Use of osbzip40 protein or its encoding gene in regulating rice seed germination time

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to application of OsbZIP40 protein or a coding gene thereof in regulating seed germination time of rice, and the present application obtains a rice osbzip40 mutant with nucleotide sequences of SEQ ID NO. 8 and SEQ ID NO. 9 by using CRISPR / Cas9 technology to target edit a target site sequence as shown in SEQ ID NO. 3 in the OsbZIP40 gene, and the rice osbzip40 mutant can effectively and quickly improve the seed germination rate of rice. The gene and the genetic engineering technical means provided by the present application provide important gene resources for solving problems such as seed germination retardation and irregular germination during direct seeding of rice, and have a relatively wide application prospect in agricultural production.

Description

Technical Field

[0001] This invention relates to the application of OsbZIP40 protein or its encoding gene in regulating rice seed germination time, and belongs to the fields of biotechnology and plant genetic engineering technology. Background Technology

[0002] Rice, as one of my country's main food crops, supports the food needs of more than half of the population, and its yield is of strategic significance to national food security. Against the backdrop of economic transformation, rising agricultural labor costs coupled with an aging population have led to severe challenges to traditional planting methods, creating an urgent need for mechanized production. Direct seeding technology for rice has been rapidly adopted due to its ease of operation and compatibility with mechanized farming. The germination ability and speed of seeds directly determine the uniformity and neatness of rice seedling emergence in direct seeding, becoming one of the key factors restricting the application of this technology. Therefore, identifying and utilizing key genes controlling rice seed germination is of significant scientific and practical value for improving the adaptability of varieties to direct seeding, reducing labor intensity, and ensuring stable and efficient rice yields.

[0003] Transcription factors play a crucial role in seed germination. Basic leucine zipper (bZIP) transcription factors are one of the most conserved and numerous families in eukaryotes. These family members are widely involved in physiological processes such as plant growth and development, flowering transition, environmental signal transduction, and stress response, and are closely related to seed germination. While rice OsbZIP40 has been shown to regulate rice flowering and drought resistance, its function in seed germination has not been reported. Therefore, in-depth investigation of the effects of the OsbZIP40 gene and its encoded protein on seed germination will contribute to the precise regulation of rice seed germination, providing strong theoretical support and technical assurance for direct-seeded rice production. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the application of OsbZIP40 protein or its encoding gene in regulating rice seed germination.

[0005] Technical solution: This invention provides the application of OsbZIP40 protein in regulating rice seed germination, and the amino acid sequence of the rice OsbZIP40 protein is shown in SEQ ID NO.2.

[0006] SEQ ID NO.2: MASSRVMAAAAASSSSSPPPPPPAAAAAGGAADLARFRSTSSGI GSMNMDDILRNIYGEAAPPPGAAGSAPAPPPAGEAAGAPVAEVAARRTAEEVWKEISSSGGLSAPAPAPAAGAAGRGGGPEMTLEDFLAREDDPRATAVEGNMVVGFPNVTE GVGTAGGGRGGGGGGRGRKRTLMDPADRAAMQRQKRMIKNRESAARSRERKQAYIAELEAQVAELEEEHAQLLREQEEKNQKRLKEIKEQAVAVVIRKKTQDLRRTNSMEW.

[0007] The present invention also provides the application of the gene encoding the above-mentioned OsbZIP40 protein in regulating rice seed germination, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008]

[0009] Furthermore, the application involves enhancing the germination rate of rice seeds by knocking out the OsbZIP40 gene.

[0010] Furthermore, the application also includes improving the uniformity of rice seedling emergence.

[0011] A method for obtaining rice seeds with a fast germination rate includes: genetically transforming rice with a pre-constructed OsbZIP40 gene knockout vector, obtaining regenerated plants, and then identifying homozygous lines with successfully knocked-out OsbZIP40 through sequencing.

[0012] Furthermore, the OsbZIP40 gene knockout vector targets the nucleotide sequence shown in SEQ ID NO.3.

[0013] Furthermore, the primer sequences used for target knockout are shown in SEQ ID NO.4-5.

[0014] Furthermore, the primer sequences used to identify genetically transformed rice are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0015] Furthermore, the two rice OsbZIP40 mutant lines obtained after genetic transformation exhibited faster seed germination rates, and their gene nucleotide sequences are shown in SEQ ID NO.8 or 9.

