Application of MYB22 gene in regulating rice flowering time during the day

Abstract

The application discloses application of a MYB22 gene in regulating daily flowering time of rice and belongs to the technical field of genetic engineering and molecular breeding. A nucleotide sequence of the MYB22 gene is shown as SEQ ID NO. 1. The application discloses the function of the MYB22 gene in regulating flowering time of rice. Experiments prove that knocking out the MYB22 gene can promote the daily flowering time of rice (especially japonica rice as a parent) to be advanced by about 0.5 hours, which is helpful to shorten the time interval of flowering of indica rice and japonica rice, thereby improving cross-pollination efficiency and seed production yield. The application lays a solid molecular foundation for cultivating hybridization parents with coordinated flowering time, reducing hybrid seed production cost and promoting industrial application of high-yield indica-japonica hybrid rice and has important breeding application value.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular breeding technology, and in particular to the application of the MYB22 gene in regulating the daytime flowering time of rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of the major food crops, and increasing its yield can ensure food security. Hybrid breeding is one of the most effective methods to increase rice yield, especially indica-japonica hybrid rice, which can increase yield by 30% compared to conventional indica-japonica hybrid rice. The mismatch in flowering time between subspecies parents severely restricts the seed production yield of indica-japonica hybrid rice, resulting in a much higher seed production cost for indica-japonica hybrid rice than for indica-japonica hybrid rice. This is one of the main factors hindering the promotion of indica-japonica hybrid rice. Therefore, shortening the flowering interval between indica and japonica rice is expected to significantly increase the seed production yield of indica-japonica hybrid rice, thereby reducing the cost of hybrid seed production.

[0003] MYB transcription factors play crucial roles in plant secondary metabolism, abiotic stress responses, and growth and development. Genome-wide analysis of the rice MYB transcription factor family revealed that 98.71% of MYB proteins are located in the nucleus, with the remainder located in mitochondria and the plasma membrane. The characteristic DNA-binding domain of the MYB protein family consists of four highly conserved repeat units (R1-R4), each containing approximately 52 amino acid residues, folding to form three α-helical structures. Between the second and third α-helices are three regularly spaced tryptophan / hydrophobic amino acids, forming a helical-turn-helical (HTH) structure. The hydrophobic core within this structure specifically recognizes and binds to target DNA sequences. Based on the number of conserved domains, these proteins can be divided into four subgroups: 1R, R2R3, 3R, and 4R. Each subgroup has its specific biological function. In plants, R2R3-MYBs are the most common type, performing functions including cell fate determination, abiotic stress responses, and regulation of secondary metabolite synthesis.

[0004] MYB family transcription factors also play important roles in the regulation of rice daytime flowering time. Knockout of OsMYB8 in indica rice TFB and japonica rice ZH11 using CRISPR / Cas9 technology delayed daytime flowering time by 1 h and 0.5 h, respectively. Further research showed that OsMYB8 can directly bind to and activate OsJAR1, a key enzyme gene involved in jasmonic acid synthesis, increasing jasmonic acid (JA-Ile) accumulation, promoting the expression of genes related to osmotic pressure and cell wall remodeling, causing swelling of the florets, and promoting floret opening. However, there are currently no reports on the regulatory role of MYB22 in rice daytime flowering time. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the MYB22 gene in regulating the daytime flowering time of rice, thereby addressing the problems existing in the prior art. This invention reveals the function of the MYB22 gene in regulating the flowering time of rice; its knockout can advance the flowering time of japonica rice by approximately 0.5 hours, effectively shortening the flowering time interval between indica and japonica subspecies. This provides a clear gene target and technical solution for creating parents with coordinated flowering periods through molecular breeding, increasing the yield of indica-japonica hybrid seed production, and reducing production costs, and has significant application value.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of knocking out the MYB22 gene in any of the following:

[0008] (1) Application in promoting earlier daytime flowering time of rice;

[0009] (2) Application in the cultivation of transgenic rice with earlier daytime flowering time;

[0010] The nucleotide sequence of the MYB22 gene is shown in SEQ ID NO.1.

[0011] This invention also provides the use of the protein encoded by the MYB22 gene in any of the following:

[0012] (1) Application in promoting earlier daytime flowering time of rice;

[0013] (2) Application in the cultivation of transgenic rice with earlier daytime flowering time.

[0014] This invention also provides the use of the recombinant vector for knocking out the MYB22 gene in any of the following:

[0015] (1) Application in promoting earlier daytime flowering time of rice;

[0016] (2) Application in the cultivation of transgenic rice with earlier daytime flowering time.

