OsEra2 gene and method for cultivating and regulating flowering period of rice by using OsEra2 gene

Abstract

The invention relates to the technical field of gene engineering, in particular to an OsEra2 gene and a method for cultivating and regulating the flowering period of rice by using the OsEra2 gene. The invention provides an OsEra2 gene, wherein the nucleotide sequence of the OsEra2 gene is as shown in SEQ ID NO: 1. Experiments prove that the OsEra2 gene in wild type rice is knocked down, and transgenic rice with phenotype change in the heading stage can be obtained. Compared with receptor rice, the expression of the OsEra2 gene in transgenic rice is reduced. The OsEra2 gene is related to rice heading stage control, and a theoretical foundation is laid for cultivating transgenic plants suitable for the heading stage.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to an OsEra2 gene and a method for cultivating and regulating the flowering period of rice. Background Art

[0002] Plant flowering represents the transition of plants from vegetative growth to reproductive growth, and plays an important role in completing offspring reproduction and adapting to specific ecological environments. The heading date of rice is a key trait index determining rice yield, and heading at an appropriate time is of great significance for rice to complete its growth cycle. Currently, the research on the heading date of rice focuses on the photoperiod and circadian clock pathways, hormone signal pathways, and chromatin regulation pathways. In the photoperiod and circadian clock pathways, plants receive light signals from the external environment through photoreceptors and then transmit them to the circadian clock system for integration of light signals, ultimately affecting downstream specific signal pathways, and further regulating the growth and development process of plants, including the flowering process of plants. In the hormone signal pathway, it has been found that abscisic acid and others participate in the control of the heading date of rice. In the process of regulating the heading date of rice involving chromatin, it has been found that epigenetic modifications such as H3K4me3 on histones affect the heading date of rice, and other types of epigenetic modification factors are being discovered one after another. However, there is no report on the research of RNA epigenetic modification regulating the heading date of rice.

[0003] The RNA m6A modification involved in the present invention belongs to RNA methylation modification. Specifically, it refers to a very conservative chemical modification type formed after the hydrogen atom (H) at the N6 position of RNA is replaced by a methyl group (CH3). The rice heading date regulator OsEra2 involved in the present invention belongs to an RNA methylation removal enzyme, which is responsible for removing the methyl group (CH3) that has been m6A-modified and restoring it to a hydrogen atom (H). The present invention discovers that after the OsEra2 gene is knocked out by CRISPR gene editing, compared with the receptor rice Zhonghua 11 (ZH11), it is observed that both lines of the OsEra2 knockout mutants show an early flowering phenotype during the heading date, proving that the OsEra2 gene plays an important role in mediating RNA m6A modification and then responding to the control of the rice heading date. Subsequent research on the OsEra2 gene and the m6A modification of its downstream regulatory genes will be of great significance for m6A modification to regulate the rice heading date. Summary of the Invention

[0004] The purpose of the present invention is to provide an OsEra2 gene and a method for cultivating and regulating the flowering period of rice.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides an OsEra2 gene, and the nucleotide sequence of the OsEra2 gene is shown as SEQ ID NO: 1.

[0007] The present invention also provides the application of the OsEra2 gene in regulating the heading stage of rice.

[0008] Preferably, knocking out or knocking down the OsEra2 gene results in rice with an early heading phenotype.

[0009] The present invention also provides an expression vector, which includes an initial vector and the OsEra2 gene described above.

[0010] The present invention also provides a host, which is transformed or transfected with the expression vector described above; the host is a microorganism.

[0011] The present invention also provides the application of the expression vector or the host in regulating the heading stage of rice.

[0012] The present invention also provides a silencing vector, which includes a primitive vector and the OsEra2 gene described above.

[0013] The present invention also provides a silencing recombinant bacterium, which is transformed or transfected with the silencing vector.

[0014] The present invention also provides the application of the silencing vector or the silencing recombinant bacterium in regulating the heading stage of rice.

