An OsJAZ7 gene for regulating rice diurnal flowering time and its application

Abstract

The present invention discloses an OsJAZ7 gene for regulating the daytime flowering time of rice and its application, belonging to the field of genetic engineering and molecular breeding. The nucleotide sequence of the OsJAZ7 gene is shown in SEQ ID NO: 1; the present invention also discloses a method for promoting the early daytime flowering time of rice, comprising the step of down-regulating the expression level of the OsJAZ7 gene in a rice receptor material. The present invention verifies through experiments that the mutant obtained by knocking out OsJAZ7 blooms about 50 minutes earlier than the wild-type rice Zhonghua 11 (ZH11), indicating that knocking out the OsJAZ7 gene can be used to prepare rice varieties with early daytime flowering time, used for hybrid rice breeding, increasing the time when the parents meet when they flower, and helping to increase the yield of indica-japonica hybrid rice seed production and reduce the cost of seed production.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and molecular breeding, and in particular to an OsJAZ7 gene for regulating the daytime flowering time of rice and an application thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the three major food crops for human beings, supporting 21% of the total calorie intake of the world's population. Meeting the growing global demand for rice has driven the continuous pursuit of rice breeding yield (Fitzgerald et al., 2009; Qian et al., 2016; Chen et al., 2022). Hybrid rice can increase rice yield. Inter-subspecies hybridization between indica and japonica rice can increase yield by 30% compared with intra-subspecies hybridization between indica and japonica rice (Cheng et al., 2007; Qian et al., 2016; Chen et al., 2020). However, there are many restrictive problems in inter-subspecies hybridization between indica and japonica rice, and one of them is the mismatch of parental flowering time (Yuan Longping, 2002; Wang et al., 2022). Generally speaking, indica rice flowers earlier and in a concentrated manner (before 10 a.m.), while japonica rice flowers later and in a dispersed manner (after 11 a.m.) (Zhong Weigong et al., 1990; Tian Dacheng et al., 1991). Therefore, studying the flowering regulation mechanism of rice and applying genes that can regulate the flowering time of rice to hybrid seed production can increase the time when the parents meet during flowering and improve the success rate of cross-pollination, thereby reducing the cost of hybrid seed production, which has potential application value in hybrid seed production.

[0003] Rice is a monocot, and its lodicule is equivalent to the petal of a dicot, and is an important organ that determines the opening and closing of rice spikelets (Ma and dePamphilis, 2000; Liu et al., 2017). The lodicule is composed of many thin-walled cells and a few vascular bundles. The thin-walled cells are connected by intercellular filaments, which are necessary for rapid expansion and transport of substances throughout the lodicule (Wang et al., 1991; Heslop-Harrison, 1996). Therefore, understanding the mechanism of how rice lodicules expand and shrink is crucial to studying rice flowering.

[0004] According to research, jasmonic acid substances (JAs) can effectively promote rice flowering (Ding Chaochen et al., 1994; Zeng Xiaochun et al., 1999; Huang Junbao et al., 2015). The jasmonic acid signal transduction pathway mainly includes the COI1-JAZ co-receptor formed by CORONATINE INSENSITIVE 1 (COI1) and Jasmonate ZIM-domain (JAZ), the perception of JA-Ile, the degradation of JAZ repressor protein and the release of transcription factors that interact with JAZ repressor protein to regulate the expression of downstream genes (Zeng et al., 1999; Chinat al., 2007). JAZ repressor protein belongs to the TIFY protein family, has a highly conserved TIF[F / Y]XG motif in the ZIM domain, and a Jas motif of about 27 amino acids near the C-terminus (Wu Hua, 2015; Wasternack et al., 2019).

[0005] The present invention discloses a gene OsJAZ7 that controls the flowering time of rice during the day. The gene knockout mutant blooms about 50 minutes earlier than the wild type ZH11. Knocking out the OsJAZ7 gene can be used to prepare rice varieties with early flowering time during the day, which can be used for hybrid rice breeding, increase the flowering time of the parents, help increase the yield of seed production, and reduce the cost of seed production. Summary of the invention

[0006] The purpose of the present invention is to provide an OsJAZ7 gene for regulating the daily flowering time of rice and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention confirms that the OsJAZ7 gene is related to the daily flowering time of rice. Knocking out the OsJAZ7 gene can promote the early flowering time of rice, thereby increasing the time when the flowering of the parents meets, which is helpful to improve the yield of indica-japonica hybrid rice seed production and reduce the cost of seed production.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an OsJAZ7 gene for regulating the diurnal flowering time of rice, and the nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0009] The present invention also provides an OsJAZ7 protein for regulating the daytime flowering time of rice, and the amino acid sequence of the protein is shown in SEQ ID NO:2.

