Application of OsABCG22 gene in regulation and control of vigor of rice seeds

Abstract

The invention provides application of an OsABCG22 gene in regulation and control of rice seed vigor, and relates to the field of rice breeding and biological genes. The key function of the OsABCG22 gene in rice seed vigor is disclosed for the first time, a function deletion mutant is created through a CRISPR-Cas9 technology, and in combination with an overexpression material, it is proved that the gene positively regulates and controls the germination rate, the germination potential and seedling growth. Experiments show that the germination rate of an OsABCG22 overexpression strain is significantly superior to that of a wild type, while a knockout mutant shows germination delay, which proves that the gene is an important target for improving the activity of rice seeds. According to the discovery, the blank of ABCG subfamily gene functions is filled, the germination rate, the germination index, the germination potential, the bud length and the root length of rice seeds are remarkably improved by adopting an OsABCG22 gene overexpression mode, and the time when the germination rate reaches 50% is remarkably lower than that of a wild type; the seed germination activity and the seedling growth activity of rice seeds overexpressed by the OsABCG22 gene are higher than those of wild rice seeds.

Description

Technical Field

[0001] The invention belongs to the field of rice breeding and biological genes, and particularly relates to the application of the OsABCG22 gene in regulating rice seed vigor. Background Art

[0002] Seed germination is the process by which viable seeds absorb water, activate DNA repair, and rebuild their cell membranes, prompting the radicle and plumule to elongate and break through the seed coat. As a critical stage in the plant life cycle, seed germination directly determines the growth of seedlings. The vigor and uniformity of seedlings are closely related to field management, mechanized production, and ultimate yield.

[0003] Rice, a globally important food crop, has a significant impact on seedling emergence, vigor, and ultimate yield. High-vigor seeds are crucial for adaptability to environmental stress, particularly under direct-seeding cultivation. However, seed vigor is regulated by multiple genes, and the underlying molecular mechanisms remain incompletely elucidated, leading to a lack of effective genetic targets for breeding. Although genes related to abscisic acid and gibberellins, such as OsVP1 and OsGA20ox, have been implicated in seed germination regulation, the role of ATP-binding cassette transporter family genes in seed vigor is less well understood. OsABCG22, a member of the ABCG subfamily, has previously had no known biological function, particularly in regulating rice seed vigor. Therefore, exploring the function of OsABCG22 and clarifying its relationship to seed vigor is crucial for uncovering the regulatory network for rice germination and for molecular breeding. Recent advances in gene editing technologies have provided new strategies for precisely regulating seed vigor, but genetic improvement of ABC transporter genes remains scarce. Summary of the Invention

[0004] The present invention provides an application of the OsABCG22 gene in regulating rice seed vigor. The present invention prepares rice seeds with overexpression of OsABCG22. The germination rate, germination index, germination potential, bud length and root length of the OsABCG22-overexpressing rice seeds are significantly higher than those of the wild type, and the time it takes for the germination rate to reach 50% is significantly lower than that of the wild type.

[0005] The present invention provides the following technical solution: application of the OsABCG22 gene in regulating rice seed vigor, the nucleotide sequence of the OsABCG22 gene is shown in SEQ ID NO: 1.

[0006] Furthermore, the amino acid sequence of the protein encoded by the OsABCG22 gene is shown in SEQ ID NO: 2.

[0007] Furthermore, the application includes improving the germination rate of rice seeds and the growth vitality of seedlings.

[0008] Furthermore, the expression level of the OsABCG22 gene in rice seeds is increased to obtain overexpression seeds, thereby improving the germination rate, germination index, germination potential, sprout length and root length of the rice seeds.

[0009] Furthermore, the time it takes for the overexpression seeds to reach 50% germination rate is lower than that of the wild-type seeds, and the time it takes for the wild-type seeds to reach 50% germination rate is lower than that of the Osabcg22 mutant seeds.

[0010] A method for regulating rice seed vigor, for preparing seeds overexpressing the OsABCG22 gene according to any one of claims 1 to 5, the method comprising:

[0011] The OsABCG22 gene of rice was cloned and the OsABCG22 gene fragment was obtained;

[0012] An overexpression vector was constructed based on the OsABCG22 gene fragment;

[0013] genetically transforming rice seeds using the overexpression vector to obtain overexpression seeds;

[0014] The germination of overexpressing seeds and the growth of seedlings were measured.

