Application of rice OsGET2 gene in regulating early maturity of rice

Abstract

The application discloses application of a rice OsGET2 gene in regulation of early maturity of rice, relates to the technical field of plant genetic engineering and molecular breeding, and realizes promotion of early heading and shortening of a growth period of rice by knocking out or reducing expression of the OsGET2 gene in the rice; the nucleotide sequence of the OsGET2 gene is shown as SEQ ID NO:1, and the amino acid sequence of a protein coded by the OsGET2 gene is shown as SEQ ID NO:2; the application of the rice OsGET2 gene in regulation of early maturity of rice can significantly promote early heading of the rice, shorten a growth period, realize early maturity, and does not affect thousand-grain weight yield traits by knocking out the OsGET2 gene through a gene editing technology; important gene resources are provided for molecular breeding of early maturity rice varieties, and the application can be widely applied to early maturity improvement of different rice varieties.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular breeding technology, specifically to the application of the rice OsGET2 gene in regulating early maturity of rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's three major staple crops, providing essential food for approximately 4 billion people globally. The heading stage is the most critical growth period for rice varieties, determining their adaptation to different regions and seasons, and plays a vital role in rice yield. Early-maturing varieties can expand planting areas, increase the multiple cropping index, and mitigate adverse climatic conditions later in the growing season. Therefore, developing new early-maturing rice varieties that do not reduce yield has always been a key objective of rice breeding. A thorough understanding of the molecular mechanisms and regulatory networks of rice growth and development is a theoretical prerequisite for creating high-yielding and high-quality rice varieties using bio-breeding techniques.

[0003] The currently identified genes regulating heading date are far from meeting the needs of breeding practice. On the one hand, there are numerous rice varieties with significant differences in genetic background, and the functional verification of reported heading date regulating genes in other varieties is insufficient, leaving their application scope and effects uncertain. On the other hand, some identified heading date regulating genes, while affecting the growth period, often have negative effects on yield traits. How to maintain or even increase yield while shortening the growth period is a pressing technical challenge in early-maturing breeding. Therefore, discovering and identifying new rice growth period regulating gene resources, especially functional genes that can achieve early maturity without reducing yield, has significant theoretical and applied value for rice variety improvement and molecular breeding. The rice OsGET2 gene (Os01g0863400; LOC4324784) and its encoded protein provided by this invention can significantly promote early heading and shorten the growth period of rice by knocking out or reducing the expression of this gene through gene editing technology, thereby achieving early maturity without reducing the thousand-grain weight. This provides a new gene resource and technical means to solve the contradiction between shortened growth period and reduced yield in early-maturing breeding. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the rice OsGET2 gene in regulating early maturity of rice, so as to solve the technical problem in existing early maturity rice breeding where some identified heading period regulating genes are difficult to balance yield traits while shortening the growth period.

[0005] To achieve the above objectives, the present invention provides the following technical solution: the application of rice OsGET2 protein or the gene encoding said rice OsGET2 protein in regulating rice growth period or breeding early-maturing rice varieties.

[0006] Furthermore, the regulation of rice growth period aims to promote early maturity and heading of rice.

[0007] The present invention also discloses a method for regulating the growth period of rice, the method comprising: knocking out or reducing the expression of the OsGET2 gene (gene number: Os01g0863400; LOC4324784) in rice.

[0008] Furthermore, the knockout or reduction of OsGET2 gene expression is achieved through gene editing technology.

[0009] Furthermore, the gene editing technology is CRISPR / Cas9 gene editing technology.

[0010] Furthermore, the nucleotide sequence of the OsGET2 gene is shown in SEQ ID NO:1, and the amino acid sequence of the OsGET2 protein is shown in SEQ ID NO:2.

