Application of an oat AsEFT1 gene and its encoded protein

Abstract

The present invention belongs to the technical field of molecular genetics, and particularly relates to the application of an oat AsEFT1 gene and its encoded protein. The oat AsEFT1 gene is characterized in that the CDS nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. The present invention discovers and clones the AsEFT1 gene for the first time. Through Arabidopsis experiments, it is confirmed that it can regulate the flowering trait, providing a new molecular marker for oat breeding and having great market application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics, and particularly relates to the application of an oat AsEFT1 gene and its encoded protein. Background Art

[0002] Oat (Avena sativa L.) is a gramineous crop that can be used for both food and forage, with characteristics such as high yield, high quality, and strong stress resistance. It is one of the key forage grasses to solve the shortage of forage supply in winter and spring pastures in northern China and occupies an important position in the development of the domestic forage industry. In recent years, with the rapid development of animal husbandry in China, the domestic demand for the yield of forage oat hay has become increasingly urgent, which in turn puts forward higher requirements for the breeding work of high-yield forage oat varieties.

[0003] Flowering is an important physiological process from reproductive growth to vegetative growth in higher plants and is crucial for plant reproduction. In agricultural production, the timely flowering of crops is of great significance for variety breeding, introduction, and breeding, as well as for stable and increased yields. By controlling the flowering time, the production cycle of crops can be optimized, and the yield and quality of crops can be improved. With the development of molecular biology and breeding techniques, identifying important genes that control plant flowering can provide potential targets for crop genetic improvement, and precisely control the flowering time of plants through molecular breeding means to develop new crop varieties to adapt to climate and environmental changes.

[0004] Based on the above needs, inventing a gene related to the flowering traits of oats to provide candidate genes and molecular markers for the field of oat molecular breeding is of great significance in the technical field of molecular genetics. Summary of the Invention

[0005] The present invention first provides an oat AsEFT1 gene, the CDS nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2.

[0006] The present invention also provides an oat AsEFT1 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides a biological material, any one of the following (B1) to (B7):

[0008] (B1) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 or a CDS composed of the nucleotide sequence shown in SEQ ID NO.1 or a nucleic acid containing SEQ ID NO.1;

[0009] (B2) A vector containing the nucleic acid molecule described in (B1);

[0010] (B3) A microorganism containing the nucleic acid molecule described in (B1);

[0011] (B4) A microorganism containing the vector described in (B2);

[0012] (B5) A transgenic plant containing the nucleic acid molecule described in (B1);

[0013] (B6) A transgenic plant containing the vector described in (B2);

[0014] (B7) A recombinant vector / recombinant microorganism / recombinant plant cell line containing the nucleic acid sequence or partial sequence described in (B1) and used for editing (or constructing Crispr / Cas9 materials) of oats;

[0015] (B8) A protein composed of the amino acid sequence shown in SEQ ID NO.2 or a protein containing this amino acid sequence;

[0016] (B9) A protein containing the amino acid SEQ ID NO. shown in SEQ ID NO.2 and with a tag.

[0017] The present invention also provides the application of the above-mentioned AsEFT1 gene, or the above-mentioned oat AsEFT1 protein, or the above-mentioned biological material in identifying or assisting in identifying oat flowering traits or breeding.

[0018] The present invention also provides the application of a substance for detecting the above-mentioned AsEFT1 gene, or the above-mentioned oat AsEFT1 protein, or the above-mentioned biological material in oat flowering traits or breeding.

[0019] In some embodiments, the substance is a primer or a kit.

[0020] The present invention also provides a kit for identifying or assisting in identifying oat flowering traits, and the kit contains primer sequences shown in SEQ ID NO.3 - SEQ ID NO.4.

[0021] The present invention also provides the application of the above-mentioned kit in any of the following:

[0022] (A1) Identifying or assisting in identifying oat flowering traits;

[0023] (A2) Identifying or assisting in identifying oat germplasm resources;

[0024] (A3) Breeding of oats.

[0025] The present invention also provides a method for identifying or assisting in identifying oat flowering traits, including the step of detecting the above-mentioned AsEFT1 gene, or the above-mentioned oat AsEFT1 protein, or the above-mentioned biological material.

[0026] Finally, the present invention provides an application of the above method in any one of the following:

[0027] (A1) Identifying or assisting in identifying the flowering traits of oats;

[0028] (A2) Identifying or assisting in identifying oat germplasm resources;

[0029] (A3) Breeding of oats.

