Application of rice PP2C06 gene knockout in regulation and control of storage time of rice seeds
Knocking out the PP2C06 gene of rice through CRISPR/Cas9 technology solves the problem of short storage time of rice seeds, significantly extends the seed storage time, provides new resources for high-quality molecular breeding of rice, and solves the short storage time of rice seeds The problem has achieved the improvement of seed storage tolerance.
Patent Information
- Application Number
- CN202411478903.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-22
- Publication Date
- 2025-01-03
AI Technical Summary
There are few reports on genes that ABA regulates seed storage time in rice, which seriously restricts the understanding of the molecular regulatory mechanism and gene network of seed storage time, resulting in a short storage time of rice seeds, affecting agricultural production.
The targeted knockout of the rice PP2C06 gene was obtained through CRISPR/Cas9 technology, which significantly extended the storage time of rice seeds.
The appearance phenotype and germination rate before storage of gene knockout mutants are consistent with the wild type, but significantly extend the storage time of rice seeds, providing new theoretical and technical resources for high-quality molecular breeding of rice, and having important agricultural utilization value. .
Smart Images
Figure CN119242694A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of genetic engineering technology, and in particular to an application of rice PP2C06 gene knockout in regulating the storage time of rice seeds. Background Art
[0002] Seeds are an important guarantee for human survival. They are the most basic means of production for agricultural production. Their rich storage materials, such as starch, protein and fat, provide an important source of food for humans. During the storage process, seeds will inevitably age, which will lead to a decline in seed quality, and ultimately affect their edibility or even make them inedible. In agricultural production, seed aging will directly lead to the inability of seeds to germinate and become seedlings, seriously affecting agricultural production. Therefore, extending the storage time of seeds is of great theoretical and practical significance.
[0003] Rice is one of the most important food crops in my country. Among crops, rice seeds have a relatively short storage time, which seriously affects their production and application value. To date, the molecular regulatory mechanism of rice seed storage time is still poorly understood, and only a few genes that control seed storage time have been cloned, including bZIP23, PER1A, Rc, GLYI3, PIMT, MSRB5, etc. ABA plays a vital role in regulating seed storage time. The Arabidopsis ABA synthesis deficiency mutant aba1-5 and the ABA-insensitive mutant abi3 have significantly reduced seed lifespan relative to the wild type. ABI3, LEC1, LEC2 and FUS3 form the LAFL transcriptional regulatory complex to regulate the seed development process, among which LEC1, like ABI3, also positively regulates seed storage time. In addition, in Arabidopsis, the seed storage time of the triple mutant of the SnRK2 protein, a core component of ABA signaling, is greatly shortened. However, there are few reports on genes regulating seed storage time by ABA in rice. Therefore, discovering and identifying the genetic resources of ABA controlling the storage time of rice seeds and deeply exploring its molecular regulatory mechanism are of great theoretical guiding significance for high-quality molecular breeding of rice.
[0004] Based on this, the present specification provides an application of rice PP2C06 gene knockout in regulating the storage time of rice seeds. Summary of the invention
[0005] The embodiments of this specification provide an application of rice PP2C06 gene knockout in regulating rice seed storage time, which is used to solve the following technical problems: At present, there are few reports on genes regulating seed storage time of ABA in rice, which seriously limits the understanding of the molecular regulatory mechanism and gene network of seed storage time.
[0006] To solve the above technical problems, the embodiments of this specification are implemented as follows:
[0007] The use of the gene knockout mutant provided in the embodiments of this specification in regulating the storage time of rice seeds is characterized in that the nucleotide sequence of the rice PP2C06 gene is as shown in SEQ ID NO: 1;
[0008] The gene knockout is to use CRISPR / Cas9 technology to perform targeted knockout of the rice PP2C06 gene to obtain a gene knockout mutant.
[0009] Furthermore, the gene knockout target site of the gene knockout mutant is located in the second exon of the rice PP2C06 gene, and the start site is located at 1092 bp.
[0010] Furthermore, the gene knockout mutant has one base inserted at 1109 bp of the rice PP2C06 gene, resulting in a change in the amino acid sequence at 258 aa and premature termination at 296 aa;
[0011] and / or
[0012] The gene knockout mutant is a mutant in which one base is knocked out at 1109 bp of the rice PP2C06 gene, resulting in premature termination of the amino acid sequence at 257 aa.