[0015] Furthermore, the cloning of the rice OsABCG22 gene to obtain the OsABCG22 gene fragment includes:

[0016] Total RNA from Zhonghua 11 was extracted using TRIZOL;

[0017] Reverse transcribing the total RNA into cDNA;

[0018] The OsABCG22 gene sequence was amplified by PCR;

[0019] Gel electrophoresis was used to detect whether the OsABCG22 gene fragment had been amplified. If so, the OsABCG22 gene fragment was cut out from the gel and recovered.

[0020] Furthermore, the construction of an overexpression vector based on the OsABCG22 gene fragment comprises:

[0021] The vector p26KcGFP was selected, and SalI and KpnI were used as restriction enzyme sites for vector construction;

[0022] The recovered vector backbone and OsABCG22 gene fragment were connected by homologous recombination;

[0023] The DH5α strain was transformed by heat shock, and the positive plasmids with correct sequencing were screened on LB plates containing kanamycin to obtain the overexpression vector.

[0024] Furthermore, the determination of the germination of the overexpressed seeds and the growth of the seedlings comprises:

[0025] Remove impurities from the surface of overexpressed seeds;

[0026] The overexpression seeds were placed in corresponding culture dishes, 12 mL of pure water was added, and the culture was placed in a tissue culture room at 25° C. with a photoperiod of 16 h / 8 h;

[0027] The number of germinated seeds was counted every 12 h, and the root length and shoot length of mature seeds were measured and recorded on the 5th day.

[0028] Furthermore, the ligation reaction system of the homologous recombination method is 1 μL of vector backbone, 3 μL of OsABCG22 gene fragment, 1 μL of homologous recombination enzyme, 2 μL of homologous recombination enzyme buffer and 3 μL of water, and the total volume of the ligation reaction system is 10 μL.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This study reveals for the first time the key role of the OsABCG22 gene in rice seed vigor. Using CRISPR-Cas9 technology to create loss-of-function mutants, combined with overexpression materials, it was confirmed that the gene positively regulates germination rate, germination potential, and seedling growth. Experiments showed that the germination rate of OsABCG22 overexpression lines was significantly better than that of the wild type, while the knockout mutant exhibited delayed germination, demonstrating that this gene is an important target for improving rice seed vigor. This discovery not only fills a gap in the functional understanding of the ABCG subfamily genes, but also significantly improves the germination rate, germination index, germination potential, sprout length, and root length of rice seeds by overexpressing the OsABCG22 gene. The time it takes for the germination rate to reach 50% is significantly lower than that of the wild type. Rice seeds overexpressing the OsABCG22 gene exhibit stronger seed germination and seedling growth vigor than the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0032] In the attached figure:

[0033] Figure 1 This is a diagram showing the vigor results of rice OsABCG22 gene overexpression seeds of the present invention;

[0034] Figure 2This is a diagram showing the identification results of the rice OsABCG22 gene overexpression material of the present invention;

[0035] Figure 3 This is a diagram of the gene editing process of the rice OsABCG22 gene knockout mutant material of the present invention;

[0036] Figure 4 This is a diagram showing the seed vigor results of the rice OsABCG22 gene knockout mutant of the present invention;

[0037] Figure 5 This is a diagram showing the direct seeding results of the rice OsABCG22 gene overexpression material of the present invention. DETAILED DESCRIPTION

[0038] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention, without departing from the spirit and substance of the present invention, fall within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, and instruments used in the examples herein are commercially available; unless otherwise specified, all technical means in the examples herein are conventional means well known to those skilled in the art.

[0039] The present invention provides an application of the OsABCG22 gene in regulating rice seed vigor. The nucleotide sequence of the OsABCG22 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the OsABCG22 gene is shown in SEQ ID NO: 2.

[0040] The application includes improving the germination rate of rice seeds and the growth vitality of seedlings. The present invention improves the germination rate, germination index, germination potential, bud length and root length of rice seeds by increasing the expression level of the OsABCG22 gene in rice seeds, that is, overexpressing the OsABCG22 gene.

