Method for efficiently obtaining apomictic plant line and related nucleic acid molecules and applications thereof

Abstract

The application discloses a method for efficiently obtaining a plant apomictic strain, comprising the following steps: A, constructing a first-embryo autonomous generation gene expression cassette E1 driven by a first egg cell specific promoter; B, constructing a parent-derived transcription factor gene expression cassette E2 driven by a second egg cell specific promoter; C, constructing a MiMe mutant gene editing vector expression cassette E3; D, introducing the above-mentioned E1, E2 and E3 linkage expression cassettes into a plant to obtain a transgenic plant; the expression cassette E1 induces the oocyte parthenogenesis to form a clone embryo while ensuring the seed setting rate of the clone plant; the gene expression cassette E2 combines the parthenogenesis efficient enhancer and the second embryo autonomous development gene to improve the induction rate of the clone embryo; and the MiMe mutant in the expression cassette E3 can replace the meiosis with mitosis. The application can efficiently obtain a clone embryo and ensure the seed setting rate of the clone plant.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and agricultural biotechnology, and in particular relates to a method for efficiently obtaining apomixis lines of plants. Background Technology

[0002] Apomixis refers to the process in which male and female cells in plants form clonal embryos without meiosis and nuclear fusion, thus producing cloned offspring that retain the superior traits of the maternal parent through seed production. Recent advancements in apomixis research have been made using molecular biology and genetic engineering techniques. In late 2018, Professor Venkatesan Sundaresan's team at the University of California, Davis, achieved apomixis in rice by editing three genes—PAIR1, REC8, and OSD1 (MiMe)—and combining them with the ectopic expression of the BBM1 gene in oocytes. In early 2019, Wang Kejian's team at the China National Rice Research Institute obtained apomixis material, Fix, in rice by editing four genes: PAIR1, REC8, OSD1 (MiMe), and MTL. In late 2022, Emmanuel Guiderdoni's team at the French Center for International Agricultural Research and Development (CIRAD) constructed an expression cassette for the ectopic expression of the OsBBM1 gene driven by an oocyte-specific promoter (AtEC1.2 / OsECA1) into a MiMe editing vector. This was simultaneously transformed into the indica hybrid rice BRS-CIRAD 302, resulting in apomixis lines with a cloning seed induction rate exceeding 95%, but with a seed setting rate of only 35%. In 2023, Wang Kejian's team at the China National Rice Research Institute used the oocyte-specific promoter AtDD45 to drive the ectopic expression of the BBM4 gene in oocytes. After combining this with a MiMe mutant, they obtained apomixis rice lines with a seed setting rate as high as 82%, but with a cloning seed induction rate of only 2%. In 2024, Professor Venkatesan Sundaresan's team at the University of California, Davis, co-expressed the OsWOX9A and BBM1 genes in oocytes, inducing a parthenogenetic frequency of up to 91% in rice. However, the seed setting rate of the transgenic plants after co-expression was only 49%. Although apomixis technology is developing rapidly in rice, it is difficult to obtain both high induction rates and high seed setting rates simultaneously, which limits the further application of apomixis technology. Summary of the Invention

[0003] The purpose of this invention is to provide an efficient method for obtaining apomixis lines of plants, as well as related nucleic acid molecules and their applications.

[0004] This invention is achieved as follows: A method for efficiently obtaining apomixis lines in plants, comprising the following steps:

[0005] A. Constructing the gene expression cassette E1, driven by a first oocyte-specific promoter for autonomous embryogenesis;

[0006] B. Constructing a second oocyte-specific promoter to drive the expression cassette E2 of paternally derived transcription factor genes;

[0007] C. Constructing the gene editing vector expression cassette E3 for the MiMe mutant;

[0008] D. Introduce the above E1, E2, and E3 linkage expression cassettes into plants to obtain transgenic plants;

[0009] The expression cassette E1 can induce parthenogenesis in oocytes to form cloned embryos, maintain plant fertility stability, and ensure the seed setting rate of cloned plants; the expression cassette E2 combines a highly efficient parthenogenesis enhancement factor with a gene for autonomous development of the second embryo to improve the cloned embryo induction rate; the expression cassette E3 is used to obtain the MiMe mutant, which achieves the substitution of meiosis with mitosis.