[0011] SEQ ID NO:1 (OsGET2 gene nucleotide sequence):

[0012] ATGGAGAGCGGCGCGAACGCACGGCGGCGGCGGCGGCTGGTGGATAGGGGATCCGATCGCCTCGCCTTCATCACCGGCCAAGCTCAGAGCCTCCCCTCCGATCCGTCCCCAGATTCACCACTTTATACTGTTGATGCTGCTTCCCTTCAGCGATCTGAACGTCAGCTAAATGAAGTTGGCATTGGTGATGACATATTCAACTACATAACTCAGTTGCAAAAATCAGAATCTGGTGTTCAACCGACTAGTGAGGCCCACCTGGAAACTCATAAAGAGAAACACCAAGGTAATGAAAGTGACCTCCAAAAGCTCAAGACAAGCAATGTGGTTCCTGAAATTCAGCCAGTAAATGAAAAGGCATTTCAAAGGCACAGTGAGGAAACCCTCAGGAAGAAGAACAGTCATGACAGATCGGCAAGTACACAGCCTATGAGGGAAATGGAAACAAGATCCAGATATGTGCCTCCAAATCAGTCTAATCAATCTGATAGTGCAGGTTGGTCTGTGGAAACTCTGAAGGAAATCCTGAATTTCGCACCACATGAGATTACCCAGGCCATATCAGCTACAGAGTATAACCGATTTTTGGCTTCCGTCATCATTGGTTTTCTGGTGGTTCTTTCAAATTGGGGGCTGGACGTTGGTGGCACTATCACCAAAGTATTAGTTGCTACAAGGCCAATTCTATTTCTTATTGTAACGAACATAACCATTGTCTTCACTTTGTTGATGGAGAACAAGGACCCCAACGTGAGGGGTAGACCAGCTGGCAGCAACCTTGGTTCTGCTGATAACTTAGGCCAAATGCTAGAGATTGGGTTGCTGCTACAGAAAGCCCTGAGCACCCTCCTGATAGACTGCAGCGTTTGCGCTGTCATCATGATCTGTTTTATTTGA.

[0013] SEQ ID NO: 2 (Amino acid sequence of OsGET2 protein):

[0014] MESGANARRRRRRLVDRGSDRLAFITGQAQSLPSDPSPDSPLYTVDAASLQRSERQLNEVGIGDDIFNYITQLQKSESGVQPTSEAHLETHKEKHQGNESDLQKLKTSNVVPEIQPVNEKAFQRHSEETLRKKNSHDRSASTQPMREMET RSRYVPPNQSNQSDSAGWSVETLKEILNFAPHEITQAISATEYNRFLASVIIGFLVVLSNWGLDVGGTITKVLVATRPILFLIVTNITIVFTLLMENKDPNVRGRPAGSNLGSADNLGQMLEIGLLLQKALSTLLIDCSVCAVIMICFI.

[0015] The present invention also discloses a rice plant with OsGET2 gene loss of function, which is obtained by the above-described method for regulating the growth period of rice.

[0016] This invention also discloses the application of a rice plant with OsGET2 gene loss of function in early-maturing rice breeding.

[0017] Compared with existing technologies, the application of the rice OsGET2 gene provided by this invention in regulating early maturity of rice has the following beneficial effects:

[0018] (1) This invention knocks out the OsGET2 gene using gene editing technology, demonstrating that the OsGET2 gene has the function of regulating the heading and growth stages of rice, providing new gene resources for the molecular regulatory network of rice growth stages.

[0019] (2) By knocking out the OsGET2 gene, this invention can significantly promote early heading and shorten the growth period of rice, while the thousand-grain weight does not decrease significantly or even increases slightly. This effectively solves the contradiction between shortened growth period and reduced yield in early-maturing breeding, achieving early maturity without reduced yield.

[0020] (3) The OsGET2 gene encodes a protein sequence that is basically consistent across rice varieties, and it is expected to promote early maturity in most rice varieties, making it widely applicable.

[0021] (4) This invention provides important theoretical basis and gene resources for the targeted improvement of rice growth period and the cultivation of new early-maturing rice varieties using gene editing technology. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0023] Figure 1 Genotype diagrams of OsGET2 in gene-edited rice and wild-type ZH11 rice provided in embodiments of the present invention;

[0024] Figure 2 Phenotypic diagrams at maturity of control group rice ZH11 and OsGET2 gene-edited rice (CR-OsGET2#1 and CR-OsGET2#2) provided in embodiments of the present invention;

[0025] Figure 3 The image shows the DNA level identification results of transgenic OsGET2 rice provided in this embodiment of the invention.

[0026] Figure 4 A bar chart showing the relative expression levels of OsGET2 in transgenic OsGET2 rice and wild-type rice, as provided in this embodiment of the invention.

