Osbb gene for regulating shattering and grain type of rice and use thereof

Abstract

The application discloses an OsBb gene for regulating rice shattering and grain type and application of the gene, and the gene has a nucleotide sequence shown in SEQ ID NO.1. The application can simultaneously regulate rice shattering and grain type, and has very important significance for breeding and improvement of rice.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an OsBb gene that regulates rice grain shattering and grain shape, and its uses. Background Technology

[0002] During the domestication of rice, the loss of strong shattering ability is a key step in the transformation from wild rice to cultivated rice. Rice shattering ability (the ease with which seeds fall from the plant) and grain shape (including grain length, width, and thickness) are core agronomic traits that affect rice yield, harvest efficiency, and commercial value. Through continuous domestication, selection, and genetic improvement, their regulation involves the synergistic effects of multiple genes and pathways.

[0003] In 2006, the first gene regulating grain shedding domestication, sh4 / SHA1, was cloned. sh4 / SHA1 inhibits the differentiation of shedding layer cells and lignin synthesis: the MYB protein encoded by the sh4 gene can bind to the promoter of lignin biosynthesis genes and inhibit their expression; natural mutations (such as the substitution of amino acid at position 261) lead to weakened protein function, incomplete development of the shedding layer, and more prone to grain shedding.

[0004] Konishi et al. used QTL mapping on the progeny of a cross between the easily shattering indica rice variety Kasalath and the difficult-to-shatter japonica rice variety Nipponbare to clone the major gene qSH1 controlling grain shattering. This gene encodes a BEL1-type homeobox transcription factor, which is highly homologous to the Arabidopsis RPL gene. A difference at a SNP site 10 kb upstream of the 5' end of the qSH1 gene led to reduced expression of this gene in the abscission layer region, preventing abscission layer formation and ultimately altering rice grain shattering. The AP2 transcription factor SHAT1 also participates in abscission layer differentiation and development, and qSH1 can maintain the expression of Sh4 and SHAT1 genes in the abscission layer. SH5 is highly homologous to qSH1 and can induce the expression of Sh4 and SHAT1 genes in the abscission layer, affecting seed detachment by regulating lignin accumulation at the grain-panicle node. The OsCPL1 gene encodes a CTD-like phosphatase protein that inhibits abscission layer development.

[0005] Wu Hao and He Qi, through a genome-wide association study, discovered that SLR1, a negative feedback regulator of gibberellin signaling, is involved in the regulation of seed shattering. They found that SLR1 interacts with rice shattering genes qSH1, OSH15, and SNB. Furthermore, these three genes, acting as transcriptional repressors, bind to the promoter of the lignin synthesis gene 4CL3. The interaction between SLR1 and qSH1, OSH15, and SNB can relieve the repression of 4CL3 by these three genes, increasing lignin deposition in the abscission zone, thereby improving tensile strength and reducing seed shattering. This study establishes a connection between hormone regulation and shattering regulation genes.

[0006] The OsBb gene contains an AIG2 domain. In Arabidopsis thaliana, AIG2A and AIG2B proteins have been reported to simultaneously regulate the tryptophan-derived secondary metabolites (TDSMs) and salicylic acid (SA) chemical defense systems, thus exploring the connection between these two chemical defense pathways. Currently, there are no reports of AIG2 participating in the regulation of grain shattering in rice. Summary of the Invention

[0007] The purpose of this invention is to provide an OsBb gene that regulates rice grain shattering and grain shape, and its application. This gene can simultaneously regulate rice grain shattering and grain shape, which is of great significance for rice breeding and improvement.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] The OsBb gene, which regulates rice grain shattering and grain shape, has the nucleotide sequence shown in SEQ ID NO.1.

[0010] A protein encoded by the OsBb gene that regulates rice grain shattering and grain shape, having the amino acid sequence shown in SEQ ID NO.2.

[0011] While studying rice grain shattering, the inventors discovered a gene that regulates grain shattering—the OsBb gene. This gene also regulates rice grain shape, exhibiting a dual regulatory function. Deletion of the OsBb gene significantly enhances rice grain shattering, while significantly reducing grain length and width.

[0012] A plasmid containing the nucleotide sequence of the OsBb gene.

[0013] A host cell containing the plasmid described above.

[0014] The application of the OsBb gene or its encoded protein in the breeding of rice varieties with reduced grain shattering and / or increased grain size.

[0015] The reduced susceptibility to grain shedding manifests as an increase in delamination strength.

[0016] The increase in particle size is manifested in an increase in both particle length and particle width.