[0016]

[0017]

[0018] Furthermore, the rice variety used for genetic transformation was Nipponbare.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This application discloses for the first time the important role of the rice OsbZIP40 gene in regulating rice seed germination, and that the OsbZIP40 gene and its encoded protein can regulate rice seed germination. By targeting and editing this gene sequence, this invention can significantly promote rice seed germination and improve seedling uniformity, providing excellent gene resources for solving problems such as delayed seed germination and uneven germination during direct seeding of rice, and has broad application prospects in agricultural production. Attached Figure Description

[0020] Figure 1 Schematic diagram of mutation types and amino acid changes of osbzip40-1 and osbzip40-2 obtained by CRISPR / Cas9 editing of OsbZIP40 under the genetic background of Nipponbare.

[0021] Figure 2 Photographs of seeds of osbzip40-1 and osbzip40-2 mutants and their wild-type seeds at 84 hours of germination under the Nipponbare genetic background;

[0022] Figure 3 Figure 1 shows the statistical results of seed germination rates of osbzip40-1 and osbzip40-2 mutants and their wild-type strains under the Nipponbare genetic background. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1: Construction of OsbZIP40 gene knockout vector and acquisition of rice gene knockout plants

[0025] Step 1: Target Primer Design: The MSU_Locus sequence of the OsbZIP40 gene was submitted to the CRISPR-GE online website. A sequence with high knockout efficiency and good specificity (SEQ ID NO.3: GGCGTCGTCGAGGGTGATGG) was selected from the first exon region of this gene, and a pair of target primers was designed based on this sequence. The forward primer F1 and the reverse primer R1 constitute a target primer pair, and their sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0026] Forward primer F1: 5'ggcaGGCGTCGTCGAGGGTGATGG 3'

[0027] Reverse primer R1: 5'aaacCCATCACCCTCGACGACGCC 3'

[0028] Step 2, primer annealing and adapter ligation: Take 20 μl each of the forward primer F1 and the reverse primer R1 at a concentration of 100 μM, add them to a centrifuge tube and mix well. Place the mixture in a 100℃ environment for denaturation for 5 min, and then allow it to cool naturally at room temperature to complete the primer annealing.

[0029] like Figure 1 As shown, the annealed targeting adapter was ligated into the AarI-digested intermediate vector SKm-gRNA (provided by Wang Kejian, China National Rice Research Institute) to obtain the ligation product SKm-gRNA-Target. The PCR program was: 22℃ for 30 min, 4℃ for 2 min; the PCR reaction system is as follows:

[0030]

[0031] Step 3, Transformation into E. coli: The ligation product is transformed into E. coli DH5α competent cells. The specific steps are as follows:

[0032] (1) Add all the ligation products obtained in step 2 into Escherichia coli DH5α competent cells. Mix the ligation products with competent cells by gently tapping the bottom of the tube with your finger, and then let it stand on ice for 5 minutes.

[0033] (2) Place the mixture in a 42℃ environment for 45s heat shock, and after the heat shock is completed, quickly transfer it to ice and place it for 2min;

[0034] (3) Add 500 μl of LB liquid culture medium to the mixture and incubate at 37°C and 220 rpm for 20 min;

[0035] (4) Spread the treated Escherichia coli evenly on solid LB medium containing 50 μg / ml ampicillin resistance.

[0036] Step 4: Positive clone screening: Pick single clones from the solid LB medium described above and inoculate them into approximately 5 ml of LB liquid medium containing 50 μg / ml ampicillin. Incubate at 37°C and 220 rpm for approximately 12 hours. After incubation, extract plasmids according to the instructions of the Vazyme (DC201) plasmid extraction kit. Sequencing the extracted plasmids using universal primers M13R or T7 is performed. Plasmids with correctly aligned sequencing results are selected for subsequent construction of the final vector.

[0037] Step 5: Final Vector Construction and Preservation: Positive SKm-gRNA-Target fragments with correct sequencing alignment were digested with KpnI / SalI, and the final vector pC1300-Cas9 (provided by Wang Kejian, China National Rice Research Institute) was digested with KpnI / BamHI. After digestion, the digested fragments were recovered using a Vazyme gel extraction kit. The recovered SKm-gRNA-Target fragment was ligated into the digested final vector pC1300-Cas9 and transformed again into *E. coli* DH5α competent cells. Single clones were picked for plasmid extraction and sequenced using universal primers M13R and M13F. The correctly sequenced plasmid was named OsbZIP40-Cas9 and sent to Hangzhou Baige Biotechnology Co., Ltd. for genetic transformation (Nipponbare background).

[0038] Example 2: Identification of OsbZIP40 homozygous mutant plants

[0039] This embodiment uses the Japonica rice variety Nipponbare as the experimental material.

[0040] Step 1: Extraction of genomic DNA from T0 generation transgenic rice plants: Genomic DNA was extracted from the T0 generation transgenic rice plants obtained in Example 1.

