Protein atgrf8 controlling seed dormancy and germination and use of the encoding gene
By regulating the expression level of the Arabidopsis thaliana GRF8 gene and using gene editing technology to regulate seed dormancy, the problem of seed germination under unsuitable conditions was solved, achieving effective control of seed dormancy and germination, and improving agricultural production efficiency and quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO AGRI UNIV
- Filing Date
- 2026-05-07
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies are insufficient to effectively control seed dormancy and germination, leading to crop germination under unsuitable conditions, which affects grain yield and quality. Furthermore, seeds are prone to premature germination during storage, resulting in a decline in quality.
By regulating the expression level of the Arabidopsis thaliana GRF8 gene, and using gene editing technology to knock out or overexpress the GRF8 gene, seed dormancy characteristics can be altered, thereby achieving the regulation of seed dormancy.
Significantly increasing or decreasing seed dormancy levels and enhancing or inhibiting germination ability provides an effective means of controlling seed dormancy and germination in agricultural production, thereby improving crop production efficiency and quality.
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Figure CN122146774A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the protein AtGRF8, which controls seed dormancy and germination, and the application of its encoding gene, and belongs to the field of molecular biology technology. Background Technology
[0002] Seed dormancy refers to the phenomenon where seeds with normal germination ability fail to germinate even under suitable germination conditions. It is an adaptive trait developed by plants during long-term evolution, preventing them from growing in unfavorable environments and ensuring the survival and continuation of the species. In agricultural production, appropriate seed dormancy is crucial. It effectively prevents sprouting on the ears of crops during prolonged periods of high temperatures and rainy weather before harvest, avoiding severe losses in grain yield and quality. Simultaneously, dormancy facilitates long-term safe storage of seeds, extending seed lifespan and ensuring the preservation of germplasm resources. Therefore, crop production always faces two demands: striving for rapid and uniform seed germination at sowing, while maintaining appropriate dormancy during grain development, maturation, and storage to reduce sprouting on the ears and during storage, preventing premature germination and subsequent quality decline.
[0003] Growth regulatory factors (GRFs) are a family of transcription factors that widely participate in the regulation of plant growth and development and have important biological functions. In recent years, GRF transcription factors have been identified in an increasing number of species, with 9 and 12 GRF members identified in Arabidopsis and rice, respectively. The core feature of GRF proteins is the presence of two highly conserved QLQ and WRC domains at the N-terminus. The QLQ domain is mainly responsible for protein-protein interaction, while the WRC domain is responsible for DNA binding. GRFs play important roles in leaf and floral organ development and stress response, and are mainly expressed in the meristematic tissues of young organs and in areas of active cell growth. For example, in floral meristems, the key factors AP1 and SEP3 can act as initiators to induce the expression of Arabidopsis GRF8 (AtGRF8), thereby participating in the formation of plant floral organs. In addition, AtGRF8 can also control embryogenic responses and participate in chlorophyll synthesis. Furthermore, Arabidopsis AtGRF8 is highly expressed under cold, heat, and waterlogging stresses, indicating its important role in plant response to abiotic stress. Based on the above functions, it is evident that AtGRF8 has strong agricultural application value. Summary of the Invention
[0004] This application first provides a use of the AtGRF8 gene in promoting seed dormancy or inhibiting seed germination.
[0005] This application also provides a method for promoting seed dormancy by increasing the expression level of the GRF8 gene in seeds. For example, the expression level of the GRF8 gene can be increased by transferring an exogenous GRF8 gene into target seeds.
[0006] This application also provides a method for promoting seed germination by selectively knocking out or gene editing the GRF8 gene in seeds, thereby silencing the gene expression, downregulating its expression level, or causing all or part of the biological function of the protein encoded by the GRF8 gene to be lost.
[0007] In some implementations, the GRF8 gene is the Arabidopsis GRF8 gene (AtGRF8).
[0008] In some implementations, the seeds are Arabidopsis thaliana seeds.