[0017] A method for cultivating rice plants with reduced grain shattering and / or increased grain size is achieved by increasing the expression level of the OsBb gene in the rice plants.

[0018] The beneficial effects of this invention are that it can simultaneously regulate rice grain shattering and grain shape, which is of great significance for rice breeding and improvement. Attached Figure Description

[0019] Figure 1This is a plot showing the predicted tissue expression analysis of the OsBb gene at different sites.

[0020] Figure 2 Subcellular localization map of OsBb protein;

[0021] Figure 3 Map of CRISPR-Cas9 knockout vectors containing the target site sequence of the rice OsBb gene;

[0022] Figure 4 This is a schematic diagram of gene editing of OsBb in the ZH11 background; where a is a schematic diagram of the gene structure and the sequences of the wild-type target site and the gene mutation target site; b is a sequencing peak diagram of the sequences of the wild-type target site and the gene mutation target site.

[0023] Figure 5 The tensile strength of mature seeds of wild-type ZH11 and mutant osbb;

[0024] Figure 6 Electron micrographs of the fracture surfaces of the delamination region of wild-type ZH11 and mutant osbb;

[0025] Figure 7 a, Figure 7 b is a statistical graph of the grain shape of mature seeds of wild-type ZH11 and mutant osbb. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0027] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0028] The OsBb gene of this invention, which regulates rice grain shattering and grain shape, has the nucleotide sequence shown in SEQ ID NO: 1:

[0029] ATGGCGCCGCCACCCGCCGCCGCCGCCGCCGTGGACGGAGTCGGGGTCGGGGTCGGGGCGCACAGCGTGTTCGTGTACGGGAGCCTGATGCAGGACGAGGTGGTGCGCACCATCATCAAGCGCGTCCCGCCCTCCTCCCCGGCGCTCCTCCCCAACTACCACAGGTTCAACATCAAGGGTAGGATTTATCCTGCAATCCTACCTGTTCAAAGCAAGAAAGTTGCTGGCAAGGTCATCACCGGTGTTACTGATGCAGAGCTCCAGATTCTGGATGAATTTGAAGATGTGGAGTACGTGAGGACAAGAGTTGAGATATCATTAACTGATACTTCAGAGACAATGCTTGCTGACACCTATGTATGGGCTGATGCAGAGGATCCAAATCTTTATGGTGAATGGGATTTTGAGGAATGGAAGAGGTTGCACATGAAAGATTTCCTTGCGATGACCCACGGGTTCATGGATGGCCTCGAACAGCCTGAATCCAAGTCCAGGGTTGAAACCTACCAATCATTCATGCAGGAAATCCAACAGCCTGGGACGACGACGACGACGACCCAGGTTGAGATTTGA (SEQ ID NO: 1).

[0030] The protein encoded by the OsBb gene has the amino acid sequence shown in SEQ ID NO: 2:

[0031] MAPPPAAAAAVDGVGVGVGAHSVFVYGSLMQDEVVRTIIKRVPPSSPALLPNYHRFNIKGRIYPAILPVQSKKVAGKVITGVTDAELQILDEFEDVEYVRTRVEISLTDTSETMLADTYVWADAEDPNLYGEWDFEEWKRLHMKDFLAMTHGFMDGLEQPESKSRVETYQSFMQEIQQPGTTTTTTQVEI* (SEQ ID NO: 2).

[0032] Analysis of the predicted tissue expression pattern of the rice OsBb gene in Example 1

[0033] Tissue expression pattern analysis was performed using the RAP-DB website by inputting the RAP_Locus number of OsBb. The prediction results showed that OsBb was highly expressed in the 2.0-2.5 cm region of rice panicles. Figure 1 ).

[0034] Example 2: Prediction of the structure of rice OsBb protein

[0035] Using the SMART website, the protein sequence corresponding to OsBb was input for domain prediction analysis. The prediction results showed that the OsBb protein contains an AIG2 domain between amino acids 23 and 140.

