Application of OsHCRF2 functional protein and its encoding gene in rice breeding

Abstract

The present invention discloses the application of OsHCRF2 functional protein and its coding gene in rice breeding and production, belonging to the technical field of plant genetic engineering and genetic improvement. The OsHCRF2 coding DNA sequence is shown in SEQ ID No: 1, and the amino acid sequence of its protein product is shown in SEQ ID No: 5. The OsHCRF2 of the present invention can regulate important agronomic traits such as the number of rice grains per panicle, the number of filled grains, the panicle length, the heading period, the maturity period, etc., and provides genes and germplasm materials with great potential utilization value for cultivating high-yield and early-maturing rice varieties.

Description

Technical Field

[0001] The invention relates to plant genetic engineering and genetic improvement, and in particular to application of an OsHCRF2 functional protein and a coding gene thereof in rice breeding. Background Art

[0002] In recent years, the rapid development of rice functional genomics research has identified a large number of genes with important utilization value for rice genetic breeding in my country, and rice breeding is moving towards a new era of design breeding (China Rice, Zhou Zhengping et al., 2019). The innovative development of rice breeding provides an important guarantee for my country's food security, and increasing yield per unit area will remain the main focus of rice breeding in my country for some time to come. Therefore, we must continue to attach importance to the discovery, cloning and identification of new regulatory genes for important agronomic traits of rice, provide components for design breeding, and serve the strategic needs of precision, dataization and intelligence of rice breeding in the new era (China Rice, Zhou Zhengping et al., 2019).

[0003] According to Wikipedia (https: / / en.wikipedia.org / wiki / Homeodomain), the typical DNA binding homeodomain is composed of a 60-amino acid long HTH (helix-turn-helix) structure, while the TALE (three aminoacid loop extension) homeodomain is composed of 63 amino acids. Based on the conserved endonucleoside-exonucleoside structure and unique codomain of these homeodomain proteins, they are divided into 14 different types, including HD-ZIP I-IV, BEL, KNOX, PLINC, WOX, PHD, etc.

[0004] Rice OsHCRF2 Homologous genes OsHCRF1 It can regulate important agronomic traits of rice such as plant height, panicle length, chloroplast development and chlorophyll content (patent ZL202010110531.1).

[0005] OsHCRF2 Encodes Homeodomain-like containing protein, not seen yet OsHCRF2 Reports on regulating important agronomic traits of rice. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a rice OsHCRF2 functional protein, a coding gene and an application thereof in rice breeding.

[0007] The technical solution provided by the present invention is: to create OsHCRF2Loss-of-function mutants, created by transgenic overexpression OsHCRF2 Overexpression materials to study their functions in regulating important agronomic traits of rice.

[0008] from OsHCRF2 A 19 bp specific editing target sequence (SEQ ID No: 2) was selected from the coding sequence (SEQ ID No: 1) and the knockout vector pHUN4c12- OsHCRF2 . Multiple rice lines with one A or T base inserted into the target sequence were obtained through Agrobacterium-mediated transformation. The mutated target sequences are shown in SEQ ID No: 3 and ID No: 4. Mutation 1 produces a frameshift, which is different from the wild type (SEQ ID No: 5) starting from the 80th amino acid at the N-terminus and terminates prematurely after the 87th amino acid. The amino acid sequence after mutation is shown in SEQ ID No: 6. Mutation 2 produces a frameshift, which is different from the wild type (SEQ ID No: 5) starting from the 79th amino acid at the N-terminus and terminates prematurely after the 87th amino acid. The amino acid sequence after mutation is shown in SEQ ID No: 7.

[0009] Will OsHCRF2 The coding sequence (SEQ ID No: 1) was deleted and the stop codon was integrated into the modified Bam HI (NEB) and Sac I (NEB) double-digested pCAMBIA1303 vector to make it Ubi-1 The 3' end of the promoter is driven by it. The japonica rice variety Kitaake was obtained by Agrobacterium-mediated transformation OsHCRF2 Overexpression transgenic lines.