[0009] In some embodiments, the amino acid sequence of the protein encoded by the GRF8 gene is shown in SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3.
[0010] The technical advantages of this application are as follows: the AtGRF8 gene has significant tissue expression specificity. Functional verification results show that the seed dormancy level of Arabidopsis thaliana is significantly reduced and the germination ability is enhanced after the gene is lost, while the seed dormancy level of the overexpressing gene line is significantly increased. This confirms that AtGRF8 plays a positive regulatory role in seed dormancy and has important application value in crop genetics and breeding and agricultural production practices. Attached Figure Description
[0011] Figure 1 Tissue-specific expression of the AtGRF8 gene. Figure A shows the expression level of the AtGRF8 gene in different tissues (roots, stems, leaves, flowers, pods, and seeds) of Arabidopsis thaliana; Figure B shows the expression level of the AtGRF8 gene in pods 10, 13, 15, 18, and 21 days after pollination.
[0012] Figure 2 AtGRF8 T-DNA insertion mutants and gene-editing mutants. Figure A shows the T-DNA insertion site and the location of the CRISPR dual target sites; Figure B shows the sequencing results of long-fragment knockout.
[0013] Figure 3 : Identification of GRF8 gene expression levels in T-DNA insertion mutant (grf8-1), knockout mutant (grf8-cr), wild-type Col, and overexpression material (35S:GRF8).
[0014] Figure 4 Germination rate determination of newly harvested seeds of AtGRF8 gene mutants and overexpression materials. Figure A shows the germination rate statistics of newly harvested seeds of the T-DNA insertion mutant grf8-1; Figure B shows the germination rate statistics of newly harvested seeds of the dual-target knockout mutant grf8-cr; Figure C shows the germination rate statistics of newly harvested seeds of the overexpression material 35S:GRF8. Detailed Implementation
[0015] Several classification methods have been proposed for seed dormancy. Based on the different stages of seed dormancy formation, it can be divided into primary dormancy and secondary dormancy. Primary dormancy gradually forms during seed maturation and dehydration, occurs in the parent plant, and is jointly regulated by the species' genetic background and environmental conditions at the seed development stage. Secondary dormancy refers to the phenomenon where non-dormant mature seeds, having detached from the parent plant and lost primary dormancy, fail to germinate and re-enter dormancy after being subjected to abiotic stress. Primary dormancy is an inherent biological characteristic of species such as Arabidopsis thaliana, rice, and wheat, while secondary dormancy is greatly affected by environmental disturbances, exhibits significant phenotypic fluctuations, and is difficult to control artificially. In this application, seed dormancy refers to primary dormancy, which is also the common meaning of dormancy.
[0016] In this application, the evaluation criterion for seed dormancy intensity is as follows: under the optimal germination conditions without stress intervention, the germination rate of newly harvested seeds is used as a quantitative indicator. The germination rate is negatively correlated with the seed dormancy intensity, that is, the lower the germination rate, the higher the degree of seed dormancy.
[0017] It should be noted that seed dormancy, plant bud (branch / flower / leaf) dormancy, and tuber dormancy are biological characteristics of different plant organs. The mechanisms of occurrence, inducing factors, phenotypic characteristics, and core regulatory genes of the three are all fundamentally different, and the relevant regulatory rules cannot be directly learned from or applied to each other.
[0018] Arabidopsis thaliana is a widely used model plant in plant functional genomics research. This application uses Arabidopsis thaliana as the research material to carry out functional verification. In the absence of contrary experimental evidence, the gene regulation rules revealed by this invention can be expected to be applicable to various plant species.
