ABA receptor for controlling storage time of rice seeds and application of ABA receptor
By identifying and utilizing the rice ABA receptor PYL10 gene, constructing a recombinant binary expression vector overexpression vector and introducing it into rice cells, the problem of unclear storage time of rice seeds was solved, the seed storage time was extended, the germination rate and seedling rate were maintained, and the storage performance of rice seeds was improved.
Patent Information
- Application Number
- CN202510932661.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-10-28
AI Technical Summary
The ABA receptor gene that regulates the storage time of rice seeds has not been identified in the existing technology, and the molecular mechanism by which ABA regulates the storage time of seeds is unclear, which leads to seed aging affecting the germination rate and seedling rate, affecting agricultural production and crop yields.
By identifying and utilizing the rice ABA receptor PYL10 gene, a recombinant binary expression vector overexpression vector was constructed and introduced into rice cells to regulate the seed storage time and improve the seed storage time.
By regulating the rice ABA receptor PYL10 gene, the seed storage time can be significantly improved, the seed germination rate and seedling rate can be kept unchanged, the seed storage time can be extended, and the storage resistance of rice seeds can be improved.
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Abstract
Description
Technical Field
[0001] This specification relates to the field of genetic engineering technology, and in particular to an ABA receptor for controlling the storage time of rice seeds and its application. Background Technology
[0002] Seeds inevitably age during storage. In agricultural production, seed aging leads to reduced germination rates and seedling failure, severely impacting crop production and yield. Seed aging also affects the starch and protein structure and composition, reducing their edibility. Therefore, extending seed storage time is of great significance for agricultural production and national food security.
[0003] ABA is an important plant hormone that controls seed storage time. Currently, only a few genes have been identified as involved in the ABA signaling pathway to regulate seed storage time, such as ABI3, ABI5, bZIP23, and bZIP42. In plants, ABA is sensed by the PYRABACTINRESISTANCE 1 / PYR1-LIKE / REGULATORY COMPONENT OF ABA RECEPTOR (PYR1 / PYL / RCAR) receptor, which initiates downstream signaling pathways. Thirteen ABA receptors exist in rice, but it is not yet clear which receptor controls seed storage time. Therefore, identifying and utilizing ABA receptor genes that control rice seed storage time, and further exploring the molecular regulatory mechanisms of ABA, has significant theoretical guiding significance for high-quality molecular breeding of rice.
[0004] Based on this, this specification provides an ABA receptor for controlling the storage time of rice seeds and its application. Summary of the Invention
[0005] This specification provides an ABA receptor for controlling the storage time of rice seeds and its application, in order to solve the following technical problems: ABA is an important plant hormone that controls the storage time of seeds, but so far no receptor gene for controlling the storage time of seeds has been identified, and the understanding of the molecular mechanism and gene network of ABA regulating the storage time of seeds is very limited.
[0006] To solve the above-mentioned technical problems, the embodiments in this specification are implemented as follows:
[0007] This specification provides an ABA receptor for controlling the storage time of rice seeds, wherein the ABA receptor for controlling the storage time of rice seeds is PYL10, the nucleotide sequence of the ABA receptor encoding gene is shown in SEQ ID NO:1, and the amino acid sequence of the ABA receptor encoding gene is shown in SEQ ID NO:2.
[0008] Furthermore, the ABA receptor PYL10 encoding gene is upregulated during seed development and storage.
[0009] The embodiments of this specification also provide an application of an ABA receptor for controlling the storage time of rice seeds, wherein biological materials are constructed using the gene of the ABA receptor PYL10 to regulate the storage time of rice seeds.
[0010] Furthermore, the biological material is an overexpression material, which increases the storage time of rice seeds.
[0011] Furthermore, the biological material is a nucleic acid molecule, a recombinant expression vector, a host cell, or a transformed plant cell.
[0012] Furthermore, the gene of the ABA receptor PYL10 is introduced into plant cells, tissues, or organs to cultivate plants, resulting in transgenic plants with improved storage time.
[0013] Furthermore, the gene of the ABA receptor PYL10 is introduced into plant cells, tissues, or organs via a plant overexpression vector, wherein the overexpression vector is a recombinant binary expression vector.
[0014] Furthermore, the transgenic plant is rice Zhonghua 11.
[0015] Furthermore, the ABA receptor PYL10 positively regulates seed storage time.
[0016] Furthermore, the aforementioned regulation does not affect the seed's appearance phenotype or the germination rate and seedling rate before storage; however, the regulation can extend the seed's storage time.
