Use of genes in increasing plant biomass and seed yield

Abstract

The present invention belongs to the field of plant genetics research, and particularly relates to gene functions for increasing plant biomass and seed yield. The present invention discloses the use of a gene that negatively regulates plant biomass and seed yield. This gene is the AT3G28990 gene whose nucleotide sequence is shown in SEQ ID NO: 1. By knocking out the AT3G28990 gene in plants, the biomass and seed yield of plants can be increased. Knocking out the AT3G28990 gene of Arabidopsis thaliana can effectively increase the biomass and yield of plants. This has important reference significance for the breeding of high-yield crops.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetics research, and particularly relates to gene functions for increasing plant biomass and seed yield.

Background Art

[0002] Plant biomass synthesis is closely related to the yield of fruits and seeds. Increasing the overall biomass of plants can effectively increase the yield of fruits and seeds, which is very important for crop breeding and production practice. Arabidopsis thaliana is a model plant for dicotyledons, and the study of its gene function in the regulation of biomass synthesis has important reference significance for other plants.

[0003] The gene AT3G28990 is publicly available in the Arabidopsis Information Resource database (https: / / www.arabidopsis.org / ), but the function and purpose of this gene are unknown.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The technical problem solved by the present invention is to provide a gene that can effectively increase plant biomass and seed yield and guide the breeding of high-yield crops.

Means for Solving the Problems

[0005] To solve the above technical problems, the present invention provides the use of a gene that negatively regulates plant biomass and seed yield. This gene is the AT3G28990 gene having the nucleotide sequence shown in SEQ ID NO: 1. Knocking out the AT3G28990 gene in plants can increase (significantly increase) plant biomass and seed yield.

[0006] As an improvement in the use of the gene that negatively regulates the plant biomass and seed yield of the present invention, the coding region sequence of the AT3G28990 gene is shown in SEQ ID NO: 2.

[0007] As a further improvement in the use of the gene that negatively regulates the plant biomass and seed yield of the present invention, the plant is Arabidopsis thaliana.

[0008] As a further improvement in the use of the gene that negatively regulates the plant biomass and seed yield of the present invention, the sgRNA sequence for targeted knockout of the AT3G28990 gene: 5′-GAAACCAGTGGACGTGATGA-3′.

[0009] The present invention also provides a method for regulating plant biomass and seed yield, that is, a method for knocking out the AT3G28990 gene in a plant to obtain a knockout line of the AT3G28990 gene. The nucleotide sequence of the AT3G28990 gene is shown in SEQ ID NO: 1.

[0010] As an improvement in the method for regulating the plant biomass and seed yield of the present invention: AT3G28990 gene knockout strains: ko-1, ko-2; The nucleotide sequence of ko-1 is shown in SEQ ID NO: 4, and the nucleotide sequence of ko-2 is shown in SEQ ID NO: 6.

[0011] The gene AT3G28990 of the model plant Arabidopsis thaliana of the present invention has the genomic nucleotide sequence shown in SEQ ID NO: 1 and the protein sequence encoded by SEQ ID NO: 2.

[0012] The present invention also provides a method for knocking out the AT3G28990 gene of Arabidopsis thaliana, which includes the following steps.

[0013] 1) Using CRISPR / Cas9 technology, design an sgRNA sequence for targeted knockout of the AT3G28990 gene: 5′-GAAACCAGTGGACGTGATGA-3′.

[0014] 2) Use the array from step 1) to synthesize primers and construct them into the CRISPR / Cas9 vector.

[0015] 3) Genetically transform the vector obtained in step 2) into the Arabidopsis wild-type variety Col-0 to obtain the corresponding transgenic plants. Two lines, ko-1 and ko-2, with different mutant forms of the AT3G28990 gene were identified from the transgenic plants.