[0017] The present invention also provides the use of recombinant microorganisms containing the recombinant vector in any of the following:

[0018] (1) Application in promoting earlier daytime flowering time of rice;

[0019] (2) Application in the cultivation of transgenic rice with earlier daytime flowering time.

[0020] Furthermore, by using gene editing technology to mutate the MYB22 gene in rice, the function of the MYB22 gene expression protein is lost, thereby advancing the daytime flowering time of the rice.

[0021] Furthermore, the method for causing loss of function of the MYB22 gene expression protein includes deletion, insertion, or mutation of bases on the target sequence.

[0022] The present invention also provides a method for advancing the daytime flowering time of rice, comprising the following steps: editing the MYB22 gene in rice to render the MYB22 gene protein expression function lost, thereby advancing the daytime flowering time of rice; the nucleotide sequence of the MYB22 gene is shown in SEQ ID NO.1.

[0023] The present invention also provides a method for cultivating transgenic rice with advanced daytime flowering time, comprising the following steps: gene editing of the MYB22 gene in rice cells to render the MYB22 gene protein expression function lost; then cultivating the rice cells and using the rice cells to regenerate rice, thereby obtaining transgenic rice with advanced daytime flowering time; the nucleotide sequence of the MYB22 gene is shown in SEQ ID NO.1.

[0024] The present invention discloses the following technical effects:

[0025] This invention reveals the function of the MYB22 gene in regulating the daytime flowering time of rice, providing an effective solution to the key technical challenge of mismatched flowering times between parental lines in indica-japonica hybrid seed production. Experiments have shown that knocking out the MYB22 gene can advance the daytime flowering time of rice (especially japonica rice as the parent) by approximately 0.5 hours. This directly helps to shorten the time interval between the flowering of indica and japonica rice, thereby improving cross-pollination efficiency and seed production yield.

[0026] Compared to existing technologies, the application of this invention is clearly defined and yields significant results. By providing specific knockout targets for the MYB22 gene, vector construction methods, and application pathways, those skilled in the art can reliably create rice germplasm resources that flower earlier. This invention lays a solid molecular foundation for breeding hybrid parents with coordinated flowering periods, reducing the production cost of hybrid seeds, and promoting the industrial application of high-yield indica-japonica hybrid rice, and has significant breeding application value. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1This is a diagram illustrating the spatiotemporal expression pattern of the MYB22 gene in wild-type rice ZH11.

[0029] Figure 2 Figures showing the mutation status and daytime flowering time of the MYB22 gene knockout mutant; a: Sequencing results of the MYB22 gene knockout mutant; bc: Phenotypic observation of the MYB22 gene knockout mutant and the wild type, scale bar: 20 cm (b), 2 cm (c); d: Statistical graph of flowering of the MYB22 gene knockout mutant and the wild type;

[0030] Figure 3 Figures showing the changes in the size of pulp segments and surface cells of MYB22 gene knockout mutant and wild-type rice ZH11 before and after flowering; a: phenotypic diagram, scale bar: 2 mm; b: phenotypic diagram of dynamic changes in surface cells of pulp segments, scale bar: 50 µm; c: cell area statistics; d: phenotypic diagram of dynamic changes in pulp segments, scale bar: 1 mm; e: surface area statistics. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example

[0037] 1. Acquisition and analysis of the target gene sequence

[0038] The coding sequence (CDS) of the rice (Oryza sativa L.) MYB22 gene was obtained from the NCBI database. Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0039] SEQ ID NO.1:

[0040] ATGGGGAGGTCGCCATGCTGCGAGAAGGCGCACACGAACAAGGGGGCGTGGACGAAGGAGGAGGACCAGCGGCTGATCGCCTACATCAAGGCGCACGGCGAGGGTTGCTGGCGGTCGCTGCCCAAGGCGGCGGGGCTCCTCCGCTGCGGCAAGAGCTGCCGCCTCCGCTGGATGAACTACCTCCGCCCCGACCTCAAGCGCGGCAACTTCACCGACGACGACGACGAGCTCATCATCAAGCTCCACGCCCTTCTCGGCAACAAGTGGTCGTTGATTGCGGGGCAGCTGCCGGGGAGGACGGACAACGAGATCAAGAACTACTGGAACACGCACATCAAGCGCAAGCTCCTGAGCCGGGGCATCGACCCGCAGACGCACCGGCCGGTCAGCGCCGGGAGCAGCGCCGCCGCGGCGAGCGGGCTGACCACGACGGCCAGCACCGCCGCCTTTCCGTCCCTTGCGCCGGCGCCGCCGCCGCAGCAGCACAGGCTACACAACCCGGTGCACGCCGCGGCGCCGAGCAATGCGAGCTTCGCCAGGTCCGCGGCGTCCCCGCCGTCGGAGGACGGCCACAGCAGCAGCGGCGGCAGCTCGGACGCGCCGCGGTGCCCCGACCTCAACCTCGACCTCGACCTCGACCTGTCCATGAGCCTGCCGAGCTCGCCGCCCAAGACGCCGGCCGCCGCGTCGTCCACGACCGCGTCGCGCCACCATCACCACCAGCAGCAGAAGACCATCTGCCTCTGCTACCACCTCGGCGTCCGCAACGGCGACGTCTGCAGCTGCAAGGCGGCCGCGCCATCGCCGGCCGGCCCACGCGCGTTCCGGTTTCTCAGGCCACTGGAGGAGGGCCAGTACATATAG。

[0041] SEQ ID NO.2:

[0042] MGRSPCCEKAHTNKGAWTKEEDQRLIAYIKAHGEGCWRSLPKAAGLLRCGKSCRLRWMNYLRPDLKRGNFTDDDDELIIKLHALLGNKWSLIAGQLPGRTDNEIKNYWNTHIKRKLLSRGIDPQTHRPVSAGSSAAAASGLTTT ASTAAFSLAPAPPPQQHRLHNPVHAAAPSNASFARSAASPPSEDGHSSSGGSSDAPRCPDLNLDLDLSMSLPSSPPKTPAAASSTTASRHHHHQQQKTICLCYHLGVRNGDVCSCKAAAPSPAGPRAFRFLRPLEEGQYI.

[0043] 2. Spatiotemporal expression pattern of the MYB22 gene

[0044] To investigate the expression pattern of the MYB22 gene, the root, stem, leaf, and pulp tissues of the japonica rice variety 'Zhonghua 11' (ZH11) were collected.

[0045] RNA was extracted from different tissues of wild-type rice ZH11 using Trizol reagent (Invitrogen). Reverse transcription was performed using the HiScript III first-strand cDNA synthesis kit (Vazyme, Nanjing) to obtain cDNA. RT-qPCR was performed using the 2×RealStar Green Fast Mixture kit (Genstar, Beijing), following the manufacturer's instructions. Each experiment was repeated three times. Actin was used as a control to normalize the cDNA amount. The results were obtained according to BioRad's "Quantitative PCR Application Guide". -ΔCt The data is processed by calculating the relative expression level of the target gene.

[0046] The primers for real-time PCR are as follows:

[0047] QPCR-MYB22-CDS-F: CCTCCGCTGGATGAACTACC, SEQ ID NO.3;

[0048] QPCR-MYB22-CDS-R:GATCTCGTTGTCCGTCCTCC, SEQ ID NO.4.

[0049] The quantitative PCR system (20 µL) is shown in Table 1.

[0050] Table 1 RT-qPCR reaction system

[0051]

[0052] The quantitative PCR reaction program was as follows: pre-denaturation at 95℃ for 3 min, 40 cycles (95℃ for 10 s, 60℃ for 10 s, 72℃ for 15 s), and extension at 60℃ for 6 s.

[0053] The results are as follows Figure 1 As shown, the MYB22 gene was most highly expressed in the stalks of wild-type rice ZH11, followed by high expression in the leaves, suggesting that MYB22 may play a key role in regulating the development of flowering-related organs such as stalks.

[0054] 3. Construction of knockout vectors and identification and phenotypic observation of transformed seedlings

[0055] (1) Target sequence design

[0056] Two specific sgRNA targets, namely target 1 (MYB22-U3) and target 2 (MYB22-U6a), were designed using an online design website (http: / / skl.scau.edu.cn / primerdesign / ) targeting conserved sequences in the coding region of the MYB22 gene. The target sequences are as follows:

[0057] MYB22-U3: TCAAGCGCAAGCTCCTGAGCGGG (SEQ ID NO.5);

[0058] MYB22-U6a: CAGCACAGGCTACACAACCAGG (SEQ ID NO. 6).