[0010] The present invention also provides a pCRISPR / Cas9 vector for knocking out the OsJAZ7 gene, which contains the target sequence of the OsJAZ7 gene.

[0011] Furthermore, the nucleotide sequence of the target sequence is shown in SEQ ID NO: 3-4.

[0012] The present invention also provides a recombinant bacterium, comprising the above-mentioned pCRISPR / Cas9 vector.

[0013] The present invention also provides an application of the OsJAZ7 gene, the pCRISPR / Cas9 vector or the recombinant bacteria in regulating the daytime flowering time of rice.

[0014] Furthermore, it is specifically for promoting the early flowering time of rice.

[0015] The present invention also provides a use of the above-mentioned OsJAZ7 gene, the above-mentioned pCRISPR / Cas9 vector or the above-mentioned recombinant bacteria in creating transgenic rice with an advanced daytime flowering time.

[0016] The present invention also provides a method for promoting the early flowering time of rice, comprising the step of down-regulating the expression level of OsJAZ7 gene in a rice receptor material; the nucleotide sequence of the OsJAZ7 gene is shown in SEQ ID NO:1.

[0017] Further, the expression level of the OsJAZ7 gene is down-regulated by knocking out the target sequence of the OsJAZ7 gene in the rice receptor material;

[0018] The nucleotide sequence of the target sequence is shown in SEQ ID NO: 3-4.

[0019] The present invention discloses the following technical effects:

[0020] The present invention provides an OsJAZ7 gene for regulating the daytime flowering time of rice. The protein encoded by the gene can form a COI1-JAZ co-receptor with CORONATINE INSENSITIVE 1 (COI1) and respond to jasmonic acid-L-isoleucine (JA-Ile) signals. Experiments have verified that the mutant obtained by knocking out OsJAZ7 blooms about 50 minutes earlier than the wild-type rice Zhonghua 11 (ZH11). The present invention shows that knocking out the OsJAZ7 gene can be used to prepare rice varieties with advanced daytime flowering time, which is applied to hybrid rice breeding, increases the time when the parents meet when they flower, and helps to increase the yield of indica-japonica hybrid rice seed production and reduce seed production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 is the spatiotemporal expression pattern of OsJAZ7 gene in wild-type rice ZH11;

[0023] Figure 2 The mutation and flowering conditions of the OsJAZ7 gene knockout mutant; A: Sequencing results of the OsJAZ7 gene knockout mutant; BC: Phenotypic observation diagram of the OsJAZ7 gene knockout mutant and the wild type; D: Statistical diagram of the flowering conditions of the OsJAZ7 gene knockout mutant and the wild type;

[0024] Figure 3 The changes in lodicule size of the OsJAZ7 gene knockout mutant and wild-type rice ZH11 before and after flowering; A: phenotypic diagram; B: statistical diagram. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Gene alignment

[0032] Obtain the coding region sequence of OsJAZ7 on NCBI, as shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0033] SEQ ID NO.1:

[0034] ATGGCGGCTTCCGCGAGGCCCGTCGGCGTCGGCGGGGAGAGGGCGACGAGCTTCGCCATGGCGTGCAGCCTGCTCAGCCGCTACGTCCGCCAGAACGGCGCCGCCGCCGCCGAGCTCGGCCTCGGCATCAGAGGTGAGGGTGAGGCTCCGAGGGCGGCGCCGGCGACGATGAGCTTGCTGCCCGGGGAGGCGGAGAGGAAGAAGGAGACCATGGAGCTCTTCCCGCAGAGCGCCGGCTTTGGCCAGCAGGATGCCATCACCGCCGATTCTGCTGCTGATGCTAGGGAACAAGAGCCTGAGAAGCGTCAGCTGACCATCTTCTATGGTGGGAAGGTGCTCGTGTTCAACGACTTCCCAGCCGACAAGGCAAAGGGCTTGATGCAGCTGGCTAGCAAGGGCAGCCCGGTGGCTCCTCAGAATGCCGCGGCACCTGCACCAGCAGCTGTTACAGACAACACCAAGGCCCCTATGGCCGTGCCGGCCCCGGTCAGTAGCTTGCCTACAGCTCAGGCCGATGCTCAGAAGCCTGCTCGCGCGAATGCTTCTGATATGCCTATTGCTAGGAAGGCATCACTCCACAGGTTCCTTGAGAAGAGAAAGGATCGTCTTAATGCAAAGACGCCATACCAGGCTTCTCCTTCAGATGCAACCCCAGTCAAGAAGGAGCCTGAGAGCCAGCCATGGCTCGGACTAGGGCCGAACGCCGTCGTGAAGCCCATAGAACGCGGCCAATGA.