[0019] This application is the first to discover that the AtGRF8 gene encodes three alternative splice variants (CDS1, CDS2, and CDS3), the amino acid sequences of which are shown in SEQ.ID.NO. 1, SEQ.ID.NO. 2, and SEQ.ID.NO. 3, respectively. These three alternative splice variant proteins are collectively named AtGRF8 proteins or proteins encoded by the AtGRF8 gene. Therefore, in this application, AtGRF8 protein or proteins encoded by the AtGRF8 gene refers to proteins with amino acid sequences shown in SEQ.ID.NO. 1, SEQ.ID.NO. 2, or SEQ.ID.NO. 3, respectively; and the CDS sequence of the AtGRF8 gene refers to the open reading frame encoding SEQ.ID.NO. 1, SEQ.ID.NO. 2, or SEQ.ID.NO. 3. This application demonstrates that overexpression of the AtGRF8 gene by introducing its CDS sequence into Arabidopsis thaliana can promote seed dormancy or inhibit seed germination.
[0020] The core experimental results of this application are summarized as follows: The Arabidopsis thaliana AtGRF8 gene exhibits a specific high expression pattern in leaves and seeds, showing significant tissue expression specificity; the seed dormancy phenotype identification results show that, compared with wild-type Col plants, the seed dormancy levels of the grf8-1 and grf8-cr mutant lines are significantly reduced, while the seed dormancy level of the 35S:GRF8 overexpression material is significantly increased.
[0021] This application further illustrates the technical solution through the following embodiments, but no embodiment or combination thereof should be construed as limiting the scope of protection or implementation of the present invention. In the following embodiments, unless otherwise specified, the experimental methods used are conventional experimental methods in the art; the experimental materials and reagents used, unless otherwise specified, can be obtained through commercial channels.
[0022] Example 1: Obtaining Experimental Materials
[0023] (1) Overexpression vector pCAMBIA1305, gene editing vector pHK2-AtCas9-U6, GT bone, can be purchased by the public from CASMA Mall.
[0024] (2) Agrobacterium GV3101 can be obtained by the public from the Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences.
[0025] (3) Columbia ecotype Arabidopsis thaliana: wild type Columbia-0 (Col-0), which can be obtained by the public from the Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences.
[0026] (4) The Arabidopsis T-DNA insertion mutant grf8-1 was purchased from the company. The insertion site was at the SAIL_90_H11 position. The T-DNA insertion in the exon region resulted in the inability to produce a complete gene transcript. It was confirmed by qRT-PCR to be a GRF8 deletion mutant.
[0027] (5) Construction of GRF8 overexpression vector (35S:GRF8): The overexpression vector pCAMBIA1305 was used to fuse the GRF8 coding region (CDS1, CDS2, or CDS3) amplified using Arabidopsis seed cDNA as a template. The expression level of the AtGRF8 gene in the transgenic material was detected by qRT-PCR.
[0028] GRF8 sequence information: Total RNA was extracted from Arabidopsis thaliana ecotype in Colombia. After reverse transcription, sequence analysis, expression level detection and functional verification revealed the existence of three alternative splice variants (CDS1, CDS2, CDS3), whose encoded protein sequences are shown in SEQ.ID.NO. 1, SEQ.ID.NO. 2 or SEQ.ID.NO. 3, respectively.
[0029] The protein sequence encoded by CDS1 (SEQ.ID.NO. 1):
[0030] MRMLLGIPYVDKSVLSNSVLERGKQDKSKLLLVDKCHYELDVEERKEDFVGGFGFGVVENSHKDVMVLPHHHYYPSYSSPSSSSLCYCSAGVSDPMFSVSSNQAYTSSHSGMFTPAGSGSAAV TVADPFFSLSSSGEMRRSMNEDAGAAFSEAQWHELERQRNIYKYMMASVPVPPELLTPFPKNHQSNTNPDVDTYRSGMFSIYADYKNLPLSMWMTVAVATGGSLQLGIASSASNNTADLEPW RCKRTDGKKWRCSRNVIPDQKYCERHTHKSRPRSRKHVESSHQSSHHNDIRTAKNDTSQLVRTYPQFYGQPISQIPVLSTLPSASSPYDHHRGLRWFTKEDDAIGTLNPETQEAVQLKVGSSR ELKRGFDYDLNFRQKEPIVDQSFGALQGLLSLNQTPQHNQETRQFVVEGKQDEAMGSSLTLSMAGGGMEETEGTNQHQWVSHEGPSWLYSTTPGGPLAEALCLGVSNNPSSSTTTSCSSRSSS.
