Dicotyledon parthenogenesis inducing gene rwp and its application

Abstract

This invention discloses a gene for inducing parthenogenesis in dicotyledonous plants. RWP Its applications, specifically, the parthenogenesis-inducing gene in dicotyledonous plants. RWP This invention is the first to discover and verify its application in cultivating haploid or haploid induction lines of dicotyledonous plants or improving haploid induction ability. RWP This gene family possesses parthenogenetic haploid induction function in dicotyledonous plants, filling a gap in the list of parthenogenetic haploid induction genes in dicotyledonous plants. This invention lays an important foundation for broadening the application of haploid breeding technology in dicotyledonous plants and elucidating the biological mechanism of parthenogenetic haploid formation. Given the widespread use of haploid breeding technology in the current breeding industry, this invention has broad application prospects in the field of bio-breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to a gene for inducing parthenogenesis in dicotyledonous plants. RWP And its applications. Background Technology

[0002] Plant haploid induction technology has significant value in crop breeding. It accelerates the breeding process by rapidly generating completely homozygous double haploid systems. These double haploid systems originate from the doubling of the genome of haploid plants, directly yielding completely homozygous genotypes. Traditionally, haploid induction relied on in vitro culture of stamen or pistil tissue containing gametes to regenerate haploid plants, but this method is inefficient and genotype-dependent. In recent years, in vivo haploid induction technology has been implemented in various crops, primarily through two different pathways: The first pathway utilizes haploid induction lines, eliminating abnormal genomes from either the fertilized egg or sperm cell, thus producing haploids carrying only chromosomes from the other parent. The second pathway involves directly expressing embryogenesis triggering factors in egg cells, causing the egg cells to develop into haploid plants without sperm fertilization, i.e., parthenogenesis.

[0003] Currently, parthenogenesis has been successfully used to achieve efficient haploid induction in monocotyledonous plants. However, genes that have been successfully induced to produce haploids through parthenogenesis in monocotyledonous plants are not applicable to dicotyledonous plants. This greatly limits the application of parthenogenesis in haploid breeding of dicotyledonous plants. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a gene for inducing parthenogenesis in dicotyledonous plants. RWP The invention also describes a technical solution for inducing haploids to address the technical challenges of inducing parthenogenetic haploids in dicotyledonous plants.

[0005] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0006] This invention provides a gene for inducing parthenogenesis in dicotyledonous plants. RWP Application in cultivating haploids or haploid induction lines of dicotyledonous plants or improving haploid induction ability, wherein the dicotyledonous plant parthenogenesis induction gene is CitRWP, AbRWP, ZbRWP, MiRWP, CpRWP, BoRWP, AtRWPsyn, AtRWPsyn-N1, AtRWPsyn-N2 Any one of them; their corresponding nucleotide sequences are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, and SEQ ID No.9, respectively.

[0007] This invention also provides a method for preparing a dicotyledonous haploid inducible line. The method involves introducing the complete or partial sequence of a dicotyledonous parthenogenesis-inducing gene into a dicotyledonous plant for ectopic expression, resulting in a transgenic plant, which is a dicotyledonous haploid inducible line. The dicotyledonous parthenogenesis-inducing gene... RWP for CitRWP, AbRWP, ZbRWP, MiRWP, CpRWP, BoRWP, AtRWPsyn, AtRWPsyn-N1, AtRWPsyn-N2 Any one of them; their corresponding nucleotide sequences are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, and SEQ ID No.9, respectively.

[0008] Furthermore, the dicotyledonous plant is any one of Arabidopsis thaliana, tomato, citrus, sage, Sichuan pepper, mango, papaya, and cabbage.

[0009] Furthermore, the importation involves introducing a recombinant expression vector containing the complete or partial sequence of a parthenogenesis-inducing gene in dicotyledonous plants into dicotyledonous plants.

[0010] Furthermore, the proteins encoded by the parthenogenesis-inducing genes of the dicotyledonous plants are any one of the following: protein CitRWP, protein AbRWP, protein ZbRWP, protein MiRWP, protein CpRWP, protein BoRWP, protein AtRWPsyn, protein AtRWPsyn-N1, and protein AtRWPsyn-N2; their corresponding amino acid sequences are as shown in SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18, respectively.