[0010] The expression cassette E1 in step A comprises the following elements from upstream to downstream: a first oocyte-specific promoter, a coding sequence of the first autogenic embryogenesis gene, and a terminator; the first oocyte-specific promoter is selected from AtDD45, Os03g0296600 pro, DCL2, AT1G74480.1, or ZmEAl pro; the first autogenic embryogenesis gene is BBM4; the terminator is selected from NOS, OCS, or PINII; the sequence of expression cassette E1 is shown in SEQ ID NO.1;

[0011] In step B, the expression cassette E2 comprises the following elements from upstream to downstream: a second oocyte promoter, a parthenogenetic enhancement factor, a coding sequence for a second embryo autonomous development gene, and a terminator; the second oocyte-specific promoter is selected from AtEC1.1, AtEC1.2, AtEC1.3, AtEC1.4, or AtEC1.5; the parthenogenetic enhancement factor is selected from OsWOX9A, OsWOX9B, OsWOX9C, or AtWOX9; the coding sequence for the second embryo autonomous development gene is selected from BBM1, WUS, SERK, LEC, CLAVATA, or MYB115; the terminator is selected from NOS, OCS, or PINII; the sequence of expression cassette E2 is shown in SEQ ID NO.2;

[0012] In step C, the expression cassette E3 includes the MiMe mutant gene. The gene edited by the MiMe mutant gene is a key gene for meiosis, enabling mitosis to replace meiosis. The genes edited by the MiMe mutant gene include OSD1, PAIR1, and REC8. The sequences of OSD1, PAIR1, and REC8 are shown in SEQ ID NO.3-SEQ ID NO.5, respectively.

[0013] A nucleic acid molecule comprising the nucleic acid sequences of expression cassettes E1, E2, and E3 as described in claim 1.

[0014] The application of the nucleic acid molecule in the cultivation of transgenic plants includes the following steps: introducing the nucleic acid molecule into a plant to obtain a transgenic plant.

[0015] The plants include monocotyledons and dicotyledons.

[0016] A recombinant vector containing the aforementioned nucleic acid molecules.

[0017] The application of the aforementioned recombinant vector,

[0018] 1) Preparation of apomixis;

[0019] 2) Obtaining parthenogenetic clone seeds;

[0020] 3) Fixation of hybrid vigor.

[0021] In the expression cassette of this invention, the first oocyte-specific expression promoter drives the first embryo autonomous development gene, which enables the oocyte to reproduce parthenogenetically to produce cloned embryos while maintaining fertility stability, thereby ensuring the seed setting rate of cloned plants; the second oocyte promoter drives the parthenogenetic high-efficiency enhancement factor and the second embryo autonomous development gene, enabling the oocyte to perform efficient parthenogenetic reproduction, thereby improving the induction rate of cloned embryos; the gene-edited meiotic key gene MiMe can make mitosis replace meiosis to produce diploid gametes, thereby producing cloned seeds containing only the maternal genotype. The cloned seeds retain heterosis and can be used for propagation.

[0022] The method for identifying the plant described in this invention involves determining whether the plant contains the nucleic acid molecules described. This method can determine whether a plant belongs to the plant species of this invention. The determination steps can be performed using conventional nucleic acid detection and sequencing. Exemplary methods include: nucleic acid sequencing, polymerase chain reaction (PCR) detection, quantitative fluorescence sorting, and probe hybridization detection.