[0027] Figure 5 Flowering and heading phenotypes of OsGET2 gene-edited rice, OsGET2 gene-transgenic rice, and wild-type rice provided in embodiments of the present invention;

[0028] Figure 6 A statistical result diagram of the heading time of OsGET2 gene-edited rice, OsGET2 gene-transgenic rice, and wild-type rice provided in the embodiments of the present invention;

[0029] Figure 7 The figure shows the statistical results of the thousand-grain weight of OsGET2 gene-edited rice, OsGET2 gene-transgenic rice, and wild-type rice provided in the embodiments of the present invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0031] Terminology Explanation:

[0032] Rice: Rice (Oryza sativa L.), commonly known as paddy rice, is an annual aquatic herbaceous plant of the Poaceae family (perennial rice varieties also exist). The culms are erect, and mature plants are generally 0.5–1.5 meters tall, varying depending on the variety. The leaf sheaths are generally covered with epidermal hairs; the ligule is lanceolate; the leaf blade is linear-lanceolate, about 1–3 cm wide, and rough. The panicle is large and loosely spreading; each spikelet contains one mature floret; the mature floret, from the outside in, consists of a pair of glumes, lemma, palea, two stigmas, six stamens, and one pistil; the glumes are very small, leaving only a crescent-shaped scar at the base of the spikelet pedicel, resembling a cone; the lemma of the lateral fertile florets is thick, with five veins, the midrib forming a ridge, and the surface covered with small, square, papillate projections, thick and papery, covered with fine hairs, awned or awnless; the palea is the same material as the lemma, with three veins and an acute apex; the stigmas are flattened; the stamens consist of prismatic anthers and filiform filaments; the pistil consists of a bottle-shaped ovary and a feathery stigma. The caryopsis is about 5–12 mm long, about 3–4 mm wide, and 2–3 mm thick, with the embryo about 1 / 4 the length of the caryopsis.

[0033] Heading: Heading is the growth stage in cereal crops (rice, wheat, corn, etc.) where the fully developed young panicle emerges from the sheath of the flag leaf. The standard for judgment is the emergence of the middle of the young panicle from the flag leaf sheath. The initial heading stage occurs when 10% of the plants in the field are heading, the heading stage when 50% are heading, and the full heading stage when 80% are heading. This stage directly affects the number of grains filled and is sensitive to external conditions. Rice heading generally lasts 7–10 days, with the main panicle emerging first, followed by the tillers.

[0034] Early maturity: In crop science, this specifically refers to a crop that takes significantly less time to complete its entire life cycle (from emergence to seed maturity) than the average time for that variety population.

[0035] Example 1:

[0036] Cloning and sequence analysis of the OsGET2 gene:

[0037] (1) Extraction of total RNA from rice and synthesis of cDNA:

[0038] Total RNA was extracted from the rice variety Zhonghua 11 (ZH11). The specific steps are as follows: Young leaf tissue of rice ZH11 was collected, ground with liquid nitrogen, and total RNA was extracted using the Trizol method. Using the extracted total RNA as a template, first-strand cDNA was synthesized by reverse transcription using a reverse transcription kit (such as the TaKaRa PrimeScript™ RT Reagent Kit).

[0039] (2) PCR amplification of the OsGET2 gene:

[0040] Using the cDNA obtained in step (1) as a template, PCR amplification was performed using primers OsGET2-F and OsGET2-R. The primer sequences are shown in Table 1:

[0041] Table 1. Primer sequences for OsGET2 gene amplification

[0042]

[0043] The nucleotide sequences of the primers OsGET2-F and OsGET2-R are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0044] The PCR reaction system was a standard system, and the PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 35 cycles; and a final extension at 72℃ for 10 minutes. After PCR product detection by 1.0% agarose gel electrophoresis, the target fragment was recovered and purified using a gel extraction kit.

[0045] (3) Sequencing and sequence analysis of PCR products:

[0046] The PCR product obtained in step (2) was sequenced. The sequencing results showed that the PCR product contained a DNA fragment of 897 bp, the nucleotide sequence of which is shown in SEQ ID NO:1. This gene was named OsGET2 gene (gene number: Os01g0863400; LOC4324784).

[0047] The protein encoded by the OsGET2 gene is named OsGET2 protein, which consists of 298 amino acids, and its amino acid sequence is shown in SEQ ID NO:2.