[0010] The beneficial effects of the present invention are as follows: 1. The present invention discovers a new functional protein and its encoding gene for regulating the number of rice grains per panicle, the number of filled grains and the length of the panicle OsHCRF2 Through gene editing and knockout, we obtained OsHCRF2 The rice mutants had significantly increased grain number and filled grain number per panicle and increased panicle length, providing genes and germplasm materials with significant potential value for breeding high-yield rice varieties.

[0011] 2. The present invention discovered a new functional protein and its encoding gene that regulates the heading and maturity period of rice OsHCRF2 Through gene editing and knockout, we obtained OsHCRF2 The heading and maturity of rice mutants are both advanced, providing genes and germplasm materials with great potential value for breeding early-maturing rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 for OsHCRF2 Identification of knockout (KO) rice lines.

[0013] Figure 2 for OsHCRF2 Identification of overexpression (OE) rice lines, M, DNA Marker.

[0014] Figure 3 This is the mature ear phenotype.

[0015] Figure 4 Statistical data for the number of grains per ear, the number of filled grains and the ear length.

[0016] Figure 5 Statistical data for effective tillering number and thousand-grain weight.

[0017] Figure 6 for OsHCRF2 The heading date of knockout rice lines is advanced.

[0018] Figure 7 for OsHCRF2 Knockout rice lines mature earlier. DETAILED DESCRIPTION

[0019] The present invention will be described in detail below through embodiments in conjunction with the accompanying drawings, but this is not intended to limit the present invention and is merely an example.

[0020] Example 1 OsHCRF2 Construction of knockout vector and OsHCRF2 Obtaining knockout rice lines

[0021] 1. Knockout vector pHUN4c12- OsHCRF2 Construction

[0022] A 19 bp specific target sequence was selected using E-CRISP (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) for the generation of sgRNA (single-guide RNA). The target sequence 5'-acctcgctatcttgaacga -3' (SEQ ID No: 2) is OsHCRF2 A segment of the 807 bp CDS sequence (SEQ ID No: 1). Synthetic primer pair OsHCRF2-CRIS (Table 1) The phosphate group was added to the 5' end of the sequence using the following system:

[0023] OsHCRF2-CRISF (10 mM) 1ml,

[0024] OsHCRF2-CRISR(10 mM) 1ml,

[0025] 10x T4 DNA Ligase Buffer (with 10 mM ATP) 2 ml,

[0026] T4 polynucleotide kinase (3' phosphatase plus; NEB) 1 ml,

[0027] H2O 15 ml.

[0028] 37 °C 40 min, 65 °C 20 min (heat inactivation of enzyme), 94 °C 5 min, 50 °C 2 min.

[0029] Plasmid pHUN4c12 (In Vitro Mutagenesis, Xu et al. 2017) was Bsa I (NEB) digestion system is as follows:

[0030] pHUN4c12 3 ml (0.3 mg / ml),

[0031] 10x CutSmart Buffer 2 ml,

[0032] Bsa I (NEB) 1 ml,

[0033] H2O 14 ml.

[0034] 3 tubes in total. 37 °C for 5 h, 65 °C for 20 min (heat inactivation of enzyme). Make up to 200 ml with 60 ml of enzyme digestion solution from 3 tubes, add 500 ml of anhydrous ethanol, mix well and place at -80 °C for 1 h. Centrifuge at 12000 rpm for 12 min to precipitate, dry and add 20 ml of H2O to dissolve.

[0035] Then add the phosphate group OsHCRF2-CRIS The primers were ligated to the Bsa I digested pHUN4c12 vector.

[0036] two, OsHCRF2 Obtaining knockout rice lines

[0037] The constructed knockout vector pHUN4c12- OsHCRF2 According to the product instructions (heat shock method), transform into Agrobacterium EHA105 (pSoup) competent cells (2 ndThe rice transformation work was entrusted to the Wuming Xingwang System Crop Design Frontier Laboratory (Beijing) Co., Ltd. OsHCRF2 The callus of the japonica rice variety Kitaake was infected with Agrobacterium EHA105 (pSoup), and then transferred to the co-culture medium for dark culture at 24 ° C for 2-4 days. The washed callus was transferred to the selection medium containing hygromycin for resistance screening. The selected resistant callus was transferred to the pre-differentiation medium for 7-10 days, and then transferred to the differentiation medium for light culture. When the seedlings grew to 2-4 cm, they were transferred to the rooting medium for growth for 3 weeks. The T0 generation seedlings were hardened for 3 days and then transplanted into the soil. The seeds were received and planted again to obtain the T1 generation.

