Application of rice OsSAP18 gene in regulating leaf angle traits of rice

Abstract

This invention discloses the application of the rice OsSAP18 gene in regulating the leaf angle trait in rice. The OsSAP18 gene has the nucleotide sequence shown in SEQ ID NO.1. Increasing the expression level of the rice OsSAP18 gene can reduce the leaf angle of rice, resulting in a more compact rice plant architecture. This invention clones the OsSAP18 gene, constructs knockout and overexpression vectors, obtains mutants and overexpression materials, and measures the traits of these materials. The results show that the leaf angle of the mutant material is increased, while the leaf angle of the overexpression material is decreased, and the yield per plant of the overexpression material is increased in the field. This invention demonstrates the important function of the OsSAP18 gene in rice plant architecture, especially in regulating the leaf angle. The gene of this invention provides a key gene resource for breeding superior rice varieties suitable for dense planting, and has significant application value and industrialization potential in the field of crop genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the application of the rice OsSAP18 gene in regulating the rice leaf angle trait. Specifically, it relates to the rice histone deacetylation complex component OsSAP18 gene and its genetic engineering applications, such as its function and application in regulating the rice leaf angle. Background Technology

[0002] The leaf angle in rice refers to the angle between the leaf and the stem, and it is a key agronomical trait determining the canopy structure of rice. The size of the leaf angle directly affects the plant's ability to capture light and the canopy's ventilation and light penetration: a smaller leaf angle is suitable for modern high-density planting. Under high-density planting conditions, upright leaves can better capture light within the canopy, thereby improving photosynthetic efficiency, ventilation, and stress resistance, ultimately increasing grain yield (Cao et al. 2022). Therefore, the leaf angle is an important trait of great interest in breeding ideal rice plant architecture. Upright leaves are one of the key traits for ensuring high and stable yields in high-density crop cultivation. Identifying the genes regulating the leaf angle and elucidating their functional mechanisms provides a basis and genetic resources for breeding superior varieties suitable for high-density planting.

[0003] The leaf angle is influenced by various plant hormones, including brassinolide, auxin, and gibberellin. Brassinolide, as a key node signal, affects both its biosynthesis and signal transduction in rice leaf angle. Examples include OsBRI1, OsBAK1, OsGSK1, and OsBZR1 (Bai et al., 2007; Hong et al., 2003, 2005; Li et al., 2009; Mori et al., 2002; Nakamura et al., 2006; Sakamoto et al., 2006; Tanabe et al., 2005; Yamamuro et al., 2000; Zhang et al., 2016). Auxin biosynthesis and signal transduction pathways also play an important regulatory role in rice leaf angle. For example, OsFIBs, OsIAA1, OsGH3, OsTIR1 and OsAFB2 (Bian et al., 2012; Du et al., 2012; Song et al., 2009; Yoshikawa et al., 2014).

[0004] Although the above studies identified several genes regulating leaf angle, there are few reports on the function of histone deacetylation complex components in this process, and it remains to be discovered whether there are other key components regulating leaf angle in rice.

[0005] Therefore, discovering and identifying new genes that regulate leaf angle and further enriching the genetic resources for rice plant architecture improvement is of great theoretical and applied value for breeding high-yielding rice varieties suitable for dense planting. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of the rice OsSAP18 gene in regulating the rice leaf angle trait.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides the application of the rice OsSAP18 gene in regulating the rice leaf angle trait, wherein the nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0009] Preferably, the application specifically involves: increasing the expression level of the rice OsSAP18 gene to reduce the leaf angle of rice, resulting in a compact rice plant type; or knocking out the rice OsSAP18 gene to increase the leaf angle of rice, resulting in a spreading rice plant type.

[0010] As a preferred method, the method for increasing the expression level of the rice OsSAP18 gene includes: constructing an overexpression vector containing the OsSAP18 gene and transforming it into rice; the overexpression vector is constructed by: amplifying the OsSAP18 gene using rice cDNA as a template, and performing homologous recombination ligation of the amplification product with the pCambia1300-35s-FLAG vector digested with XbaI and SalI.