[0011] Furthermore, the recombinant expression vector is based on the expression vector FASTRFP-pEC-ccdB-NOS, with the complete or partial sequence of the parthenogenesis-inducing gene of dicotyledonous plants inserted into it, and the expression vector FASTRFP-pEC-ccdB-NOS contains the oocyte-specific expression promoters EC1.2-EC1.1.

[0012] Furthermore, the recombinant expression vector is introduced into dicotyledonous plants via Agrobacterium-mediated genetic transformation, gene gun method, or PEG-mediated protoplast transformation.

[0013] Furthermore, the Agrobacterium-mediated Agrobacterium contains a recombinant expression vector, and the Agrobacterium is Agrobacterium GV3101.

[0014] The beneficial effects of this invention are:

[0015] (1) This invention is the first to discover and verify that the RWP gene family has the function of inducing parthenogenetic haploids in dicotyledonous plants, filling the gap in parthenogenetic haploid induction genes in dicotyledonous plants.

[0016] (2) This invention provides nine RWP genes from different plant species, including CitRWP from citrus, AbRWP from sage, ZbRWP from Sichuan pepper, MiRWP from mango, CpRWP from papaya, BoRWP from cabbage, AtRWPsyn from Arabidopsis thaliana but through synonymous mutation, and AtRWPsyn-N1 and AtRWPsyn-N2 from Arabidopsis thaliana with a partial sequence of synonymous mutation combined with the original partial sequence of Arabidopsis thaliana, providing rich gene resources for application in different dicotyledonous plants.

[0017] (3) This invention demonstrates that these RWP genes can induce haploids in Arabidopsis thaliana through oocyte-specific expression, providing valuable gene resources for haploid induction in dicotyledonous plants through genetic engineering.

[0018] (4) This invention establishes a complete technical system from gene cloning, vector construction, plant transformation to haploid identification, providing a technical solution for the application of RWP gene in dicotyledonous crop breeding.

[0019] (5) This invention lays an important foundation for expanding the application of haploid breeding technology in dicotyledonous plants and revealing the biological mechanism of parthenogenetic haploid formation. Given the widespread use of haploid breeding technology in the current breeding industry, this invention has broad application space and prospects in the field of biological breeding. Attached Figure Description

[0020] Figure 1 for pEC::CitRWP, pEC::AbRWP, pEC::ZbRWP, pEC::MiRWP, pEC::CpRWP, pEC:: BoRWP, pEC::AtRWPsyn, pEC::AtRWPsyn-N1, pEC::AtRWPsyn-N2 A schematic diagram of the carrier structure;

[0021] Figure 2 For diploid Col 0 wild-type Arabidopsis and pEC::CitRWP, pEC::AbRWP, pEC::ZbRWP, pEC:: MiRWP, pEC::CpRWP, pEC::BoRWP, pEC::AtRWPsyn, pEC::AtRWPsyn-N1, pEC::AtRWPsyn-N2 Comparison of haploid plants.

[0022] Figure 3 For diploid Col 0 wild-type Arabidopsis and pEC::CitRWP, pEC::AbRWP, pEC::ZbRWP, pEC:: MiRWP, pEC::CpRWP, pEC::BoRWP, pEC::AtRWPsyn, pEC::AtRWPsyn-N1, pEC::AtRWPsyn-N2 Comparison of flow cytometry detection of haploids;

[0023] In the figure, A is a flow cytometry diagram of diploid Col 0 wild-type Arabidopsis thaliana;

[0024] B is pEC::CitRWP A schematic diagram of flow cytometry detection of the generated haploids;

[0025] C for pEC::AbRWP A schematic diagram of flow cytometry detection of the generated haploids;

[0026] D is pEC::ZbRWP A schematic diagram of flow cytometry detection of the generated haploids;

[0027] E is pEC::MiRWP A schematic diagram of flow cytometry detection of the generated haploids;

[0028] F is pEC::CpRWP A schematic diagram of flow cytometry detection of the generated haploids;

[0029] G is pEC::BoRWP A schematic diagram of flow cytometry detection of the generated haploids;

[0030] H is pEC::AtRWPsyn A schematic diagram of flow cytometry detection of the generated haploids;

[0031] I am pEC::AtRWPsyn-N1 A schematic diagram of flow cytometry detection of the generated haploids;

[0032] J is pEC::AtRWPsyn-N2 A schematic diagram of flow cytometry detection of the generated haploids.

