Cloning and Breeding Application of Novel Semi-dominant Dwarf Gene OsSD-D1 in Rice

Abstract

The present application relates to the field of plant genetic breeding and molecular biology, and particularly relates to cloning, function verification and application in rice dwarf breeding of a rice semi-dominant dwarf gene OsSD-D1 Oryza sativa Semi-Dominant- Drawf1 .​

Description

Technical Field

[0001] This invention relates to the fields of plant genetics and breeding and molecular biology, and particularly to a semi-dominant dwarf gene in rice. OsSD-D1 ( Oryza sativa Semi-Dominant-Drawf1 Cloning, functional verification and application of ) in rice dwarfing breeding. Background Technology rice sd1 It is a recessive gene, which means that to express the dwarf trait, a homozygous mutation site must be obtained. In hybrid rice breeding, in order to obtain homozygous dwarf hybrid rice, both the male and female parents need to undergo multiple generations of screening and identification, which greatly prolongs the breeding cycle and increases the breeding cost.

[0002] Dominant dwarfing genes have a dominant inheritance effect, and the dwarf trait can be expressed in the heterozygous state. This can greatly shorten the time for fixing the dwarfing trait in hybrid rice breeding and improve breeding efficiency. Summary of the Invention

[0003] This patent discovers, clones, and verifies a novel semi-dominant dwarfing gene in rice derived from EMS mutagenesis. OsSD-D1 ( Oryza sativa Semi-Dominant-Drawf1 They also developed a complete set of molecular breeding application schemes based on this gene. This technology can effectively reduce plant height and enhance lodging resistance, while having no significant negative impact on important agronomic traits and yield per plant of rice. It provides a brand-new gene resource and solution for overcoming the existing breeding bottlenecks based on the sd1 gene.

[0004] The specific technical solution is as follows: 1. A novel semi-dominant dwarfing gene in rice OsSD-D1, Its features ， Its nucleotide sequence is shown in Seq ID No. 1.

[0005] 2. The above-mentioned novel semi-dominant dwarfing gene in rice OsSD-D1 The coding region sequence is characterized in that its nucleotide sequence is as shown in Seq ID No. 5; compared with the coding region sequence of the LOC_Os08g07740 gene on chromosome 8 of the wild-type Nipponbare genome as shown in Seq ID No. 9, the 196th base is mutated from G to A, and the corresponding amino acid codon is changed from alanine to threonine, constituting a single base mutation.

[0006] 3. A method for constructing OsSD-D1 The primer pair for the gene complementation vector is characterized by having nucleotide sequences as shown in Seq ID No. 6 and Seq ID No. 7; Seq ID No. 6 Upstream primer: 5'-cggggatcctctagagtcgacGAAAGGTGGACACCGACGATG-3'; Seq ID No. 7 Downstream primer: 5'-cttgcatgcctgcaggtcgacTGCAAAGTAATGGAACGTTGGA-3'; The underlined areas represent SalI restriction sites.

[0007] 4. A kind OsSD-D1 A gene complementation vector, characterized in that it carries the nucleotide sequence shown in Seq ID No. 8 on a backbone expression vector.

[0008] 5. Preferably, the OsSD-D1 The backbone vector for the gene complementation vector is pCAMBIA1300.

[0009] 6. A molecular breeding method for improving plant height of rice, characterized in that it includes precise gene editing of the target rice material, such that the coding region of the gene LOC_Os08g07740 on chromosome 8 is mutated from G to A at position 196 as shown in the nucleotide sequence as shown in SeqID No. 9, so that the protein encoded by the gene is mutated at the corresponding amino acid site to obtain dwarf material; The plant height improvement refers to dwarfing and lodging resistance.

[0010] 7. Preferably, the molecular breeding method includes the following steps: (1) The LOC_Os08g07740 gene is edited by introducing the vector described in claim 4 or 5 into the target rice material through transgenic operation; (2) The transgenic material is cultivated and screened to obtain transgenic positive T0 seedlings with the target rice material as the background.

[0011] This invention obtained a semi-dominant dwarf mutant from a mutant library obtained by chemical mutagenesis of the japonica rice variety Nipponbare using ethyl methane sulfonate (EMS), and identified it as follows: ossd-d1 A plant height survey of this mutant showed that... ossd-d1 The mutant had an average plant height of 90 cm, while the Nipponbare plant height was 110 cm, indicating that... ossd-d1 The plant height was 20 cm shorter than the control group, Nipponbare. ossd-d1The mutant was crossed with wild-type Nipponbare, and then self-crossed. Analysis of the plant height in the F1 and F2 progeny populations revealed that the dwarfing trait was controlled by a pair of semi-dominant genes. Whole-genome sequencing and MutMap analysis of the F2 population identified the LOC_Os08g07740 gene on chromosome 8. OsSD-D1 As shown in Seq ID No.1, its coding region contains a high-variance SNP site ( Figure 2 B), which is converted from guanine (G) to adenine (A) ( Figure 2 C).