[0038] three, OsHCRF2 Identification of knockout rice lines

[0039] First, we identified whether the T0 transgenic rice lines had integrated the OsHCRF2-CRIS Using wild-type and T0 generation leaf genomic DNA as template, U3F / UbiR (Table 1) PCR (polymerase chain reaction) amplification was performed, and the plants with specific amplification bands were transgenic positive plants. Specific primers were designed on both sides of the editing target sequence. OsHCRF2 - target (Table 1), using the genomic DNA of the positive plants as templates, the PCR amplification products were sequenced to detect the target sequence of the T0 generation. Using the wild type as the control, two homozygous mutations were identified in the T0 generation, such as Figure 1 As shown, denoted as KO1 and KO2. The mutated target sequences are shown in SEQ ID No: 3 and SEQ ID No: 4. KO1 produces a frameshift, which is different from the wild type (SEQ ID No: 5) starting from the 80th amino acid at the N-terminus and terminates prematurely after the 87th amino acid. The amino acid sequence after mutation is shown in SEQ ID No: 6. KO2 produces a frameshift, which is different from the wild type starting from the 79th amino acid at the N-terminus and terminates prematurely after the 87th amino acid. The amino acid sequence after mutation is shown in SEQ ID No: 7. The T1 generation mutants were detected by the same sequencing method.

[0040] Table 1 Primers used in the examples

[0041]

[0042] Example 2 OsHCRF2 Obtaining and identifying overexpressing rice lines

[0043] To build OsHCRF2 Overexpression vector, OsHCRF2The coding sequence (SEQ ID No: 1) was PCR amplified using primer pair OsHCRF2-OE The amplified fragments verified by sequencing were integrated into the modified and Bam HI (NEB) and Sac I (NEB) double-digested pCAMBIA1303 vector to make it Ubi-1 The 3' end of the promoter is driven by it. As in Example 1, Agrobacterium-mediated transformation was used to obtain an overexpression transgenic strain with japonica rice variety Kitaake as the background. Using genomic DNA as a template, primers OsHCRF2-OE (Table 1) PCR amplification of exogenous CDS fragments to identify positive transgenic lines ( Figure 2 ).

[0044] Example 3 OsHCRF2 There are significant differences in the number of grains per panicle, number of filled grains, and panicle length between knockout and overexpression rice lines

[0045] Field phenotypic observations and agronomic trait statistics showed that compared with the wild type, OsHCRF2 After knockout, the number of grains per ear and the number of filled grains increased significantly, and the ear length increased ( Figure 3 and Figure 4 ), the effective tillering number is not much different ( Figure 5 ), thousand-grain weight slightly decreased ( Figure 5 ). OsHCRF2 The number of grains per panicle, number of filled grains, panicle length and number of effective tillers in overexpressed rice were significantly reduced ( Figure 3-5 ), thousand-grain weight slightly decreased ( Figure 5 ).

[0046] Example 4 OsHCRF2 Knockout rice lines have advanced heading and maturity

[0047] Field phenotypic observations, OsHCRF2 Heading period of knockout rice lines ( Figure 6 ) and maturity ( Figure 7 ) are in advance.

Claims

1. Application of rice OsHCRF2 functional protein and its encoding gene in rice breeding, characterized in that: The amino acid sequence of the rice OsHCRF2 functional protein is shown in SEQ ID No: 5 in the sequence list, and the nucleotide sequence of the gene is shown in SEQ ID No: 1 in the sequence list; The OsHCRF2 functional protein and its encoding gene are used to regulate the number of grains per ear and the number of filled grains; The OsHCRF2 functional protein and its encoding gene are used to regulate spike length; The OsHCRF2 functional protein and its encoding gene are used to regulate the heading period and the maturity period.

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