Rice osdne1 protein and its coding gene in regulating plant growth and development

Abstract

The present application belongs to the field of plant genetic engineering, and relates to a rice OsDNE1 protein, a coding gene thereof and application. The protein and the coding gene thereof can increase plant height, increase tiller number, increase seed number, increase seed length and / or increase yield. The protein and the coding gene thereof can be used for increasing rice yield and improving appearance quality of rice, have important significance in rice breeding, and can provide important biological resources for increasing rice yield and improving grain type.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to the application of rice OsDNE1 protein and its encoding gene in regulating plant growth and development. Background Technology

[0002] Rice is one of the world's most important food crops, with more than half of the global population relying on it as a primary food source. Given the increasing population and decreasing arable land, high yield remains a crucial agricultural objective, and increasing rice production is of significant positive importance for ensuring my country's food security. The main components of rice yield include grain weight, effective tiller number, and grains per panicle, with the number of grains per panicle determined by the number of primary and secondary branches. Increasing the number of effective tillers and grains per panicle can effectively improve rice yield. Therefore, studying yield-related traits such as grains per panicle and tiller number is of great significance for high-yield rice breeding. Furthermore, quality is also a major goal in rice breeding, with grain shape being an important appearance quality trait. Grain shape includes grain length, width, thickness, and length-to-width ratio, which directly affect grain weight and thus rice yield. Therefore, identifying genes related to grain shape regulation is helpful in breeding high-yielding and high-quality rice varieties. In recent years, significant progress has been made in the molecular genetics and functional genomics of rice, utilizing modern molecular genetic theories and methods to uncover new genes regulating rice yield traits and elucidate their mechanisms of action. Rice has thus become an important model plant for molecular biology research on monocotyledonous development. Gene editing technologies, such as CRISPR / Cas9, can generate precise mutations in the genome and have been widely applied to site-specific editing of plant genes, providing revolutionary tools for functional research of plant genes and crop genetic improvement. Summary of the Invention

[0003] The purpose of this invention is to provide an application of the OsDNE1 protein derived from rice and its protein-coding gene in regulating plant growth and development. The protein and its encoding gene described in this invention can be used to increase rice yield and improve the appearance quality of rice, which is of great significance in rice breeding and can provide important biological resources for improving rice yield and grain shape.

[0004] The technical solution provided by this invention is as follows:

[0005] A rice OsDNE1 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The amino acid sequence of the OsDNE1 protein (SEQ ID No. 1)

[0007] MARRQQQKRSSRPHLLPVTAAEGVAVSASQIWLEGMWPVASELVVEAVARLLQWWALFSLSLLHCRQLPSPPLPWAVVASTAVSSGPAGSGRHDNGGGRWPDPPPLLTGAAPR.

[0008] This invention also provides a rice OsDNE1 gene, which encodes the aforementioned rice OsDNE1 protein, and the nucleotide sequence of the rice OsDNE1 gene is shown in SEQ ID NO. 15. (SEQ ID No. 15)

[0009] ATGGCGCGGCGACAACAGCAGAAGAGGTCCTCGCGGCCTCACCTCCTGCCAGTGACAGCAGCGGAAGGGGTAGCGGTCGGCTTCCCAGATATGGCTGGAGGGGATGTGGCCAGTGGCTAGTGAGCTCGTCGTGGAGGCGGTCGCGCGACTGCTCCAGTGGTGGGCTCTC TTCTCCCTCTCCCTCCTCCACTGCCGCCAGCTGCCATCGCCACCACTGCCATGGGCTGTCGTCGCCTCTACCGCCGTGAGCTCGGGGCCGGCCGGATCCGGCCGCCACGACAACGGGGGTGGCCGGTGGCCGGATCCACCACCGCTGCTGACGGGGGCAGCGCCGCGATGA.

[0010] The present invention also provides expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing the above-mentioned genes.

[0011] The present invention also provides the application of the above-mentioned proteins or genes in regulating plant growth and development.

[0012] Furthermore, the regulation of plant growth and development includes increasing plant height, increasing the number of tillers, increasing the number of seeds, increasing seed length, and / or increasing yield.

[0013] The present invention also provides a method for cultivating transgenic rice, the method comprising increasing the content and / or activity of the protein in the target plant, wherein the resulting transgenic rice has a taller plant height and / or an increased number of tillers and / or an increased number of seeds and / or a larger seed length and / or a higher yield compared with the target rice.

[0014] Furthermore, using CRISPR / Cas9 gene editing technology, a recombinant plasmid pCas9-OsDNE1 was constructed and introduced into the target rice to obtain transgenic rice.