[0033] Figure 4 For diploid Col 0 wild-type Arabidopsis and pEC::CitRWP, pEC::AbRWP, pEC::ZbRWP, pEC:: MiRWP, pEC::CpRWP, pEC::BoRWP, pEC::AtRWPsyn, pEC::AtRWPsyn-N1, pEC::AtRWPsyn-N2 A comparison chart of chromosome counts for haploids. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0035] Example 1: Plant parthenogenesis-inducing gene RWP get

[0036] 1. CitRWP Acquisition of genes

[0037] Through sweet oranges ( Citrus sinensisUsing the reference genome sequence of the sweet orange genome (Citrussinensis genome assembly ASM1810434v1 - NCBI - NLM) (version 2.0), primer pairs were designed:

[0038] CitRWP primer F: 5'-ATGGCGGATCCTAGGGCC-3', as shown in SEQ ID No. 19.

[0039] CitRWP primer R: 5'-TCATCTAGTGAGTCCAGCACA-3', as shown in SEQ ID No. 20;

[0040] Using sweet orange ovule cDNA as a template, PCR amplification was performed using Vazyme Phanta Max Super-Fidelity DNApolymerase.

[0041] Reaction system (50 μl): 2×Phanta Max Buffer 25 μl, dNTP Mix 1 μl, CitRWP primer F 2 μl, CitRWP primer R 2 μl, Phanta Max Super-Fidelity DNA Polymerase 1 μl, template 1 μl, ddH2O 18 μl.

[0042] Reaction program: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 60 s, 35 cycles; 72℃ for 5 min.

[0043] After obtaining the PCR products, agarose gel electrophoresis was performed, and the target fragment was recovered using the BIOMIGA gel extraction kit. The fragment was ligated into the pMD19-T vector (TaKaRa), transformed into *E. coli* DH5α competent cells, and positive clones were selected for sequencing. CitRWP Gene( CitRWP The full-length CDS sequence is shown in SEQ ID No. 1, and the encoded protein amino acid sequence is shown in SEQ ID No. 10.

[0044] 2. AbRWP Acquisition of genes

[0045] Through the wine cake basket ( Atalantia buxifolia Using the reference genome sequence (Atalantia buxifolia genome assembly ASM201393v1 - NCBI - NLM) (genome assembly version 1.0), primer pairs were designed:

[0046] AbRWP primer F: 5'-ATGGCAGATCCTAGGGCCAT-3', as shown in SEQ ID No. 21.

[0047] AbRWP primer R: 5'-TCATCTAGTGAGTCCAGCACA-3', as shown in SEQ ID No. 22;

[0048] Using *Gnaphalium affine* pollen cDNA as a template, PCR amplification, cloning, and sequencing were performed according to the same method described above to obtain... AbRWP Gene( AbRWP The full-length CDS sequence is shown in SEQ ID No. 2, and the encoded protein amino acid sequence is shown in SEQ ID No. 11.

[0049] 3. ZbRWP Acquisition of genes

[0050] Through Sichuan pepper ( Zanthoxylum bungeanum Using the reference genome sequence (sp version of the Sichuan pepper genome (Item - The chromosome-level genome assembly of Sichuan pepper - figshare - Figshare)), primer pairs were designed:

[0051] ZbRWP primer F: 5'-ATGGCAGATCCTATGGCCAT-3', as shown in SEQ ID No. 23.

[0052] ZbRWP primer R: 5'-TCATCTAGAGAGTCCAGCACAT-3', as shown in SEQ ID No. 24;

[0053] Using the reference genome sequence of Sichuan pepper as a reference, a biotechnology company was commissioned to synthesize the ZbRWP gene. Using the synthesized plasmid as a template, PCR amplification, cloning, and sequencing were performed according to the same methods described above, resulting in... ZbRWP Gene( ZbRWP The full-length CDS sequence is shown in SEQ ID No. 3, and the encoded protein amino acid sequence is shown in SEQ ID No. 12.

[0054] 4. MiRWP Acquisition of genes

[0055] Through mango ( Mangifera indicaUsing the reference genome sequence (Mango genome TA4 version (Mangifera indica cultivar Tommy-Atkins, whole genome shotgun sequencin - Nucleotide - NCBI)), primer pairs were designed:

[0056] MiRWP primer F: 5'-ATGGCAGATTCCAAGTCCAAT-3', as shown in SEQ ID No. 25.

