CDS-F as shown in SEQ ID NO.3, CDS-R as shown in SEQ ID NO.4.

[0044] CDS-F:ATGAAGAAGGCCAAGGAGATC (SEQ ID NO.3)

[0045] CDS-R:TTAGCCGAGACGCAGGTCGTG (SEQ ID NO.4)

[0046] The amplification system is as follows: 50 μL reaction system, containing 20 ng DNA and Mg. 2+ 2 μL, PCR buffer 5 μL, dNTP 5 μL, KOD enzyme 1.5 μL, and forward and reverse primers 1.5 μL each.

[0047] The PCR amplification program was as follows: 34 PCR cycles were started after a 3-minute pre-denaturation at 95℃. The cycle consisted of denaturation at 95℃ for 35 seconds, annealing at 58℃ for 35 seconds, and extension at 72℃ for 2 minutes. After the final cycle, a final extension at 72℃ for 10 minutes was performed. Following amplification, 1.5% agarose gel electrophoresis was performed. The amplified products were recovered and sequenced using Sanger sequencing to obtain the desired results. AsTN6 The nucleotide sequence of the gene (shown in SEQ ID NO.1).

[0048] Using the online analytics platform Expasy AsTN6 The gene CDS sequence (shown in SEQ ID NO.1) was translated into a protein sequence (shown in SEQ ID NO.2). The AsTN6 protein consists of 201 amino acid residues, with a molecular weight of 22.77 KD and an isoelectric point of 6.31. The primary structure functional domains of the AsTN6 protein were analyzed using the online analysis platform SMART, and it was found that 1aa-32aa is a conserved MADS domain, indicating that the AsTN6 protein belongs to the MADS transcription factor.

[0049] Example 2 AsTN6 Creation of Gene Overexpression Transgenic Feed Oats

[0050] (1) AsTN6 Construction of gene overexpression vectors

[0051] 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. AsTN6 The CDS region is used to obtain the carrier arm. AsTN6 The CDS region of a gene.

[0052] pUBI-FLAG-F: (SEQ ID NO.5) TGTTACTTCTGCAGAAGCTTATGAAGAAGGCCAAGGAG

[0053] pUBI-FLAG-R: (SEQ ID NO.6)

[0054] TCTTTGTAGTCCATGGATCCGCCGAGACGCAGGTCGTG

[0055] 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 agar containing 50 mg / L Kan, and incubated upside down at 37°C for 10 h. Positive single clones were then picked. Sequencing confirmed the presence of the recombinant product. AsTN6 Gene overexpression vector pUBI-FLAG:: AsTN6 .

[0056] (2) AsTN6 Creation of feed oats through overexpression

[0057] The built AsTN6 Gene overexpression vector pUBI-FLAG:: AsTN6 Transformed 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.

[0058] (3) In genetically modified feed oat plants AsTN6 Gene expression level detection

[0059] 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. AsTN6 Gene expression levels.

[0060] RT-F: (SEQ ID NO.7)

[0061] GGAGGTTCGCCAGAGGAAGTATC

[0062] RT-R: (SEQ ID NO.8)

[0063] CGTGTGAGCCGCCGTAGG

[0064] 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.

[0065] 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).

[0066] Exploit 2 -ΔΔCt Method calculation AsTN6 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-10, OE-19, OE-21, OE-22, OE-23) showed... AsTN6 Gene expression levels were significantly increased in all strains, with the highest expression level in the OE-19 line (approximately 10.34-fold). The relative expression levels of the OE-23, OE-10, OE-22, and OE-21 lines were approximately 9.98-fold, 9.61-fold, 6.43-fold, and 7.98-fold, respectively, indicating that... AsTN6 The gene was successfully overexpressed in transgenic feed oats.

[0067] Example 3 AsTN6 Phenotypic analysis of transgenic feed oats overexpressing

[0068] Wild type (CK) and two AsTN6 Transgenic feed oat lines (OE-19 and OE-23) expressing the gene were grown 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 the differences were analyzed for significance.

[0069] The results are shown in Table 1 and Figure 2 As shown, compared to the wild type (CK), AsTN6 The total number of tillers and the number of effective tillers in transgenic feed oats overexpressed significantly increased. The OE-19 line with high expression level showed the largest increase (total tiller number increased by 185.71% and effective tiller number increased by 278.57% compared to CK). The OE-19 line with slightly lower expression level also showed a significant tillering promotion effect (tiller number increased by 176.19% and effective tiller number increased by 271.43% 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.

[0070] Table 1 AsTN6Phenotypic comparison between overexpression transgenic feed oat lines and wild-type lines

[0071]

[0072] The above results indicate that AsTN6 Overexpression of the gene significantly promoted tillering ability in feed oats without affecting other yield-related traits, confirming... AsTN6 Genes are key positive regulators of oat tillering traits and can be used for targeted improvement of tillering traits in feed oats.

[0073] 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. AsTN6 protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the AsTN6 protein of claim 1.

3. The gene according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

4. A biomaterial containing the gene of claim 2 or 3, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable oat cells or tissues.

5. The application of the AsTN6 protein of claim 1, the gene of claim 2 or 3, or the biological material of claim 4 in regulating oat tillering traits; the application is achieved by introducing a recombinant plasmid containing the gene of claim 2 or 3 to increase the expression level of the AsTN6 protein or its encoding gene, thereby increasing the total number of oat tillers or the number of effective tillers.

6. The application according to claim 5, characterized in that, By introducing a recombinant plasmid containing the gene described in claim 2 or 3, the expression level of AsTN6 protein or its encoding gene is increased, thereby increasing the total number of tillers or the number of effective tillers in oats.

7. The application of the AsTN6 protein of claim 1, the gene of claim 2 or 3, or the biological material of claim 4 in oat breeding; wherein the purpose of oat breeding is to increase the number of oat tillers.

8. The application of the AsTN6 protein of claim 1, the gene of claim 2 or 3, or the biological material of claim 4 in the improvement of oat germplasm resources; the purpose of the oat germplasm resource improvement is to increase the number of oat tillers.

9. A method for regulating the number of oat tillers, characterized in that, include: By introducing a recombinant plasmid containing the gene described in claim 2 or 3, the expression level of AsTN6 protein or its encoding gene in oats is increased, thereby increasing the total number of tillers or the number of effective tillers in oats.

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