[0035] SEQ ID NO: 2:

[0036] MAASARPVGVGGERATSFAMACSLLSRYVRQNGAAAAELGLGIRGEGEAPRAAPATMSLLPGEAERKKETMELFPQSAGFGQQDAITADSAADAREQEPEKRQLTIFYGGKVLVFNDFPADKA KGLMQLASKGSPVAPQNAAAPAPAAVTDNTKAPMAVPAPVSSLPTAQADAQKPARANASDMPIARKASLHRFLEKRKDRLNAKTPYQASPSDATPVKKEPESQPWLGLGPNAVVKPIERGQ*.

[0037] 2. Temporal and spatial expression patterns of OsJAZ7 gene

[0038] RNA was extracted from different tissues of wild-type rice ZH11 using Trizol reagent (Invitrogen), and reverse transcription was performed using HiScript III First-Strand cDNA Synthesis Kit (Vazyme, Nanjing) to obtain cDNA. RT-qPCR was performed using 2× RealStar Green Fast Mixture Kit (Genstar, Beijing) according to the instructions. Each experiment was repeated three times. Actin was used as a control to standardize the amount of cDNA. The results were analyzed by 2 according to the “Fluorescence Quantitative PCR Application Guide” of BioRad. -ΔCt The relative expression levels of the target genes were calculated to process the data. Fluorescence quantitative PCR primers (QPCR-OsJAZ7-CDS-F: AAGACGCCATACCAGGCTTC, QPCR-OsJAZ7-CDS-R: TTCTATGGGCTTCACGACGG);

[0039] Fluorescence quantitative PCR system (20 μl):

[0040] Table 1

[0041]

[0042]

[0043] Fluorescence quantitative PCR reaction program: pre-denaturation at 95°C for 3 min, 40 cycles (95°C for 10 s, 60°C for 10 s, 72°C for 15 s), and extension at 60°C for 6 s.

[0044] The results are as follows Figure 1As shown, the OsJAZ7 gene was expressed at the highest level in leaves of wild-type rice ZH11; in lodicules, the expression level was relatively low in the morning on the day of flowering, and gradually increased as the flowering time of wild-type rice ZH11 (ZH11 generally blooms at 11:00) was approached.

[0045] 3. Construction of knockout vector, identification of transformed seedlings and phenotypic observation

[0046] (1) Target sequence design (website: http: / / skl.scau.edu.cn / primerdesign / )

[0047] OsJAZ7-U3: GAGGGCGACGAGCTTCGCCATGG (SEQ ID NO: 3);

[0048] OsJAZ7-U6a:GGGGAGGCGGAGAGGAAGAAGG (SEQ ID NO: 4).

[0049] (2) Construction of pYL-pU3-gRNA and pYL-pU6a-gRNA vectors containing OsJAZ7-U3 and U6a fragments

[0050] Referring to the research of Zeng Dongchang et al. (Zeng Dongchang, Ma Xingliang, Xie Xianrong, et al. Operational methods for construction of plant CRISPR / Cas9 multi-gene editing vector and mutation analysis [J]. Chinese Science: Life Sciences, 2018, 48(07): 783-794.), target primers with sticky ends were synthesized (OsU3T1F: GGCAGAGGGCGACGAGCTTCGCCA, OsU3T1R: AAACTGGCGAAGCTCGTCGCCCTC, OsU6aT2F: GCCGTTCTTCCTCTCCGCCTCCCC, OsU6aT2R: AAACGGGGAGGCGGAGAGGAAGAA), and the target linker was heated at 90°C for 30s and then cooled at room temperature. At the same time, about 1 μg of pYL-U3 / U6a-gRNA plasmids were taken for enzyme digestion (37°C for 15 min, 70°C for 5 min), recovered, and the recovered enzyme-cut vectors were ligated with their corresponding linkers (16°C for 30 min).