[0016] The technical solution of the present invention is as follows. Using the CRISPR / Cas9 gene editing technology, based on the nucleotide sequence of the AT3G28990 gene (SEQ ID NO: 1), an sgRNA sequence specifically targeting the AT3G28990 gene was designed in its coding region, and the corresponding CRISPR / Cas9 vector was constructed. It was genetically transformed into the Arabidopsis wild-type variety Col-0 to obtain transgenic plants. The AT3G28990 gene in the transgenic plants was PCR amplified and sequenced, and two non-mutant strains of the AT3G28990 gene, ko-1 (1-base insertion) and ko-2 (28-base deletion + 6-base insertion), were identified (Figure 1). Both caused frameshift mutations in the AT3G28990 gene, resulting in premature termination of protein translation, that is, the gene was knocked out. In the plants of the ko-1 mutant strain, the coding region sequence of the AT3G28990 gene is SEQ ID NO: 4, and the encoded protein sequence is SEQ ID NO: 5. In the plants of the ko-2 mutant strain, the coding sequence of the AT3G28990 gene is SEQ ID NO: 6, and the encoded protein sequence is SEQ ID NO: 7.

[0017] Compared with the wild-type control of Arabidopsis, the fresh plant weight (Figure 1), dry plant weight (Figure 2), number of fruits per plant (Figure 3), and seed weight (Figure 4) of the mutant strains ko-1 and ko-2 all increased significantly. This indicates that knocking out the AT3G28990 gene in Arabidopsis can effectively increase plant biomass and yield, which is of important reference value for the breeding of high-yield crops.

Brief Description of the Drawings

[0018] Hereinafter, with reference to the accompanying drawings, specific embodiments of the present invention will be described in more detail.

Figure 1

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Modes for Carrying Out the Invention

[0019] Hereinafter, the present invention will be further described in connection with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0020] (Example 1) Construction of Arabidopsis thaliana AT3G28990 Gene Knockout Vector According to the AT3G28990 gene sequence (SEQ ID NO: 1), a target editing sgRNA sequence was designed for its coding region: 5′-GAAACCAGTGGACGTGATGA-3′. Except for the construction method of the knockdown vector of the AT3G28990 gene, other procedures were carried out according to the product manual using the CRISPR / Cas9 kit (Biogle, China).

[0021] (Example 2) Genetic transformation of Arabidopsis thaliana using the AT3G28990 gene knockout vector The CRISPR / Cas9 vector constructed in Example 1 was genetically transformed into the wild-type Arabidopsis thaliana variety Col-0, and the transformation method was as described in the literature (Plant Journal, 1998, 16(6): 735-743). The corresponding transgenic Arabidopsis thaliana plants (including ko-1 and ko-2).

[0022] (Example 3) Identification of Arabidopsis thaliana AT3G28990 gene knockout plants Collect 0.1 g of fresh leaves from Arabidopsis thaliana wild-type variety Col-0 and transgenic plants, grind them in liquid nitrogen, add 300 μL of extraction solution (0.1 mol / L Tris-HCl pH8.0, 500 mmol / L NaCl, 1.25 g / L SDS), incubate at 65 °C for 1 hour, shake 2-3 times during this period, add 100 μL of 5 mol / L KAC, shake well, leave it in an ice bath for 10 minutes, add 250 μL of chloroform, mix well, and leave it for 5 minutes. Then, centrifuge at 8000 r / min for 10 minutes, transfer 250 μL of the supernatant to a new 1.5 ml tube, add 250 μL of pre-cooled isopropyl alcohol, shake well until a cotton-like precipitate appears, and refrigerate for 10 minutes. Then, centrifuge at 12000 r / min at 4 °C for 5 minutes, discard the supernatant, add 1 mL of 70% ethanol, centrifuge at 12000 r / min for 7 minutes, discard the supernatant, invert it and air-dry at room temperature, add 80 μL of ddH2O, mix well, and store at -20 °C for later use.

[0023] Primers for PCR amplification of the AT3G28990 gene were synthesized. The upstream primer was 5′-CATGTGCATGCCGTTTCTTCT-3′, and the downstream primer was 5′-GCTTCTGCTCGTACGTCTCA-3′. DNA from Arabidopsis wild-type variety Col-0 and transgenic plants was used as a template, and PCR amplification was carried out using 2× Taq PCR reagent (Tiangen, Beijing). The PCR amplification system consisted of 1 μL of template DNA, 10 μL of 2× Taq PCR MasterMixII, 1 μL of primer (upstream + downstream mix, 10 μM), and ddH2O was added to make a total volume of 20 μL. The PCR amplification program was pre-denaturation at 94°C for 2 minutes, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, followed by extension at 72°C for 2 minutes.