[0012] Based on the above findings, this invention verifies the application of this gene in improving rice plant height and protects the mutant gene, along with the corresponding molecular breeding methods and transgenic vectors. Transgenic experiments demonstrate that the transgenic plants obtained are positive. ossd-d1-c Plant height and mutant ossd-d1 The same height indicates ossd-d1 The LOC_Os08g07740 mutation significantly reduced plant height. Furthermore, measurements of agronomic and yield traits revealed that the wild type and... ossd-d1 There were no significant differences in tiller number, flag leaf length, leaf width, primary branch number, and thousand-grain weight between the two varieties, and the yield per plant was also not significantly different, meaning it did not affect other agronomic traits and yield of the rice varieties. This mutant gene is very suitable for application in rice plant height improvement breeding. Attached Figure Description

[0013] Figure 1 . ossd-d1 Phenotypic and plant height statistics; Figure 2 . ossd-d1 Gene cloning; Figure 3 Functionally complementary vector pCAMBIA1300- ossd-d1-C Atlas; Figure 4 Functional verification plants ossd-d1-C Mutation site sequencing peak diagram; Figure 5 .wild type, ossd-d1 and ossd-d1-c Plant height comparison; Figure 6 .wild type and ossd-d1 Statistics on agronomic traits of mutants Detailed Implementation The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0014] Example 1. The Discovery of Genes From the mutant library obtained by chemically mutagenesis of the japonica rice variety Nipponbare using ethyl methane sulfonate (EMS), a semi-dominant dwarf mutant was screened and identified, and named [name missing]. ossd-d1 .

[0015] ossd-d1 Plant height survey statistics A survey conducted in Chengdu, Sichuan in 2022 revealed that... ossd-d1 The mutant had an average plant height of 90 cm, while the Nipponbare plant height was 110 cm, indicating that... ossd-d1 The plant height was 20 cm lower than that of the control group, Nipponbare. Figure 1 The overall trend of the statistical results for the following three years (2023-2025) is consistent with that of 2022.

[0016] Example 2. ossd-d1 Genetic analysis of mutant traits and MutMap cloning 2.1 ossd-d1 Genetic analysis of mutant traits Table 1 ossd-d1 plant height segregation ratio of F2 generation after hybridization with wild type

[0017] When df=2, x20.05(2)=5.99.

[0018] Will ossd-d1 The plants were hybridized with wild-type Nipponbare, and then self-pollinated. The plant height of the F1 and F2 progeny populations was analyzed.

[0019] The results showed that ossd-d1 The F1 generation plants obtained by crossing with wild-type Nipponbare exhibited plant height characteristics intermediate between the wild type and... ossd-d1 Among them, three plant height phenotypes segregated, namely wild type, intermediate type and dwarf type, with a wild type: intermediate type: dwarf type segregation ratio of 1:2:1 (Table 1), indicating that the dwarf trait of this mutant is controlled by a pair of semi-dominant genes.

[0020] exist ossd-d1 From the F2 generation population produced by crossing with WT, leaves from 20-30 dwarf plants were selected and mixed for whole-genome sequencing and MutMap analysis. The results revealed the presence of the LOC_Os08g07740 gene on chromosome 8. OsSD-D1 As shown in Seq ID No.1, its coding region contains a high-variance SNP site ( Figure 2 B), guanine (G) is converted to adenine (A) ( Figure 2 C).