[0030] Compared with the prior art, the present invention has at least the following beneficial effects:

[0031] The present invention first discovered and cloned the AsEFT1 gene. Through Arabidopsis experiments, it was confirmed that it can regulate flowering traits, providing a new molecular marker for oat breeding and having great market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Heat map of transcription factor genes;

[0033] Figure 2 Expression of the AsEFT1 gene in different leaves;

[0034] Figure 3 Effect of overexpressing the AsEFT1 gene on plant flowering;

[0035] Figure 4 Effect of overexpressing the AsEFT1 gene on the number of rosette leaves. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0037] The experimental methods involved in this invention are all conventional methods, and the reagent consumables are ordered from reagent consumable companies.

[0038] 1. The CDS (nucleic acid) sequence is as follows:

[0039] ATGCTGGCCGTGTCGTCCGCGAGGTGCGCCGCCGCCGACGATGCCGCGGATCAGCACAGC

[0040] GCTGGAGCGGATGTCGCCGGGAGCGTGGAGACGGCGGCGGTAGTGACTGAACTAGACATG

[0041] AACTTCGACTTCACCGTCGACGACATAGACTTCGGGGACTTCTTCCTCAGGCTAGAGGAC

[0042] GGAGACGCGCTGCCGGACCTCGAGGTCGACCCGGCCGAGATCTTCGCCGAATTCGAGGCC

[0043] GTGGTGGCGGGCGGCGGCGTGGAGGAGCTGCAGGACCAGCAGGTGCCCTGTGCGGAGATC

[0044] TTGGCTTCCGTCGAGGACGTCGGTTCGGTGAGCCCGACCGGTGGTGCCATCGGCGTCCAG

[0045] AACACGGCGTTTGCAGCTGAGTCTGGCGGAGACGACAAGGGGGAGTGCAATAGTCTAGCC

[0046] GAGGAAGAAGGCAACATGCTGAGTGGCGACCGAGCCACTGTGCCGGACGCGAAATCTCCC

[0047] TCTTCGTCGACCACTTCATCGTCCACCGAGGCCGAAAGCCGCCGGCACAAGTCGTCAGGC

[0048] AAGAGCTCCCACGGAAAGAAGAAAGCCAAGGTGGACTGGACTCCGGATCTTCACCGGAGG

[0049] TTCGTTCAGGCCGTAGAGCAGCTCGGCATCGATAAGGCCGTGCCATCTAGGATACTGGAG

[0050] ATCATGGGGATCAACTCACTCACTAGGCACAACATAGCAAGCCATTTGCAGAAGTACCGG

[0051] TCTCACCGGAAGCACATGATTGCACGGGAGGCGGAGGCGGCGAGTTGGACCCAACGGCGG

[0052] CAGATGTACGCTGCCGGAGGACCTGCTGCCCCCGCGAAGAGGCAGGACCCGAACATGTGG

[0053] ACCGTGCCAACCGTGGGCTACTCGCCGACACCGGCTCCTCCTCCTCCGCCGCCGCCGCCG

[0054] CATGCTATGCAGCACTTTGCTCGGCCGCTGCACGTCTGGGGCCACCCGACGATGGACTCG

[0055] CCTCGGATGCCAATGTGGCCGAGGCATCATCCGATGGCGCCCCGTGCCCCCATGCCGTCA

[0056] TGGGCTCCCCCGCCGCCGCCGCCATCCGACCCGGCTTTCTGGCACCACCACCACCCGTAC

[0057] ATGAGGCCGGCGTACATGCCGACTCACGGGACGCCTTGCATGGCAATGCCGATGGCGCCT

[0058] GCGAAATTCCCTGCGCCGCCTCTAGTCCCCGTTGCTATGCCGTGTCCGACTCCGGCCTAC

[0059] ACGGCCCCCTCCCCGCAAGCACTATCAAGCAAGAGCCAACATGACAGTTCGCAGCTCCAA

[0060] CTACAATCACAACCATCTAACGAGAGCATAGACGCGGCCATCGGTGACGTTTTATCCAAA

[0061] CCGTGGCTGCCGCTGCCGCTTGGGCTGAAGCCTCCTTCGTTGGGCAGCGTCATGGGCGAG

[0062] CTCGAGAGGCAAGGCGTAGCTAATGTCCCTCAAGCGTGTGGATAG(SEQ ID NO.1);

[0063] 2. The amino acid sequence of AsEFT1 is as follows:

[0064] MLAVSSARCAAADDAADQHSAGADVAGSVETAAVVTELDMNFDFTVDDIDFGDFFLRLED

[0065] GDALPDLEVDPAEIFAEFEAVVAGGGVEELQDQQVPCAEILASVEDVGSVSPTGGAIGVQNTAFAAESGGDD

[0066] KGECNSLAEEEGNMLSGDRATVPDAKSPSSSTTSSSTEAESRRHKSSGKSSHGKKKAKVDWTPDLHRRFVQ

[0067] AVEQLGIDKAVPSRILEIMGINSLTRHNIASHLQKYRSHRKHMIAREAEAASWTQRRQMYAAGGPAAPAKRQ

[0068] DPNMWTVPTVGYSPTPAPPPPPPPPHAMQHFARPLHVWGHPTMDSPRMPMWPRHHPMAPRAPMPSWAPPP

[0069] PPPSDPAFWHHHHPYMRPAYMPTHGTPCMAMPMAPAKFPAPPLVPVAMPCPTPAYTAPSPQALSSKSQHDSS

[0070] QLQLQSQPSNESIDAAIGDVLSKPWLPLPLGLKPPSLGSVMGELERQGVANVPQACG(SEQ IDNO.2);

[0071] Example 1. Obtaining of Avena sativa AsEFT1 gene

[0072] Using Avena sativa variety "Qingyin 2" as the material, apical tissues at different growth and development stages and young spikes at different stages were cut, quickly frozen in liquid nitrogen, and RNA was extracted for sequencing. By analyzing the sequencing data, transcription factor-encoding genes whose expression levels changed in apical tissues and flowers during the growth and development of Avena sativa were screened out. GARP family transcription factors play important roles in plant growth and development and in response to biotic and abiotic stresses, but there is no report on the participation of family members in regulating plant flowering (Safi, A., Medici, A., Szponarski, W., Ruffel, S., Lacombe, B., Krouk, G., 2017. The world according to GARP transcription factors. Curr. Opin. Plant Biol. 39, 159–167.). The results of expression abundance analysis showed that with the growth and development of Avena sativa, the expression abundance of AsEFT1 gradually increased and reached the highest level in flowers (Appended Figure 1)。The flag leaf plays an important role in regulating plant flowering. As oats grow and develop, the expression abundance of the AsEFT1 gene in the flag leaf is significantly higher than that in young leaves (Appendix Figure 2 )。

[0073] Example 2. Cloning of the oat AsEFT1 gene

[0074] The gene number of AsEFT1 is VESA.00001b.r3.7Ag0002785. The CDS sequence of this gene was downloaded using Tbtools. Primers Primer1 and Primer2 were designed at the 5' end and 3' end of the CDS sequence respectively. Using the cDNA of "Qingyin No. 2" as a template, the CDS sequence of the AsEFT1 gene was amplified.

[0075] Amplification system (50 μL): 5 μL of 10× Taq Buffer, 0.5 μL of ExTaq, 4 μL of dNTPs, 2.5 μL of primer Primer1, 2.5 μL of primer Primer2, 35 μL of ddH2O, 1 μL of template.

[0076] Primer1: 5'-ATGCTGGCCGTGTCGTCCG-3' (SEQ ID NO.3);

[0077] Primer2: 5'-CTATCCACACGCTTGAGGGACAT-3' (SEQ ID NO.4);

[0078] Amplification program: Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles of amplification, and extension at 72°C for 10 min.

[0079] Example 3. Construction of overexpression vector

[0080] The overexpression vector used to construct the overexpression material of the AsEFT1 gene is Super1300. Using the cDNA of "Qingyin No. 2" as a template, the CDS sequence of the AsEFT1 gene was amplified with primers Primer3 and Primer4 with adapters, so that both ends of the recovered target CDS carried adapters (the adapter is the sequence on the vector, located at both ends of the SmaI restriction site). At the same time, the vector was digested with SmaI to linearize the vector. The CDS sequence with adapters was ligated to the linearized vector by homologous recombination.

[0081] Primer3: 5'- TCTAGA AAGCTT CTGCAGGG ATGCTGGCCG TGTCGTCCG-3' (SEQ IDNO.5); Primer4: 5'- GCTCCTC GCCCTTGCTCCCATCTATCCACACGCTTGAGGGACAT-3’(SEQ ID NO.6); the underlined part is the adapter sequence.

[0082] Cloning of the gene: The cloned AsEFT1 gene with the adapter CDS sequence was amplified using the same amplification system and procedure as in Example 1 above.

[0083] Vector linearization system: 5 μL of 10×QuickCut Buffer, 1 μL of QuickCut SmaI, plasmid ≤1 μg, supplemented with ddH2O to 50 μL.

[0084] Vector linearization condition: Incubate at 37 °C for 5 min.