[0017] The above-mentioned at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects: by regulating the rice ABA receptor PYL10 gene and protein, the storage time of rice seeds can be regulated, providing new gene resources for basic research on rice seed storage and high-quality molecular breeding practices, and can be applied to production practices. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments or prior art of this specification, the drawings used in the description of the embodiments or prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this specification. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 This diagram illustrates the expression changes of the rice ABA receptor PYL10 encoding gene during seed development and high-temperature and high-humidity storage, as provided in the embodiments of this specification.
[0020] Figure 2 A schematic diagram of the structure of the rice ABA receptor PYL10 gene overexpression vector pCAMBIA2300-PYL10 provided in the embodiments of this specification;
[0021] Figure 3 This diagram illustrates the results of PYL10 transcription levels in transgenic rice (PYL10-ox) that overexpresses the rice ABA receptor PYL10, as provided in the embodiments of this specification.
[0022] Figure 4 The image shows the morphological appearance of transgenic rice seeds overexpressing the rice ABA receptor PYL10 gene, as provided in the embodiments of this specification.
[0023] Figure 5 Statistical results of germination and seedling growth of freshly harvested seeds of transgenic rice with overexpression of the rice ABA receptor PYL10 provided in the embodiments of this specification;
[0024] Figure 6 Germination morphology of freshly harvested transgenic rice seeds overexpressing the rice ABA receptor PYL10, as provided in the embodiments of this specification.
[0025] Figure 7 This diagram illustrates the germination changes of transgenic rice seeds overexpressing the rice ABA receptor PYL10 gene during storage under high temperature and high humidity conditions, as provided in the embodiments of this specification.
[0026] Figure 8 Statistical results of germination and seedling growth of transgenic rice seeds overexpressing the rice ABA receptor PYL10 gene provided in the embodiments of this specification after storage under high temperature and high humidity conditions;
[0027] Figure 9 Germination morphology of transgenic rice seeds overexpressing the rice ABA receptor PYL10 gene, provided in the embodiments of this specification, after storage under high temperature and high humidity conditions;
[0028] Figure 10 The storage time calculation results of transgenic rice seeds overexpressing the rice ABA receptor PYL10 gene provided in the embodiments of this specification. Detailed Implementation
[0029] 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 with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this specification, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.
[0030] Unless otherwise specified, the experimental methods used in the embodiments of this specification are conventional methods. Unless otherwise specified, all experimental materials used are commercially available.
[0031] Example 1
[0032] Using wild-type rice Zhonghua 11 (Oryza sativa ssp. japonica cv. Zhonghua 11) as material, seeds were harvested and embryos extracted at 20, 25, 35, and 45 days after flowering. After seed maturity, the seeds were harvested, dried, and stored under high temperature and humidity conditions for 0, 4, 8, 10, 12, and 15 days, respectively, and embryos were extracted. Subsequently, the gene expression level of the rice ABA receptor PYL10 was identified using the above materials. Specifically, RNA was extracted from the seed embryos, reverse transcribed into cDNA, and GAPDH was set as the internal control. Quantitative real-time PCR amplification was performed using the internal control primers GAPDH-F (SEQ ID NO:3) and GAPDH-R (SEQ ID NO:4), and the PYL10 gene-specific primers PYL10-qRT-F and PYL10-qRT-R, respectively, to detect the expression level of the PYL10 gene during development and storage under high temperature and humidity conditions. Figure 1 As shown, PYL10 expression is upregulated during seed development and further upregulated during high temperature and high humidity storage, suggesting that PYL10 may control seed storage tolerance.
[0033] In the above embodiments, RNA was extracted using the DiSpin Polysaccharide-Polyphenol Plant RNA Rapid Extraction Kit, reverse transcription was performed using the FastKing RT Kit, and RT-qPCR was conducted using the 2X Fast qPCR Master Mixture Kit. All embodiments were performed according to the kit instructions, and the primers for quantitative real-time PCR amplification were PYL10-qRT-F (SEQ ID NO:5) and PYL10-qRT-R (SEQ ID NO:6).
[0034] Example 2
[0035] Construction of an overexpression vector for the rice ABA receptor PYL10 gene
[0036] (1) Obtaining the rice ABA receptor PYL10 encoding gene
[0037] Using the seeds of Zhonghua 11 as material, RNA was extracted and reverse transcribed into cDNA as a template. PCR primers were designed and PCR amplification was performed to obtain the rice ABA receptor PYL10 encoding gene.
[0038] In the embodiments described in this specification, the PCR amplification primers are primer1 (SEQ ID NO:7) and primer2 (SEQ ID NO:8).
[0039] In one embodiment of this specification, PCR amplification was performed using KOD FX Neo DNA polymerase, and the PCR amplification was performed according to the KOD FX Neo DNA polymerase instructions.