[0041] Step 2: Sequence detection of the target sequence in the transgenic plant: Using the crude extracted DNA as a template, PCR amplification was performed using identification primers F2 and R2, following the instructions for Vazyme's 2XTaq Master Mix (P112-03-AA). The sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively. The amplified PCR product was then sent to Qingke Biotechnology Co., Ltd. for sequencing. The identification primer sequences are as follows:

[0042] F2:5'CTCGCAAAAACACAAAAGCA 3'

[0043] R2:5'TAGGGAACCCCACAACCATA3'

[0044] PCR program: 95℃ for 3 min, {95℃ for 30 s; 60℃ for 45 s, 72℃ for 30 s} – 35 cycles, 72℃ for 5 min, 4℃ for 2 min; and the amplification system is as follows:

[0045]

[0046]

[0047] Sequence alignment and screening successfully yielded two homozygous mutants, named osbzip40-1 and osbzip40-2. The nucleotide sequence of osbzip40-1 is shown in SEQ ID NO.8, with an insertion of base A at the target site. The nucleotide sequence of osbzip40-2 is shown in SEQ ID NO.9, with a deletion of base A at the target site. Both homozygous mutant systems resulted in frameshift mutations, ultimately causing premature termination of the encoded protein (see [link to relevant documentation]). Figure 1 ).

[0048] Example 3: Phenotypic Analysis of OsbZIP40 Homozygous Mutant - Transgenic Plant Seed Germination Analysis Experiment

[0049] Seeds from wild-type rice plants WT and OsbZIP40 gene knockout mutants osbzip40-1 and osbzip40-2 were dehulled and placed in transparent round petri dishes covered with two layers of moist filter paper for dark incubation at 28℃. Four biological replicates were set up, with 50 rice seeds selected for each replicate. Seed germination was observed and counted every 12 hours, with complete embryo breakthrough of the seed coat as the standard. The germination rate was calculated using the formula: Germination rate = (Number of germinated seeds / Total number of seeds) × 100%. Results after 84 hours of seed germination (see...) Figure 2 This indicates that, compared to the wild type, the OsbZIP40 gene knockout mutant lines exhibit faster seed germination rates. Furthermore, the mutant seeds show higher germination rates and a more uniform germination process in the early stages of germination (see...). Figure 3 ).

[0050] This invention successfully obtained two homozygous mutants, osbzip40-1 and osbzip40-2, by editing the OsbZIP40 gene in the rice variety Nipponbare. The germination rate of wild-type and mutant seeds was examined, showing that the mutant seeds had a faster germination rate and higher uniformity of emergence, fully demonstrating that the OsbZIP40 gene participates in regulating the rice seed germination process and belongs to the rice germination-related genes. The results of this invention provide excellent gene resources for solving problems such as slow seed germination and uneven germination during direct seeding of rice.

Claims

1. OsbZIP40 The use of the protein in regulating seed germination of rice, characterized in that, The rice OsbZIP40 The amino acid sequence of the protein is shown as SEQ ID NO. 2, and the application is to improve the germination rate of rice seeds by targeted knockout OsbZIP40 of the gene.

2. The gene encoding the protein of claim 1 OsbZIP40 application of the gene encoding the protein in regulating rice seed germination, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the application is for improving the germination rate of rice seeds by targeted knockout OsbZIP40 a gene to improve the germination rate of rice seeds.

3. Use according to claim 1 or 2, characterized in that, The application also includes improving the uniformity of rice emergence.

4. A method of increasing the germination rate of rice seeds, characterized by, Comprise: The already constructed OsbZIP40 Gene knockout vectors were used for genetic transformation of rice. After regenerating plants, sequencing confirmed the successful knockout. OsbZIP40 The homozygous strain; OsbZIP40 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. OsbZIP40 Gene-encoded OsbZIP40 The amino acid sequence of the protein is shown in SEQ ID NO.

2.

5. The method of claim 4, wherein, The OsbZIP40 The gene knockout vector is targeted to the nucleotide sequence shown in SEQ ID NO.

3.

6. The method of claim 5, wherein, The primer sequence for target knockout is shown as SEQ ID NO. 4~5.

7. The method of claim 4, wherein, The primer sequence for identifying rice after genetic transformation is shown as SEQ ID NO. 6 and SEQ ID NO.

7.

8. The method of claim 4, wherein, Two rice plants obtained after genetic transformation OsbZIP40 The mutant lines showed faster seed germination rate ， and the gene nucleotide sequence thereof is shown as SEQ ID NO. 8 or 9.

9. The method according to any one of claims 4 to 8, characterized in that, The rice variety for genetic transformation is Nipponbare.

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