[0011] Preferably, the knockout is performed using the CRISPR-Cas9 method, with the target sequence being AGGGCCGCCGATGCAGTACCGGG, and the editing type including the deletion of a base A or the addition of a base T.

[0012] Preferably, the OsSAP18 gene regulates the leaf angle of rice through the brassinolide signaling pathway.

[0013] Secondly, the present invention provides the application of the rice OsSAP18 gene in regulating rice yield, wherein the nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0014] Preferably, the application specifically involves: increasing the expression level of the rice OsSAP18 gene to increase the yield per plant, or knocking out or inhibiting the expression level of the rice OsSAP18 gene to reduce the yield per plant.

[0015] Thirdly, the present invention provides a reagent for detecting the expression level of the rice OsSAP18 gene in the screening of rice leaf angle traits or in rice breeding, wherein the nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1 and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0016] Fourthly, the present invention provides the application of the rice OsSAP18 gene in the creation of transgenic rice with large or small leaf angles, wherein the nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1 and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0017] Fifthly, the present invention provides a method for regulating the leaf angle of rice or improving the plant architecture of rice, including regulating the expression level of the OsSAP18 gene in rice, wherein the nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention reveals for the first time the biological function of the rice histone deacetylation complex component OsSAP18 gene in regulating leaf angle. The OsSAP18 gene was created using CRISPR-Cas9 technology, resulting in two mutant materials with different editing types. Compared with wild-type rice, the mutant materials showed a significantly increased leaf angle and decreased yield; while the overexpressed materials showed a significantly decreased leaf angle and increased yield compared with wild-type rice, providing a new gene target for rice plant architecture improvement.

[0020] The genetic evidence provided by this invention indicates that the OsSAP18 gene participates in the fine regulation of rice leaf angle through the brassinolide signaling pathway. The discovery of this gene provides a novel gene resource with independent intellectual property rights for rice plant architecture improvement. It can be applied to the breeding of high-yielding rice varieties with moderate or small leaf angles suitable for dense planting, and has significant application value and industrialization prospects in the fields of crop genetic improvement and molecular breeding. Attached Figure Description

[0021] Figure 1 Obtained from the OsSAP18 mutant.

[0022] Figure 2 : Plasmid map of overexpression lines.

[0023] Figure 3 Molecular detection of OsSAP18 transcription and protein expression levels in overexpression lines.

[0024] Figure 4Phenotypic and statistical data of leaf angle at the heading stage of OsSAP18 mutant and overexpression material.

[0025] Figure 5 Statistical analysis of plant type and leaf angle phenotype of OsSAP18 mutant and overexpression material at maturity.

[0026] Figure 6 The yield of single plants of OsSAP18 mutant and overexpression material.

[0027] Figure 7 The OsSAP18 mutant exhibits reduced leaf angle sensitivity to BL. Detailed Implementation

[0028] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] As mentioned earlier, through long-term research, the inventors discovered that a mutant of OsSAP18, a component of a histone deacetylation complex in rice, exhibits a phenotype of increased leaf angle, while overexpression results in decreased leaf angle. Field surveys of leaf angle phenotypes showed that the leaf angle phenotype at maturity could be stably reproduced. Further treatment of leaf angle with brassinolide analogs confirmed that OsSAP18 is indeed involved in the brassinolide-regulated leaf angle pathway in rice.