[0048] SEQ ID NO:1 (OsGET2 gene nucleotide sequence):

[0049] ATGGAGAGCGGCGCGAACGCACGGCGGCGGCGGCGGCTGGTGGATAGGGGATCCGATCGCCTCGCCTTCATCACCGGCCAAGCTCAGAGCCTCCCCTCCGATCCGTCCCCAGATTCACCACTTTATACTGTTGATGCTGCTTCCCTTCAGCGATCTGAACGTCAGCTAAATGAAGTTGGCATTGGTGATGACATATTCAACTACATAACTCAGTTGCAAAAATCAGAATCTGGTGTTCAACCGACTAGTGAGGCCCACCTGGAAACTCATAAAGAGAAACACCAAGGTAATGAAAGTGACCTCCAAAAGCTCAAGACAAGCAATGTGGTTCCTGAAATTCAGCCAGTAAATGAAAAGGCATTTCAAAGGCACAGTGAGGAAACCCTCAGGAAGAAGAACAGTCATGACAGATCGGCAAGTACACAGCCTATGAGGGAAATGGAAACAAGATCCAGATATGTGCCTCCAAATCAGTCTAATCAATCTGATAGTGCAGGTTGGTCTGTGGAAACTCTGAAGGAAATCCTGAATTTCGCACCACATGAGATTACCCAGGCCATATCAGCTACAGAGTATAACCGATTTTTGGCTTCCGTCATCATTGGTTTTCTGGTGGTTCTTTCAAATTGGGGGCTGGACGTTGGTGGCACTATCACCAAAGTATTAGTTGCTACAAGGCCAATTCTATTTCTTATTGTAACGAACATAACCATTGTCTTCACTTTGTTGATGGAGAACAAGGACCCCAACGTGAGGGGTAGACCAGCTGGCAGCAACCTTGGTTCTGCTGATAACTTAGGCCAAATGCTAGAGATTGGGTTGCTGCTACAGAAAGCCCTGAGCACCCTCCTGATAGACTGCAGCGTTTGCGCTGTCATCATGATCTGTTTTATTTGA.

[0050] SEQ ID NO: 2 (Amino acid sequence of OsGET2 protein):

[0051] MESGANARRRRRRLVDRGSDRLAFITGQAQSLPSDPSPDSPLYTVDAASLQRSERQLNEVGIGDDIFNYITQLQKSESGVQPTSEAHLETHKEKHQGNESDLQKLKTSNVVPEIQPVNEKAFQRHSEETLRKKNSHDRSASTQPMREMET RSRYVPPNQSNQSDSAGWSVETLKEILNFAPHEITQAISATEYNRFLASVIIGFLVVLSNWGLDVGGTITKVLVATRPILFLIVTNITIVFTLLMENKDPNVRGRPAGSNLGSADNLGQMLEIGLLLQKALSTLLIDCSVCAVIMICFI.

[0052] Example 2:

[0053] Obtaining and identifying OsGET2 gene-edited rice:

[0054] (1) Obtaining OsGET2 gene-edited rice:

[0055] Using the nucleotide sequence of the rice OsGET2 gene (SEQ ID NO:1) as a target, a specific target was designed using the CRISPR / Cas9 target design tool, and an sgRNA oligonucleotide chain was synthesized. The sgRNA was cloned into a CRISPR / Cas9 plant expression vector to obtain a recombinant vector.

[0056] The recombinant vector described above was transformed into the rice variety ZH11 using Agrobacterium-mediated transformation. Specifically, the recombinant vector was transformed into Agrobacterium strain (such as EHA105) to infect the callus tissue of rice ZH11. After co-culture and screening on a selection medium, resistant callus tissue was obtained, which was then differentiated and regenerated to obtain T0 generation transgenic plants.

[0057] Total DNA was extracted from the leaves of T0 generation plants. Using the extracted DNA as a template, PCR amplification was performed using specific primers OsGET2-CRISPR-F and OsGET2-CRISPR-R. The PCR products were sequenced to identify whether the target sequence had been edited. Positive T0 generation plants were self-crossed, and after multiple generations of propagation and selection, homozygous OsGET2 gene loss-of-function mutant plants were finally obtained, named CR-OsGET2#1 and CR-OsGET2#2, respectively.