[0021] [Seq ID No.1 LOC_Os08g07740 gene ( OsSD-D1 )】 2.2 ossd-d1 Sequencing validation of the LOC_Os08g07740 variant site The gene was retrieved using data from the National Rice Data Center, according to Example 1. ossd-d1 Gene design and synthesis of a pair of primers, 1F / 1R, spanning the mutation site, for... ossd-d1 Sequencing validation of the LOC_Os08g07740 variant site: Upstream primer 1F (Seq ID No. 2): 5'-TATGGGCACTTGCTGAGC-3'; Downstream primer 1R (Seq ID No. 3): 5'-GCGGTAGCGGTTGAGGTA-3'. With mutant ossd-d1 Using genomic DNA as a template, PCR amplification was performed. OsSD-D1 The gene fragment (Seq ID No. 4) was compared and analyzed with the CDS sequence of the same gene from Nipponbare (Seq ID No. 9), and it was found that... Figure 2 As shown, in the mutant, this OsSD-D1 gene A single-base mutation occurred at position 196 of the CDS (such as Seq ID No. 5), where a G was changed to an A. The corresponding codon encodes an amino acid that changes from alanine to threonine, suggesting that this gene may be responsible for the mutation. ossd-d1 A mutant gene that causes stunted plant height. This mutation at this site leads to semi-dominant dwarfing in rice, and it is a novel functional locus that has not been reported to date.

[0022] Seq ID No. 4 OsSD-D1 gene fragment containing the mutation site Tatgggcacttgctgagcccggtgggcagcccgccgttggacaacgagtccggcgaggcggcggcggcggctgcggctggcggcggcggctgcgggagcagcgccgggtatgtcgtctacggcggcggcggcggtggggactcgccggcgaaggagcaggacaggttcctgccgatcacgaacgtgagccgcatcatgaagcggtcgctgccggcgaacgccaagatctccaaggagtcgaaggagacggtgcaggagtgcgtgtcggagttcatcagcttcgttacaggcgaggcctccgacaagtgccagcgcgagaagcggaagaccatcaacggcgacgacctcctctgggccatgaccacgctggggttcgaggcctacgtcggcccgctcaagtcctacctcaaccgctaccgc In the mutant of Seq ID No.5 OsSD-D1 The CDS sequence of the gene Atgaagagtaggaagagctatgggcacttgctgagcccggtgggcagcccgccgttggacaacgagtccggcgaggcggcggcggcggctgcggctggcggcggcggctgcgggagcagcgccgggtatgtcgtctacggcggtggcggcggtggggactcgccggcgaaggagcaggacaggttcctgccgatcacgaacgtgagccgcatcatgaagcggtcgctgccggcgaacgccaagatctccaaggagtcgaaggagacggtgcaggagtgcgtgtcggagttcatcagcttcgttacaggcgaggcctccgacaagtgccagcgcgagaagcggaagaccatcaacggcgacgacctcctctgggccatgaccacgctggggttcgaggcctacgtcggcccgctcaagtcctacctcaaccgctaccgcgaggccgagggcgagaaggccgacgtgctcggcggcgccggcggcgccgccgcggcgcgccacggcgagggcggttgctgcggcggcggcggcggcggcgccgatggcgtcgtcatcgacgggcattacccgctcgccggcggcctgtcacactcacaccatggtcatcagcagcaggacggcggcggcgacgtcgggctcatgatgggcggcggcgacgccggcgtcgggtacaacgccggggccgggtcgacgacgacggcgttctacgcgccggcggcgacggcggcgtcagggaacaaggcgtactgcggcggcgacgggtcgagggtgatggagttcgagggcatcggcggcgaggaggagagcggaggcggcggcggcggcggcgagagggggttcgccggccacctccatggcgtgcaatggtttagactaaagaggaatactaattag Seq ID No.9 CDS of LOC_Os08g07740 gene in Nipponbare Atgaagagtaggaagagctatgggcacttgctgagcccggtgggcagcccgccgttggacaacgagtccggcgaggcggcggcggcggctgcggctggcggcggcggctgcgggagcagcgccgggtatgtcgtctacggcggtggcggcggtggggactcgccggcgaaggagcaggacaggttcctgccgatcgcgaacgtgagccgcatcatgaagcggtcgctgccggcgaacgccaagatctccaaggagtcgaaggagacggtgcaggagtgcgtgtcggagttcatcagcttcgttacaggcgaggcctccgacaagtgccagcgcgagaagcggaagaccatcaacggcgacgacctcctctgggccatgaccacgctggggttcgaggcctacgtcggcccgctcaagtcctacctcaaccgctaccgcgaggccgagggcgagaaggccgacgtgctcggcggcgccggcggcgccgccgcggcgcgccacggcgagggcggttgctgcggcggcggcggcggcggcgccgatggcgtcgtcatcgacgggcattacccgctcgccggcggcctgtcacactcacaccatggtcatcagcagcaggacggcggcggcgacgtcgggctcatgatgggcggcggcgacgccggcgtcgggtacaacgccggggccgggtcgacgacgacggcgttctacgcgccggcggcgacggcggcgtcagggaacaaggcgtactgcggcggcgacgggtcgagggtgatggagttcgagggcatcggcggcgaggaggagagcggaggcggcggcggcggcggcgagagggggttcgccggccacctccatggcgtgcaatggtttagactaaagaggaatactaattag Example 3. ossd-d1 Semidominant function genetic verification Using the mutant ossd-d1Using the genome as a template, a fragment of the LOC_Os08g07740 gene was amplified, a complementary vector was constructed, and it was transformed into wild-type Nipponbare corn, resulting in 20 positive plants. The genetic analysis of these transgenic plants... OsSD-D1 Genetic testing was performed.