[0030] In this invention, the nucleotide sequence of the OsSAP18 gene is shown in SEQ ID NO.1 below:

[0031] ATGGCGGGAAGGGGCGAGATGCCGATGAGGCCTGTGCGGCCAGGGCCGCCGATGCAGTACCGGGGCCTCCTCCCATGGCTCGCGCGCGTCGAACCCGTCGATCGCGAAAGACGTGCCCTCTCCTGCTCCGGGTTTTTCACAAAGGTTGGTGGCCATCACCAAAATGAAGAGTTTGCCGTGAGAGGAAAGGAGCCGAAAGATGAAGTGCAGATATACACGTGGAAGG ATGCCACACTCCGAGAGCTCACTGATCTTGTGAAAGAGGTTGCTCTTGCAGCAAGAAAAAGAAATGCAAGGCTTTCTTTTGCTTTCGTGTACCCTGATAAGCATGGTCGTTCGTTGTCAAAGAGGTGGGTTCAACCTTCTCTTATGGCCATGGAAGGGGAGATGATGCTAAAACTCTTGCAGAGCTAGGATTCCAGATAGGGGATTACTTGAGGTTGTGCAATCTATTGA

[0032] The amino acid sequence of the protein encoded by the OsSAP18 gene is shown in SEQ ID NO.2 below:

[0033] MAGRGEMPMRPVRPGPPMQYRGPPPMARVEPVDREKTCPLLLRVFTKVGGHHQNEEFAVRGKEPKDEVQIYTWKDATLRELTDLVKEVALAARKRNARLSFAFVYPDKHGRFVVKEVGSTFSYGHGRGDDAKTLAELGFQIGDYLSVAIY

[0034] The present invention will be further described below.

[0035] Example 1: Obtaining the OsSAP18 mutant in rice

[0036] like Figure 1As shown, a suitable target site was selected at the first exon position of the gene OsSAP18 (Os02g0122000). The target sequence is AGGGCCGCCGATGCAGTACCGGG. Based on the target, the corresponding sgRNA was designed and synthesized. The forward primer for the target was synthesized by Youkang Biotechnology Co., Ltd.: CACCGAGGGCCGCCGATGCAGTACC; the reverse primer was synthesized by AAACGGTACTGCATCGGCGGCCCT. First, the dimer was formed by annealing at 95℃ for 10 min and then at 55℃ for 10 min, allowing the two single strands to pair complementaryly to form a double-stranded DNA fragment. Then, the sgRNA was ligated to the pC1300-Cas9 backbone vector, which had been linearized by BsaI digestion. The successfully constructed plasmid was then transformed into rice by Weimi Biotechnology Co., Ltd. Progeny sequencing identified two mutants with different editing types: a mutant lacking one base A was named sap18-1, and a mutant with one base T was named sap18-2.

[0037] Example 2 Obtaining OsSAP18 overexpression material

[0038] Using the cDNA of Zhonghua 11 as a template, primers with recombinant arms were designed for amplification. The primer sequences are as follows:

[0039] Primer 1: gagagaacacgggggactctagaATGGCGGGAAGGGGC

[0040] Primer 2:ttgtagtccatgtcgacATAGATTGCAACACTCAAGTAATCCCC

[0041] Amplification was performed using KOD enzyme. The PCR program was as follows: 94℃ for 2 min pre-denaturation, 98℃ for 10 s denaturation, and 68℃ for annealing and extension for 4 min. The obtained PCR products were then detected by electrophoresis on a 1% agarose gel. After electrophoresis, the target band was recovered under UV excision gel extraction and purified using a gel extraction kit. The pCambia1300-35S-FLAG vector was double-digested with XbaI and SalI, followed by agarose gel electrophoresis and purification using a gel extraction kit. Homologous recombination was then performed with the amplified product, followed by ligation and transformation into *E. coli*. After screening with KNA's antibiotic plate and sequencing confirmation, the correctly sequenced plasmid was sent to Weimi Biotechnology Co., Ltd. for rice transformation.

[0042] The constructed plasmid map is as follows Figure 2 As shown.