[0041] like Figure 2 As shown, the present invention identifies rice seeds with overexpression of OsABCG22 gene by detecting the expression level of OsABCG22 gene. There are two types of rice seeds with overexpression of OsABCG22 gene, the first type is OsABCG22-1 and the second type is OsABCG22-2. Figure 2 It can be seen that the expression level of OsABCG22 gene in OsABCG22-1 is higher than that in OsABCG22-2.

[0042] like Figure 1 and Figure 4As shown, the present invention sets up a control group for rice seed germination experiments. The control group includes wild-type rice seeds and Osabcg22 mutant seeds. The Osabcg22 mutant seeds are rice seeds in which the OsABCG22 gene is knocked out. The Osabcg22 mutants include two types: the first type is Osabcg22-1, and the second type is Osabcg22-2. The germination rate, germination index, germination potential, sprout length, and root length of rice seeds overexpressing the OsABCG22 gene are significantly higher than those of the wild type, while the germination rate, germination index, germination potential, sprout length, and root length of the Osabcg22 mutant seeds are significantly lower than those of the wild type. The time it takes for the overexpression seeds to reach 50% germination is lower than that of the wild-type seeds, and the time it takes for the wild-type seeds to reach 50% germination is lower than that of the Osabcg22 mutant seeds.

[0043] like Figure 5 As shown in the direct seeding experiment of rice, it was found that the seed germination and seedling growth vitality of rice seeds OsABCG22-1 and OsABCG22-2 overexpressing the OsABCG22 gene were stronger than those of the wild type.

[0044] This example reveals for the first time the key role of the OsABCG22 gene in rice seed vigor. By creating a loss-of-function mutant using CRISPR-Cas9 technology and combining it with overexpression materials, it was confirmed that the gene positively regulates germination rate, germination potential, and seedling growth. Experiments showed that the germination rate of the OsABCG22 overexpression strain was significantly better than that of the wild type, while the knockout mutant showed delayed germination, proving that this gene is an important target for improving rice seed vigor. This discovery not only fills the gap in the function of ABCG subfamily genes, but also significantly improves the germination rate, germination index, germination potential, bud length, and root length of rice seeds by overexpressing the OsABCG22 gene. The time it takes for the germination rate to reach 50% is significantly lower than that of the wild type. The seed germination and seedling growth vigor of rice seeds overexpressing the OsABCG22 gene are stronger than those of the wild type.

[0045] Example 2

[0046] This example provides a method for regulating rice seed vigor, which is used to prepare seeds overexpressing the OsABCG22 gene in Example 1. The method comprises:

[0047] S1: Clone the OsABCG22 gene of rice and obtain the OsABCG22 gene fragment.

[0048] S2: Construct an overexpression vector based on the OsABCG22 gene fragment.

[0049] S3: genetically transforming rice seeds using the overexpression vector to obtain overexpression seeds.

[0050] S4: Determine the germination of overexpressing seeds and the growth of seedlings.

[0051] The OsABCG22 gene of rice is cloned to obtain an OsABCG22 gene fragment, comprising:

[0052] S11: Total RNA from S. cerevisiae 11 was extracted using TRIZOL.

[0053] S12: Reverse transcribing the total RNA into cDNA.

[0054] S13: PCR amplification of the OsABCG22 gene sequence.

[0055] S14: using gel electrophoresis to detect whether the OsABCG22 gene fragment has been amplified; if so, cutting the OsABCG22 gene fragment from the gel to recover the OsABCG22 gene fragment.

[0056] The OsABCG22 gene was searched through the Rice Annotation Project (http: / / rice.uga.edu / index.shtml), and the nucleotide sequence of the OsABCG22 gene was SEQ ID NO: 1, and the amino acid sequence was SEQ ID NO: 2.