[0023] The specific steps are as follows: Step 1 ossd-d1-c Carrier construction Primers containing SalI restriction sites (Seq ID No. 6 and Seq ID No. 7) were designed and synthesized to amplify the mutant. ossd-d1 Artificial fragments were obtained from genomic DNA (Seq ID No. 8). Seq ID No. 6 is the upstream primer for constructing the vector; 5'-cggggatcctctagagtcgacGAAAGGTGGACACCGACGATG-3'; Seq ID No. 7 Downstream primers for constructing the vector: 5'-cttgcatgcctgcaggtcgacTGCAAAGTAATGGAACGTTGGA-3'; The vector pCAMBIA1300 was digested with SalI, and the artificial fragment (shown in Seq ID No. 8) was ligated into the vector pCAMBIA1300 to obtain the complementary vector, the structure of which is shown below. Figure 3 As shown; verified by single enzyme digestion and sequencing.

[0024] Seq ID No. 8 is an artificial fragment used to construct the vector. Step 2. Plant Transformation 1. The constructed complementary vector was introduced into Agrobacterium EHA105 (a product of Shanghai Weidi Biotechnology Co., Ltd., CAT#: AC1010) to obtain recombinant Agrobacterium.

[0025] 2. Recombinant Agrobacterium was cultured in YEP medium (containing 50 µg / ml kanamycin and 25 µg / ml rifampin) at 28°C with shaking at 150 rpm until OD was reached. 600 At room temperature, centrifuge at 10,000 rpm for 1 min, and resuspend the cells in infection buffer (replacing the sugar in N6 liquid medium with glucose and sucrose, with glucose and sucrose concentrations of 10 g / L and 20 g / L, respectively) and dilute to OD 1.0-2.0. 600 The concentration was 0.2, and the Agrobacterium infection solution was obtained.

[0026] 3. Remove the husks and thresh the mature seeds of the wild-type rice variety Nipponbare, place them in a 100 mL Erlenmeyer flask, add 70% (v / v) ethanol aqueous solution and soak for 30 seconds, then place them in 25% (v / v) sodium hypochlorite aqueous solution, shake at 120 rpm for 30 min to sterilize, rinse 3 times with sterile water, blot dry with filter paper, then place the seed embryo-side down on N6 solid culture medium and incubate in the dark at 28℃ for 4-6 weeks to obtain rice callus.

[0027] 4. After completing step 3, soak the rice callus in Agrobacterium infection solution A (Agrobacterium infection solution A is a liquid obtained by adding acetosyringone to Agrobacterium infection solution, and the amount of acetosyringone added is such that the volume ratio of acetosyringone to Agrobacterium infection solution is 25 µl: 50 ml) for 10 min. Then, place it on a petri dish (containing about 200 ml of infection solution without Agrobacterium) lined with two layers of sterile filter paper and incubate in the dark at 21°C for 1 day.

[0028] 5. Place the rice callus obtained in step 4 onto a recovery culture medium and incubate in the dark at 25-28℃ for 3 days.

[0029] 6. Take the rice callus obtained in step 5, place it on the screening medium, and incubate it in the dark at 28°C for 2 weeks.

[0030] 7. Take the rice callus obtained in step 6 and place it on the screening medium again. Incubate it in the dark at 28°C for 2 weeks to obtain resistant rice callus.

[0031] 8. Take the rice resistant callus obtained in step 7 and place it on the differentiation medium. Culture it at 25°C under light for about 1 month. Then, transfer the differentiated seedlings to the rooting medium and culture them at 25°C under light for 2 weeks to obtain rice T0 seedlings.