[0040] After amplifying the target gene using cDNA as a module, the PCR amplification product is recovered, purified, ligated into a sequencing vector, transformed into competent cells, and positive clones are selected. After plasmid extraction, DNA sequencing is performed.
[0041] In one embodiment of this specification, the sequencing vector is the Blunt3 vector, and the competent cells are Trans-T1 competent cells.
[0042] Sequencing results showed that the amplified PCR product had a nucleotide sequence as shown in SEQ ID No:1, with a length of 639 bp, and was named the PYL10 gene. The amino acid sequence of the protein encoded by the PYL10 gene (rice ABA receptor PYL10) is shown in SEQ ID NO:2.
[0043] The plasmid used for DNA sequencing and which was correctly sequenced was named Blunt3-PYL10 plasmid.
[0044] (2) Construction of overexpression vector for rice ABA receptor PYL10 gene
[0045] In the embodiments described in this specification, the overexpression vector is selected as a recombinant binary expression vector, which is started by a 35S promoter. In a specific embodiment, the overexpression vector is a pCAMBIA series vector. In one embodiment of this specification, the overexpression vector is pCAMBIA2300.
[0046] The construction of the rice ABA receptor PYL10 encoding gene overexpression vector specifically includes: using a correctly sequenced plasmid as a template, performing PCR amplification with primers primer3 (SEQ ID NO:9) and primer4 (SEQ ID NO:10), and inserting the PCR amplified fragment into the overexpression vector pCAMBIA2300 through seamless cloning to form a 35S promoter-driven PYL10 gene overexpression vector.
[0047] Continuing from the previous example, using the Blunt3-PYL10 plasmid as a template, PCR amplification was performed using KOD FX Neo DNA polymerase with primers primer3 and primer4. The PCR amplification was performed according to the KOD FX Neo DNA polymerase instructions. The PCR amplified fragment was then seamlessly cloned into the plant overexpression vector pCAMBIA2300, forming a 35S promoter-driven PYL10 gene overexpression structure, and the recombinant plasmid vector pCAMBIA2300-PYL10 was obtained.
[0048] Sequencing of the obtained recombinant plasmid vector pCAMBIA2300-PYL10 revealed that the recombinant vector pCAMBIA2300-PYL10 had a nucleotide sequence as shown in SEQ ID No. 1 inserted forward at the KpnI restriction site of the expression vector pCAMBIA2300. This successfully replaced the DNA sequence between the KpnI restriction endonuclease and BamHI recognition sites (recognition sequence) of pCAMBIA2300 with the DNA sequence shown in SEQ ID No. 1. Figure 2 ).
[0049] Example 3
[0050] The rice ABA receptor PYL10 gene overexpression vector was transformed into the rice receptor variety Zhonghua 11.
[0051] Continuing from the previous example, the plasmid of the constructed and correctly sequenced PYL10 overexpression vector pCAMBIA2300-PYL10 was transformed into competent Agrobacterium cells. In this example, competent Agrobacterium EHA105 cells were used, and the transformation method is as follows:
[0052] Remove EHA105 competent cells from the -80℃ freezer and thaw them on ice. Add 2 μL of plasmid to a 100 μL tube of competent cells and incubate on ice for 30 minutes. Freeze in liquid nitrogen for 1 minute. Incubate in a 37℃ water bath for 5 minutes. Incubate on ice for 2-3 minutes. Immediately afterward, add 1 mL of antibiotic-free LB liquid medium and incubate at 28℃ for 2-3 hours at 180 rpm. Centrifuge at 4000 rpm for 2 minutes and then resuspend in 100 μL of LB liquid medium. Spread the suspension onto LB agar plates containing rifampicin and kanamycin resistance and incubate upside down at 28℃ for 2-3 days. Pick single clones growing on the plates, culture them in liquid medium, extract the recombinant plasmid, identify positive clones by enzyme digestion, and store the positive clones at -80℃ for later use.
[0053] The above-mentioned Agrobacterium containing the overexpression vector was used to transform the wild-type rice variety Zhonghua 11 using an infection method. The specific operation steps are as follows:
[0054] (1) Induction of callus: Remove the glumes from mature seeds of rice variety Zhonghua 11, disinfect them by soaking in 75% alcohol for no more than 1 minute; then wash them with sterile water more than 5 times; soak them in 50% sodium hypochlorite for 25 minutes and shake them gently at room temperature; wash them with sterile water more than 8 times; blot dry with sterile filter paper and place them evenly on the rice callus induction medium; finally, place them in a 30℃ light incubator for 2-3 weeks until callus grows; after that, subculture for 1-2 generations and select callus with good growth for Agrobacterium infection experiments.