[0023] This invention involves self-pollinating transformed plants and detecting whether they meet the following three conditions: (a1) a MiMe gene mutation, where mitosis replaces meiosis, producing diploid gametes; and (a2) the genotype of the resulting seeds is identical to that of the maternal parent. Exemplary methods include: screening marker observation, flow cytometry analysis, and whole-genome sequencing analysis. This allows for the screening of apomixis lines.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention utilizes gene editing technology to knock out the MiMe (OSD1, PAIR1, REC8) genes in recipient plants, while simultaneously introducing into the recipient plants an expression cassette E1, driven by a promoter specifically expressed in the first oocyte; an expression cassette E2, driven by a promoter specifically expressed in the second oocyte, representing a highly efficient parthenogenetic enhancement factor and a second embryonic autonomous development gene; and an expression cassette E3, representing a gene editing vector for creating MiMe mutants. The specific expression of the first embryonic autonomous development gene in the first oocyte enables parthenogenesis, forming cloned embryos while maintaining fertility stability, thus ensuring the seed setting rate of cloned plants. The specific expression of paternal transcription factors in the second oocyte efficiently induces parthenogenesis, thereby increasing the induction rate of cloned seeds. Fixing heterosis reduces the complex seed production process of hybrid seeds, lessens labor intensity, lowers production costs, and meets the industrialization requirements for utilizing heterosis, ultimately improving the overall efficiency of the hybrid seed industry. Attached Figure Description

[0026] Figure 1 The present invention provides an apomixis vector p29C containing expression cassettes E1, E2 and E3. Detailed Implementation

[0027] The technical solution of the present invention will be described in detail below with reference to the embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional molecular biology methods. The primers used were synthesized by a biotechnology company.

[0028] Construction of the apomixis expression vector p29C

[0029] 1. Synthesis expression cassettes E1 and E2

[0030] Synthetic expression cassette E1: The encoding sequence of the embryo-autogenetic gene BBM4 carries the oocyte-specific expression promoter AtDD45 and the NOS terminator. The sequence of the embryo-autogenetic gene expression cassette E1 is shown in SEQ ID NO.1.

[0031] SEQ ID NO.1:

[0032] 1 AAATGTTCCT CGCTGACGTA AGAAGACATT AGTAATGGTT ATAATATATA GCTTTCTATG

[0033] 61 AATGTATGGT GAGAAAATGT CTGTTCACTG ATTTTGAGTT TGGAATAAAA GCATTTGCGT

[0034] 121 TTGGTTTATC ATTGCGTTTA TACAAGGACA GAGATCCACT GAGCTGGAAT AGCTTAAAAC

[0035] 181 CATTATCAGA ACAAAATAAA CCATTTTTTG TTAAGAATCA GAGCATAGTA AACAACAGAA

[0036] 241 ACAACCTAAG AGAGGTAACT TGTCCAAGAA GATAGCTAAT TATATCTATT TTATAAAAGT

[0037] 301 TATCATAGTT TGTAAGTCAC AAAAGATGCA AATAACAGAG AAACTAGGAG ACTTGAGAAT

[0038] 361 ATACATTCTT GTATATTGT ATTCGAGATT GTGAAATTT GACCATAAGT TTAAATTCTTT

[0039] 421 AAAAAGATAT ATCTGATCTA GATGATGGTT ATAGACTGTA ATTTTACCAC ATGTTTAATG

[0040] 481 ATGGATAGTG ACACACATGA CACATCGACA ACACTATAGC ATCTTATTTA GATTACAACA

[0041] 541 TGAAATTTTT CTGTAATACA TGTCTTTGTA CATAATTTAA AAGTAATTCC TAAGAAATAT

[0042] 601 ATTTATACAA GGAGTTTAAA GAAAACATAG CATAAAGTTC AATGAGTAGT AAAAACCATA

[0043] 661 TACAGTATAT AGCATAAAGT TCAATGAGTT TATTACAAAA GCATTGGTTC ACTTTCTGTA

[0044] 721 ACACGACGTT AAACCTTCGT CTCCAATAGG AGCGCTACTG ATTCAACATG CCAATATATA

[0045] 781 CTAAATACGT TTCTACAGTC AAATGCTTTA ACGTTTCATG ATTAAGTGAC TATTTACCGT

[0046] 841 CAATCCTTTC CCATTCCTCC CACTAATCCA ACTTTTTAAT TACTCTTAAA TCACCACTAA

[0047] 901 GCTTCGAATC CATCCAAAAC CACAATATAA AAACAGAACT CTCGTAACTC AATCATCGCA

[0048] 961 AAACAAAACA AAACAAAACA AAAACCCCAA AAAGAAAGAA TAATGGCTTC TGCAGATAAC

[0049] 1021 TGGCTAGGCT TCTCGCTCTC CGGCCAAGGC AACCCACAGC ATCACCAGAA CGGCTCGCCG

[0050] 1081 TCTGCCGCCG GCGACGCCGC CATCGACATC TCCGGCTCAG GCGACTTCTA TGGTCTGCCA

[0051] 1141 ACGCCGGACG CACACCACAT CGGCATGGCG GGCGAAGACG CGCCCTATGG CGTCATGGAT

[0052] 1201 GCTTTCAACA GAGGCACCCA TGAAACCCAA GATTGGGCGA TGAGGGGTTT GGACTACGGC

[0053] 1261 GGCGGCTCCT CCGACCTCTC GATGCTCGTC GGCTCGAGCG GCGGCGGGAG GAGGACGGTG

[0054] 1321 GCCGGCGACG GCGTCGGCGA GGCGCCGAAG CTGGAGAACT TCCTCGACGG CAACTCATTC

[0055] 1381 TCCGACGTGC ACGGCCAAGC CGCCGGCGGG TACCTCTACT CCGGAAGCGC TGTCGGCGGC

[0056] 1441 GCCGGTGGTT ACAGTAACGG CGGATGCGGC GGCGGAACCA TAGAGCTGTC CATGATCAAG

[0057] 1501 ACGTGGCTCC GGAGCAACCA GTCGCAGCAG CAGCCATCGC CGCCGCAGCA CGCTGATCAG

[0058] 1561 GGCATGAGCA CCGACGCCAG CGCGAGCAGC TACGCGTGCT CCGACGTGCT GGTGGGGAGC

[0059] 1621 TGCGGCGGCG GCGGCGCCGG GGGCACGGCG AGCTCGCATG GGCAGGGCCT GGCGCTGTCG

[0060] 1681 ATGAGCACGG GGTCGGTGGC CGCCGCCGGA GGGGGCGGCG CCGTCGTCGC GGCCGAGAGC