[0032] Step 3. Sanger sequencing detection Genomic DNA was extracted from the obtained rice T0 seedlings and used as a template. PCR amplification was performed using primer pair 1F (Seq ID No. 2) 5'-TATGGGCACTTGCTGAGC-3' and primer 1R (Seq ID No. 3) 5'-GCGGTAGCGGTTGAGGTA-3' to obtain the PCR amplification product. The PCR amplification product was subjected to agarose gel electrophoresis, and then the following judgment was made: If the PCR amplification product contained a DNA fragment of approximately 420 bp (e.g., the length of Seq ID No. 4), the result was determined as follows: The samples were then sent to a biotechnology company for sequencing. If the mutation site showed a bimodal distribution, the corresponding rice T0 seedling was identified as a positive T0 seedling. If the mutation site in the PCR amplification product did not show a bimodal distribution, the corresponding rice T0 seedling was identified as a negative T0 seedling.

[0033] Results Analysis All PCR amplification products were sequenced, and the sequencing results showed that positive plants were... ossd-d1 The mutation sites show a bimodal distribution, with wild-type Nipponbare and [other variants present]. ossd-d1 Two bases, such as Figure 4 As shown, this demonstrates that a background image of a sunny Japanese day was successfully obtained. ossd-d1-c Positive-positive plants.

[0034] Example 4. ossd-d1 Survey of transgenic plant height For the positive plants obtained after transformation ossd-d1 - c and mutants ossd-d1 The plant height was statistically analyzed. The results showed that the positive plants... ossd-d1-c Plant height and mutant ossd-d1 The same height indicates ossd-d1 The LOC_Os08g07740 mutation significantly reduced plant height. Figure 5 AE).

[0035] Example 5. Wild type and ossd-d1 Agronomic traits and yield survey Wild-type Nippon Haruwa was grown under Chengdu field conditions. ossd-d1 When sown at the same time, measurements of agronomic and yield traits revealed that wild-type and ossd-d1 There were no significant differences in the number of tillers, sword leaf length, leaf width, primary branch length, and thousand-grain weight between the two species, and there was also no significant difference in yield per plant between them. Figure 6 .

[0036] In summary, this invention has fully demonstrated through complementary experiments that... ossd-d1 The influence of genes on plant height phenotype was investigated and demonstrated. ossd-d1 The dwarfing phenotype is caused by OsSD-D1 A single base mutation causes a change in a single amino acid. Therefore, for the target material OsSD-D1 The gene is edited to change guanine (G) to adenine (A), causing a base substitution in its CDS region. ossd-d1 This method can quickly obtain dominant rice breeding materials with dwarfed plant height and apply them to rice breeding.

Claims

1. A novel semi-dominant dwarfing gene of rice OsSD-D1, characterized in that ， the nucleotide sequence of which is shown in Seq ID No.

1.

2. The coding region sequence of the novel semi-dominant dwarfing gene of rice as described above OsSD-D1 characterized in that, Its nucleotide sequence is shown in Seq ID No. 5; compared with the coding region sequence of the LOC_Os08g07740 gene on chromosome 8 of the wild-type Nipponbare genome, as shown in Seq ID No. 9, the 196th base was mutated from G to A, and the corresponding amino acid codon was changed from alanine to threonine, constituting a single base mutation.

3. A method for constructing OsSD-D1 Primer pairs for gene complementation vectors are characterized by, Its nucleotide sequence is shown in Seq ID No. 6 and Seq ID No. 7; Seq ID No. 6 Upstream primer: 5'-cggggatcctctagagtcgacGAAAGGTGGACACCGACGATG-3'; Seq ID No. 7 Downstream primer: 5'-cttgcatgcctgcaggtcgacTGCAAAGTAATGGAACGTTGGA-3'; 4. An OsSD-D1 gene complementary vector, characterized in that, The backbone expression vector carries the nucleotide sequence shown in Seq ID No.

8.

5. The OsSD-D1 gene complementation vector according to claim 4, wherein the backbone vector is pCAMBIA1300.

6. A molecular breeding method for improving plant height of rice, characterized by, This includes precise gene editing of the target rice material, causing the coding region of the LOC_Os08g07740 gene on chromosome 8, as shown in the nucleotide sequence as shown in Seq ID No. 9, to mutate the 196th base from G to A, so that the protein encoded by this gene mutates at the corresponding amino acid site, thus obtaining dwarf material; The plant height improvement refers to dwarfing and lodging resistance.

7. The molecular breeding method according to claim 6, comprising the following steps: (1) The LOC_Os08g07740 gene is edited by introducing the vector described in claim 4 or 5 into the target rice material through transgenic operation; (2) The transgenic material is cultivated and screened to obtain transgenic positive T0 seedlings with the target rice material as the background.

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