Genetic use 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 dicotyledonous plants, and the research on 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 of a plant can increase (significantly increase) the plant biomass and seed yield.

[0006] As an improvement to the use of genes for negatively controlling plant biomass and seed yield according to the present invention, the coding region sequence of the AT3G28990 gene is shown in Sequence ID No. 2.

[0007] As a further improvement to the use of genes for negatively controlling plant biomass and seed yield in the present invention, the plant is Arabidopsis thaliana.

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

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

[0010] As an improvement to the method for regulating 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, according to the present invention, has the genomic base sequence shown in Sequence ID No. 1 and the protein sequence encoded in Sequence ID No. 2.

[0012] The present invention also provides a method for knocking out the AT3G28990 gene in Arabidopsis thaliana, comprising the following steps:

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

[0014] 2) Synthesize primers using the sequence from step 1) and construct them in a CRISPR / Cas9 vector.

[0015] 3) The vector obtained in step 2) was used to genetically transform the wild-type Arabidopsis thaliana variety Col-0 to obtain the corresponding transgenic plants. Two lines, ko-1 and ko-2, possessing different mutants of the AT3G28990 gene, were identified from the transgenic plants.

[0016] The technical solution of the present invention is as follows: Using CRISPR / Cas9 gene editing technology, an sgRNA sequence specifically targeting the AT3G28990 gene was designed in its coding region based on the nucleotide sequence of the AT3G28990 gene (SEQ ID NO: 1), and a corresponding CRISPR / Cas9 vector was constructed. These vectors were genetically transformed into the wild-type Arabidopsis thaliana variety Col-0 to obtain transgenic plants. The AT3G28990 gene in these transgenic plants was amplified by PCR and sequenced, identifying two non-mutant strains of the AT3G28990 gene: ko-1 (1 nucleotide insertion) and ko-2 (28 nucleotide deletion + 6 nucleotide insertion) (Figure 1). Both strains induced frameshift mutations in the AT3G28990 gene, resulting in premature termination of protein translation, i.e., gene knockout. In the ko-1 mutant plant, the coding region sequence of the AT3G28990 gene is SEQ ID NO: 4, and the coding protein sequence is SEQ ID NO: 5. In the ko-2 mutant plant, the coding sequence for the AT3G28990 gene is sequence number 6, and the coding protein sequence is sequence number 7.

[0017] Compared to the wild-type control of Arabidopsis thaliana, the mutants ko-1 and ko-2 showed significant increases in fresh plant weight (Figure 1), dry plant weight (Figure 2), number of fruits per plant (Figure 3), and seed weight (Figure 4). This indicates that knocking out the AT3G28990 gene in Arabidopsis thaliana can effectively increase plant biomass and yield, which is of significant importance for breeding high-yielding crops. [Brief explanation of the drawing]

[0018] Hereinafter, specific embodiments of the present invention will be described in more detail with reference to the attached drawings. [Figure 1] This shows the editing status of the target site of the AT3G28990 gene in Arabidopsis thaliana ko-1 and ko-2 mutant plants. Arrows or underlines indicate the location of the mutation. [Figure 2] This shows measurements of fresh plant weights of the above-ground parts of Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type control. [Figure 3] This shows the measurement of the dry plant weight of the above-ground parts of Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type controls. [Figure 4] This shows statistics on the number of siliques per plant in Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type controls. [Figure 5] This shows the thousand-grain weight measurements of Arabidopsis thaliana ko-1 and ko-2 mutant plants and their wild-type controls. The values ​​in Figures 2-5 are mean ± standard deviation. ** indicates a highly significant difference (P<0.01) between the ko-1 or ko-2 mutant plants and the wild-type control, as determined by t-test analysis. [Modes for carrying out the invention]

[0019] The present invention will be further described below in relation to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0020] (Example 1) Construction of an Arabidopsis thaliana AT3G28990 gene knockout vector According to the AT3G28990 gene sequence (SEQ ID NO: 1), a targeted editing sgRNA sequence was designed in its coding region: 5′-GAAACCAGTGGACGTGATGA-3′. The construction method, other than the knockdown vector of the AT3G28990 gene, was carried out using a CRISPR / Cas9 kit (Biogle, China) according to the product instructions.