[0015] Furthermore, the recombinant plasmid pCas9-OsDNE expresses the gene for Cas9 protein and the gene for sgRNA, and the target sequence of sgRNA is shown in the sequence listing as SEQ ID No. 2.

[0016] sgRNA target sequence (SEQ ID No.2): CCTCCTGCCAGTGACAGCAG.

[0017] Furthermore, single-stranded DNA molecules OsDNE1-pCas9-F and OsDNE1-pCas9-R were synthesized, with the sequence of OsDNE1-pCas9-F shown in SEQ ID No. 11 and the sequence of OsDNE1-pCas9-R shown in SEQ ID No. 12.

[0018] The present invention also provides primers for the above-mentioned rice OsDNE1 gene, as shown in SEQ ID No. 3-4.

[0019] In embodiments of the present invention, the plant is specifically rice, a grass of the Poaceae family. The growth and development are reflected in plant height and / or the number of tillers and / or the number and / or seed morphology.

[0020] Another object of the present invention is to provide a method for cultivating transgenic rice with increased plant height and / or increased number of tillers and / or increased number of seeds and / or larger seed length and / or increased yield.

[0021] The method includes the following steps: using CRISPR / Cas9 gene editing technology, constructing a recombinant plasmid pCas9-OsDNE1, introducing it into the target rice to obtain transgenic rice; compared with the target rice, the transgenic rice has a taller plant and / or a greater number of tillers and / or a greater number of seeds and / or a larger seed length and / or a higher yield.

[0022] The protein provided by this invention is obtained from rice and named OsDNE1 protein, which is as follows (a) or (b) or (c).

[0023] (a) A protein consisting of the amino acid sequence shown in SEQ ID No. 1;

[0024] (b) A protein derived from sequence 1 with one or more amino acid residues substituted and / or added and / or deleted, relating to plant height and / or tiller number and / or seed number and / or seed morphology.

[0025] (c) Proteins derived from SEQ ID No. 1 that are related to plant growth and development, by substitution and / or addition and / or deletion of one or more amino acid residues of the amino acid sequence of SEQ ID No. 1.

[0026] The transgenic rice is a transgenic rice that has undergone site-directed mutation of the OsDNE1 protein-coding gene using CRISPR / Cas9 gene editing technology.

[0027] In an embodiment of the present invention, the rice is specifically the rice variety Zhonghua 11.

[0028] Beneficial effects

[0029] This invention utilizes CRISPR / Cas9 gene editing to site-directedly mutate the OsDNE1 protein-encoding gene, thereby regulating rice plant height and / or tiller number and / or seed number and / or seed morphology. Compared to wild-type controls, transgenic rice plants exhibit increased plant height, tillering, grains per panicle, grain length, and yield. Therefore, this invention can be used to increase rice yield and improve rice appearance quality, holding significant importance in rice breeding and providing crucial biological resources for improving rice yield and grain shape. Attached Figure Description

[0030] Figure 1 This is a diagram showing the expression analysis of the OsDNE1 protein-coding gene of the present invention in different tissues of japonica rice Zhonghua 11.

[0031] Figure 2 Subcellular localization analysis of the OsDNE1 protein for the present invention.

[0032] Figure 3 This invention provides an in vitro expression analysis of the OsDNE1 protein. Figure 3 A shows the OsDNE1-GST protein isolated and purified by SDS-PAGE, photographed after Coomassie blue staining. Figure 3 B represents the purified OsDNE1-GST protein as analyzed by Western blot. M indicates the protein marker; lane 1: GST protein control; lane 2: OsDNE1-GST recombinant protein.

[0033] Figure 4 The mutation sites and sequencing peaks of OsDNE1 in two homozygous knockout mutant lines (KO1 and KO2) are shown. Figure 4A sequencing peak diagram, Figure 4 B is a diagram of mutation sites.

[0034] Figure 5 Comparison of plant morphology characteristics between rice OsDNE1 mutant lines and wild-type Zhonghua 11. Figure 5 A and Figure 5 B shows the growth of the OsDNE1 mutant rice line and the wild-type Zhonghua 11. Figure 5 C is a section of the third intersegmental cell tissue. Figure 5 D shows a comparison of plant height between the OsDNE1 mutant rice line and the wild-type Zhonghua 11. Figure 5 E represents a comparison of internode length between the OsDNE1 mutant rice line and the wild-type Zhonghua 11. Figure 5 F represents a comparison of cell length between the OsDNE1 mutant rice line and the wild-type Zhonghua 11. Figure 5 G represents a comparison of the number of tillers in the OsDNE1 mutant rice line and the wild-type Zhonghua 11.