[0061] 1741 TCGTCGTCGG AGAACAAGCG GGTGGATTCG CCGGGCGGCG CCGTGGACGG CGCCGTCCCG

[0062] 1801 AGGAAATCCA TCGACACCTT CGGGCAAAGG ACGTCTATAT ACCGAGGTGT AACAAGGCAT

[0063] 1861 AGATGGACAG GAAGATATGA AGCTCATCTG TGGGATAATA GCTGTAGGAG AGAAGGCCAA

[0064] 1921 AGTCGCAAGG GGAGACAGGT TTATTTGGGC GGTTATGACA AAGAAGATAA GGCGGCTCGG

[0065] 1981 GCTTATGATT TGGCAGCTCT AAAATACTGG GGCACGACCA CAACAACAAA TTTCCCAATG

[0066] 2041 AGTAATTATG AAAAGGAGCT AGAGGAATG AAACACATGA CCAGGCAGGA GTACATTGCA

[0067] 2101 CATCTTAGAA GGAATAGCAG TGGATTTTCT CGTGGTGCAT CCAAATATCG TGGTGTTACT

[0068] 2161 AGGCATCATC AGCATGGGAG ATGGCAGGCA AGGATAGGGC GAGTTGCAGG CAACAAGGAT

[0069] 2221 ATCTACCTAG GCACCTTCAG CACCGAGGAG GAGGCCGCCG AGGCGTACGA CATCGCCGCC

[0070] 2281 ATCAAGTTCC GCGGGCTCAA CGCCGTCACC AACTTCGACA TGAGCCGGTA CGACGTCAAG

[0071] 2341 AGCATCCTGG ACAGCAGCAC GCTGCCGGTC GGCGGCGCGG CGCGGCGGCT CAAGGAGGCG

[0072] 2401 GAGGTCGCCG CCGCCGCCGC GGGCGGCGGC GTGATCGTCT CCCACCTGGC CGACGGCGGT

[0073] 2461 GTGGGTGGGT ACTACTACGG GTGCGGCCCG ACCATCGCGT TCGGCGGCGG CGGCCAGCAG

[0074] 2521 CCGGCGCCGC TCGCCGTGCA CTACCCGTCG TACGGCCAGG CCAGCGGGTG GTGCAAGCCG

[0075] 2581 GAGCAGGACG CGGTGATCGC GGCCGGGCAC TGCGCGACGG ACCTCCAGCA CCTGCACCTC

[0076] 2641 GGGAGCGGCG GCGCCGCCGC CACCCACAAC TTCTTCCAGC AGCCGGCGTC AAGCTCGGCC

[0077] 2701 GTCTACGGCA ACGGCGGCGG CGGCGGCGGC AACGCGTTCA TGATGCCGAT GGGCGCCGTG

[0078] 2761 GTGGCCGCCG CCGATCACGG CGGGCAGAGC AGCGCCTACG GCGGTGGCGA CGAGAGCGGG

[0079] 2821 AGGCTCGTCG TGGGGTACGA CGGCGTCGTC GACCCGTACG CGGCCATGAG AAGGCGTAC

[0080] 2881 GAGCTCTCGC AGGGCTCGTC GTCGTCGTCG GTGAGCGTCG CGAAGGCGGC GAACGGGTAC

[0081] 2941 CCGGACAACT GGAGCTCGCC GTTCAACGGC ATGGGATGAG GTGACCAGCT CGAATTTCCCC

[0082] 3001 CGATCGTTCA AACATTTGGC AATAAAGTTT CTTAAGATTG AATCCTGTTG CCGGTCTTGC

[0083] 3061 GATGATTATC FATHERATTTC TGTTGAATTA CGTTAAGCAT GTAATAATTA ACATGTAATG

[0084] 3121 CATGACGTTA TTTATGAGAT GGGTTTTTAT GATTAGAGTC CCGCAATTAT ACATTTAATA

[0085] 3181 CGCGATAGAA AACAAAATAT AGCGCGCAAA CTAGGATAAA TTATCGCGCG CGGTGTCATC

[0086] 3241 TATGTTACTA GATCGGG

[0087] Synthetic expression cassette E2: The coding sequences for the parthenogenesis enhancement factor OsWOX9A and the embryo autogenesis gene BBM1 carry the oocyte-specific expression promoter AtEC1.2 and the PINII terminator. The sequence of expression cassette E2 is shown in SEQ ID NO.2.

[0088] SEQ ID NO.2:

[0089] 1 ATGGCTTCTA ACACAAGTTT CCTCTTTGCC ACCATCGCTA TCCTCCTCGT TCTCAACATC

[0090] 61 TCCGGAAGAA CTCTCCCGGA GACGGAAGAT TCCACAAACA TAGCGGCAAG ACTCAACGGA

[0091] 121 GGAGGACTAA TGGAGTGTTG GAACGCACTT TATGAGCTCA AATCATGCAC CAACGAAATC

[0092] 181 GTTCTCTTCT TCCTCAACGG TGAAACCAAA CTCGGCGTCG ATTGCTGTCA AGCCGTCGAG

[0093] 241 GTCATCACCA CCGATTGTTG GCCTGCGATG CTCACGTCTC TAGGCTTTAC CTCTGATGAA

[0094] 301 ACCAACGTTC TTCGTGGTTT CTGTCAATCT CCAAATTCCG GTGGTTCTTC TCCGGCGCCT

[0095] 361 TCCTCTGTGA AACTTTGAAT GGCGTCGTCG AACAGGCACT GGCCGAGCAT GTTCAGGTCG

[0096] 421 AAGCACGCCA CGCAGCCGTG GCAGACGCAG CCTGACATGG CCGGGTCGCC GCCCTCCCTC

[0097] 481 CTCTCCGGCT CCTCCGCCGG CAGCGCCGGC GGCGGCGGCT ACTCCCTCAA GTCGTCGCCC

[0098] 541 TTCTCGTCAG TGGGCGAGGA GAGGGTTCCG GACCCGAAGC CGCGGTGGAA CCCGCGGCCG

[0099] 601 GAGCAGATCC GGATCCTGGA GGCGATCTTC AACTCCGGCA TGGTCAACCC GCCGCGCGAC

[0100] 661 GAGATCCCGC GCATCCGCAT GCAGCTGCAG GAGTACGGCC AGGTCGGCGA CGCCAACGTC

[0101] 721 TTCTACTGGT TCCAGAACCG CAAGTCCCGC TCCAAGAACA AGCTGCGCTC CGGCGGGACA

[0102] 781 GGCCGCGCGG GGCTCGGCCT CGGCGGCAAC CGGGCCTCCG CGCCGGCGGC GGCGCACCGG

[0103] 841 GAGGCCGTGG CGCCGTCGTT CACGCCGCCG CCACCAATCC TCCCGGCGCC CCAGCCGGTG

[0104] 901 CAGCCGCAGC AGCAGCTTGT CTCGCCTGTG GCGGCGCCTA CCTCGTCGTC GTCTTCCTCC

[0105] 961 TCCGACCGTT CGTCCGGGTC CAGCAAGCCT GCGAGGGCTA CGTCGACGCA GGCGATGTCC

[0106] 1021 GTGACGACGG CCATGGACCT GCTCTCGCCG CTCGCCGCGG CGTGCCACCA GCAGATGCTC

[0107] 1081 TATCAAGGCC AGCCACTGGA GTCGCCGCCG GCGCCTGCTC CCAAAGTGCA CGGCATCGTG

[0108] 1141 CCACACGACG AGCCGGTCTT CCTGCAGTGG CCGCAGAGCC CCTGCCTGTC GGCCGTCGAC

[0109] 1201 CTCGGCGCCG CCATTCTTGG CGGCCAGTAC ATGCACCTGC CGGTGCCCGC TCCGCAGCCA

[0110] 1261 CCGTCGTCGC CGGGCGCGGC GGGCATGTTC TGGGGGCTCT GCAACGACGT GCAAGCGCCA

[0111] 1321 AACAACACCG GCCACAAGAG CTGCGCCTGG AGCGCCGGGC TCGGCCAGCA CTGGTGCGGC

[0112] 1381 TCCGCCGATC AGCTCGGCCT CGGCAAGAGC AGCGCGGCGT CGATCGCCAC CGTGTCTAGG

[0113] 1441 CCGGAGGAGG CGCACGACGT CGACGCCACG AAGCACGGTC TGCTACAGTA CGGCTTTGGC

[0114] 1501 ATCACCACGC CGCAAGTGCA CGTGGACGTT ACCTCCTCGG CTGCTGGCGT TCTGCCTCCT

[0115] 1561 GTTCCGTCCT CGCCGTCGCC GCCGAACGCC GCCGTCACCG TCGCGAGCGT GGCCGCCACC

[0116] 1621 GCTAGCCTGA CTGATTTTGC TGCAAGTGCT ATATCTGCTG GCGCCGTCGC TAACAATCAG

[0117] 1681 TTTCAAGGTC TCGCGGATTT CGGGCTCGTC GCCGGCGCCT GCTCCGGCGC CGGAGCCGCC

[0118] 1741 GCCGCCGCCG CCGCGCCCGA GGCGGGCAGT TCCGTGGCCG CGGTTGTGTG CGTCAGCGTC

[0119] 1801 GCGGGCGCCG CGCCGCCGCT CTTCTACCCG GCCGCGCACT TCAACGTGAG GCACTACGGC

[0120] 1861 GACGAGGCCG AGCTGCTCCG CTACAGAGGA GGCAGCCGCA CGGAGCCTGT GCCCGTCGAC

[0121] 1921 GAGTCGGGCG TCACCGTCGA GCCGCTCCAG CAGGGCGCCG TCTACATTGT TGTCATGTAA

[0122] 1981 ATGGCCTCCA TCACCAACTG GCTCGGCTTC TCCTCCTCCT CCTTCTCCGG CGCCGGCGCC

[0123] 2041 GACCCCGTCC TGCCCCACCC GCCGCTGCAA GAGTGGGGGA GCGCTTATGA GGGCGGCGGC