[0057] MiRWP primer R: 5'-TCATGATTTTCTGCCAGTACAAG-3', as shown in SEQ ID No. 26;

[0058] Using the reference genome sequence of mango as a reference, a biotechnology company was commissioned to synthesize the MiRWP gene. Using the synthesized plasmid as a template, PCR amplification, cloning, and sequencing were performed according to the same methods described above, resulting in... MiRWP Gene( MiRWP The full-length CDS sequence is shown in SEQ ID No. 4, and the encoded protein amino acid sequence is shown in SEQ ID No. 13.

[0059] 5. CpRWP Acquisition of genes

[0060] Through papaya ( Carica papaya Using the reference genome sequence (Papaya genome sunup version (Genome Warehouse)), primer pairs were designed:

[0061] CpRWP primer F: 5'-ATGGCGGATCCTGCAGCA-3', as shown in SEQ ID No. 27.

[0062] CpRWP primer R: 5'-TCATGCACCTCCAGCACG-3', as shown in SEQ ID No. 28;

[0063] Using the reference genome sequence of papaya as a reference, a biotechnology company was commissioned to synthesize the CpRWP gene. Using the synthesized plasmid as a template, PCR amplification, cloning, and sequencing were performed according to the same methods described above, resulting in... CpRWP Gene( CpRWP The full-length CDS sequence is shown in SEQ ID No. 5, and the encoded protein amino acid sequence is shown in SEQ ID No. 14.

[0064] 6. BoRWP Acquisition of genes

[0065] Through cabbage ( Brassica oleracea Using the reference genome sequence (Boleracea HDEM version of the Brassica oleracea genome (BoleraceaHDEM | Brassica oleracea | Assembly)), primer pairs were designed:

[0066] BoRWP primer F: 5'-ATGGCGGGTGATGATGAT-3', as shown in SEQ ID No. 29.

[0067] BoRWP primer R: 5'-TTAAGCGTATTTGAGTGCTTCA-3', as shown in SEQ ID No. 30;

[0068] Using the reference genome sequence of cabbage as a reference, a biotechnology company was commissioned to synthesize the BoRWP gene. Using the synthesized plasmid as a template, PCR amplification, cloning, and sequencing were performed according to the same methods described above, resulting in... BoRWP Gene( BoRWP The full-length CDS sequence is shown in SEQ ID No. 6, and the encoded protein amino acid sequence is shown in SEQ ID No. 15.

[0069] 7. AtRWPsyn, AtRWPsyn-N1, AtRWPsyn-N2 Acquisition of genes

[0070] Through Arabidopsis thaliana ( Arabidopsis thaliana Using the reference genome sequence of AtRWP (Arabidopsis genome TAIR10 version (TAIR-Arabidopsis)), synonymous mutations were performed on the full-length CDS sequence of AtRWP, and primer pairs were designed as follows:

[0071] AtRWPsyn primer F: 5'-ATGGCGGGCGATGATCCG-3', as shown in SEQ ID No. 31.

[0072] AtRWPsyn primer R: 5'-TTACATATTCTTAAGGATGTCGG-3', as shown in SEQ ID No. 32;

[0073] AtRWPsyn-N1 primer F: 5'-ATGGCGGGCGATGATCCG-3', as shown in SEQ ID No. 33.

[0074] AtRWPsyn-N1 primer R: 5'-TTACATATTTTTGAGTATGTCACTGC-3', as shown in SEQ ID No. 34;

[0075] AtRWPsyn-N2 primer F: 5'-ATGGCGGGCGATGATCCG-3', as shown in SEQ ID No. 35.

[0076] AtRWPsyn-N2 primer R: 5'-TTACATATTTTTGAGTATGTCACTGC-3', as shown in SEQ ID No. 36;

[0077] Using the reference genome sequence of Arabidopsis thaliana as a reference, a biotechnology company was commissioned to perform synonymous mutations on AtRWP. AtRWPsyn, AtRWPsyn-N1, AtRWPsyn-N2 Gene synthesis was performed, and using the gene synthesis plasmid as a template, PCR amplification, cloning, and sequencing were carried out according to the same method as described above to obtain... AtRWPsyn, AtRWPsyn-N1, AtRWPsyn-N2 ( AtRWPsyn, AtRWPsyn-N1, AtRWPsyn-N2 (Full-length CDS sequence). AtRWPsyn The nucleotide sequence is shown in SEQ ID No. 7, and the encoded protein amino acid sequence is shown in SEQ ID No. 16; AtRWPsyn-N1 The nucleotide sequence is shown in SEQ ID No. 8, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 17; AtRWPsyn-N2 The nucleotide sequence is shown in SEQ ID No. 9, and the encoded protein amino acid sequence is shown in SEQ ID No. 18.