[0035] Figure 6 This study compares the panicle type and single-plant grain characteristics of the OsDNE1 mutant rice line with those of the wild-type Zhonghua 11. Figure 6 A is a comparison of the appearance of the main stem and spikelet. Figure 6 B shows a comparison of the entire plant's seeds. Figure 6 C represents the comparison of the number of primary branches. Figure 6 D represents the comparison of the number of secondary branches. Figure 6 E represents the number of grains per ear. Figure 6 F represents the yield comparison per plant.

[0036] Figure 7 Comparison of grain morphology characteristics between rice OsDNE1 mutant lines and wild-type Zhonghua 11. Figure 7 A shows a comparison of the appearance of the seeds. Figure 7 B represents a comparison of grain length. Figure 7 C represents the particle width comparison. Figure 7 D represents the comparison of grain thickness. Detailed Implementation

[0037] The technical solutions provided by the present invention will be described in detail below with reference to embodiments, but these embodiments are not limited to the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. All experimental materials, reagents, and carriers used in the following embodiments can be obtained commercially.

[0038] Example 1

[0039] OsDNE1 gene expression pattern analysis

[0040] The expression patterns of the OsDNE1 gene in different tissues of rice variety Zhonghua 11 were analyzed as follows: Using the RNA-easy Isolation Reagent kit (Vazyme), total RNA was extracted from leaves, leaf sheaths, roots, and stems at different developmental stages of the wild-type rice variety Zhonghua 11, including the tillering stage (leaves, leaf sheaths, roots, stems, and young panicles), the heading stage (leaves, leaf sheaths, roots, stems, and young panicles), and the mature stage (leaves, leaf sheaths, roots, stems, and panicles). Following the instructions of the cDNA synthesis kit (HiScript Q RTSuperMix for qPCR (Vazyme), cDNA was synthesized and used for tissue expression pattern analysis. The results are as follows: Figure 1 As shown.

[0041] Primers used for real-time quantitative PCR (qRT-PCR) analysis of the OsDNE1 gene were designed based on its full-length cDNA. The upstream primer sequence is SEQ ID No. 3, and the downstream primer sequence is SEQ ID No. 4. OsActin was used as an internal reference gene, with the upstream primer sequence being SEQ ID No. 5 and the downstream primer sequence being SEQ ID No. 6.

[0042] SEQ ID No.3: 5'-CAGTGGCTAGTGAGCTCGTC-3';

[0043] SEQ ID No.4: 5'-CGGTAGAGGCGACGACAG-3';

[0044] SEQ ID No.5: 5'-GATGACCCAGATCATCGTTTG-3';

[0045] SEQ ID No.6: 5'-GGGCGATGTAGGAAAGC-3';

[0046] The results are as follows Figure 1 The OsDNE1 gene is expressed in rice roots, stems, leaves, leaf sheaths, and panicles, with relatively high expression levels in young panicles and stems during the heading stage. OsActin is used as an internal reference gene, and the error bars represent the standard deviation of three biological replicates.

[0047] Example 2

[0048] OsDNE1 subcellular localization analysis

[0049] To investigate the subcellular localization of the OsDNE1 protein, a transient expression system in rice protoplasts was used. The coding sequence of the OsDNE1 gene was fused into the pUN1301-eGFP vector, and transient expression was performed in rice protoplasts via PEG-mediated expression. Both the pUN1301-eGFP and OsDNE1-eGFP vector plasmids were transformed into rice protoplasts for transient expression, and the expression was observed and photographed under a laser confocal microscope.

[0050] The upstream primer sequence for constructing the pUN1301-eGFP vector with OsDNE1 subcellular localization is SEQ ID No. 7, and the downstream primer sequence is SEQ ID No. 8.

[0051] SEQ ID No.7: 5'

[0052] -CAGGTCGACTCTAGAGGATCCATGGCGCGGCGACAACAG-3';

[0053] SEQ ID No. 8: 5'

[0054] -CTCGCCCTTGCTCACGGTACCTCGCGGCGCTGCCCCCGT-3';

[0055] The results are as follows Figure 2 The subcellular localization results of the OsDNE1 protein were similar to those of the empty vector control, showing localization in the nucleus, cytoplasm, and cell membrane. N represents the nucleus, PM represents the cell membrane, and CS represents the cytoplasm. The scale bar is 10 μm.