[0058] In addition, OsGET2 gene-edited rice can be obtained by purchasing seeds of the mutant with the ZH11 background from germplasm resource banks and conducting multi-generation propagation and screening.

[0059] (2) Genotyping of OsGET2 gene-edited rice:

[0060] Based on the DNA fragment near the gene-editing target site in rice, a pair of specific primers, OsGET2-CRISPR-F and OsGET2-CRISPR-R, were designed. The primer sequences are shown in Table 2.

[0061] Table 2 Primer sequences for identifying OsGET2 gene-edited rice

[0062]

[0063] The nucleotide sequences of the primers OsGET2-CRISPR-F and OsGET2-CRISPR-R are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0064] Total DNA was extracted from leaves of each gene-edited rice line (CR-OsGET2#1 and CR-OsGET2#2) and the wild-type control rice ZH11. Using the extracted total DNA as a template, PCR amplification was performed using primers OsGET2-CRISPR-F and OsGET2-CRISPR-R. The PCR products were sequenced to identify the genotypes of the OsGET2 gene-edited rice. The genotypes of OsGET2 in gene-edited rice and wild-type ZH11 rice are as follows: Figure 1 As shown.

[0065] The target sequence results of each gene-edited line were compared with the target sequence of wild-type ZH11. The results showed that the target sequence of the CR-OsGET2#1 line was missing one base, and the target sequence of the CR-OsGET2#2 line was missing two bases "AG" compared with the wild type. Both lines showed base deletion mutations in their target sequences, indicating that the OsGET2 gene in CR-OsGET2#1 and CR-OsGET2#2 was effectively edited, resulting in the loss of function of the OsGET2 gene.

[0066] (3) Analysis of the early-maturing phenotype of OsGET2 gene-edited rice:

[0067] The plant materials used were as follows: control group rice ZH11, and gene-edited rice CR-OsGET2#1 and CR-OsGET2#2.

[0068] Photographs were taken of control rice ZH11 and gene-edited rice CR-OsGET2#1 and CR-OsGET2#2 at the mid-maturity stage in a field in Zhengzhou, Henan Province, for observation. Figure 2 ).

[0069] The results are as follows Figure 2As shown, CR-OsGET2#1 and CR-OsGET2#2 matured significantly earlier than the wild type, indicating that OsGET2 deficiency can promote early maturity in rice.

[0070] Example 3:

[0071] Obtaining and identifying OsGET2 transgenic rice:

[0072] (1) Obtaining transgenic OsGET2 rice:

[0073] The intermediate vector carrying the correctly sequenced OsGET2 gene was given to Wuhan Boyuan Biotechnology Co., Ltd., who were commissioned to construct an overexpression vector and perform genetic transformation on rice. The recipient rice plant was the rice variety ZH11. The T1 generation OsGET2 transgenic rice was finally obtained and named OsGET2-OE1 and OsGET2-OE2, respectively.

[0074] (2) Genotyping of transgenic rice:

[0075] Genotyping of transgenic rice identified as generation T1 was performed. Based on the sequence of the inserted fragment in the transgenic rice, two pairs of primers, OsGET2-OE-F and OsGET2-OE-R, were designed. The primer sequences are shown in Table 3.

[0076] Table 3. Primer sequences for identifying OsGET2 transgenic rice

[0077]

[0078] The nucleotide sequences of the primers OsGET2-OE-F and OsGET2-OE-R are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0079] Total DNA was extracted from leaves of each transgenic rice line (OsGET2-OE1 and OsGET2-OE2) and the wild-type control rice ZH11. Using the extracted total DNA as a template, PCR amplification was performed using primers OsGET2-OE-F and OsGET2-OE-R. The PCR amplification products were detected by agarose gel electrophoresis to identify the genotype of each OsGET2 transgenic rice line. Wild-type rice (WT) was used as a negative control.

[0080] The results are as follows Figure 3 As shown, both OsGET2 gene transgenic rice lines, OsGET2-OE1 and OsGET2-OE2, can amplify the target fragment on the inserted fragment, while the wild-type control has no corresponding band. This indicates that the OsGET2 gene overexpression element has been successfully inserted into OsGET2-OE1 and OsGET2-OE2, and the transgenic plants have been successfully constructed.