[0015] The present invention also provides a method for cultivating rice with regulated flowering period, which regulates the expression of proteins related to the heading stage in rice; the proteins related to the heading stage are proteins as follows in A1), A2) or A3):

[0016] A1) The protein encoded by SEQ ID NO: 3;

[0017] A2) A protein having more than 90% identity with the protein described in A1);

[0018] A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein described in A1) or A2).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention proves through experiments that knocking down the OsEra2 gene in wild-type rice can obtain transgenic rice with altered heading stage phenotypes. Compared with the recipient rice, the expression of the OsEra2 gene is reduced in the transgenic rice. The OsEra2 gene is related to the control of the heading stage of rice, which can lay a theoretical foundation for cultivating transgenic plants with appropriate heading stages.

[0021] The present invention has important theoretical significance for further clarifying the molecular mechanism of plant regulation of heading date and cultivating new varieties with regulated rice heading date by genetic engineering means. Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0023] Figure 1 Phenotypes of heading date of wild-type ZH11 and homozygous mutants OsEra2-L1 and OsEra2-L2 under natural long-day conditions.

[0024] Figure 2 Statistical results of heading date of wild-type ZH11 and homozygous mutants OsEra2-L1 and OsEra2-L2 under natural long-day conditions.

[0025] Figure 3 Mutation types of OsEra2 mutants in the ZH11 background obtained by CRISPR / Cas9 technology, named OsEra2-L1 and OsEra2-L2 respectively, both of which are loss-of-function mutants. Detailed Embodiments

[0026] The following will elaborate on the technical solutions provided by the present invention in combination with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0027] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0028] The experimental material used in this study is the common japonica rice variety Zhonghua 11 (ZH11).

[0029] The CRISPR / Cas9 vector system in the following examples is described in the following literature (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y-G (2015) A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8: 1274-1284).

[0030] Example 1: Cloning of the rice salt stress response gene OsEra2

[0031] The inventors of the present invention isolated and cloned a rice heading date regulation gene OsEra2 from the rice variety Zhonghua 11, as shown in SEQ NO: 1 of the sequence listing, and named its encoded protein OsEra2 protein, as shown in SEQ NO: 2 of the sequence listing.

[0032] Total RNA of the rice variety Zhonghua 11 was extracted and reverse transcribed into cDNA, and PCR amplification was performed using F: ATGATCATACTGACCAAGGA and R: TTATTTTGCGGCATTCGGCT as primers (SEQ NO: 4 and 5). The high-fidelity enzyme used in the amplification reaction was TOYOBO high success rate PCR enzyme KOD FX, and the reaction system was 2x PCR buffer for KOD FX 25 μL, 2 mM dNTPs 10 μL, 10 pmol / μL Primer#1 1.5 μL, 10 pmol / μL Primer#2 1.5 μL, Template DNA ≧ 1 μL (cDNA ∼ 200 ng), KOD FX (1.0 U / μL) 1 μL, ddH 2 O water up to 50 μL.

[0033] The PCR reaction procedure was as follows: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 30 s, annealing at 56°C for 30 s, extension at 68°C for 1 min, for 35 cycles; extension at 68°C for 5 min. The PCR products were subjected to Sanger sequencing. The sequencing results showed that the nucleotide sequence of the PCR amplification product was SEQ NO:1 in the sequence listing, and its coding sequence was nucleotides 1-765 of SEQ NO:2 in the sequence listing, encoding the protein OsEra2 shown in SEQ NO:2 (amino acid residues 1-254 of SEQ NO:3 in the sequence listing); the DNA shown in SEQ NO:1 in the sequence listing was named the OsEra2 gene.