[0078] Example 2 RWP Construction of gene oocyte-specific expression vector

[0079] Based on the expression vector FASTRFP-pEC-ccdB-NOS, GoldenGate was used to... CitRWP, AbRWP, ZbRWP, MiRWP, CpRWP, BoRWP, AtRWPsyn, AtRWPsyn-N1, AtRWPsyn-N2 The CDS fragments were inserted downstream of the EC promoter and between the NOS terminator, respectively, and expression was driven by the oocyte-specific expression promoters EC1.2-EC1.1. Figure 1 Electrophoresis and sequencing analysis confirmed the successful acquisition of the oocyte-specific expression vector for the RWP gene, which was named... pEC::CitRWP, pEC:: AbRWP, pEC::ZbRWP, pEC::MiRWP, pEC::CpRWP, pEC::BoRWP, pEC::AtRWPsyn, pEC:: AtRWPsyn-N1, pEC::AtRWPsyn-N2 The specific experimental steps are as follows:

[0080] 1. RWP Gene amplification:

[0081] according to RWP GoldenGate primers were designed based on the CDS sequence of the gene for PCR amplification. The primer sequences are as follows:

[0082] BsaI-AGAA-CitRWP primer F:

[0083] 5'-GTGGTCTCAAGAAATGGCGGATCCTAGGGCC-3', as shown in SEQ ID No. 37,

[0084] BsaI-GATC-CitRWP primer R:

[0085] 5'-GTGGTCTCAGATCTCATCTAGTGAGTCCAGCACA-3', as shown in SEQ ID No. 38.

[0086] BsaI-AGAA-AbRWP primer F:

[0087] 5'-GTGGTCTCAAGAAATGGCAGATCCTAGGGCCAT-3', as shown in SEQ ID No. 39,

[0088] BsaI-GATC-AbRWP primer R:

[0089] 5'-GTGGTCTCAGATCTCATCTAGTGAGTCCAGCACA-3', as shown in SEQ ID No. 40.

[0090] BsaI-AGAA-ZbRWP primer F:

[0091] 5'-GTGGTCTCAAGAAATGGCAGATCCTATGGCCAT-3', as shown in SEQ ID No. 41,

[0092] BsaI-GATC-ZbRWP primer R:

[0093] 5'-GTGGTCTCAGATCTCATCTAGAGAGTCCAGCACAT-3', as shown in SEQ ID No. 42.

[0094] BsaI-AGAA-MiRWP primer F:

[0095] 5'-GTGGTCTCAAGAAATGGCAGATTCCAAGTCCAAT-3', as shown in SEQ ID No. 43,

[0096] BsaI-GATC-MiRWP primer R:

[0097] 5'-GTGGTCTCAGATCTCATGATTTTCTGCCAGTACAAG-3', as shown in SEQ ID No. 44.

[0098] BsaI-AGAA-CpRWP primer F:

[0099] 5'-GTGGTCTCAAGAAATGGCGGATCCTGCAGCA-3', as shown in SEQ ID No. 45,

[0100] BsaI-GATC-CpRWP primer R:

[0101] 5'-GTGGTCTCAGATCTCATGCACCTCCAGCACG-3', as shown in SEQ ID No. 46.

[0102] BsaI-AGAA-BoRWP primer F:

[0103] 5'-GTGGTCTCAAGAAATGGCGGGTGATGATGAT-3', as shown in SEQ ID No. 47,

[0104] BsaI-GATC-BoRWP primer R:

[0105] 5'-GTGGTCTCAGATCTTAAGCGTATTTGAGTGCTTCA-3', as shown in SEQ ID No. 48.

[0106] BsaI-AGAA-AtRWPsyn primer F:

[0107] 5'-GTGGTCTCAAGAAATGGCGGGCGATGATCCG-3', as shown in SEQ ID No. 49,

[0108] BsaI-GATC-AtRWPsyn primer R:

[0109] 5'-GTGGTCTCAGATCTTACATATTCTTAAGGATGTCGG-3', as shown in SEQ ID No. 50.