[0036] Example 3: Subcellular localization of the rice OsBb gene

[0037] Recombinant primers were designed based on the nucleotide sequence of the OsBb gene (upstream primer: OsBbGFPF (SEQ ID NO.3): 5′ACTCTAGAGGATCCGGTACCATGGCGCCGCCACCCGCC3′; downstream primer: OsBbGFPR (SEQ ID NO.4): 5′TGCTCACCATGTCGACAATCTCAACCTGGGTCGTCG3′). PCR amplification was performed using cDNA from rice Zhonghua 11 (ZH11) as a template. Agarose gel electrophoresis showed a single band indicating specific amplification. Therefore, the PCR product was recovered and purified to obtain the purified PCR product. The pBI221-GFP vector with a green fluorescent GFP tag was double-digested with KpnI and SalI to obtain the purified linearized vector, which was then set aside for later use. Further, the PCR product of the target gene was ligated into the linearized pBI221 vector using 2×MultiF Seamless Assembly Mix homologous recombinase (Aibotek, catalog number: RK21020) and transformed into E. coli. The correctly sequenced protoplasts were then subjected to high-concentration plasmid extraction (high-concentration plasmids are required for protoplast transformation; the kit used was Tiangen Biotech, catalog number: DP117) and transformed into rice protoplasts. Fluorescent expression sites were observed and photographed using laser confocal microscopy (LSM880, Zeiss). The specific operational steps are as follows:

[0038] Rice seedling culture: Select plump ZH11 seeds, soak them in water for two days to promote germination, and sow the sprouted seeds into 96-well exposed-bottom PCR plates. Then place them in a 30 ℃ incubator for cultivation. Use distilled water for the first four days, replace it with 1 / 2 rice nutrient solution (purchased from Cooler Master Technology Co., Ltd.) on the fourth day, and replace it with complete nutrient solution (purchased from Cooler Master Technology Co., Ltd.) on the fifth day. Change the nutrient solution every two days thereafter. Seedlings can be used for protoplast extraction after 7-14 days of growth.

[0039] Protoplast extraction: Seedlings aged 7-14 days were harvested and cut into fragments smaller than 0.5 mm on clean A4 paper. These fragments were immediately transferred to 0.6 M mannitol solution. After all seedlings were cut, the 0.6 M mannitol was removed through a 100-mesh cell sieve. Room temperature enzymatic hydrolysis buffer was added, and the mixture was incubated at 28°C and 50 rpm for 4-5 hours on a shaker. After hydrolysis, the hydrolysate was removed through a 200-mesh cell sieve, and the fragments were transferred to a 50 mL centrifuge tube. The centrifuge was performed at 150 g at room temperature for 5 minutes using a horizontal rotor, with the speed set to 1, and the supernatant was slowly discarded.

[0040] The room temperature enzymatic hydrolysate formulation was as follows: mannitol: 0.6 M, 4-morpholinoethanesulfonic acid: 10 mM, cellulase RS: 1.5%, and cleavage enzyme: 0.75%. The solution was heated at 65 °C for 10 minutes, followed by the addition of 0.1% BSA, 3.4 mM CaCl2, 5 mM β-mercaptoethanol, and 50 μg / mL carbenicillin. All reagents were purchased from Sigma-Aldrich.

[0041] Post-centrifugation procedures: Add 10 mL of W5 solution to suspend the protoplasts. Gently agitate the centrifuge tube to prevent protoplasts from clumping together. Centrifuge at 150 g for 5 minutes at room temperature using a horizontal rotor, with the speed set to 1. Slowly wash away the supernatant. Repeat the post-centrifugation procedures once. Pivot 10 μL of the protoplast suspension and count the protoplasts. Adjust the protoplast concentration to 0.51 × 10⁻⁶. 7 Protoplasts / mL; centrifuge at 150 g at room temperature for 5 minutes, discard the supernatant, resuspend the protoplasts in MMG medium, and adjust the concentration to 0.51 × 10⁻⁶. 7 Prepare 5 μg (or 10 μg) of plasmid to be transformed, dilute it to 10 μL, add 200 μL of protoplast suspension to each round-bottom centrifuge tube, gently tap to mix, then add 210 μL of 40% PEG solution and gently tap to mix. Incubate at 28 ℃ for 15 minutes to induce transformation; then add 1 mL of W5 solution to stop transformation; transfer the centrifuge tube to a centrifuge (horizontal rotor, 150 g) and centrifuge for 5 minutes, discard the supernatant, add 1 mL of W5 solution to resuspend the protoplasts, and incubate overnight at 28 ℃ for 14-16 hours. Centrifuge the cultured protoplasts for 5 minutes (horizontal rotor, 150 g), discard the supernatant, and keep approximately 100 μL for fluorescence signal observation.

[0042] W5 solution formulation: 154 mM NaCl, 125 mM KCl, 2 mM 4-morpholine ethanesulfonic acid;

[0043] MMG medium formulation: 0.6 mM mannitol, 15 mM MgCl2, 4 mM 4-morpholinoethanesulfonic acid;

[0044] 40% PEG solution formulation: 0.6M mannitol, 100 mM CaCl2, 40% v / v PEG4000.