[0031] The protein sequence encoded by CDS2 (SEQ ID NO. 2):
[0032] MRMLLGIPYVDKSVLSNSVLERGKQDKSKLLLVDKCHYELDVEERKEDFVGGFGFGVVENSHKDVMVLPHHHYYPSYSSPSSSSLCYCSAGVSDPMFSVSSNQAYTSSHSGMFTPAGSGSAAVTVADPFFSLSSSGEMRRSMNEDAGAAFSEAQWHELERQRNIYKYMMASVPVPPELLTPFPKNHQSNTNPDVTVAVATGGSLQLGIASSASNNTADLEPWRCKRTDGKKWRCSRNVIPDQKYCERHTHKSRPRSRKHVESSHQSSHHNDIRTAKNDTSQLVRTYPQFYGQPISQIPVLSTLPSASSPYDHHRGLRWFTKEDDAIGTLNPETQEAVQLKVGSSRELKRGFDYDLNFRQKEPIVDQSFGALQGLLSLNQTPQHNQETRQFVVEGKQDEAMGSSLTLSMAGGGMEETEGTNQHQWVSHEGPSWLYSTTPGGPLAEALCLGVSNNPSSSTTTSSCSRSSS。
[0033] Protein sequence encoded by CDS3 (SEQ.ID.NO. 3):
[0034] MGTRAERKEDFVGGFGFGVVENSHKDVMVLPHHHYYPSYSSPSSSSLCYCSAGVSDPMFSVSSNQAYTSSHSGMFTPAGSGSAAVTVADPFFSLSSSGEMRRSMNEDAGAAFSEAQWHELERQRNIYKYMMASVPVPPELLTPFPKNHQSNTNPDVTVAVATGGSLQLGIASSASNNTADLEPWRCKRTDGKKWRCSRNVIPDQKYCERHTHKSRPRSRKHVESSHQSSHHNDIRTAKNDTSQLVRTYPQFYGQPISQIPVLSTLPSASSPYDHHRGLRWFTKEDDAIGTLNPETQEAVQLKVGSSRELKRGFDYDLNFRQKEPIVDQSFGALQGLLSLNQTPQHNQETRQFVVEGKQDEAMGSSLTLSMAGGGMEETEGTNQHQWVSHEGPSWLYSTTPGGPLAEALCLGVSNNPSSSTTTSSCSRSSS。
[0035] Example 2 Construction of AtGRF8 gene knockout material
[0036] Considering the existence of three alternative splicing variants in the AtGRF8 gene, we constructed a dual-target knockout material, grf8-cr, using gene editing technology. The knockout of a long fragment between the two gRNAs simultaneously affects transcripts from all three alternative splicing variants. Sanger sequencing confirmed the long fragment deletion (e.g., Figure 2 A, B).
[0037] Example 3 Construction of AtGRF8 overexpression materials
[0038] Step 1: Extract total RNA from Arabidopsis thaliana ecotype in Colombia and reverse transcribe it into cDNA. Using cDNA as a template, perform PCR amplification of the AtGRF8 gene using primer pairs containing XbaI and SalI restriction sites, and recover the PCR amplification product.
[0039] Step 2: Digest the vector pCAMBIA1305 with the restriction endonucleases XbaI and SalI, and recover the vector backbone.
[0040] Step 3: Homologous recombination ligation of the purified product obtained in Step 1 and the vector backbone obtained in Step 2 yields the recombinant expression vector Pro35S-AtGRF8.
[0041] Step 4: Based on the sequencing results, the structure of the recombinant expression vector Pro35S-AtGRF8 is described as follows: The small fragment between the XbaI and SalI restriction sites of the overexpression vector pCAMBIA1305 is replaced with the sequence of the AtGRF8 gene.