[0051] (3) Construction of pCRISPR / Cas9 vector containing OsJAZ7 fragment

[0052] Referring to the study by Zeng Dongchang et al. (Zeng Dongchang et al., 2018), the vector connected in the previous step was PCR amplified with primers (Pps-L: TTCAGAGGTCTCTCTCGCCCGGGCCCAATCGGCAGCAAAGGA, Pgs-2: AGCGTGGGTCTCGTCAGTCCATCCACTCCAAGCTC, Pps-2: TTCAGAGGTCTCTCTGAGGCCCAATCGGCAGCAAAGGA, Pgs-R: AGTGTAGGTCTCTACCGTCCCGGGTCCATCCACTCCAAGCT, the product was recovered and purified, and connected to the pCRISPR / Cas9 vector containing the Cas9 expression cassette by cutting and ligating with T4 DNA ligase and BsaI-HF restriction endonuclease. Escherichia coli was transformed, a single colony was picked for culture, and the plasmid was extracted.

[0053] PCR amplification reaction system (25 μl):

[0054] Table 2

[0055]

[0056]

[0057] Reaction program: pre-denaturation at 95°C for 1 min, 28 PCR cycles (95°C for 10 s, 58°C for 15 s, 72°C for 20 s), extension at 72°C for 1 min.

[0058] Cutting and ligating system (15μl):

[0059] Table 3

[0060]

[0061] Ligation procedure: Use PCR instrument to perform enzyme digestion and ligation reaction: 37℃10min; 10-12 cycles (37℃5min, 10℃3min, 20℃5min); 37℃3min.

[0062] (4) Obtaining transgenic seedlings

[0063] The target-containing pCRISPR / Cas9 vector plasmid was sent to a biological company for genetic transformation of wild-type rice ZH11. After the transformed seedlings were obtained, they were planted in the Qilin North experimental field of South China Agricultural University.

[0064] (5) Identification of mutations

[0065] The DNA of the plant was extracted and amplified using the following primers: JAZ7-OsTIFY10b-F: TCGTCTCCGTCTCCTCTCCTT, JAZ7-OsTIFY10b-R: CCGAAGCACACCCACAAA. The DNA was sent to the company for sequencing. The results were as follows: Figure 2 As shown in A.

[0066] (6) Phenotypic observation of mutant plants

[0067] The knockout mutants obtained above were named OsJAZ7-CAS1 and OsJAZ7-CAS2. All rice was planted in the experimental field of South China Agricultural University (in Guangzhou, where the average summer temperature is 28-30°C). Field observations showed that the knockout mutants of the OsJAZ7 gene flowered about 50 minutes earlier than the wild type ZH11. Figure 2 As shown in BD.

[0068] (7) Observation of mutant lodicules

[0069] Florets of ZH11, OsJAZ7-CAS1 and OsJAZ7-CAS2 plants were selected at different times of the day, and the lemma and palea were peeled off to expose a pair of lodicules. The dissected florets were observed and photographed under a dissecting microscope. Subsequently, a pair of lodicules on the florets were peeled off and photographed under a dissecting microscope. The surface area of ​​the lodicules was calculated using ImageJ software, and it was found that the lodicules of ZH11, OsJAZ7-CAS1 and OsJAZ7-CAS2 were basically the same size before flowering. When OsJAZ7-CAS1 and OsJAZ7-CAS2 bloomed, the lodicules were larger than those of ZH11 at the same period. After the flowers were closed, the lodicules of ZH11, OsJAZ7-CAS1 and OsJAZ7-CAS2 were all wrinkled, as shown in the figure. Figure 3 As shown in AB.

[0070] The results of the above examples show that by knocking out the OsJAZ7 gene in the wild-type rice ZH11, the OsJAZ7 gene knockout mutant has a daytime flowering time that is about 50 minutes earlier than the wild-type ZH11. Therefore, by knocking out the OsJAZ7 gene, rice varieties with early daytime flowering time can be cultivated, which helps to solve the problem of inconsistent flowering time of parents in hybrid breeding, increase the yield of seed production, and reduce the cost of hybrid seed production.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Knockout OsJAZ7 Use of genes in any of the following: (1) Application in promoting the early flowering time of rice; (2) Application in the creation of transgenic rice with early flowering time; Said OsJAZ7 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; the knockout OsJAZ7 Knockout OsJAZ7 The target sequence of the gene; the nucleotide sequence of the target sequence is shown in SEQ ID NO: 3-4.

2. A method for promoting early flowering time of rice, characterized in that: Including knockout rice receptor material OsJAZ7 The target sequence of the gene is down-regulated OsJAZ7 The step of measuring the expression level of a gene; OsJAZ7 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; the nucleotide sequence of the target sequence is shown in SEQ ID NO: 3-4.

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