[0110] BsaI-AGAA-AtRWPsyn-N1 primer F:

[0111] 5'-GTGGTCTCAAGAAATGGCGGGCGATGATCCG-3', as shown in SEQ ID No. 51,

[0112] BsaI-GATC-AtRWPsyn-N1 primer R:

[0113] 5'-GTGGTCTCAGATCTTACATATTTTTGAGTATGTCACTGC-3', as shown in SEQ ID No. 52.

[0114] BsaI-AGAA-AtRWPsyn-N2 primer F:

[0115] 5'-GTGGTCTCAAGAAATGGCGGGCGATGATCCG-3', as shown in SEQ ID No. 53,

[0116] BsaI-GATC-AtRWPsyn-N2 primer R:

[0117] 5'-GTGGTCTCAGATCTTACATATTTTTGAGTATGTCACTGC-3', as shown in SEQ ID No. 54.

[0118] Gene amplification was performed using Vazyme PhantaMax Super-Fidelity DNA polymerase to obtain PCR products. The reaction system was as follows (total volume 50 μL):

[0119]

[0120] The reaction program was as follows: 95℃ for 3 min; 95℃ for 15 s; 55℃ for 15 s; 72℃ for 60 s; 72℃ for 5 min; 35 cycles.

[0121] 2. PCR product detection and recovery: PCR products were detected by agarose gel electrophoresis. The target fragment was recovered using the BIOMIGA gel recovery kit.

[0122] 3. GolenGate digestion and ligation reaction: The GolenGate digestion and ligation reaction was performed using Thermofisher 10X T4 DNA ligase buffer, T4 DNA ligase, FastDigest Eco31I, and New England Biolabs rCutSmart™ buffer; the reaction system is as follows:

[0123]

[0124] The reaction program was: 16℃ for 10 min; 16℃ for 2 min; 37℃ for 2 min; 26 cycles;

[0125] 4. Transformation of GolenGate digestion and ligation products: Transform DH5α competent cells with the GolenGate digestion and ligation products, following the guidelines for molecular cloning experiments.

[0126] 5. Sequencing and Identification: After PCR identification of positive clones, sequencing analysis is performed. Successfully sequenced single clones undergo plasmid extraction, which is then used as the sequencing result. pEC::CitRWP, pEC::AbRWP, pEC::ZbRWP, pEC::MiRWP, pEC::CpRWP, pEC::BoRWP,pEC::AtRWPsyn, pEC::AtRWPsyn-N1, pEC::AtRWPsyn-N2 Vector; PCR and sequencing primers are as follows:

[0127] pEC primer F: 5'-GAGCTTCCCTCGTAATTACTTG-3', as shown in SEQ ID No. 55.

[0128] FASTRFP primer R: 5'-AGCTCAAGCTAAGCTTACCT-3', as shown in SEQ ID No. 56.

[0129] Example 3: Obtaining transgenic Arabidopsis plants

[0130] The built pEC::CitRWP, pEC::AbRWP, pEC::ZbRWP, pEC::MiRWP, pEC::CpRWP, pEC::BoRWP, pEC::AtRWPsyn, pEC::AtRWPsyn-N1, pEC::AtRWPsyn-N2 The vector was transformed into Agrobacterium strain GV3101.

[0131] Agrobacterium strain GV3101 carrying the target vector was streaked onto a plate and incubated upside down at 28°C for 2 days. The Agrobacterium was then resuspended in 5% sucrose solution to OD0.05. 600nm =0.6.

[0132] The vector was transferred into the full-blown Arabidopsis thaliana Col-0 using the dip-in infection method. The inflorescence of Arabidopsis thaliana was completely immersed in Agrobacterium for 1 minute and then removed. The Arabidopsis thaliana treated with Agrobacterium was covered with a black plastic bag and grown in the dark for 24 hours. Then it was placed in a light-cured culture room and grown for 3-4 weeks before harvesting.

[0133] T0 generation seeds were placed under a fluorescence microscope, and seeds with red fluorescence were selected and sown on MS medium. The seeds were then placed in a 22°C light incubator for one week. The plants that germinated from the red fluorescent seeds were then transplanted into pots filled with nutrient soil for planting and harvesting.

[0134] Leaves of Arabidopsis thaliana that had grown for two weeks were taken, and DNA was extracted using the CTAB method. Positive lines were identified using the PCR and sequencing primers in Example 2, and T1 generation transgenic Arabidopsis thaliana plants were obtained.