[0055] (2) Agrobacterium activation: Take 20 μL of Agrobacterium containing the PYL10 overexpression plasmid vector stored at -80℃ and inoculate it into 3 mL of liquid YEP medium containing kanamycin and rifampicin. Then, culture it in a shaker at 200 rpm (28℃) until the OD600 is about 0.3-0.5. Take 1 mL of bacterial culture and inoculate it into 50 mL of fresh liquid YEP medium containing kanamycin and rifampicin. Continue to culture it in a shaker at 28℃ and 200 rpm until the OD600 is about 0.3-0.5. Finally, centrifuge to collect the bacterial culture and resuspend the bacterial cells in AAM medium containing 100 μM acetylsyl syringone (AS).
[0056] (3) Co-culture of callus with Agrobacterium, screening of callus resistance and differentiation and rooting: The callus obtained in step a was immersed in the AAM bacterial solution obtained in step b for 30 minutes and gently shaken; then the excess bacterial solution was removed with sterile filter paper, and the immersed callus was transferred to a solid culture medium for co-culture and cultured in the dark at 28°C for 3 days; then transferred to a screening medium for culture; transferred to a differentiation medium for differentiation and regeneration, and then transferred to MS medium for rooting and seedling growth; after seedling growth, the seedlings were transplanted to the field and managed according to conventional field management to obtain transgenic plants overexpressing PYL10, namely T0 generation seedlings of pCAMBIA2300-PYL10 plants.
[0057] Example 4
[0058] Identification and phenotypic analysis of rice plants overexpressing the ABA receptor PYL10 gene
[0059] To identify and phenotypically analyze rice plants overexpressing the ABA receptor PYL10, PCR identification was first performed on pCAMBIA2300-PYL10 transgenic plants with increased PYL10 gene expression. Specifically, genomic DNA was extracted from leaves of T0 generation seedlings of the obtained pCAMBIA2300-PYL10 transgenic plants and seedlings of the recipient control parent (referred to as CK, i.e., seedlings of Zhonghua 11 plants). PCR was used with primers primer 5 (SEQ ID NO:11) and primer 6 (SEQ ID NO:12) to identify positive seedlings. Plants yielding a 300bp G418 PCR product were considered positive.
[0060] After obtaining positive seedlings, the gene expression level of the rice ABA receptor PYL10 was further identified. Specifically, RNA was extracted from positive seedlings and CK seeds, reverse transcribed into cDNA, and GAPDH was set as the internal control. Real-time quantitative PCR amplification was performed using the internal control primers GAPDH-F and GAPDH-R, and the PYL10 gene-specific primers PYL10-qRT-F and PYL10-qRT-R, respectively, to detect the expression level of the PYL10 gene in different transgenic plants. Positive transformants PYL10-ox#1 and #2, with significantly increased expression levels, were obtained. Figure 3 As shown, in positive plants transformed with the recombinant vector pCAMBIA2300-PYL10, the gene expression level of PYL10 was significantly higher than that of CK.
[0061] After obtaining positively transformed plants PYL10-ox#1 and #2 with significantly increased expression levels, the obtained PYL10-ox#1, #2 and CK plants were further planted, mature seeds were harvested, and the phenotypic differences of PYL10-ox and CK seeds stored under high temperature and high humidity conditions were observed.
[0062] Comparative observations of seed morphology in PYL10-overexpressing transgenic rice and CK rice revealed that PYL10 overexpression did not affect seed appearance phenotype, meaning that seed and embryo development were normal. Specifically, as shown below... Figure 4 As shown. Seed germination rate and seedling emergence rate of PYL10-overexpressing transgenic and CK rice before storage were measured. It was found that PYL10 overexpression did not alter seed germination or seedling emergence. Figure 5 and Figure 6 As shown. Figure 5 As shown, the vertical axis represents seed germination rate and seedling survival rate, respectively. Compared with the control (CK), PYL10-ox#1 and #2 showed no significant changes in germination rate and seedling survival rate. Figure 6 As shown, compared with CK, the germination and seedling morphology of PYL10-ox#1 and #2 did not change. During high-temperature and high-humidity storage, the seed germination rate gradually decreased, and the rate of decrease in transgenic PYL10-ox#1 and #2 rice seeds was significantly slower than that in CK rice seeds. Figure 7 As shown. Observation of seed germination rate and seedling rate after storage for a period of time revealed that the germination rate and seedling rate of transgenic PYL10-ox#1 and #2 rice seeds were significantly higher than those of CK rice seeds, such as... Figure 8 and Figure 9 As shown. Figure 8 As shown, compared with CK, PYL10-ox#1 and #2 had significantly higher germination rates and seedling rates after storage under high temperature and humidity conditions. Figure 9 As shown, compared with CK, PYL10-ox#1 and #2, after being stored under high temperature and high humidity conditions, showed significantly better germination morphology and seedling morphology than CK.