[0081] (3) Analysis of OsGET2 gene expression in transgenic rice:

[0082] The expression level of OsGET2 in the two transgenic rice lines, OsGET2-OE1 and OsGET2-OE2, which were identified as positive, was detected. The specific steps are as follows: Total RNA was extracted from the leaves of the two transgenic rice lines and the wild-type control rice ZH11, and the extracted total RNA was reverse transcribed into cDNA. Using the cDNA obtained from reverse transcription as a template, real-time quantitative PCR (qPCR) experiments were performed using primers RT-OsGET2-F and RT-OsGET2-R, and primers OsUbi-F and OsUbi-R (OsUbi gene was used as an internal reference gene). The primer sequences are shown in Table 4.

[0083] Table 4 Primer Sequences for Real-Time Quantitative PCR

[0084]

[0085] The nucleotide sequences of the primers RT-OsGET2-F, RT-OsGET2-R, OsUbi-F and OsUbi-R are shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0086] The real-time quantitative PCR reaction system was the standard SYBR Green system, and the reaction conditions were: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds; 60℃ annealing for 30 seconds, for a total of 40 cycles. (-ΔΔCT) The relative expression level was calculated. Using the expression level of the OsGET2 gene in wild-type rice (WT) as a baseline, the relative expression levels of the OsGET2 gene in each transgenic rice line were compared.

[0087] The results are as follows Figure 4 As shown, with the expression level of the OsGET2 gene in wild-type rice (WT) as a baseline, the expression levels of the OsGET2 gene in the transgenic OsGET2-OE1 and OsGET2-OE2 rice lines were significantly higher than those in the wild type, indicating that the OsGET2 gene has been successfully overexpressed in the overexpression lines. Subsequent experiments were conducted using the aforementioned OsGET2 gene-overexpressing rice lines.

[0088] Example 4:

[0089] Analysis of heading phenotype in OsGET2 gene-edited materials and transgenic OsGET2 rice:

[0090] The plant materials are as follows: wild-type rice ZH11, OsGET2 gene-edited rice CR-OsGET2#1 and CR-OsGET2#2, and positive T3 generation transgenic OsGET2 rice OsGET2-OE1 and OsGET2-OE2.

[0091] Wild-type rice ZH11, transgenic OsGET2 rice plants OsGET2-OE1 and OsGET2-OE2, and OsGET2 gene-edited plants CR-OsGET2#1 and CR-OsGET2#2, all grown in open fields in Hainan Province, were selected. The heading phenotype of each line was observed at the heading stage, and the heading time of each line was recorded in the field. The emergence of the flag leaf sheath from the middle of the young panicle was used as the criterion for determining heading. At least 10 plants were investigated for each line, and the date when 50% of the plants began to head was recorded; this date is considered the heading stage of that line.

[0092] Flowering and heading phenotypes such as Figure 5 As shown in the figure, the statistical results of heading time are as follows: Figure 6 And as shown in Table 5. From Figure 5 and Figure 6 It can be seen that the heading time of CR-OsGET2#1 and CR-OsGET2#2 is significantly earlier than that of the wild type, and the growth period is significantly shorter than that of the wild type, showing an early-maturing phenotype; while the heading time of the overexpression lines OsGET2-OE1 and OsGET2-OE2 is later than that of the wild type.

[0093] Table 5. Statistical results of heading time for each line

[0094]

[0095] As shown in Table 5, the heading days of CR-OsGET2#1 and CR-OsGET2#2 were 70.5 days and 71.7 days, respectively, which were shortened by 8 days and 6.8 days, respectively, compared with the wild type (78.5 days), and the differences were highly significant (P < 0.0001); while the heading days of the overexpression lines OsGET2-OE1 and OsGET2-OE2 were 83.7 days and 84.1 days, respectively, which were delayed by 5.2 days and 5.6 days, respectively, compared with the wild type.

[0096] The above results indicate that OsGET2 negatively regulates the heading date of rice. Loss of OsGET2 gene function significantly promotes earlier heading and shortens the growth period by 7–8 days; while overexpression of OsGET2 delays heading and prolongs the growth period. This demonstrates that OsGET2 can affect the rice growth period, and gene editing to knock out OsGET2 can lead to earlier maturity in rice.

