Oat AsEFT1 gene and application of encoded protein thereof

Abstract

The invention belongs to the technical field of molecular genetics, and particularly relates to an oat AsEFT1 gene and application of an encoding protein thereof. The oat AsEFT1 gene is characterized in that a CDS nucleotide sequence of the gene is as shown in SEQ ID NO.1, and an amino acid sequence coded by the gene is as shown in SEQ ID NO.2. The AsEFT1 gene is found and cloned for the first time, arabidopsis thaliana experiments prove that the AsEFT1 gene can regulate and control the flowering character, a new molecular marker is provided for oat breeding, and the AsEFT1 gene has very high market application value.

Description

Technical Field

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

[0002] Oats (Avena sativa L.), a dual-purpose grain and forage crop in the grass family, boast high yield, high quality, and strong stress tolerance. They are a key forage for addressing the shortage of forage grass in winter and spring pastures in northern my country and play a vital role in the development of the domestic forage industry. In recent years, with the rapid development of my country's animal husbandry industry, the demand for forage oat hay has become increasingly urgent, placing higher demands on the breeding of high-yield forage oat varieties.

[0003] Flowering is an important physiological process in higher plants, from reproductive growth to vegetative growth, and is crucial to plant reproduction. In agricultural production, timely flowering of crops is of great significance for variety breeding, introduction and breeding, as well as stable and increased yields. By controlling the timing of flowering, 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 technology, the identification of important genes that control plant flowering can provide potential targets for crop genetic improvement. Through molecular breeding methods, the flowering time of plants can be precisely controlled, and new crop varieties can be developed to adapt to climate and environmental changes.

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

[0005] The present invention first provides an oat AsEFT1 gene, the CDS nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown as 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 biomaterial, 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 consisting of the base sequence shown in SEQ ID NO. 1 or a nucleic acid comprising 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 of (B1) for editing (or constructing Crispr / Cas9 materials) oats;

[0015] (B8) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 or a protein comprising the amino acid sequence;

[0016] (B9) A protein comprising the amino acids shown in SEQ ID NO. 2 and having a tag.

[0017] The present invention also provides the use 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 the flowering traits of oats or breeding.

[0018] The present invention also provides a method for detecting the aforementioned AsEFT1 gene, or the aforementioned oat AsEFT1 protein, or the aforementioned biological material in oat flowering traits or breeding.

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

[0020] The present invention also provides a kit for identifying or assisting in identifying the flowering traits of oats, wherein the kit contains primer sequences as shown in SEQ ID NO.3 to SEQ ID NO.4.

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

[0022] (A1) Identify or assist in identifying flowering traits of oats;

[0023] (A2) Identify or assist in identifying oat germplasm resources;

[0024] (A3) Breeding of oats.

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

[0026] The present invention finally provides an application of the above method in any of the following:

[0027] (A1) Identify or assist in identifying flowering traits of oats;

[0028] (A2) Identify or assist 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 discovered and cloned the AsEFT1 gene for the first time. Experiments on Arabidopsis thaliana 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 Transcription factor gene heatmap;

[0033] Figure 2 The expression of AsEFT1 gene in different leaves;

[0034] Figure 3 Effects of overexpression of the AsEFT1 gene on plant flowering;

[0035] Figure 4 Effect of overexpression of AsEFT1 gene on the number of rosette leaves. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

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

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

[0039] ATGCTGGCCGTGTCGTCCGCGAGGTGCGCCGCCGCCGACGATGCCGGATCAGCACAGC

[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: Acquisition of the oat AsEFT1 gene

[0072] Using the oat variety "Qingyin No. 2" as the material, apical tissues and young panicles at different growth and development stages were cut, quick-frozen in liquid nitrogen, and RNA was extracted for sequencing. By analyzing the sequencing data, transcription factor encoding genes whose expression levels in apical tissues and flowers changed with the growth and development of oats were screened. GARP family transcription factors play an important role in plant growth and development and in responding to biotic and abiotic stresses, but there have been no reports of members of this family being involved 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.). Expression abundance analysis results showed that the expression abundance of AsEFT1 gradually increased with the growth and development of oats, reaching the highest expression level in flowers (Appendix). Figure 1Flag leaves play an important role in regulating plant flowering. As oats grow and develop, the expression abundance of the AsEFT1 gene in flag leaves is significantly higher than that in tender leaves (see attached). Figure 2 ).

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

[0074] The gene ID 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' and 3' ends of the CDS sequence, respectively, and the CDS sequence of the AsEFT1 gene was amplified using the cDNA of "Qingyin 2" as a template.

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

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

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

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

[0079] Example 3: Construction of overexpression vector

[0080] The overexpression vector used to construct the AsEFT1 gene overexpression material was Super1300. Using the "Qingyin 2" cDNA as a template, the CDS sequence of the AsEFT1 gene was amplified using primers Primer3 and Primer4 with adapters. The recovered target CDS sequence was then terminated with adapters (i.e., sequences on the vector located at either end of the SmaI restriction site). The vector was then linearized by digesting it with SmaI. The adapter-containing CDS sequence was then ligated to the linearized vector using 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 sequence is the linker sequence.

[0082] Gene cloning: The AsEFT1 gene with the linker CDS sequence was cloned, and the amplification system and procedure were the same as those in Example 1 above.

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

[0084] Vector linearization conditions: incubate at 37°C for 5 min.