[0057] Total RNA from Zhonghua 11 was extracted using TRIZOL and reverse-transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Vazyme, China). The rice gene OsABCG22 was cloned, and a SalI I restriction site was introduced upstream and a Kpn I restriction site was introduced downstream into the OsABCG22 amplification primer. The OsABCG22 gene sequence was amplified by PCR using PrimeSTAR MaxDNA Polymerase (TaKaRa, Japan). The PCR reaction conditions were: 98°C for 30 s, followed by 35 cycles of 98°C for 15 s, 60°C for 15 s, and 72°C for 2 min, and finally 72°C for 10 min.

[0058] Amplification primers are:

[0059] ABCG22-F:5'-cttgcatgcctgcaggtcgacATGGCGTCCGGGC-3';

[0060] ABCG22-R:5'-cctttactcattttttctaccggtaccGTAAGGCTTCCTGCCGAG-3'.

[0061] After PCR amplification, gel electrophoresis was used to detect amplification of the OsABCG22 gene fragment, which revealed a distinct band near 2166 bp. The OsABCG22 gene fragment was excised from the gel and recovered using a DNA recovery kit (Sangon, model B518131-0100).

[0062] The method of constructing an overexpression vector based on the OsABCG22 gene fragment comprises:

[0063] S21: Select vector p26KcGFP, SalⅠ and KpnI as restriction enzyme sites for vector construction.

[0064] S22: The recovered vector backbone and the OsABCG22 gene fragment were connected using homologous recombination.

[0065] S23: Heat shock transformation of DH5α strain, and screening of positive plasmids with correct sequencing using LB plates containing kanamycin to obtain overexpression vectors.

[0066] The construction of the overexpression vector includes the following steps:

[0067] (1) Based on the cloned OsABCG22 gene sequence, the vector p26KcGFP was selected, SalⅠ and KpnI were used as restriction enzyme sites for vector construction, and the recovered vector backbone and the OsABCG22 amplified product were connected using homologous recombination.

[0068] (2) Construct the ligation reaction system: 1 μL vector backbone, 3 μL OsABCG22 amplification product, 1 μL homologous recombinase, 2 μL homologous recombinase buffer, 3 μL water. The total volume of the ligation reaction system is 10 μL. Incubate at 37°C for 30 min.

[0069] (3) Screening: Heat shock transformed DH5α strains were screened on kanamycin-containing LB plates. Colony PCR primers were 35S-F and ABCG22-R. The PCR program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 60°C annealing for 30 s, and 72°C extension for 45 s, for a total of 25 cycles. 72°C extension for 5 min, and storage at 4°C. Positive plasmids with correct sequencing results were selected and named p26KcGFP-OsABCG22. They were stored at -20°C until ready for use.

[0070] The knockout vector was constructed as follows:

[0071] like Figure 3 As shown, using the CRISPR design tool (http: / / tools.genome-engineering.org), the OsABCG22 gene sequence was input, suitable target sites were identified and aligned, and off-target sites were calculated and predicted for each designated target. Finally, two target sites, SG25312 and SG15314, were selected for knockout, respectively, to generate the CRISPR-cas9 plasmid.

[0072] The target sequence is:

[0073] SG25312: 5'-CCGTGCGCAGCACCGACGGGTTC-3';

[0074] SG15314: 5'-TCCCTCGCGTGGGAGGACCTGTGG-3'.

[0075] The determination of the germination of overexpressed seeds and the growth of seedlings comprises:

[0076] S41: Removing impurities on the surface of overexpressed seeds.

[0077] S42: Place the overexpression seeds into a corresponding culture dish, add 12 mL of pure water, and culture in a tissue culture room at 25° C. with a photoperiod of 16 h / 8 h.

[0078] S43: Count the number of germinated seeds every 12 hours, and measure and record the root length and shoot length of mature seeds on the 5th day.

[0079] 90 plump, normal seeds of each of the wild-type, overexpression lines OsABCG22-1 and OsABCG22-2, and knockout lines Osabcg22-1 and Osabcg22-2 were collected. The seeds were washed 2-3 times with pure water to remove all surface impurities and then filtered to remove as much surface moisture as possible. Three replicates of 30 seeds were placed in corresponding Petri dishes, each containing 12 mL of pure water. The dishes were then incubated at 25°C in a tissue culture room with a 16-hour / 8-hour photoperiod. Germinated seeds (i.e., seeds with a bud ≥2 mm above the seed coat) were counted every 12 hours. Root and shoot lengths of established seeds were measured and recorded on the fifth day.