[0013] Furthermore, the knockout target site sequence of the gene knockout mutant is shown in SEQ ID NO:2.
[0014] Furthermore, the primer sequences for detecting the gene knockout mutants are shown in SEQ ID NO: 3-4.
[0015] Furthermore, the gene knockout mutant is obtained by the following method:
[0016] Design knockout targets based on the sgRNA expression cassette and rice PP2C06 gene, and construct the target sgRNA expression cassette;
[0017] Based on the target sgRNA expression cassette and CRISPR / Cas9 gene knockout system, constructing a gene knockout expression vector;
[0018] The gene knockout expression vector is transformed into rice varieties by Agrobacterium-mediated method to obtain transgenic rice plants;
[0019] The primer sequences are detected to identify T0 generation transgenic rice plants and obtain the gene knockout mutants.
[0020] Furthermore, the detection primer sequence is as shown in SEQ ID NO: 5-6.
[0021] Furthermore, the rice variety is Zhonghua 11.
[0022] Furthermore, the appearance phenotype and germination rate before storage of the seeds of the gene knockout mutant are consistent with those of the wild-type recipient parent rice plant.
[0023] Furthermore, the seed storage time of the gene knockout mutant is longer than that of the wild-type recipient parent rice plant.
[0024] At least one of the above technical solutions adopted in the embodiments of this specification can achieve the following beneficial effects: using CRISPR / Cas9 technology to knock out the rice PP2C06 gene to obtain a gene knockout mutant, the appearance phenotype and the germination rate before storage of the gene knockout mutant do not change, but the storage time of rice seeds can be significantly extended, providing new theories and technologies for solving the problem of short storage time of rice seeds, and also providing new gene resources for high-quality molecular breeding practices, which can be applied to production practices and have important practical significance and agricultural utilization value. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of this specification or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this specification. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
[0026] Figure 1 A schematic diagram of the rice PP2C06 gene structure, knockout target sites and genotype provided in the examples of this specification;
[0027] Figure 2 A schematic diagram of the structure of the rice PP2C06 gene knockout vector provided in the examples of this specification;
[0028] Figure 3 The appearance morphology of rice panicles and seeds of rice PP2C06 gene knockout and wild-type rice provided in the examples of this specification;
[0029] Figure 4 The germination morphology of freshly harvested seeds of rice PP2C06 gene knockout and wild-type rice provided in the examples of this specification;
[0030] Figure 5 The statistical results of germination of freshly harvested seeds of rice PP2C06 gene knockout and wild-type rice provided in the examples of this specification;
[0031] Figure 6 The germination morphology of rice PP2C06 gene knockout and wild-type rice seeds after storage under high temperature and high humidity conditions provided in the examples of this specification;
[0032] Figure 7 The germination statistics of rice PP2C06 gene knockout and wild-type rice seeds after storage under high temperature and high humidity conditions provided in the examples of this specification;
[0033] Figure 8 The storage time calculation results of rice PP2C06 gene knockout and wild-type rice seeds provided in the examples of this specification. DETAILED DESCRIPTION
[0034] In order to enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below in conjunction with the drawings in the embodiments of this specification. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments of this specification, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.
[0035] The experimental methods used in the examples of this specification are conventional methods unless otherwise specified. The experimental materials used are commercially available unless otherwise specified.
[0036] There is a close connection between PP2C (Protein Phosphatase 2C) proteins and the signaling of the plant hormone abscisic acid (ABA). ABA is a plant hormone that plays an important role in plant growth and development, especially in response to drought, salinity and other abiotic stresses. ABA can regulate stomatal closure, promote seed dormancy, inhibit plant growth to reduce water loss, etc.
[0037] In the ABA signaling pathway, PP2C proteins usually act as negative regulators. In the absence of ABA, PP2C proteins inhibit the activity of downstream SNF1-related protein kinase (SnRK2) type protein kinases, thereby preventing the activation of the ABA response pathway. When plants perceive ABA, ABA binds to PP2C proteins through PYR / PYL / RCAR receptors, resulting in the inactivation of PP2C. In this way, SnRK2 protein kinases can be activated, which in turn initiates a series of downstream reactions, including the phosphorylation of transcription factors, ultimately affecting changes in gene expression, enabling plants to adapt to adverse conditions.