[0042] Step 5: Transform the Pro35S-AtGRF8 recombinant expression vector into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Transform Columbia ecotype Arabidopsis thaliana using the inflorescence infection method to harvest T1 generation seeds. Sow the seeds on MS solid medium containing 50 mg / L kanamycin and screen for T1 generation resistant plants. Self-pollinate the T1 generation plants to obtain the T2 generation, i.e., the seeds produced by T1 generation self-pollination and the plants grown from them, and analyze the segregation ratio of the T2 generation; self-pollinate the T2 generation to obtain the T3 generation (the seeds produced by T2 generation self-pollination and the plants grown from them), and identify the lines in the T3 generation in which all plants showed resistance, thus obtaining homozygous transgenic Arabidopsis thaliana with a single copy insertion of the AtGRF8 gene.
[0043] Example 4 RNA Extraction and Reverse Transcription
[0044] Plant samples were aliquoted into grinding tubes and rapidly ground into powder in liquid nitrogen. RNA was extracted using the Novizan Polysaccharide-Polyphenol Plant RNA Extraction Kit (see instructions). After extraction, RNA concentration and OD260 / 280 ratio were measured using Nanodrop. Reverse transcription was performed using a Novizan Biotech reverse transcription kit. The steps are as follows:
[0045] (1) Genomic DNA removal
[0046] Based on the measured RNA concentration, the reaction system was prepared in RNase-free centrifuge tubes (Table 1). After gently mixing with a pipette, the mixture was incubated at 42°C for 2 min.
[0047] Table 1. Preparation of the reverse transcription system
[0048] reagents Dosage 5 × gDNA wiper Mix 2 μL Total RNA 1 ug <![CDATA[RNase-free ddH2O]]> To 10 μL
[0049] (2) Preparation of reverse transcription reaction system
[0050] Add the prepared reaction solution to the mixture after the above reaction, and gently mix by blowing. The formulation of the reaction system is shown in Table 2 below.
[0051] Table 2 Preparation of reverse transcription reaction system
[0052] reagents Dosage 10 × RT Mix 2 μL Hiscript Ⅲ Enzyme Mix 2 μL <![CDATA[Olig(dT) 20 VN]]> 1 μL Random hexamers 1 μL <![CDATA[RNase-free ddH2O]]> 4 μL
[0053] (3) Perform reverse transcription reaction
[0054] Place the mixture from step (2) (total 20 μL) into a PCR instrument, react at 37°C for 15 min, react at 85°C for 5 s, and store at 4°C. The product from the reaction is immediately used for qPCR.
[0055] Example 5: Detection of AtGRF8 gene expression level (qRT-PCR)
[0056] The expression level of the GRF8 gene in tissues was detected by qRT-PCR using reverse-transcribed cDNA as a template. The ChamQ Universal SYBR qPCR Master Mix kit from Novizan was used. The method is as follows:
[0057] (1) Take 5 μL of the product after the reaction into an octet, add 45 μL of ddH2O, dilute 10 times and place on ice.
[0058] (2) Prepare the reaction system according to Table 3, add it to the Hard-Shell PCR plate, place a layer of aluminum foil under the plate, and add the sample as accurately as possible.
[0059] (3) After adding the samples, cover the PCR plate with a layer of Micro-seal® 'B'seal membrane. Perform the qPCR reaction on the Bio-Red qRT-PCR instrument according to the reaction procedure in Table 4.
[0060] (4) The CT values obtained in the experiment are expressed as 2 -ΔΔCT The algorithm performs conversions to obtain the relative expression of the gene being measured, with REF as the internal reference gene.