[0097] Example 5:

[0098] Analysis of yield traits in OsGET2 gene-edited materials and transgenic OsGET2 rice:

[0099] The plant materials are as follows: wild-type rice ZH11, OsGET2 gene-edited rice CR-OsGET2#1 and CR-OsGET2#2, and positive T3 generation transgenic OsGET2 rice OsGET2-OE1 and OsGET2-OE2.

[0100] Wild-type rice ZH11, OsGET2 gene-transgenic rice plants OsGET2-OE1 and OsGET2-OE2, and OsGET2 gene-edited plants CR-OsGET2#1 and CR-OsGET2#2, all grown in Hainan field, were selected. Seeds were harvested after each line matured, and the thousand-grain weight was calculated. Thousand-grain weight is an important indicator of rice grain plumpness and yield, and can effectively reflect differences in yield traits among different lines. The specific method was as follows: 1000 plump seeds were randomly selected from each line and weighed using a 0.01% balance. The weight was then converted to the thousand-grain weight (i.e., the weight of 1000 seeds, in grams). Each line was repeated 5 times, with 1000 plump seeds randomly selected and weighed again in each repetition. The average of the 5 repetitions was taken as the thousand-grain weight result for that line. The thousand-grain weight statistics are as follows: Figure 7 As shown in Table 6.

[0101] Table 6 Statistical results of thousand-grain weight for each strain

[0102]

[0103] from Figure 7 As shown in Table 6, the thousand-grain weights of CR-OsGET2#1 and CR-OsGET2#2 were 23.996 g and 25.362 g, respectively, both higher than the wild type (22.242 g); while the thousand-grain weights of the overexpression materials OsGET2-OE1 and OsGET2-OE2 were 21.948 g and 21.168 g, respectively, slightly lower than the wild type. The difference between CR-OsGET2#2 and the wild type was P = 0.0128 (< 0.05); the differences between the other lines and the wild type were not significant (P > 0.05). These results indicate that the deletion of the OsGET2 gene does not lead to reduced rice yield, and knocking out the OsGET2 gene can maintain yield while promoting early maturity. This discovery is of great significance for the breeding of early-maturing rice varieties, effectively resolving the contradiction between shortened growth period and reduced yield in early-maturing breeding, and providing important theoretical basis and genetic resources for creating new early-maturing, high-yielding and stable-yielding rice varieties using OsGET2 gene editing.

[0104] Example 6:

[0105] Application of the OsGET2 gene in other rice varieties:

[0106] Rice varieties are abundant, and different varieties exhibit significant differences in genetic background, ecological adaptability, and agronomic traits. Currently identified genes regulating heading time have often had their functions validated only in a few varieties, and their application in other varieties remains uncertain. The recipient rice material used in this invention is the japonica rice variety Zhonghua 11 (ZH11).

[0107] Given that the OsGET2 gene encodes a largely consistent protein sequence across rice varieties, it is reasonable to expect that knocking out the OsGET2 gene could promote early maturity and heading in most rice varieties. In practical applications, gene editing technologies such as CRISPR / Cas9 can be used to design sgRNAs targeting specific sites of the OsGET2 gene in different rice varieties. Through Agrobacterium-mediated transformation or gene gun transformation, mutant plants lacking OsGET2 gene function can be obtained, thereby improving the early maturity of different rice varieties. The specific steps are the same as described in Example 2, requiring only the design of specific targets within the conserved regions of the OsGET2 gene sequence for different rice varieties. The OsGET2 gene sequences of different rice varieties can be obtained by searching public databases (such as NCBI) or by amplifying the genomic sequence of the OsGET2 gene from the variety using homologous cloning methods. Then, corresponding CRISPR / Cas9 editing targets can be designed based on the specific sequence information of the variety. Using the above method, OsGET2 gene editing technology can be extended to rice varieties of different ecotypes, such as indica rice varieties, japonica rice varieties, or hybrid rice parent materials, to achieve early maturity improvement of rice varieties under different genetic backgrounds. Therefore, this invention is not only applicable to the rice variety ZH11, but can also be widely applied to the early maturity breeding improvement of different rice varieties, demonstrating good versatility and application prospects.