[0038] In the examples of this specification, a knockout mutant was constructed by using the rice PP2C gene, and the knockout mutant was studied.
[0039] The gene knockout mutant was obtained by the following method:
[0040] Design knockout targets based on the sgRNA expression cassette and rice PP2C06 gene, and construct the target sgRNA expression cassette;
[0041] Based on the target sgRNA expression cassette and CRISPR / Cas9 gene knockout system, constructing a gene knockout expression vector;
[0042] The gene knockout expression vector is transformed into rice varieties by Agrobacterium-mediated method to obtain transgenic rice plants;
[0043] The primer sequences are detected to identify T0 generation transgenic rice plants and obtain the gene knockout mutants.
[0044] Example 1
[0045] Construction of CRISPR / Cas9 vector for gene knockout mutants
[0046] (1) Analysis of knockout target sites based on rice PP2C06 gene design
[0047] The rice PP2C06 gene sequence is shown in SEQ ID NO: 1. The rice PP2C06 gene structure is shown in Figure 1 As shown; the full length of rice PP2C06 gene is 6368bp, containing 4 exons and 3 intron sequences.
[0048] The rice PP2C06 gene was analyzed and the CRISPR / Cas9 knockout target was designed using the online software CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / ), and the knockout target site sequence of the gene knockout mutant was obtained as shown in SEQ ID NO:2.
[0049] The gene location of the knockout target site is as follows Figure 1 As shown, it is located in the second exon and the starting site is located at 1092bp.
[0050] Based on the knockout target site, gene knockout mutants are constructed.
[0051] (2) Construction of target sgRNA expression cassette
[0052] In this example, the sgRNA expression cassette was constructed using the CRISPR / sgRNA vector pYLgRNA-OsU6a as the backbone. First, the primer sequence for connecting the target and the sgRNA expression cassette was synthesized according to SEQ ID NO: 3-8.
[0053] Then, the pYLgRNA-OsU6a plasmid was used as a template, and four primers SEQ ID NO: 3-6 were added at the same time, and the first round of PCR was performed at 25-28 cycles: 94°C 10s, 58°C 15s, 68°C 20s; then, the first round of PCR products were taken, and primers SEQ ID NO: 7-8 were added, and the second round of PCR was performed at 17-20 cycles: 95°C 10s, 58°C 15s, 68°C 20s. Finally, the second round of PCR products were recovered by agarose gel electrophoresis to obtain the target sgRNA expression cassette.
[0054] (3) Target sgRNA and CRISPR / Cas9 vector connection
[0055] In this example, the CRISPR / Cas9 vector used is pYLCRISPR / Cas9Pubi-H. The target sgRNA constructed above, pYLCRISPR / Cas9Pubi-H, DNA ligase and endonuclease BsaI were mixed, and the sgRNA was connected to the CRISPR / Cas9 vector pYLCRISPR / Cas9Pubi-H by using the enzyme cutting and ligation method for 10-15 cycles: 37°C for 5 minutes; 10°C for 5 minutes, 20°C for 5 minutes; and finally 37°C for 5 minutes.
[0056] The ligation product was transformed into Escherichia coli, and high-purity plasmids were extracted, followed by restriction digestion identification, and plasmids with the correct product size were sequenced to obtain the CRISPR / Cas9 vector for rice PP2C06 gene knockout.
[0057] Example 2: Rice PP2C06 gene knockout CRISPR / Cas9 vector transformation of rice
[0058] Continuing with the previous example, the constructed rice PP2C06 gene knockout CRISPR / Cas9 vector plasmid was transformed into Agrobacterium competent cells.
[0059] In this example, competent cells of Agrobacterium EHA105 were used for transformation by heat shock method, and strains containing positive clones were obtained by enzyme digestion identification of plasmids. Subsequently, wild-type rice variety Zhonghua 11 was transformed by infection method, and the specific operation steps were as follows:
[0060] 1) Induction of callus
[0061] The mature seeds of the rice variety Zhonghua 11 were dehulled, disinfected and cleaned in 75% alcohol and 50% sodium hypochlorite, and then planted on a rice callus induction medium; then, they were cultured in a 30°C light incubator for 2-3 weeks until callus tissue grew; thereafter, they were subcultured for 1-2 generations, and callus tissue with good growth condition was selected for Agrobacterium infection.