[0043] Example 3: Expression levels and protein identification of overexpression materials

[0044] After harvesting the seeds of the transgenic positive seedlings, they were sown in hydroponic boxes. Ten days after the seedlings matured, leaves were harvested, flash-frozen in liquid nitrogen, and ground. After grinding, 1 mL of TRIzol (Invitrogen) was quickly added, and the mixture was vortexed until the liquid was clear. Chloroform was then added, and the mixture was inverted and mixed thoroughly before centrifugation at low temperature. 300 µL of the supernatant was then gently transferred to a new 1.5 mL centrifuge tube, and 300 µL of isopropanol was added. The mixture was gently inverted and mixed thoroughly, then placed at -20°C for 2 hours. After centrifugation for 10 minutes, the supernatant was discarded, revealing a white precipitate. 1 mL of 70% ethanol was then added to wash the precipitate. The ethanol precipitate was dried in a clean bench until it became translucent, and then 50 µL of sterile water was added to dissolve the precipitate. The dissolved RNA was quality tested, and the concentration was measured using NanoDrop after passing quality control. 2 μg of RNA was reverse-transcribed in a 20 μL system using a TransGen reverse transcription kit: 1 μL of oligo DT was added, and the mixture was incubated at 65°C for 5 min, then immediately placed on ice. 2×TS Reaction Mix, TransScript RT / RI Enzyme Mix, and gDNA Remover were then added, followed by reverse transcription on a PCR instrument. The program was: 42°C for 15 min, 85°C for 5 s. The cDNA obtained after reverse transcription was diluted 10-fold and added to a 20 μL system with SYBR, primers, and template. Data were obtained using a Bio-Rad quantitative PCR instrument. The specific reaction program was: pre-denaturation 95°C for 3 min, denaturation 95°C for 10 s, annealing 60°C for 30 s, extension 72°C for 30 s, and melting curve analysis: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s. After the reaction, the expression level of the rice housekeeping gene OsUBQ was calculated using it as an internal reference. Lines with higher expression levels were named OsSAP18-OE-1 and OsSAP18-OE-2.

[0045] After cooling the mortar with liquid nitrogen, the leaves collected above were ground. After the sample was ground sufficiently, 0.2 g of the sample was weighed into a 2 mL low-adsorption centrifuge tube, and 600 μL of protein extraction solution was quickly added. The mixture was then vortexed on ice and centrifuged. The supernatant was taken as the total protein extracted.

[0046] The total protein was analyzed by Western blotting, including the following steps:

[0047] The separating and stacking gels were prepared using separating and stacking gel buffers from Kangwei Reagents Co., Ltd., with a separation gel concentration of 10% and a stacking gel concentration of 5%. First, a 5 mL separating gel was prepared. The liquid was poured between two glass plates, and 2 mL of alcohol was added to flatten the surface. After waiting 30 minutes, the top layer of alcohol was discarded, and the stacking gel preparation began. After the stacking gel was prepared, a comb of appropriate size was inserted between the glass plates. After approximately 30 minutes, the comb was removed, and sample loading and electrophoresis began. After adding the sample to the wells, a new electrophoresis buffer was added between the two plates, and the recovered electrophoresis buffer was added to the outer layer. Electrophoresis was started at 90 V. Once the sample entered the separating gel, the voltage was increased to 120 V, and electrophoresis continued until the bromophenol blue reached the bottom edge of the glass plates. The bands on the protein gel were then transferred to a PVDF membrane using a semi-dry transfer method, specifically the sandwich method, following a filter paper-membrane-filter paper sequence. After transfer, the PVDF membrane was blocked by immersing it in 5% milk. The flag antibody was diluted 1:1000 and incubated with primary antibody at room temperature for 1 hour. The membrane was then washed with TBST, changing the solution every 5 minutes for a total of three washes, followed by secondary antibody incubation. After secondary antibody incubation, the membrane was washed three times with TBST. Finally, development was performed using Femto ECL peroxide A / B developing solution from Novizan.

[0048] The results are as follows Figure 3 As shown.

[0049] Example 4: Phenotypic identification of heading stage mutants and overexpression materials

[0050] The obtained rice mutants and overexpression materials were planted in the field. At the heading stage, the main panicle flag leaves of no less than 10 materials were randomly collected and photographed. The angle was statistically analyzed using the Wanshen LA-S series plant image analysis system with the leaf pulvinus as the intersection point. Figure 4 The OsSAP18 mutant was found to have a significantly increased leaf angle compared to the wild type, while the overexpression material showed a significantly decreased leaf angle.