[0085] The steps for recovering the target gene and the linearized vector are as follows:

[0086] (1) Cut out the agarose gel bands with the AsEFT1 gene CDS band and the linearized vector Super1300 band respectively, and place them in clean centrifuge tubes;

[0087] (2) Add three volumes of GSB, place in a clean EP tube, and dissolve the gel at 55 °C until the gel is completely melted;

[0088] (3) Add the melted gel to the centrifugal column, let it stand for 1 min, centrifuge at 10,000 g for 1 min, and discard the effluent;

[0089] (4) Add 650 μL of WB (Wash Buffer) to the centrifugal column, centrifuge at 10,000 g for 1 min, and discard the effluent;

[0090] (5) Centrifuge at 10,000 g for 1 - 2 min to completely remove the residual WB;

[0091] (6) Place the centrifugal column in a clean centrifuge tube, open the lid and let it stand for 1 min. Add 30 - 50 μL of ddH2O to the center of the centrifugal column and let it stand for 1 min;

[0092] (7) Centrifuge at 10,000 g for 1 min, and store the eluted DNA at -20 °C.

[0093] Ligation system: 5 μL of 2×Cloning Master Mix, 10 - 500 ng of linearized vector, 30 - 1500 ng of the recovered product of the target gene, and the reaction system was supplemented to 10 μL with ddH2O.

[0094] Ligation condition: Mix the system and then place it in a 50 °C air bath for ligation for 30 min.

[0095] Example 4. Obtaining of transgenic Arabidopsis thaliana

[0096] The overexpression vector Super:EFT1 was transformed into Agrobacterium tumefaciens GV3101, and the method referred to the Agrobacterium transformation method of Beijing Huayuanyang Biotechnology Co., Ltd. Referring to the infection method in the published article (Clough, S.J., and Bent, A.F. (1998). Floraldip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16:735–743.), Arabidopsis wild-type Col-0 was infected with recombinant Agrobacterium, and transgenic lines Super:EFT1-1, Super:EFT1-2, and Super:EFT1-3 were obtained through glufosinate resistance screening.

[0097] Example 5. Phenotypic detection of transgenic Arabidopsis

[0098] Seeds of Col-0, Super:EFT1-1, Super:EFT1-2, and Super:EFT1-3 were placed in 1.5 ml tubes, soaked in 6% sodium hypochlorite for 10 minutes to fully disinfect the seeds, and then the seeds were washed 6 times with sterile double-distilled water to wash off the sodium hypochlorite. Finally, an appropriate amount of double-distilled water was added to the 1.5 ml tubes, and the tubes were placed in a 4°C refrigerator. Two days later, the seeds were taken out of the refrigerator, sown on MS medium, and then the petri dishes were placed vertically in a light incubator for the seeds to germinate and grow. Seven days later, the Arabidopsis seedlings were transplanted into the soil and transferred to the greenhouse to continue growing and observe the phenotype. After 21 days, the Super:EFT1 transgenic materials showed an early-flowering phenotype (attached Figure 3 ). The number of rosette leaves is closely related to the flowering time of Arabidopsis, and the number of leaves is widely used to quantify the time of flower transition. Therefore, the number of rosette leaves was also counted at the same time. The statistical results showed that the number of rosette leaves of the Super:EFT1 transgenic materials was significantly less than that of the wild-type control (attached Figure 4 ). Light incubator and greenhouse culture conditions: photoperiod 16 h (light) / 8 h (dark), humidity 70%, temperature 22°C.

[0099] MS medium formula: 1650 mg of NH4NO3, 1900 mg of KNO3, 370 mg of MgSO4·7H2O, 170 mg of KH2PO4, 440 mg of CaCl2·2H2O, 22.3 mg of MnSO4·4H2O, 0.83 mg of KI, 0.025 mg of CuSO4·5H2O, 6.25 mg of H3BO5, 0.025 mg of CoCl·6H2O, 8.65 mg of ZnSO4·7H2O, 0.25 mg of Na2Mo4·2H2O, 27.8 mg of FeSO4·7H2O, 37.3 mg of Na2·EDTA. Dissolve it thoroughly with double-distilled water and make up the volume to 1 liter, and then add 8 g of agar powder. Sterilize it at 115 °C for 15 minutes with a pressure cooker. When the temperature drops to about 40 °C, pour the medium into glass petri dishes. Soil culture soil formula: Mix imported soil, vermiculite and Northeast black soil in a ratio of 1:1:1, and divide them into flower pots for packaging.

[0100] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. AsEFT1 The application of a gene in plant breeding is characterized in that: Build contains AsEFT1 Transgenic strains of gene overexpression vectors; Wherein, the nucleotide sequence of the gene is shown as SEQ ID NO.1; Wherein, the plant is oat or Arabidopsis thaliana.

2. AsEFT1 Application of gene overexpression vector in plant breeding, the AsEFT1 The gene is shown in SEQ ID NO.1, and the plant is oat or Arabidopsis thaliana.

3. Use of a microorganism containing the overexpression vector according to claim 2 in plant breeding, wherein the plant is oat or Arabidopsis thaliana.

4. Use of a transgenic plant containing the overexpression vector according to claim 2 in plant breeding, wherein the plant is oat or Arabidopsis thaliana.

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