[0062] 2) Agrobacterium activation
[0063] Take the Agrobacterium containing positive clones that was pre-stored in a -80°C refrigerator, inoculate it into a liquid YEP medium containing kanamycin and rifampicin, and culture it in a shaker at 200 rpm at 28°C until OD600 is about 0.3-0.5; then, take 1 mL of the bacterial solution and inoculate it again into a fresh liquid YEP medium containing kanamycin and rifampicin, and culture it under the same conditions until OD600 is about 0.3-0.5; finally, centrifuge to collect the bacterial solution, and resuspend the bacteria in AAM medium containing acetosyringone (AS).
[0064] 3) Co-cultivation of callus and Agrobacterium, screening of callus resistance and rooting differentiation
[0065] The obtained callus tissue was immersed in AAM bacterial solution, infected for 30 minutes, and gently shaken; then the excess bacterial solution was absorbed with sterilized filter paper, and the infected callus tissue was transferred to a co-culture solid culture medium and cultured in the dark at 28°C for 3 days; then transferred to a screening culture medium for culture; transferred to a differentiation culture medium for differentiation and regeneration, and then transferred to an MS culture medium for rooting and seedling growth; after the seedlings were mature, they were transplanted to the field and managed according to conventional field management to obtain the T0 generation plants with CRISPR / Cas9 knockout of the rice PP2C06 gene.
[0066] Example 3
[0067] Identification and phenotypic analysis of rice PP2C06 gene knockout by CRISPR / Cas9
[0068] In order to obtain rice PP2C06 gene knockout homozygous plants for phenotypic analysis, the leaves of the T0 generation plants with PP2C06 gene knockout obtained above were taken to extract genomic DNA. At the same time, forward and reverse primers SEQ ID NO: 9-10 were designed on the left and right sides according to the target sequence, and a product of about 540 bp was amplified for sequencing.
[0069] Comparison of the sequencing results with the rice PP2C06 gene sequence revealed that there were two genotypes of rice PP2C06 gene knockout, such as Figure 1As shown: one is to insert a base at 1109bp of the rice PP2C06 gene, causing the amino acid sequence to change at 258aa and terminate prematurely at 296aa; the other is to knock out a base at 1109bp of the rice PP2C06 gene, causing the amino acid sequence to terminate prematurely at 257aa.
[0070] After harvesting seeds from the two rice PP2C06 gene knockout and homozygous plants pp2c06#1 and #2, as well as the wild-type recipient parent rice plants identified above, they were planted further. After harvesting mature T1 generation seeds, the effect of rice PP2C06 gene knockout on the storage resistance of seeds under high temperature and high humidity conditions was observed.
[0071] The obtained rice PP2C06 gene knockout and homozygous plants pp2c06#1 and #2 were compared with the wild-type recipient parent rice plants, and it was observed that the appearance phenotype and germination rate of the seeds of the gene knockout mutants before storage were consistent with those of the wild-type recipient parent rice plants. In the examples of this specification, the wild-type recipient parent rice plant is Zhonghua 11 (abbreviated as WT).
[0072] Figure 3 The morphological diagrams of rice panicles and seeds of rice PP2C06 gene knockout and wild-type rice provided in the examples of this specification are shown in FIG. Figure 3 As shown, by comparing the seed morphology of rice PP2C06 gene knockout and WT rice, it was found that rice PP2C06 gene knockout did not affect seed setting and seed plumpness, that is, it did not affect the seed development process.
[0073] The seed germination rates of rice PP2C06 gene knockout and WT rice before storage were measured, and it was found that rice PP2C06 gene knockout did not change the germination of seeds before storage, such as Figure 4 and Figure 5 As shown. Figure 4 As shown in Figure 2, before seed storage, the germination morphology of pp2c06#1 and #2 did not change compared with WT. Figure 5 As shown, the ordinate represents the seed germination rate. Before seed storage, compared with WT, there was no difference in the germination rate of pp2c06#1 and #2, and both could germinate 100%.