[0021] (Example 2) Genetic transformation of Arabidopsis thaliana with 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 flocculent 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. Using upstream 5′-CATGTGCATGCCGTTTCTTCT-3′, downstream 5′-GCTTCTGCTCGTACGTCTCA-3′, and DNA from the Arabidopsis thaliana wild-type variety Col-0 and transgenic plants as templates, PCR amplification was performed using 2× Taq PCR reagent (Tiangen, Beijing). The PCR amplification system consisted of 1 μL of template DNA, 10 μL of 2× Taq PCR Master Mix II, 1 μL of primers (upstream + downstream mix, 10 μM), and ddH2O to make a total volume of 20 μL. The PCR amplification program consisted of 35 cycles of pre-denaturation at 94°C for 2 minutes, 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 coding region of the AT3G28990 gene in WT plants is shown in SEQ ID NO: 2, and it codes for an 88-amino acid protein, the sequence of which is shown in SEQ ID NO: 3. Sequencing and analysis of PCR products from transgenic plants identified two mutant plants possessing the AT3G28990 gene.

[0025] In the ko-1 plant, a single base was inserted into the AT3G28990 gene (Figure 1), resulting in the premature termination of the translation of the gene encoding only 53 amino acids. The resulting coding sequence is Sequence ID No. 4, and the encoded protein sequence is Sequence 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 gene's translation protein terminated prematurely, encoding only 44 amino acids. The mutated coding sequence is sequence number 6, and the encoded protein sequence is sequence number 7.

[0027] (Example 4) Biomass statistics of Arabidopsis thaliana AT3G28990 gene knockout plants On the same day of maturation, ten plants each of the wild-type, ko-1, and ko-2 Arabidopsis thaliana AT3G28990 gene were randomly selected. After removing the roots, the fresh weight of the above-ground portion of each plant was measured. The plants were then dried at 80°C, and the dry weight of the above-ground portion of each plant was weighed. Significant differences were analyzed using the t-test method.

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

[0029] (Example 5) Measurement of 1000 grain weight of Arabidopsis thaliana On the same day of maturation, ten 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. All siliques were then removed, and the seeds from each individual plant were collected separately. The weight of 1,000 seeds from each plant was measured using an electronic balance. Significant differences were analyzed using the t-test.

[0030] The results are shown in Figures 4 and 5. The number of siliques and the weight of 1,000 seeds in AT3G28990 knockout ko-1 and ko-2 plants were significantly higher than those of the wild-type control. Knocking out this gene effectively increased the number of fruits and seed weight in Arabidopsis thaliana, resulting in improved seed yield.

[0031] Finally, it should be noted that the above enumeration represents only some specific embodiments of the present invention. The present invention is not limited to these embodiments, and various modifications are possible. All modifications that a person skilled in the art can directly derive or associate with the disclosure of the present invention should be considered to fall within the scope of protection of the present invention.

Claims

1. A method for increasing the biomass and seed yield of Arabidopsis thaliana by knocking out the AT3G28990 gene, the nucleotide sequence of which is shown in Sequence ID No. 1, The protein coding region sequence of the AT3G28990 gene is shown in Sequence ID No.

2. A method for increasing the biomass and seed yield of Arabidopsis thaliana, characterized in that the sgRNA sequence for targeting and knocking out the AT3G28990 gene is 5′-GAAACCAGTGGAACGTGATATGA-3′, and a CRISPR / Cas9 vector is constructed using the sgRNA.

2. A method for increasing the biomass and seed yield of Arabidopsis thaliana according to Claim 1, characterized in that, in Arabidopsis thaliana, gene transformation is performed using the CRISPR / Cas9 vector to obtain two lines, ko-1 and ko-2, having mutant forms of the AT3G28990 gene, the nucleotide sequence of ko-1 is shown as SEQ ID NO: 4, and the nucleotide sequence of ko-2 is shown as SEQ ID NO: 6.

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