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

[0086] (1) Cut the agarose gel containing the AsEFT1 gene CDS band and the linearized vector Super1300 band separately and place them in a clean centrifuge tube;

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

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

[0089] (4) Add 650 μL of WB (Wash Buffer) to the centrifuge column, centrifuge at 10,000 g for 1 min, and discard the flow-through.

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

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

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

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

[0094] Connection conditions: Mix the system and place it in a 50℃ air bath for 30 minutes.

[0095] Example 4: Obtaining transgenic Arabidopsis

[0096] The overexpression vector Super:EFT1 was transformed into Agrobacterium GV3101 using the Agrobacterium transformation method described by Beijing Huayueyang Biotechnology Co., Ltd. Arabidopsis wild-type Col-0 was infected with recombinant Agrobacterium, following the infection method described in a published article (Clough, SJ, and Bent, AF (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16:735–743.). 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] Col-0, Super:EFT1-1, Super:EFT1-2 and Super:EFT1-3 seeds were placed in 1.5 ml tubes and soaked in 6% sodium hypochlorite for 10 minutes to fully disinfect the seeds. The seeds were then washed 6 times with sterilized double distilled water to clean 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. After 2 days, the seeds were taken out of the refrigerator and spotted on MS culture medium. The culture dishes were then placed upright in a light incubator to allow the seeds to germinate and grow. After 7 days, the Arabidopsis seedlings were transplanted into the soil and transferred to the greenhouse for continued growth and phenotype observation. After 21 days, the Super:EFT1 transgenic material showed an early flowering phenotype (see attached). Figure 3 The number of rosette leaves is closely related to the flowering time of Arabidopsis thaliana. The number of leaves is widely used to quantify the time of floral transition. Therefore, the number of rosette leaves was also counted. The statistical results showed that the number of rosette leaves in the Super:EFT1 transgenic material was significantly less than that in the wild-type control (Appendix Figure 4 The culture conditions in the light incubator and greenhouse were as follows: a photoperiod of 16 h (light) / 8 h (darkness), a humidity of 70%, and a temperature of 22°C.

[0099] MS medium formula: 1650 mg NH₄NO₃, 1900 mg KNO₃, 370 mg MgSO₄.7H₂O, 170 mg KH₂PO₄, 440 mg CaCl₂.2H₂O, 22.3 mg MnSO₄.4H₂O, 0.83 mg KI, 0.025 mg CuSO₄.5H₂O, 6.25 mg H₃BO₅, 0.025 mg CoCl₂.6H₂O, 8.65 mg ZnSO₄.7H₂O, 0.25 mg Na₂Mo₄.2H₂O, 27.8 mg FeSO₄.7H₂O, 37.3 mg Na₂EDTA. Dissolve thoroughly in double-distilled water and dilute to 1 liter. Add 8 g agar powder. Sterilize in an autoclave at 115°C for 15 minutes, then cool to approximately 40°C before pouring the medium into a glass Petri dish. Soil formula for soil cultivation: Mix imported soil, vermiculite and Northeast black soil in a ratio of 1:1:1 and divide them into flower pots.

[0100] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An oat AsEFT1 gene, characterized in that The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. An oat AsEFT1 protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A biomaterial, any one of the following (B1) to (B7): (B1) a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 or a CDS consisting of the base sequence shown in SEQ ID NO.1 or a nucleic acid comprising the SEQ ID NO.1; (B2) a vector containing the nucleic acid molecule described in (B1); (B3) a microorganism containing the nucleic acid molecule described in (B1); (B4) a microorganism containing the vector described in (B2); (B5) a transgenic plant containing the nucleic acid molecule described in (B1); (B6) a transgenic plant containing the vector described in (B2); (B7) A recombinant vector / recombinant microorganism / recombinant plant cell line containing the nucleic acid sequence or a partial sequence of (B1) for editing (or constructing Crispr / Cas9 materials) oats; (B8) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 or a protein comprising the amino acid sequence; (B9) A protein comprising the amino acids shown in SEQ ID NO. 2 and having a tag.

4. Use of the AsEFT1 gene according to claim 1, or the oat AsEFT1 protein according to claim 2, or the biological material according to claim 3 in identifying or assisting in identifying the flowering traits of oats or breeding.

5. Use of a substance for detecting the AsEFT1 gene according to claim 1, or the oat AsEFT1 protein according to claim 2, or the biological material according to claim 3 in oat flowering traits or breeding.

6. The use according to claim 5, characterized in that: The substance is a primer or a kit.

7. A kit for identifying or assisting in identifying flowering traits of oats, characterized in that: The kit contains primer sequences as shown in SEQ ID NO.3-SEQ ID NO.

4.

8. Use of the kit according to claim 7 in any of the following: (A1) Identify or assist in identifying flowering traits of oats; (A2) Identify or assist in identifying oat germplasm resources; (A3) Breeding of oats.

9. A method for identifying or assisting in identifying flowering traits of oats, characterized in that: The method comprises the step of detecting the AsEFT1 gene according to claim 1, or the oat AsEFT1 protein according to claim 2, or the biological material according to claim 3.

10. Use of the method according to claim 9 in any of the following: (A1) identifying or assisting in identifying flowering traits of oats; (A2) Identify or assist in identifying oat germplasm resources; (A3) Breeding of oats.

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