[0045] The results showed that fluorescent signals were distributed in both the cytoplasm and nucleus of rice protoplasts (negative control) transformed with the empty vector plasmid (pBI221-GFP). The green fluorescence of OsBb::GFP overlapped with the red fluorescence of the nuclear Mark. The green fluorescence of OsBb::GFP also showed faint luminescence in the cytoplasm, indicating that OsBb is localized in the nucleus and possibly also in the cytoplasm (e.g., Figure 2 (As shown).

[0046] Example 4: Construction of OsBb gene knockout transgenic rice

[0047] Selection of gRNA target sequence: Based on CRISPR / Cas9 related experimental methods, the 5′GCCGCCGTGGACGGAGTCGGGG3′ sequence (SEQ ID NO.5) containing NGG as the recognition site was selected as the knockout target site on the exon of the OsBb gene, and the PAM sequence is GGG;

[0048] Design of upstream and downstream primers for gRNA oligonucleotide chains:

[0049] The upstream primer is OsBb_gRNA_F:

[0050] 5′ GCCGGCCGCCGTGGACGGAGTCG3′ (SEQ ID NO. 6);

[0051] The downstream primer is OsBb_gRNA_R:

[0052] 5' AAACCGACTCCGTCCACGGCGGC3' (SEQ ID NO. 7).

[0053] CRISPR / Cas9 vector construction:

[0054] After the target sequence is loaded, a recombinant vector containing the OsBb gene target site is formed. Figure 3 ).

[0055] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar.

[0056] The specific operating method is as follows:

[0057] (1) Preparation of primer dimers: Take 2 μL of each of the upstream and downstream primers of 10 μM target site and add 16 μL of double-distilled water. After treatment at 95℃ for 30 s, slowly reduce the temperature to 25℃ at about 0.5 ℃ / s to obtain the double-stranded sequence containing the knockout target site.

[0058] (2) Subsequent steps included cutting and ligating simultaneously, one round of PCR, and two rounds of PCR.

[0059] For specific methods, please refer to the following reference: Zeng Dongchang, Ma Xingliang, Xie Xianrong, et al. Operational methods for construction and mutation analysis of plant CRISPR / Cas9 multi-gene editing vectors [J]. Science in China: Life Sciences, 2018, 48(07):783-794.

[0060] (3) The second round of PCR products were ligated into the pYLCRISPR / Cas9pUbi-N vector and transformed into E. coli using 2×MultiF seamless cloning premix (Aibotek, catalog number: RK21020);

[0061] (4) E. coli transformation: Take 10 μL of the final product from step (3) and add it to 50 μL of freshly thawed DH10B competent cells. Gently mix, incubate on ice for 30 minutes, heat shock at 42 ℃ for 90 s, stand on ice for 2 minutes, then add 700 μL of antibiotic-free LB liquid medium, and incubate at 37 ℃ in a constant temperature shaker at 220 rpm for one hour. Then plate the LB plate with kanamycin resistance (25 μg / mL).

[0062] Preparation of 25 μg / mL kanamycin liquid: 0.25 g kanamycin powder + 10 mL sterile water, filter and sterilize, then store at -20 ℃.

[0063] (5) PCR detection of bacterial culture: The next day, single clones were picked and cultured in LB liquid medium with kanamycin resistance (50 μg / mL) at 37 ℃ until the bacterial culture became turbid. They were then amplified and sequenced using primers SP1 (CCCGACATAGATGCAATAACTTC, SEQ ID NO.8) and SP2 (GCGCGGTGTCATCTATGTTACT, SEQ ID NO.9). The plasmids of the positive clones were extracted and kept for later use.

[0064] (6) The plasmid was sent to Weimi Biotechnology Co., Ltd. for genetic transformation of rice.

[0065] Example 5: Phenotypic Analysis of Rice OsBb Gene Knockout Lines

[0066] To identify the knockout transgenic lines obtained in Example 4, the transgenic seedlings were cultured in a room temperature and light incubator for about a week, and then the positive seedlings were identified. The specific steps are as follows:

[0067] Detection of knockout transgenic material: Twenty T0 generation transgenic seedlings were obtained and cultured in a room temperature light incubator for about two weeks. DNA from the 20 seedlings was then collected and amplified by PCR using OsBb-GF1 and OsBb-GR1, followed by sequencing. Analysis of the sequencing results revealed a transgenic plant with premature termination of protein translation. The nucleotide sequence of a homozygous mutant of the OsBb gene knockout is shown in SEQ ID NO.10, and its encoded protein is shown in SEQ ID NO.11.