[0108] Example 7:

[0109] Breeding applications of OsGET2 gene-editing materials:

[0110] The OsGET2 gene-edited rice CR-OsGET2#1 or CR-OsGET2#2 obtained in Example 2 were hybridized with superior rice varieties. By using molecular marker-assisted selection, the early-maturing superior allele of the OsGET2 gene was introduced into the background of superior varieties, thereby cultivating new rice varieties with early-maturing traits and excellent agronomic traits.

[0111] The specific breeding process is as follows:

[0112] Using OsGET2 gene-edited rice (CR-OsGET2#1 or CR-OsGET2#2) as the donor parent and rice varieties with excellent overall traits but longer growth periods as the recipient parent, artificial hybridization was performed to obtain F1 generation hybrid seeds. F1 generation plants were planted and self-pollinated to obtain a segregating F2 generation population. In the F2 generation population, the OsGET2 gene editing site segregated, with some individual plants carrying homozygous OsGET2 gene editing sites, some being heterozygous, and some being wild-type.

[0113] Genotyping of the F2 generation population was performed using specific molecular markers for the OsGET2 gene, such as OsGET2-CRISPR-F or OsGET2-CRISPR-R. Individual plants carrying the OsGET2 gene editing site were screened through PCR amplification and sequencing. Homozygous mutant plants (those with the expected OsGET2 gene editing on both homologous chromosomes) were preferentially selected from the F2 generation to ensure the stable inheritance of the early-maturing trait in offspring. The selected homozygous plants were backcrossed with the recipient parent (a superior rice variety) to obtain the BC1F1 generation. Genotyping of the backcross progeny was further performed using molecular markers, and individuals carrying the OsGET2 gene editing site and exhibiting agronomic traits similar to the recipient parent were screened. After multiple generations of backcrossing and self-pollination, combined with field phenotypic selection (including assessment of agronomic traits such as heading date, plant height, number of grains per panicle, thousand-grain weight, and seed setting rate), a stable new rice variety with early maturity and excellent agronomic traits was finally obtained.

[0114] During the backcrossing process, molecular markers were used to track and identify the OsGET2 gene editing site in each generation to ensure that the target gene site was not lost. At the same time, field surveys of agronomic traits were conducted to evaluate the overall performance of each individual plant, and plants with agronomic traits closest to the recipient parent and significant early maturity were selected for the next generation of backcrossing or self-pollination. After 4 to 6 generations of backcrossing or self-pollination, near-isogenic lines with a genetic background highly consistent with the recipient parent (the genome recovery rate of the recurrent parent can reach over 95%) and carrying only the OsGET2 gene editing site can be obtained. These materials retain the high-yield, high-quality, and other comprehensive traits of excellent varieties, and also acquire the characteristic of early maturity and heading.

[0115] Using the above breeding method, the early-maturing trait conferred by OsGET2 gene editing can be efficiently introduced into different superior rice varieties, accelerating the breeding process of new early-maturing rice varieties and providing efficient technical means and important genetic resources for the cultivation of early-maturing rice varieties. This method combines the advantages of gene editing technology and traditional hybridization breeding, and has good operability and wide applicability.

[0116] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The application of rice OsGET2 protein or the gene encoding said rice OsGET2 protein in regulating rice growth period or breeding early-maturing rice varieties, characterized in that... The amino acid sequence of the OsGET2 protein is shown in SEQ ID NO:2, and the nucleotide sequence of the gene encoding the OsGET2 protein is shown in SEQ ID NO:

1. The application is to regulate the growth period of rice and promote early maturity and heading by knocking out the OsGET2 gene in rice. The knockout of the OsGET2 gene in rice is achieved by CRISPR / Cas9 gene editing technology.

2. A method for regulating the growth period of rice, characterized in that, The method includes: knocking out the OsGET2 gene in rice to regulate the rice growth period and promote early maturity and heading of rice; the nucleotide sequence of the OsGET2 gene is shown in SEQ ID NO:

1.

3. The method for regulating the growth period of rice according to claim 2, characterized in that, The OsGET2 gene knockout was achieved using CRISPR / Cas9 gene editing technology.

4. A rice plant with loss of function of the OsGET2 gene, characterized in that, The plant was obtained by a method for regulating the growth period of rice as described in claim 2 or 3.

5. The application of the OsGET2 gene-deficient rice plant as described in claim 4 in early-maturing rice breeding.

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