[0061] Table 3 qRT-PCR reaction system
[0062] reagents Added amount (10 μL) cDNA 1 μL Forward primer (10 μM) 0.2 μL Reverse Peimer (10 μM) 0.2 μL 2 × ChamQ Universal SYBR qPCR Master Mix 5 μL <![CDATA[ddH2O]]> 3.6 μL
[0063] Table 4 qRT-PCR reaction procedure
[0064] Reaction steps Reaction temperature (°C) time Cycle number 1 95 30 s 1 2 95 10 s 2 to 3 runs 40 cycles 3 60 30 s 4 95 15 s 1 5 60 60 s 1 6 95 15 s 1
[0065] Experimental results: ① The expression levels of the AtGRF8 gene in different tissues (roots, stems, leaves, flowers, pods, and seeds) of Arabidopsis thaliana were detected as follows: Figure 1 As shown: This gene is highly expressed in leaves and seeds (e.g. Figure 1 A), indicating that it may play an important role in the establishment of seed dormancy. Furthermore, by detecting the expression level of the AtGRF8 gene in fruit pods of Arabidopsis at different DAP stages, we found that the AtGRF8 gene expression level reached its highest at 21 DAP (e.g., ...). Figure 1 B), at this time, Arabidopsis seeds are basically mature. ② The results of detecting AtGRF8 expression levels after gene silencing mutation and gene overexpression treatment are as follows: Figure 3 As shown, compared with the expression level of GRF8 gene in newly harvested seeds of wild-type Col, almost no expression of GRF8 gene was detected in the two mutants grf8-1 and grf8-cr seeds, while the expression level of 35S:GRF8 was about 60 times that of Col, indicating that gene silencing mutation and gene overexpression treatment were both successful.
[0066] Example 6: Determination of Arabidopsis thaliana seed germination rate
[0067] Line the bottom of the germination box with 2-3 layers of pre-drawn grids of filter paper, moisten with ddH2O, and then evenly scatter Arabidopsis seeds in each grid, 50-80 seeds per grid. Place transgenic and control materials in the same germination box. Cover and place in a light incubator with the following parameters: temperature 23℃, 16 h light / 8 h dark. Germination is recorded every 7 days for 3 consecutive cycles. Finally, Excel and Prism software are used for statistical analysis of the germination data.
[0068] The germination experiment results of newly harvested seeds are as follows: Figure 4As shown: the dormancy level of both mutant materials was significantly reduced, and the germination rate of newly harvested seeds was significantly increased. Figure 4 A, B), while the dormancy level of the overexpressed material (taking CDS1 overexpressing AtGRF8 as an example) was significantly enhanced. Figure 4 C). Therefore, AtGRF8 inhibits seed germination and positively regulates seed dormancy, making it an effective target for regulating seed dormancy in production applications and possessing significant agricultural application value.
[0069] This application has made every effort to describe the inventive concept and evidence of its effects. The scope of this invention is defined by the appended claims, and those skilled in the art will clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the field. Without departing from the spirit and scope of this invention, those skilled in the art can make any modifications or alterations to the technical solutions of this invention, and such modifications and alterations are also included within the scope of this invention.
Claims
1. The use of the GRF8 gene in promoting seed dormancy or inhibiting seed germination.
2. Use according to claim 1, characterized in that, The GRF8 gene is the Arabidopsis thaliana GRF8 gene.
3. The use as described in claim 2, wherein the amino acid sequence of the protein encoded by the GRF8 gene is shown in SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3.
4. A method for promoting seed dormancy, characterized in that, Increase the expression level of the GRF8 gene in seeds.
5. The method as described in claim 4, characterized in that, The expression level of the GRF8 gene was increased by transferring the exogenous GRF8 gene into the target seed.
6. The method as described in claim 5, characterized in that, The exogenous GRF8 gene is the Arabidopsis thaliana GRF8 gene.
7. The method of claim 6, wherein the amino acid sequence of the protein encoded by the exogenous GRF8 gene is shown in SEQ ID NO1, SEQ ID NO2, or SEQ ID NO3.
8. A method for promoting seed germination, characterized in that, Targeted knockout or gene editing of the GRF8 gene in seeds can silence its expression, downregulate its expression level, or cause the loss of all or part of the biological function of the protein encoded by the GRF8 gene.
9. The method as described in claim 8, characterized in that, The amino acid sequence of the protein encoded by the GRF8 gene is shown in SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3.
10. The seed as described in any of the preceding claims, characterized in that, The seeds are Arabidopsis thaliana seeds.