[0063] To further determine whether storage of transgenic PYL10-ox#1 and #2 rice seeds affects seed quality, the examples in this specification further calculate the time it takes for the germination rate to decrease to meet seed quality standards after storage. Figure 10 As shown, the vertical axis represents the time required for the seed germination rate to reach 85%, derived from... Figure 10 It was found that, compared with the control (CK), the storage time of PYL10-ox#1 and #2 increased by 20.8% and 29.9%, respectively. This indicates that the rice stress-activating protein PYL10 can positively regulate seed storage time. It should be noted that the germination rate meeting the seed quality standards is no less than 85%.
[0064] In conclusion, the rice stress-activated protein PYL10 can positively regulate seed storage time and can be used to improve the storage tolerance of rice, showing significant potential for high-quality breeding.
[0065] The embodiments of this specification also provide an application of an ABA receptor for controlling the storage time of rice seeds, wherein biological materials are constructed using the gene of the ABA receptor PYL10 to regulate the storage time of rice seeds.
[0066] In the embodiments of this specification, the biological material is an overexpression material, which improves the storage time of rice seeds.
[0067] In the embodiments described in this specification, the biological material is a nucleic acid molecule, a recombinant expression vector, a host cell, or a transformed plant cell.
[0068] In the embodiments of this specification, the gene of the ABA receptor PYL10 is introduced into plant cells, tissues or organs to cultivate plants, resulting in transgenic plants with improved storage time.
[0069] In the embodiments of this specification, the gene of the ABA receptor PYL10 is introduced into plant cells, tissues or organs via a plant overexpression vector, wherein the overexpression vector is a recombinant binary expression vector.
[0070] In the embodiments described in this specification, the transgenic plant is rice Zhonghua 11.
[0071] In the embodiments described in this specification, the ABA receptor PYL10 positively regulates seed storage time.
[0072] In the embodiments described in this specification, the regulation does not affect the seed's appearance phenotype or the germination rate and seedling rate before storage; however, the regulation can increase the seed's storage time.
[0073] This specification provides an ABA receptor for controlling rice seed storage time and its application. The ABA receptor for controlling rice seed storage time is PYL10. The nucleotide sequence of the ABA receptor encoding gene is shown in SEQ ID NO:1, and the amino acid sequence of the ABA receptor encoding gene is shown in SEQ ID NO:2. By increasing the expression of the rice ABA receptor gene, the storage time of rice seeds can be increased, providing a novel gene resource for basic research on rice seed storage and high-quality molecular breeding practices, and it can be applied to production practices.
[0074] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.
[0075] The above description is merely an embodiment of this specification and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.
Claims
1. An ABA receptor for controlling the storage time of rice seeds, characterized in that, The ABA receptor controlling the storage time of rice seeds is PYL10, the nucleotide sequence of the ABA receptor encoding gene is shown in SEQ ID NO:1, and the amino acid sequence of the ABA receptor encoding the gene is shown in SEQ ID NO:
2.
2. The ABA receptor as described in claim 1, characterized in that, The ABA receptor PYL10 encoding gene is upregulated during seed development and storage.
3. An application of an ABA receptor for controlling the storage time of rice seeds, characterized in that, Biomaterials were constructed using the gene of the ABA receptor PYL10 described in claim 1 to regulate the storage time of rice seeds.
4. The application as described in claim 3, characterized in that, The biomaterial is an overexpression material, which increases the storage time of rice seeds.
5. The application as described in claim 3, characterized in that, The biomaterials are nucleic acid molecules, recombinant expression vectors, host cells, or transformed plant cells.
6. The application as described in claim 5, characterized in that, The ABA receptor PYL10 gene is introduced into plant cells, tissues, or organs to cultivate transgenic plants with improved storage time.
7. The application as described in claim 6, characterized in that, The gene of the ABA receptor PYL10 is introduced into plant cells, tissues, or organs via a plant overexpression vector, wherein the overexpression vector is a recombinant binary expression vector.
8. The application as described in claim 7, characterized in that, The transgenic plant is rice variety Zhonghua 11.
9. The application as described in claim 3, characterized in that, The ABA receptor PYL10 positively regulates seed storage time.
10. The application as described in claim 3, characterized in that, The regulation does not affect the appearance phenotype of the seeds or the germination rate and seedling rate before storage, but it can increase the storage time of the seeds.