[0024] The sequence of the coding region of the AT3G28990 gene in WT plants is shown in SEQ ID NO: 2, which encodes a protein of 88 amino acids, and its sequence is shown in SEQ ID NO: 3. The PCR products of transgenic plants were sequenced and analyzed. As a result, two types of mutant plants carrying the AT3G28990 gene were identified.

[0025] In the ko-1 plant, one base was inserted into the AT3G28990 gene (Figure 1). As a result, the translation of the protein encoded by the gene, which only encodes 53 amino acids, ended prematurely. The coding sequence after the mutation is SEQ ID NO: 4, and the encoded protein sequence is SEQ ID NO: 5.

[0026] In the ko-2 plant, 28 bases were lost and 6 bases were inserted in the AT3G28990 gene (Figure 1). As a result, the translation of the protein encoded by the gene ended prematurely, encoding only 44 amino acids. The coding sequence after the mutation is SEQ ID NO: 6, and the encoded protein sequence is SEQ ID NO: 7.

[0027] (Example 4) Biomass statistics of Arabidopsis AT3G28990 gene knockout plants On the same day of maturity, 10 wild-type, ko-1, and ko-2 plants of the Arabidopsis thaliana AT3G28990 gene were randomly selected. After removing the roots, the fresh weight of the above-ground parts of individual plants was measured. Subsequently, the plants were dried at 80 °C, and the dry weight of the above-ground parts of individual plants was weighed. The t-test method was used to analyze the significant differences.

[0028] The results are shown in Figures 2 and 3. The fresh weight and dry weight of the above-ground parts of ko-1 and ko-2 plants with the AT3G28990 gene knocked out were significantly higher than those of the wild-type control with this gene knocked out. Knocking out this gene can effectively increase the biomass synthesis of Arabidopsis thaliana plants.

[0029] (Example 5) Measurement of the 1000-seed weight of Arabidopsis thaliana On the same day of maturity, 10 Arabidopsis thaliana AT3G28990 gene knockout ko-1, ko-2 plants and their wild-type controls were randomly selected, and the number of siliques on the main stem of each plant was counted. Next, all siliques were removed, and the seeds of individual plants were collected separately, and the weight of each 1,000 seeds was measured using an electronic balance. The t-test method was used to analyze the significant differences.

[0030] The results are shown in Figures 4 and 5. The number of siliques and the 1000-seed weight of ko-1 and ko-2 plants with AT3G28990 knocked out were significantly higher than those of the wild-type control. It was found that knocking out this gene can effectively increase the number of fruits and the weight of seeds of Arabidopsis thaliana, and improve the seed yield.

[0031] Finally, it should also be noted that the above enumeration is only some specific embodiments of the present invention. It goes without saying that the present invention is not limited to the above embodiments, and various modifications are possible. All changes that can be directly derived or associated by those skilled in the art from the disclosure of the present invention should be considered to be within the protection scope of the present invention.

Claims

1. Use of a gene, wherein the gene is the AT3G28990 gene having the nucleotide sequence shown in SEQ ID NO: 1, and knocking out the AT3G28990 gene of a plant to increase the biomass and seed yield of the plant, characterized in that it is a use of a gene for negatively controlling plant biomass and seed yield.

2. The use of the gene for negatively controlling plant biomass and seed yield according to claim 1, characterized in that the coding region sequence of the AT3G28990 gene is shown in SEQ ID NO:

2.

3. The use of the gene for negatively controlling plant biomass and seed yield according to claim 1 or 2, characterized in that the plant is Arabidopsis thaliana.

4. The use of the gene for negatively controlling plant biomass and seed yield according to any one of claims 1 to 3, characterized in that the sgRNA sequence for targeted knockout of the AT3G28990 gene is 5′-GAAACCAGTGGACGTGATGA-3′.

5. In a plant, knocking out the AT3G28990 gene to obtain a knockout line of the AT3G28990 gene, characterized in that the nucleotide sequence of the AT3G28990 gene is shown in SEQ ID NO: 1, a method for regulating plant biomass and seed yield.

6. The method for adjusting plant biomass and seed yield according to claim 5, characterized in that the AT3G28990 gene knockout strains are ko-1 and ko-2, the nucleotide sequence of ko-1 is shown in SEQ ID NO: 4, and the nucleotide sequence of ko-2 is shown in SEQ ID NO: 6.

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