[0124] 2101 ACGGTGGCGG CCGCCGGCGG GGAGGAGACG GCGGCGCCGA AGCTGGAGGA CTTCCTCGGC

[0125] 2161 ATGCAGGTGC AGCAGGAGAC GGCCGCCGCG GCGGCGGGGC ACGGCCGTGG AGGCAGCTCG

[0126] 2221 TCGGTCGTTG GGCTGTCCAT GATCAAGAAC TGGCTACGCA GCCAGCCGCC GCCCGCGGTG

[0127] 2281 GTTGGGGGAG AAGACGCTAT GATGGCGCTC GCGGTGTCGA CGTCGGCGTC GCCGCCGGTG

[0128] 2341 GACGCGACGG TGCCGGCCTG CATTTCGCCG GATGGGATGG GGTCGAAGGC GGCCGACGGC

[0129] 2401 GGCGGCGCGG CCGAGGCGGC GGCGGCGGCG GCGGCGCAGA GGATGAAGGC GGCCATGGAC

[0130] 2461 ACGTTCGGGC AGCGGACGTC CATCTACCGG GGTGTCACCA AGCACAGGTG GACAGGAAGG

[0131] 2521 TATGAAGCCC ATCTTTGGGA TAACAGCTGC AGAAGAGAAG GTCAGACTCG CAAAGGCAGA

[0132] 2581 CAAGTATATC TTGGAGGATA TGATAAGGAA GAAAAAGCTG CTAGGGCTTA TGATTTGGCT

[0133] 2641 GCCCTTAAAT ACTGGGGCAC TACAACGACG ACGAATTTTC CGGTAAGCAA CTACGAAAAA

[0134] 2701 GAGTTGGATG AAATGAAGCA CATGAATAGG CAGGAATTTG TTGCATCCCT TAGAAGAAAA

[0135] 2761 AGCAGTGGAT TTTCACGTGG TGCTTCCATA TATCGTGGTG TTACAAGACA CCATCAGCAT

[0136] 2821 GGAAGGTGGC AAGCAAGGAT AGGACGGGTG GCAGGAAACA AGGATCTGTA TTTGGGCACA

[0137] 2881 TTTGGCACCC AAGAGGAAGC TGCAGAGGCA TATGATATCG CTGCAATCAA ATTCCGTGGT

[0138] 2941 CTCAATGCTG TGACAAACTT TGACATGAGC CGGTACGATG TCAAGAGCAT CATTGAAAGC

[0139] 3001 AGCAATCTCC CAATTGGTAC TGGAACCACC CGGCGATTGA AGGACTCCTC TGATCACACT

[0140] 3061 GATAATGTCA TGGACATCAA TGTCAATACC GAACCCAATA ATCGTGTATC ATCCCACTTC

[0141] 3121 ACCAATGGG TTGGCAACTA TGGTTCGCAG CATTATGGTT ACAATGGATG GTCGCCAATT

[0142] 3181 AGCATGCAGC CGATCCCCTC GCAGTACGCC AACGGCCAGC CCAGGGCATG GTTGAAACAA

[0143] 3241 GAGCAGGACA GCTCTGTGGT TACAGCGGCG CAGAACCTGC ACAATCTACA TCATTTTAGT

[0144] 3301 TCCTTGGGCT ACACCCACAA CTTCTTCCAG CAATCTGATG TTCCAGACGT CACAGGTTTC

[0145] 3361 GTTGATGCGC CTTCGAGGTC CAGTGACTCA TACTCCTTCA GGTACAATGG AACAAATGGC

[0146] 3421 TTTCATGGTC TCCCGGGTGG AATCAGCTAT GCTATGCCGG TTGCGACAGC GGTGGACCAA

[0147] 3481 GGTCAGGGCA TCCATGGCTA TGGAGAAGAT GGTGTGGCAG GCATTGACAC CACACATGAC

[0148] 3541 CTGTATGGCA GCCGTAATGT GTACTACCTT TCCGAGGGTT CGCTTCTTGC CGATGTCGAA

[0149] 3601 AAAGAAGGCG ACTATGGCCA ATCTGTGGGG GGCAACAGCT GGGTTTTGCC GACACCGTAG

[0150] 3661 CGGCCCATGG ATATTCGAAC GCGTAGACTT GTCCATCTTC TGGATTGGCC AACTTAATTA

[0151] 3721 ATGTATGAAA TAAAAGGATG CACACATAGT GACATGCTAA TCACTATAAT GTGGGCATCA

[0152] 3781 AAGTTGTGTG TTATGTGTAA TTACTAGTTA TCTGAATAAA AGAGAAAGAG ATCATCCATA

[0153] 3841 TTTCTTATCC TAAATGAATG TCACGTGTCT TTATAATTCT TTGATGAACC AGATGCATTT

[0154] 3901 CASE ATCCATATAC APPENDIX TAATTCATATA APPENDIX CAATTTGGGTT

[0155] 3961 AGCAAAACAA ATCTAGTCTA GGTGTGTTTT GCGAATGCGG CC

[0156] 2. Construction of the MiMe gene editing vector

[0157] Three targets were designed, one each in the coding regions of the OSD1, PAIR1, and REC8 genes, with the sequences shown in SEQ ID NO.3-SEQ ID NO.5, respectively.