[0034] SEQ ID NO:1

[0035] Genomic nucleotide sequence of the OsEra2 gene

[0036] TTCCCCTTCACCGTTCGCGAGATGATGGCCTCGCGGTCGCGGCTCCGCCTCGCCGCCGCC

[0037] GGCGAGAACCCTATCCCACACTCCAAGTCCGGCGGGGAGGGAGGAACGGAGAGGAAGCCG

[0038] GAGGAGGCGCTGCGGCGGGAGGTGACGGACCTGGGCGGCGGCAGCGAGGTGGTGCACGTG

[0039] CCGCGGTTCGTGCCCCGGGAGGCGGCGTGGGGGTGGTTCGACTACCTCGACAAGCGCATC

[0040] CCATGGACGCGCCCCACCATCCGCGTATTCGGCCGCTCCGCCGTCCAGGTACAGAGAGGT

[0041] CACCGCCGCCGCCCGCGCCTCTCATTGAACCCTTCATTTTAATCTTTGTGGGGCTTCCCT

[0042] AATCGGTCGCAATTTTTGCTCTGTTGTTCATTTGATAAATTTCTACTAGTGCTAAGATTT

[0043] CGCTATAGGTTGGTGCATTATCGTTGATTCTTGGAGGAAAAACCATAGTACTATCTTGCT

[0044] TGGTGAGATTCTGAATATGGTGCTGAGGTTGTGGTACTTCGGTGAGTCTGAAGAATGGTT

[0045] ACTGCAGGCAGTGTTGCTTACGATTTAGCTGTGGCCCTTCGGCCTGGGGCTTACAAATTT

[0046] ACACTAGGCATTTGCGTTTCCCCGGTAAAAGCTGGTTGAATTCATAGGAAATTGGCATAA

[0047] TGATAGTGACTGCAGCTGATCCGGATCCTAAGAAATCATAGTCGGCAGAAATAGCATCGT

[0048] GCATGTAATTGCAGTATTGGTGAGTTGTTCTCTTTAACGTCTGAGCTGAATTTTAATTTG

[0049] TGGGCAGAAATAAACTATATAAGTACATATACATTAGTGGGATATAAACAAACTGATAGG

[0050] TACACCAGTGGCTCTGATAAATATTACTCCCTCCATCCCAAAATATAACAACTTTTGGGT

[0051] GGATGAGACATATTCTAGTACTATGAATCTGGATAGGGGTTATGTCCAGATCCATGGTAC

[0052] TATGATACGTTCCATCCACCCTAAAATCGTTATATTTTATGACGGAGGGAGTAACTGCTA

[0053] AGTTTCTGATGTCTTATGTCCCAAACGATGGTAGCTCCTAAGCTCTGTAATACCATTGTC

[0054] TTTCCAGAAAACCACGGTTACCATTGTCTTCCAATTGGAGACACATTGATAGTGTAACTG

[0055] TTGTGAAAGATTCTAGCTCAAGTATCCCATTCTCTTACTGTTCTGCAGCCGAGAGATACA

[0056] TGCTATGTCGCGGACGAAGGGCTAACAGATTTGAGATATAGTGGCCATCAGCCTCATGCA

[0057] CATTCTTGGGATGAATTCCCTGTGCTCAAGGATATCCTGAAGGCGGTGAGAGCTTTGCCA

[0058] TGATTATTCTTTGCAATGCTATATATGATTTGCAGTTAATTTCAAGCATTAGTATTCTAA

[0059] AATAGTATCAACTAGTTTGTATTTGATGATGGGCATCTCAAAGCTCTCATTCTATCTAGT

[0060] GATTTGCTGATTAATGTATGTTCAATAGGTTCATGAAGCCCTCCCTGGGAGCCATTTTAA

[0061] CAGCTTGCTCCTAAACAGATACAAGACCGGTTCAGATTACGTCTCATGGCATGCTGATGA

[0062] CGAGCCGCTGTATGGACCTACCCCAGAGATAGCATCTGTCACCCTCGGATGCGAACGAGA

[0063] GTTCTTACTTAGAAAGAAGCCGACGAAATCGCAAGGTAAGCGGTGCACACACTAGGAAAA

[0064] TTTTTGGACTGGCAGCCTCACTATCATTTGTAGATTTTGGAGTTTAGATCACATCAACTC

[0065] CGAAATCGATCCCTATTATTTCCGTCGAAGAAAAGATTGATCCCTTTTAATCTACCATCC

[0066] AGCTTCACTTGGATCTGGGGAAGTTGCGCCGAAGCGGCTCAAGGTCAGTGCTCCTCAGCA

[0067] GCATTCTTTCCTCCTGAAGCATGGGTCGCTGCTTGTGATGAGAGGCTATACCCAACGGGA

[0068] CTGGCAGCACTCGGTCCCGAAACGAGCTAAAGCAAGCTCACCGAGGATCAATCTGACTTT

[0069] CCGGCGAGTGCTGTAG

[0070] SEQ ID NO:2

[0071] CDS sequence of OsEra2 gene

[0072] ATGATGGCCTCGCGGTCGCGGCTCCGCCTCGCCGCCGCCGGCGAGAACCCTATCCCACAC