[0056] Example 3

[0057] In vitro expression analysis of OsDNE1 protein

[0058] To investigate the in vitro expression of OsDNE1 protein, we fused the coding sequence of the OsDNE1 gene to the pGEX-6p-1 vector with a GST tag. The recombinant plasmid OsDNE1-GST was transformed into *E. coli* strain BL21. Protein expression was induced for 12 h at 16°C with 1 mM IMPTG. The GST-tagged recombinant protein was purified using a glutathione agarose gel purification kit (Sangon). For Western blot analysis, the purified protein was separated by SDS-PAGE, transferred to a PVDF membrane, and detected after electrophoresis. The recombinant protein was detected using a GST antibody (Abcam).

[0059] The upstream primer sequence for the OsDNE1-GST vector used in the in vitro expression analysis of OsDNE1 is SEQ ID No. 9, and the downstream primer sequence is SEQ ID No. 10.

[0060] SEQ ID No. 9: 5'

[0061] -TTCCAGGGGGCCCCTGGGATCCATGGCGCGGCGACAACAG-3';

[0062] SEQ ID No. 10: 5'

[0063] -CTCGAGTCGACCCGGGAATTCTCATCGGCGGCGCTGCCCC-3';

[0064] The results are as follows Figure 3 The purified recombinant protein OsDNE1-GST produced two bands on an SDS-PAGE gel, one of which was consistent with its molecular weight, approximately 39 kDa. Western blot analysis further confirmed the molecular weight of the OsDNE8-GST fusion protein. This indicates that OsDNE1 has protein-coding capabilities and can be expressed in vitro.

[0065] Example 4

[0066] Site-directed mutation of the OsDNE1 gene

[0067] I. Construction of recombinant plasmids

[0068] 1. Based on the full-length cDNA sequence of the rice gene OsDNE1, primer fragments were designed using the CRISPR-P online website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR). The designed primer sequences, along with a portion of the sgRNA backbone sequence, were pasted into the RNAfold Web server (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) for RNA structure prediction. The appropriate sgRNA target sequence is shown in SEQ ID No. 2 of the sequence listing.

[0069] SEQ ID No. 2: 5'--3';TCCTGCCAGTGACAGCAG.

[0070] 2. Single-stranded DNA molecules OsDNE1-pCas9-F (SEQ ID No. 11) and OsDNE1-pCas9-R (SEQ ID No. 12) were synthesized separately. SEQ ID No. 11 and SEQ ID No. 12 primers were added to a 10 μL system, and annealing was performed using a PCR instrument according to the program: 94℃ for 10 min, 15℃ for 10 min.

[0071] SEQ ID No. 11: 5'

[0072] -AGATGATCCGTGGCATCCTGCCAGTGACAGCAGGTTTTAGAGCTATGC-3';

[0073] SEQ ID No.12:5'

[0074] -GCATAGCTCTAAAACCTGCTGTCACTGGCAGGATGCCACGGATCATCT-3';

[0075] 3. The pCas9 vector was digested with the restriction endonuclease AarI (purchased from ThermoFisher), and the linearized vector backbone was recovered.

[0076] 4. Using the In-Fusion kit (purchased from TaKaRa), the annealed product obtained in step 2 was recombined and ligated with the linearized vector backbone obtained in step 3 to obtain the recombinant plasmid pCas9-OsDNE1.

[0077] II. Obtaining homozygous mutant lines

[0078] The recombinant plasmid pCas9-OsDNE1 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium, which was then used to infect embryogenic callus tissue of the japonica rice variety Zhonghua 11. Plants were then cultured from this tissue. Transgenic plants were identified by PCR and sequencing as homozygous knockout mutants with the OsDNE1 mutation and no vector backbone.

[0079] Specific primers (SEQ ID No. 13 and SEQ ID No. 14) were designed upstream and downstream of the OsDNE1 mutation target site, respectively. After PCR amplification, sequencing was performed for identification. The mutation site and sequencing peak diagram are shown below. Figure 4 As shown, the two homozygous mutants obtained are homozygous mutant lines with a deletion of 1 base G and a deletion of 2 bases AG, respectively. Figure 4 A), whose encoded amino acid sequences all terminate prematurely ( Figure 4 B);

[0080] SEQ ID NO.13: 5'-TTCTCTCCGGCCCTTAACTG-3';

[0081] SEQ ID No.14: 5'-AGGAGGGAGAGGGAGAAGAG-3';