[0135] Example 4: Ploidy determination of transgenic Arabidopsis offspring

[0136] 1. Identification through plant morphological phenotype

[0137] A typical characteristic of haploid plants is their inability to undergo normal meiosis, leading to sterility. Therefore, the pods of haploid Arabidopsis thaliana fail to elongate. Morphological observations were conducted on the progeny of transgenic Arabidopsis thaliana. pEC::CitRWP, pEC:: AbRWP, pEC::ZbRWP, pEC::MiRWP, pEC::CpRWP, pEC::BoRWP, pEC::AtRWPsyn, pEC:: AtRWPsyn-N1, pEC::AtRWPsyn-N2 In all of their offspring, there were plants whose pods could not elongate. Figure 2 These plants exhibit typical haploid characteristics such as dwarfism, male sterility, and inability of pods to elongate.

[0138] 2. Identification was performed by detecting the DNA content in the leaves using flow cytometry.

[0139] The plants with pods that could not elongate, obtained in step 1 above, were subjected to flow cytometry analysis. The specific method is as follows:

[0140] Cell nuclei were extracted from young leaves of the plants to be tested, with diploid Col-0 wild-type Arabidopsis leaves as a control, and the signals were detected by flow cytometry.

[0141] First, the nuclear signal of diploid Col-0 wild-type Arabidopsis thaliana cells was detected, and the nuclear signal peaks of diploid cells were set around 50 and 100, since the genetic material in diploid cells is twice that in haploid cells.

[0142] Therefore, the nuclear signal peaks in haploid cells appear around 25 and 50. Flow cytometry confirmed that the plants with pods that could not elongate obtained in step 1 were all haploid. Figure 3 ).

[0143] 3. Identification by chromosome counting of pedicel cells

[0144] The pedicels of plants whose pods could not elongate, obtained in step 1 above, were digested with enzymes and stained with DAPI for chromosome counting. The specific method is as follows:

[0145] Fresh flower stalks were subjected to enzymatic hydrolysis by placing them in a mixture of cellulase and pectinase and incubating at 37°C for 2 hours.

[0146] After enzymatic digestion, wash the material several times with PBS buffer, transfer the material to a glass slide containing DAPI staining solution, and stain in the dark for 10-15 minutes.

[0147] Finally, the slides were mounted with anti-quenching mounting medium, and the chromosome number was counted under a fluorescence microscope. Diploid Col-0 wild-type Arabidopsis cells had 10 chromosomes, while haploid plant cells had only 5 chromosomes. Chromosome counting confirmed that the plants with pods that could not elongate obtained in step 1 were all haploid. Figure 4 ).

[0148] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for breeding Arabidopsis thaliana haploids or for breeding Arabidopsis thaliana haploid inducer lines or for increasing the haploid induction capacity of Arabidopsis thaliana, characterized in that: CitRWP a gene is used. The CitRWP nucleotide sequence of the gene is shown as SEQ ID No. 1; the application is to introduce the CitRWP complete sequence of the gene into Arabidopsis thaliana for ectopic expression.

2. A method for preparing an Arabidopsis thaliana haploid induction line, characterized by: The method is to CitRWP The complete gene sequence was introduced into Arabidopsis thaliana for ectopic expression, resulting in transgenic Arabidopsis plants, which are Arabidopsis haploid inducible lines; wherein, the... CitRWP The nucleotide sequence corresponding to the gene is shown in SEQ ID No.

1.

3. The method of claim 2, wherein: The introduction is to introduce into Arabidopsis thaliana a recombinant expression vector containing CitRWP the complete sequence of the gene.

4. The method of claim 2, wherein: The CitRWP The gene encodes a protein, the amino acid sequence of which is shown as SEQ ID No.

10.

5. The preparation method according to claim 3, characterized in that: The recombinant expression vector is a FASTRFP-pEC-ccdB-NOS expression vector into which a gene is inserted CitRWP the complete sequence of the gene, and the expression vector FASTRFP-pEC-ccdB-NOS contains an egg cell-specific expression promoter EC1.2-EC1.

1.

6. The production method according to claim 3 or 5, characterized by: The recombinant expression vector was introduced into Arabidopsis thaliana via Agrobacterium-mediated genetic transformation, gene gun method, or PEG-mediated protoplast transformation.

7. The method of claim 6, wherein: The Agrobacterium-mediated Agrobacterium contains a recombinant expression vector, and the Agrobacterium is Agrobacterium GV3101.

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