[0073] TCCAAGTCCGGCGGGGAGGGAGGAACGGAGAGGAAGCCGGAGGAGGCGCTGCGGCGGGAG

[0074] GTGACGGACCTGGGCGGCGGCAGCGAGGTGGTGCACGTGCCGCGGTTCGTGCCCCGGGAG

[0075] GCGGCGTGGGGGTGGTTCGACTA CCTCGACAAGCGCATCCCATGG ACGCGCCCCACCATC

[0076] CGCGTATTCGGCCGCTCCGCCGTCCAGCCGAGAGATACATGCTATGTCGCGGACGAAGGG

[0077] CTAACAGATTTGAGATATAGTGGCCATCAGCCTCATGCACATTCTTGGGATGAATTCCCT

[0078] GTGCTCAAGGATATCCTGAAGGCGGTTCATGAAGCCCTCCCTGGGAGCCATTTTAACAGC

[0079] TTGCTCCTAAACAGATACAAGACCGGTTCAGATTACGTCTCATGGCATGCTGATGACGAG

[0080] CCGCTGTATGGACCTACCCCAGAGATAGCATCTGTCACCCTCGGATGCGAACGAGAGTTC

[0081] TTACTTAGAAAGAAGCCGACGAAATCGCAAGCTTCACTTGGATCTGGGGAAGTTGCGCCG

[0082] AAGCGGCTCAAGGTCAGTGCTCCTCAGCAGCATTCTTTCCTCCTGAAGCATGGGTCGCTG

[0083] CTTGTGATGAGAGGCTATACCCAACGGGACTGGCAGCACTCGGTCCCGAAACGAGCTAAA

[0084] GCAAGCTCACCGAGGATCAATCTGACTTTCCGGCGAGTGCTGTAG

[0085] SEQ ID NO:3

[0086] Amino acid sequence of OsEra2 protein

[0087] MMASRSRLRLAAAGENPIPHSKSGGEGGTERKPEEALRREVTDLGGGSEVVHVPRFVPRE

[0088] AAWGWFDYLDKRIPWTRPTIRVFGRSAVQPRDTCYVADEGLTDLRYSGHQPHAHSWDEFP

[0089] VLKDILKAVHEALPGSHFNSLLLNRYKTGSDYVSWHADDEPLYGPTPEIASVTLGCEREF

[0090] LLRKKPTKSQASLGSGEVAPKRLKVSAPQQHSFLLKHGSLLVMRGYTQRDWQHSVPKRAK

[0091] ASSPRINLTFRRVL

[0092] Example 2 Construction of OsEra2 - deleted mutant rice

[0093] (1) Construction of vector and recombinant bacteria

[0094] Use the DNA sequence shown in SEQ NO:1 to screen for appropriate target sites on the E-CRISPR website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html). Considering the score and target site location, select CCTCGACAAGCGCATCCCATGG located on the first exon of OsEra2 as the CRISPR / Cas9 target site. Synthesize primer sequences F: ggcaCCTCGACAAGCGCATCCCA and R: aaacTGGGATGCGCTTGTCGAGG (SEQ NO:6 and 7).

[0095] Construct the pCRISPR / Cas9 vector system according to the literature (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y-G (2015) A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274-1284) to obtain the new vector pCRISPR / Cas9-OsEra2. Introduce the plasmid pCRISPR / Cas9-OsEra2 into the competent cells of Agrobacterium tumefaciens EHA105 to obtain the recombinant Agrobacterium pCRISPR / Cas9-OsEra2.

[0096] (2) Transformation of rice callus with pCRISPR / Cas9-OsEra2 and screening of positive callus

[0097] The genetic transformation of rice was completed by Wuhan Boyuan Biotechnology Co., Ltd., and the T0 generation transgenic lines of CRISPR / Cas9-OsEra2 under the ZH11 background were obtained, that is, the T0 generation of rice OsEra2 mutants.