[0074] After being stored under high temperature and high humidity conditions for a certain period of time, as the seeds aged, the germination rate of rice seeds of PP2C06 gene knockout plants pp2c06#1 and #2 was significantly higher than that of WT rice seeds. Figure 6 and Figure 7 As shown. Figure 6 As shown, compared with WT, the germination morphology of pp2c06#1 and #2 after storage under high temperature and high humidity conditions was significantly better than that of WT. Figure 7As shown, the ordinate is the seed germination rate. Compared with WT, the germination rates of pp2c06#1 and #2 after storage under high temperature and high humidity conditions were significantly higher than that of WT.
[0075] In order to further clarify the regulation of rice PP2C06 gene knockout on rice seed storage time, the examples of this specification further calculate the time for the germination rate of seeds to drop to the national standard for rice seed quality after storage.
[0076] Figure 8 The storage time calculation results of rice PP2C06 gene knockout and wild-type rice seeds provided in the examples of this specification are as follows. Figure 8 As shown, the vertical axis is the time required for the seed germination rate to reach 85%. Figure 8 It can be seen that compared with WT, the seed storage time of pp2c06#1 and #2 increased by 20.0% and 21.8%, respectively. This indicates that the PP2C06 gene negatively regulates the seed storage time, and knocking out the gene improves the storage resistance of seeds. It should be noted that the germination rate that meets the national standard for rice seed quality is not less than 85%.
[0077] In summary, the rice PP2C06 gene negatively regulates seed storage time and can be knocked out using CRISPR / Cas technology to improve rice storability, which has important potential for high-quality breeding.
[0078] The application of the gene knockout mutant provided in the embodiments of this specification in regulating the storage time of rice seeds is to knock out the rice PP2C06 gene using CRISPR / Cas9 technology to obtain a gene knockout mutant. The appearance phenotype and the germination rate before storage of the gene knockout mutant do not change, but the storage time of rice seeds can be significantly extended, which provides new theories and technologies for solving the problem of short storage time of rice seeds, and also provides new gene resources for high-quality molecular breeding practice, which can be applied to production practice and has important practical significance and agricultural utilization value.
[0079] Each embodiment in this specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.
[0080] The above is only an embodiment of this specification and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims
1. Application of rice PP2C06 gene knockout in regulating rice seed storage time, characterized in that: The nucleotide sequence of the rice PP2C06 gene is shown in SEQ ID NO: 1; The gene knockout is to use CRISPR / Cas9 technology to perform targeted knockout of the rice PP2C06 gene to obtain a gene knockout mutant.
2. The use according to claim 1, characterized in that The gene knockout target site of the gene knockout mutant is located in the second exon of the rice PP2C06 gene, and the start site is located at 1092 bp.
3. The use according to claim 1, characterized in that The gene knockout mutant has one base inserted at 1109 bp of the rice PP2C06 gene, causing the amino acid sequence to change at 258 aa and terminate prematurely at 296 aa; and / or The gene knockout mutant is a mutant in which one base is knocked out at 1109 bp of the rice PP2C06 gene, resulting in premature termination of the amino acid sequence at 257 aa.
4. The use according to claim 1, characterized in that The knockout target site sequence of the gene knockout mutant is shown in SEQ ID NO:
2.
5. The use according to claim 1, characterized in that The primer sequences used to detect the gene knockout mutants are shown in SEQ ID NO: 3-4.
6. The use according to claim 1, characterized in that The gene knockout mutant is obtained by the following method: Design knockout target sites based on sgRNA expression cassette and rice PP2C06 gene, and construct target sgRNA expression cassette; Based on the target sgRNA expression cassette and CRISPR / Cas9 gene knockout system, constructing a gene knockout expression vector; The gene knockout expression vector is transformed into rice varieties by Agrobacterium-mediated method to obtain transgenic rice plants; The primer sequences are detected to identify T0 generation transgenic rice plants and obtain the gene knockout mutants.
7. The use according to claim 6, characterized in that The detection primer sequence is as shown in SEQ ID NO: 5-6.
8. The use according to claim 6, characterized in that The rice variety is Zhonghua 11.
9. The use according to claim 1, characterized in that The appearance phenotype and germination rate before storage of the seeds of the gene knockout mutant are consistent with those of the wild-type recipient parent rice plant.
10. The use according to claim 1, characterized in that The knockout mutant has a longer seed storage time than the wild-type recipient parent rice plant.