[0082] III. Analysis of the traits of mutant plants

[0083] 1. Plant type analysis of mutant plants

[0084] The growth of wild-type plants and two homozygous OsDNE1 mutant lines was observed, and the results are as follows: Figure 5 A (scale bar is 20cm), it was found that compared with wild-type plants, the mutant lines had a significantly increased plant height, with KO1 and KO2 increasing in height by 5.38% and 6.60%, respectively. Figure 5 D) Further analysis of the length between the first and fifth nodes of the stem yielded the following results: Figure 5 B (scale bar is 5cm), it was also found that the internode lengths of the OsDNE1 mutant lines were significantly longer than those of the wild type. Figure 5 E). To further investigate the reasons for the changes in stem internode length, this study observed cell tissue sections of the third internode. The results are as follows: Figure 5 C (scale bar is 200 μm), the cell length of the third intersegment in the mutant lines was significantly increased compared to the wild type. Figure 5 F). These results indicate that OsDNE1 controls stem internode length by regulating cell elongation, thereby affecting plant height. Furthermore, the number of tillers in the mutant lines was significantly increased compared to the wild type, with KO1 and KO2 showing increases of 30.77% and 29.49%, respectively. Figure 5 G).

[0085] 2. Analysis of spike type and yield per plant in mutant plants

[0086] When performing phenotypic identification on ear-related traits of OsDNE1 mutant plants, the results were as follows: Figure 6 A (scale bar is 5cm), it was found that compared with the wild type, the number of primary branches in the mutant plants did not change significantly, while the number of secondary branches increased significantly compared with the wild type, resulting in an increase of 21.26% and 16.11% in the number of grains per ear in the mutant lines KO1 and KO2, respectively. Figure 6 CE). Furthermore, when assessing the weight of the whole plant's seeds, the results were as follows: Figure 6 B (scale bar is 2.5cm), it was found that the yield per plant of the mutant lines KO1 and KO2 increased by 20.52% and 21.60% respectively compared with the wild-type plants. Figure 6F). Since the thousand-grain weight of the mutant plants did not change significantly compared to the wild-type plants, it indicates that the site-directed mutation of OsDNE1 mainly increases the yield per rice plant by increasing the number of grains per panicle and tillers.

[0087] 3. Granule shape analysis of mutant plants

[0088] When the morphology of the seeds from the OsDNE1 mutant plants was identified, the results were as follows: Figure 7 (Scale bar 1cm) Significant changes in grain shape were observed in the mutants. Compared to the wild type, the mutant grains became more elongated, specifically with a significant increase in grain length; the grain length of KO1 and KO2 increased by 4.08% and 3.84%, respectively; while the grain width decreased significantly; the grain width of KO1 and KO2 decreased by 6.10% and 6.75%, respectively. Figure 7 AC). However, the particle thickness of the mutant did not change significantly. Figure 7 D). This indicates that site-directed mutations in OsDNE1 can regulate grain morphology.

Claims

1. The application of knocking out the rice OsDNE1 gene in regulating rice growth and development, characterized in that, The nucleotide sequence of the rice OsDNE1 gene is shown in SEQ ID NO.15; the regulation of rice growth and development includes increasing rice plant height, increasing the number of rice tillers, increasing the number of grains per panicle, and increasing the length of rice grains.

2. A method for increasing rice plant height, characterized in that, The method includes knocking out the rice OsDNE1 gene to obtain transgenic plants with increased plant height; the nucleotide sequence of the rice OsDNE1 gene is shown in SEQ ID NO.

15.

3. A method for increasing the number of tillers in rice, characterized in that, The method includes knocking out the rice OsDNE1 gene to obtain transgenic plants with increased tillering; the nucleotide sequence of the rice OsDNE1 gene is shown in SEQ ID NO.

15.

4. A method for increasing the number of grains per panicle in rice, characterized in that, The method includes knocking out the rice OsDNE1 gene to obtain transgenic plants with more grains per panicle; the nucleotide sequence of the rice OsDNE1 gene is shown in SEQ ID NO.

15.

5. A method for increasing the length of rice grains, characterized in that, The method includes knocking out the rice OsDNE1 gene to obtain transgenic plants with increased rice grain length; the nucleotide sequence of the rice OsDNE1 gene is shown in SEQ ID NO.

15.

6. The method according to any one of claims 2 to 5, characterized in that, Using CRISPR / Cas9 gene editing technology, a recombinant plasmid pCas9-OsDNE1 was constructed and introduced into target rice to obtain transgenic rice. The recombinant plasmid pCas9-OsDNE1 expresses the genes for Cas9 protein and sgRNA.

7. The method according to claim 6, characterized in that, The target sequence of sgRNA is shown in SEQ ID No.

2.

8. The method according to claim 7, characterized in that, Single-stranded DNA molecules OsDNE1-pCas9-F and OsDNE1-pCas9-R were synthesized, respectively. The sequence of OsDNE1-pCas9-F is shown in SEQ ID No. 11, and the sequence of OsDNE1-pCas9-R is shown in SEQ ID No. 12.

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