[0051] Example 5: Plant type observation and leaf angle statistics of mature mutants and overexpression materials

[0052] After grain filling is completed, the overall plant type of the mutant and the overexpression material will be observed. Figure 5 The study found that the mutants exhibited a more spread-out plant type, while the overexpressing mutants showed a relatively upright plant type. In situ angle measurements using a protractor revealed that the leaf angles of the mutants were significantly increased, while those of the overexpressing mutants were significantly decreased.

[0053] Example 6: Yield per plant of OsSAP18 mutant and overexpressing plants.

[0054] After the field-grown materials are harvested, threshed, dried, and their yield per plant is measured. Figure 6 The average yield per wild-type plant was 16 g, while the average yield per mutant plant was 3 g, a significant decrease of 80% compared to the wild type. The average yield per overexpression line was 19 g, a significant increase of 17% compared to the wild type. For each statistical line (25 plants), one-way ANOVA combined with Tukey multiple comparisons was used for significance analysis, with a p-value <0.05 considered statistically significant.

[0055] Example 7: The OsSAP18 mutant exhibits reduced sensitivity to exogenous BR.

[0056] The response of detached leaf pulvinus angle to exogenous BR was observed. The pulvinus angle of the second leaf from the inverted leaf of the ZH11, Ossap18 mutant cultured normally for seven days was cut off and placed in 0, 50 nM, and 100 nM BL solutions at 28°C in the dark for 24 hours. Afterward, the leaves were removed, photographed, and the angle was statistically analyzed using the Wanshen LA-S series plant image analysis system. The detached leaf pulvinus suspension treatment method followed that of Guo et al. (Guo et al., 2021). Nine to ten biological replicates were placed in each culture dish. Figure 7 As shown in the figure, observation and statistics revealed that BL treatment can significantly increase the leaf angle of rice, and the mutants are less sensitive to BL treatment.

[0057] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be included in the appended claims.

Claims

1. The application of the rice OsSAP18 gene in regulating the rice leaf angle trait, characterized by, The nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The specific applications are: increasing the expression level of the rice OsSAP18 gene to reduce the leaf angle of rice, resulting in a compact rice plant; and knocking out the rice OsSAP18 gene to increase the leaf angle of rice, resulting in a spreading rice plant.

3. The application according to claim 2, characterized in that, The method for increasing the expression level of the rice OsSAP18 gene includes: constructing an overexpression vector containing the OsSAP18 gene and transforming it into rice; the overexpression vector is constructed by: amplifying the OsSAP18 gene using rice cDNA as a template, and then performing homologous recombination ligation between the amplification product and the pCambia1300-35s-FLAG vector digested with XbaI and SalI.

4. The application according to claim 2, characterized in that, The knockout was performed using the CRISPR-Cas9 method, with the target sequence being AGGGCCGCCGATGCAGTACCGGG. The editing type included either deleting an A base or adding a T base.

5. The application according to claim 1, characterized in that, The OsSAP18 gene regulates the leaf angle in rice through the brassinolide signaling pathway.

6. The application of the rice OsSAP18 gene in regulating rice yield, characterized in that... The nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

7. The application according to claim 6, characterized in that, The specific applications are: increasing the expression level of the rice OsSAP18 gene to increase the yield per plant, and knocking out or inhibiting the expression level of the rice OsSAP18 gene to reduce the yield per plant.

8. The application of a reagent for detecting the expression level of the rice OsSAP18 gene in the screening of rice leaf angle traits or in rice breeding, characterized in that... The nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

9. The application of the rice OsSAP18 gene in the creation of transgenic rice with large or small leaf angles, characterized in that... The nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

10. A method for regulating the leaf angle of rice or improving the plant architecture of rice, characterized in that, This includes regulating the expression level of the OsSAP18 gene in rice, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

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