[0059] (2) Construction of pYL-pU3-gRNA and pYL-pU6a-gRNA vectors containing MYB22-U3 and U6a fragments

[0060] Synthesize target primers with BsaI-cleaved sticky ends, as follows:

[0061] OsU3T1F: ggcaTCAAGCGCAAGCTCCTGAGC, SEQ ID NO.7;

[0062] OsU3T1R: aaacGCTCAGGAGCTTGCGCTTGA, SEQ ID NO.8;

[0063] OsU6aT2F: gccgCAGCACAGGCTACACAACC, SEQ ID NO.9;

[0064] OsU6aT2R: aaacGGTTGTGTAGCCTGTGCTG, SEQ ID NO. 10.

[0065] The target adapter was heated at 90℃ for 30 s in a PCR instrument, and then cooled to room temperature to complete the annealing step. Simultaneously, approximately 1 μg of each pYL-U3 / U6a-gRNA plasmid was digested with enzymes. The digestion conditions for pYL-U3-gRNA and pYL-U6a-gRNA plasmids were 37℃ for 15 min and 70℃ for 5 min. The digestion system is shown in Table 2.

[0066] Table 2 Enzyme digestion system

[0067]

[0068] The enzyme digestion vectors were ligated with their corresponding adapters at 16℃ for 20 min. The ligation reaction system is shown in Table 3.

[0069] Table 3 Connection Reaction System

[0070]

[0071] (3) Construction of pCRISPR / Cas9 vector containing MYB22 fragment

[0072] The ligated vector (sgRNA expression cassette with adapter target sequence) from the previous step was subjected to the first round of PCR amplification. The amplification primers are as follows:

[0073] UF: ctccgttttacctgtggaatcg, SEQ ID NO.11;

[0074] gR-R: cggaggaaaattccatccac, SEQ ID NO. 12.

[0075] The PCR amplification reaction system (15 µL) is shown in Table 4.

[0076] Table 4 PCR amplification reaction system

[0077]

[0078] PCR amplification reaction program: pre-denaturation at 95 ℃ for 1 min, 28 PCR cycles (95 ℃ for 10 s, 58 ℃ for 15 s, 72 ℃ for 20 s), extension at 72 ℃ for 1 min.

[0079] The amplification products from the first round were diluted 10-fold for a second round of PCR amplification. The amplification primers are as follows:

[0080] Pps-L: TTCAGAGGTCTCTCTCGCCCGGGCCCAATCGGCAGCAAAGGA, SEQ ID NO.13;

[0081] Pgs-2: AGCGTGGGTCTCGTCAGTCCATCCACTCCAAGCTC, SEQ ID NO.14;

[0082] Pps-2: TTCAGAGGTCTCTCTGAGGCCCAATCGGCAGCAAAGGA, SEQ ID NO.15;

[0083] Pgs-R: AGTGTAGGTCTCTACCGTCCCGGGTCCATCCACTCCAAGCT, SEQ ID NO. 16.

[0084] The PCR amplification reaction system (25 µL) is shown in Table 5.

[0085] Table 5 PCR amplification reaction system

[0086]

[0087] PCR amplification reaction program: pre-denaturation at 95 ℃ for 1 min, 28 PCR cycles (95 ℃ for 10 s, 58 ℃ for 15 s, 72 ℃ for 20 s), extension at 72 ℃ for 1 min.

[0088] The product was recovered and purified, and ligated into the pCRISPR / Cas9 vector containing the Cas9 expression cassette using a digest-and-ligate method with T4 DNA ligase and BsaI restriction endonuclease. The product was then transformed into *E. coli*, single colonies were picked and cultured, and plasmids were extracted.

[0089] The cut-and-connect system (15 µL) is shown in Table 6.

[0090] Table 6. Edge-cutting and edge-connecting system

[0091]

[0092] Ligation procedure: Enzyme digestion and ligation reaction was performed using a PCR instrument: 37 ℃ for 10 min; 10-12 cycles (37 ℃ for 5 min, 10 ℃ for 3 min, 20 ℃ for 5 min); 37 ℃ for 3 min.

[0093] (4) Obtaining transgenic seedlings

[0094] The pCRISPR / Cas9 vector plasmid containing the target was sent to a biotechnology company for genetic transformation of wild-type rice ZH11. After obtaining the transformed seedlings, they were planted in the Qilinbei experimental field of South China Agricultural University, and the transgenic plants were identified as positive for hygromycin resistance.