[0068] The nucleotide sequence of OsBb gene deletion homozygous mutant 1 (SEQ ID NO.10):

[0069] ATGGCGCCGCCACCCGCCGCCGCCGCCGCCGTGGACGGATCGGGGTCGGGGTCGGGGCGCACAGCGTGTTCGTGTACGGGAGCCTGATGCAGGACGAGGTGGTGCGCACCATCATCAAGCGCGTCCCGCCCTCCTCCCCGGCG CTCCTCCCCAACTACCACAGGTTCAACATCAAGGGTAGGATTTATCCTGCAATCCTACCTGTTCAAAGCAAGAAAGTTGCTGGCAAGGTCATCACCGGTGTTACTGATGCAGAGTCCCAGATTCTGGATGAATTTGAAGATGT GGAGTACGTGAGGACAAGAGTTGAGATATCATTAACTGATACTTCAGAGACAATGCTTGCTGACACCTATGTATGGGCTGATGCAGAGGATCCAAATCTTTATGGTGAATGGGATTTTGAGGAATGGAAGAGGTTGCACATGA AAGATTTCCTTGCGATGACCCACGGGTTCATGGATGGCCTCGAACAGCCTGAATCCAAGTCCAGGGTTGAAACCTACCAATCATTCATGCAGGAAATCCAACAGCCTGGGACGACGACGACGACGACCCAGGTTGAGATTTGA

[0070] The protein sequence encoded by the OsBb gene deletion homozygous mutant (SEQ ID NO.11):

[0071] MAPPPAAAAAVDGSGSGSGRTACSCTGA*.

[0072] Upstream primer OsBb-GF1 (SEQ ID NO.12): 5′ TTCGTTCCATCTCCCTTCGC3′;

[0073] Downstream primer OsBb-GR1 (SEQ ID NO.13): 5′ GCGCTTACTAGTTGGGGAGG3′.

[0074] Phenotypic identification: After obtaining a stably inherited knockout mutant, further sequencing of the target site is performed to obtain a homozygous mutant (e.g., Figure 4 As shown), mature rice seeds were harvested. The shattering performance of the mature seeds was assessed using a digital push-pull force meter (Model: ZMF-10) to measure the pull strength (BTS) of the mature seeds. Specifically, 10 panicles were randomly selected from wild-type and Osbb knockout mutants, and 10 seeds were extracted from each panicle, for a total of 100 seeds. A small clamp was fixed to the pull rod of the force meter, clamping the middle part of the seed glume. Peak mode was selected, and the seed was pulled horizontally to separate from the spikelet rachis. The BTS value was recorded. Compared with wild-type Zhonghua 11 (ZH11), the BTS level of Osbb was significantly lower (e.g., ...). Figure 5 (As shown).

[0075] Example 6: Cross-sectional observation of the abscission zone in rice OsBb knockout lines

[0076] To accurately distinguish the differences in the abscission region, we used scanning electron microscopy (SEM) to observe the fracture interface of the abscission region at the base of the Osbb spikelet. The seeds were first pulled off the peduncle using a tensile testing machine, fixed on the SEM platform, and then sputtered with gold before being placed in the instrument for observation at magnifications of ×50, ×250, and ×1000. SEM images of the fracture interface showed that, compared to the wild-type ZH11, the mutant Osbb has a relatively smooth fracture surface (e.g., ...). Figure 6 (As shown). Based on the combined results of phenotype determination and electron microscopy, rice seeds with the OsBb gene knocked out were more prone to falling off.

[0077] Example 7: Grain type analysis of OsBb knockout rice lines

[0078] Ten plants were randomly selected from both the wild-type and OsBb mutant varieties, and 20 seeds were randomly chosen from each variety to measure seed length and width. Seed length and width were measured using electronic vernier calipers (Shanghai Hengliang Measuring Instruments Co., Ltd.) at the longest diameter of each seed's major axis perpendicular to its minor axis, and these measurements were recorded as seed length and width values, respectively, in mm. Figure 7(As shown). Measurement data show that, compared with ZH11, the rice material with the OsBb gene knocked out has significantly shorter and narrower grains, with no significant difference, while the grain thickness has not changed significantly.

[0079] Based on the above experimental results, it can be concluded that the loss of OsBb function leads to rice seeds falling off more easily, and a significant reduction in grain length and width.

[0080] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. The application of OsBb gene knockout in improving rice grain shattering and / or reducing grain size, characterized in that, The nucleotide sequence of the OsBb gene is shown in SEQ ID NO.

1.

2. A method for cultivating rice plants with increased shattering tendency and / or reduced grain size, characterized in that, This was achieved by knocking out the OsBb gene in rice plants, the nucleotide sequence of which is shown in SEQ ID NO.1.

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