[0098] Example 3 Identification of T0 generation plants of transgenic rice CRISPR / Cas9-OsEra2

[0099] Extract DNA from the leaves of the T0 generation of rice OsEra2 mutants. Using the extracted DNA as a template and specific primers for OsEra2, perform PCR amplification. The primer sequences are F: GAGGGAGGAACGGAGAGGAA; R: GTACCACAACCTCAGCACCA (SEQ NO: 8 and 9). The high-fidelity enzyme used in the amplification reaction is Vazyme 2×Taq Plus MasterMix, and the reaction system is 25 μL of 2×Taq Plus MasterMix, 2 μL of Primer1 (10 μM), 2 μL of Primer2 (10 μM), Template DNA ≥ 1 μL (Genomic DNA 0.1 μg - 1 μg), ddH 2 O up to 50 μL.

[0100] The PCR reaction program is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, for 35 cycles; extension at 72°C for 5 min. Perform Sanger sequencing on the PCR products to check whether mutations occur at the target site.

[0101] Among them, the extraction of leaf DNA is carried out according to the following method: Put rice leaves into a 2.0 mL centrifuge tube, add steel beads and grind them into powder with a grinder. Add 400 μL of extraction buffer to the centrifuge tube and mix well by shaking. Add an equal volume of a chloroform and phenol (1:1) mixture, mix well by shaking, and centrifuge at 12000 rpm for 10 min. Take 200 μL of the supernatant, add an equal volume of chloroform, mix well by vigorous shaking, and centrifuge at 12000 rpm for 10 min. Take 100 μL of the supernatant, add twice the volume of absolute ethanol and precipitate at 0°C for 10 min. Centrifuge at 4°C, 12000 rpm, for 10 min. Discard the supernatant, add 500 μL of 75% ethanol and wash twice, dry and then add 30 μL of ddH 2 O to dissolve.

[0102] The identification results show that two independent transgenic plants with mutations in the OsEra2 gene (OsEra2-L1 and OsEra2-L2 respectively) are finally identified. By comparing the DNA sequences, the mutation sites are as Figure 3 shown. Among them, OsEra2-L1 lacks 17 bp, causing a frameshift mutation in OsEra2 and resulting in a complete loss of its function; OsEra2-L2 lacks 5 bp, also causing a frameshift mutation and a complete loss of function.

[0103] Example 4 Observation of the heading date of OsEra2 mutants under long-day conditions

[0104] Under the natural long-day conditions in summer in Beijing, the heading date differences among OsEra2-L1, OsEra2-L2 and the wild type ZH11 were observed and counted, and each rice line for counting was greater than or equal to 15 plants. From the attached Figure 1 , 2 It can be seen that both lines of the OsEra2 mutant showed an earlier heading phenotype compared with the wild type.

[0105] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An OsEra2 gene, characterized in that: The nucleotide sequence of the OsEra2 gene is shown in SEQ ID NO:

1.

2. Use of the OsEra2 gene according to claim 1 in regulating the heading period of rice.

3. The use according to claim 2, characterized in that: Knocking out or knocking down the OsEra2 gene can obtain rice with early heading phenotype.

4. An expression vector, characterized in that: It comprises an initial vector and the OsEra2 gene as claimed in claim 1.

5. A host, characterized in that The host is transformed or transfected with the expression vector as claimed in claim 4; the host is a microorganism.

6. Use of the expression vector according to claim 4 or the host according to claim 5 in regulating the heading period of rice.

7. A silencing vector, characterized in that: It comprises an original vector and the OsEra2 gene according to claim 1.

8. A silent recombinant bacterium transformed or transfected with the silencing vector according to claim 7.

9. Use of the silencing vector according to claim 7 or the silencing recombinant bacteria according to claim 8 in regulating the heading period of rice.

10. A method for cultivating and regulating the flowering period of rice, characterized in that: Regulating the expression of heading period-related proteins in rice; the heading period-related proteins are the following proteins A1), A2) or A3): A1) a protein encoded by SEQ ID NO: 3; A2) a protein having more than 90% identity with the protein described in A1); A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein described in A1) or A2).

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