[0095] (5) Identification of mutation status

[0096] DNA was extracted from positive plants and amplified using the following primers: cas-myb22-F: ACGGATTTCAATGCGCTGTC (SEQ ID NO. 17), cas-myb22-R: TCCTCCAGTGGCCTGAGAAA (SEQ ID NO. 18). The amplified DNA was then sent to the company for sequencing. Sequence alignment analysis showed that homozygous mutant lines with deletion or insertion mutations in the MYB22 target region were successfully obtained. Figure 2 (a) Two independent homozygous mutant lines were selected and named myb22-1 and myb22-2, respectively, for subsequent phenotypic analysis.

[0097] (6) Phenotypic observation of mutant plants

[0098] The two independent homozygous mutant lines (myb22-1 and myb22-2) obtained above, as well as the wild-type ZH11, were planted in the experimental field of South China Agricultural University (where the average summer temperature in Guangzhou is 28-30℃).

[0099] A. Observation of daytime flowering time: Under natural field conditions, the daytime flowering time of wild-type ZH11, myb22-1, and myb22-2 was observed. The results showed that the daytime flowering time of the two myb22 gene knockout mutants was approximately 0.5 h earlier than that of wild-type ZH11. Figure 2 (bd).

[0100] B. Cytological analysis of floret development: To explore the cytological basis of early flowering, samples were taken from the spikelets of wild-type ZH11 1.5 hours before flowering, 0.5 hours before flowering, at flowering, and 1 hour after flowering, and microscopic observation and image analysis were performed. Figure 3 (a).

[0101] Cell area of ​​sclerotia: 1.5 hours before flowering of ZH11 spikelets, the cell area of ​​sclerotia surface of myb22-1 and myb22-2 was significantly larger than that of ZH11. 0.5 hours before flowering of ZH11, the cell area of ​​sclerotia surface of myb22-1 and myb22-2 further increased. At the time of flowering of ZH11, the cell area of ​​sclerotia surface of myb22-1 and myb22-2 began to decrease. Figure 3 As shown in bc.

[0102] Plate area: Consistent with changes in cell area, 1.5 h before flowering of ZH11 spikelets, the plate areas of myb22-1 and myb22-2 were significantly larger than those of ZH11. 0.5 h before flowering of ZH11, the plate areas of myb22-1 and myb22-2 further increased. During flowering of ZH11, the plate areas of myb22-1 and myb22-2 began to decrease, showing no significant difference from ZH11. Figure 3 As shown in the middle.

[0103] These results from the above embodiments demonstrate that by knocking out the MYB22 gene in wild-type rice ZH11, the daytime flowering time of the myb22 gene knockout mutant is approximately 0.5 h earlier than that of wild-type ZH11. Therefore, by knocking out the MYB22 gene, it is possible to breed rice varieties with earlier daytime flowering, which helps to solve the problem of parental flowering time mismatch in rice hybridization breeding, increase seed production yield, and reduce hybrid seed production costs.

[0104] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of MYB22 gene knockout in any of the following: (1) Application in promoting earlier daytime flowering time of rice; (2) Application in the cultivation of transgenic rice with earlier daytime flowering time; in, The nucleotide sequence of the MYB22 gene is shown in SEQ ID NO.

1.

2. The use of the recombinant vector for knocking out the MYB22 gene as described in claim 1 in any of the following: (1) Application in promoting earlier daytime flowering time of rice; (2) Application in the cultivation of transgenic rice with earlier daytime flowering time.

3. The use of recombinant microorganisms containing the recombinant vector as described in claim 2 in any of the following: (1) Application in promoting earlier daytime flowering time of rice; (2) Application in the cultivation of transgenic rice with earlier daytime flowering time.

4. The application as described in any one of claims 1-3, characterized in that, By mutating the MYB22 gene in rice using gene editing technology, the function of the MYB22 gene expression protein is lost, thereby advancing the daytime flowering time of the rice.

5. The application as described in claim 4, characterized in that, The methods for causing loss of function of the MYB22 gene expression protein include deletion, insertion, or mutation of bases on the target sequence.

6. A method for advancing the daytime flowering time of rice, characterized in that, Includes the following steps: By editing the MYB22 gene in rice, the function of the protein expressed by the MYB22 gene is lost, thereby promoting the earlier daytime flowering time of the rice; the nucleotide sequence of the MYB22 gene is shown in SEQ ID NO.

1.

7. A method for cultivating transgenic rice with advanced daytime flowering time, characterized in that, The method includes the following steps: gene editing of the MYB22 gene in rice cells to render the MYB22 gene protein expression function lost; then culturing the rice cells and using the rice cells to regenerate rice, thus obtaining transgenic rice with advanced daytime flowering time; the nucleotide sequence of the MYB22 gene is shown in SEQ ID NO.1.

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