[0080] The process of direct-seeding rice assay for OsABCG22 overexpression lines is as follows:

[0081] (1) Take 50 normal and plump seeds of the wild type and OsABCG22-1 and OsABCG22-2 overexpressing lines, place them in a seed soaking bag, add water, and soak the seeds at 37°C for 2 days.

[0082] (2) Soaked seeds of the wild type and OsABCG22-1 and OsABCG22-2 overexpressing lines for 2 days were sown in the same pot containing soil. The seeds were gently patted into the soil, covered with a transparent film, and incubated at room temperature (22-29°C). Seedling emergence and growth were recorded on the 5th and 10th days after sowing.

[0083] This example uses CRISPR-CAS9 technology to edit the OsABCG22 gene and identify OsABCG22 mutant strains, resulting in homozygous OsABCG22 loss-of-function mutants. Overexpression of the OsABCG22 gene improves rice seed vigor, while loss of OsABCG22 gene function inhibits rice seed vigor.

[0084] It is understandable that the above embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can be made, all of which fall within the scope of protection of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should fall within the scope of coverage of the claims of the present invention.

Claims

1. Application of the OsABCG22 gene in regulating rice seed vigor, characterized in that: The nucleotide sequence of the OsABCG22 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the OsABCG22 gene is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that The application includes improving the germination rate of rice seeds and the growth vitality of seedlings.

4. The use according to claim 1, characterized in that The expression level of the OsABCG22 gene in rice seeds is increased to obtain overexpression seeds, thereby improving the germination rate, germination index, germination potential, sprout length and root length of the rice seeds.

5. The use according to claim 1, characterized in that The time it took for the overexpressing seeds to reach 50% germination rate was lower than that of the wild-type seeds, and the time it took for the wild-type seeds to reach 50% germination rate was lower than that of the OsABCG22 mutant seeds.

6. A method for regulating rice seed vitality, characterized in that: The method for preparing seeds overexpressing the OsABCG22 gene according to any one of claims 1 to 5 comprises: The OsABCG22 gene of rice was cloned and the OsABCG22 gene fragment was obtained; An overexpression vector was constructed based on the OsABCG22 gene fragment; genetically transforming rice seeds using the overexpression vector to obtain overexpression seeds; The germination of overexpressing seeds and the growth of seedlings were measured.

7. The method according to claim 6, characterized in that The OsABCG22 gene of rice is cloned to obtain an OsABCG22 gene fragment, comprising: Total RNA from Zhonghua 11 was extracted using TRIZOL; Reverse transcribing the total RNA into cDNA; The OsABCG22 gene sequence was amplified by PCR; Gel electrophoresis was used to detect whether the OsABCG22 gene fragment had been amplified. If so, the OsABCG22 gene fragment was cut out from the gel and recovered.

8. The method according to claim 6, characterized in that The method of constructing an overexpression vector based on the OsABCG22 gene fragment comprises: The vector p26KcGFP was selected, and SalI and KpnI were used as restriction enzyme sites for vector construction; The recovered vector backbone and OsABCG22 gene fragment were connected by homologous recombination; The DH5α strain was transformed by heat shock, and the positive plasmids with correct sequencing were screened on LB plates containing kanamycin to obtain the overexpression vector.

9. The method according to claim 6, characterized in that The determination of the germination of overexpressed seeds and the growth of seedlings comprises: Remove impurities from the surface of overexpressed seeds; The overexpression seeds were placed in corresponding culture dishes, 12 mL of pure water was added, and the culture was placed in a tissue culture room at 25° C. with a photoperiod of 16 h / 8 h; The number of germinated seeds was counted every 12 h, and the root length and shoot length of mature seeds were measured and recorded on the 5th day.

10. The method according to claim 8, characterized in that The ligation reaction system of the homologous recombination method comprises 1 μL of vector backbone, 3 μL of OsABCG22 gene fragment, 1 μL of homologous recombination enzyme, 2 μL of homologous recombination enzyme buffer and 3 μL of water, and the total volume of the ligation reaction system is 10 μL.

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