[0158] OSD1:

[0159] D1: GCGCTCGCCGACCCCTCGGGTGG SEQ ID NO.3;

[0160] PAIR1:

[0161] R1: CACCGCCACCGCCACGGAACCGG SEQ ID NO.4;

[0162] REC8:

[0163] C1: GTTGGCGATCGTGTACGAGAGG SEQ ID NO.5.

[0164] All components were sequentially introduced into the Cas9 vector to construct a gene editing vector named pMiMe, a process completed by Wuhan Aidijing Biotechnology Co., Ltd. Expression cassettes E1 and E2 were inserted into the gene editing vector. The ratio of the target fragment to the backbone vector was set to 1:5, following the NEB T4 DNA ligase instruction manual. The final vector was renamed p29C. A complete vector diagram is attached. Figure 1 .

[0165] 3. Transformation of recombinant plasmids

[0166] (1) Take a tube of 200 μL of competent Escherichia coli DH5α cells and mix it with 5 μL of ligation product, and incubate on ice for 30 min;

[0167] (2) Quickly place it in a 42 ℃ constant temperature water bath, heat shock for 90 s, then ice bath for 2 min;

[0168] (3) Add 500 μL of LB liquid culture medium and mix well;

[0169] (4) Incubate at 37 ℃ and 200 rpm for 45 min to allow the cells to return to normal growth.

[0170] (5) Spread the bacterial solution evenly on LB solid medium plates;

[0171] (6) After 30 min, place in a 37 ℃ constant temperature incubator and incubate overnight;

[0172] (7) Select the correct single clones for inoculation, extract plasmids, and verify by enzyme digestion.

[0173] 4. Obtaining recombinant bacteria

[0174] The correct plant expression vector was constructed and transformed into Agrobacterium EHA105 using electroporation. The electroporation method was employed, primarily following the instructions for the Bio-Rad electroporation system. The specific steps are as follows:

[0175] Remove EHA105 competent cells stored at -80 ℃ and freeze-thaw them on ice. Pre-cool the 1 mm electroporation cuvette on ice and thaw the frozen SOC at 37 ℃. Pre-cool clean EP centrifuge tubes on ice. Generally, use two more EP centrifuge tubes than the sample to be transformed: one for a negative control (without DNA) and the other for a positive control (with 1 μL of 10 ng / μL pUC19). Pipette 1 μL of the DNA sample to be transformed into each pre-cooled EP centrifuge tube. Then, gently remove 20 μL of the thawed EHA105 competent cells and place them at the bottom of the pre-cooled centrifuge tube. Gently mix the two, avoiding air bubbles. Do not touch the bottom of the centrifuge tube with your hands to prevent temperature changes from affecting the transformation efficiency. Perform the operation as quickly as possible. Set the transformation parameters: resistance 200 Ω, capacitance 25 μF, voltage 1800 V, and electroporation cuvette 1 mm. BioRad electroporation instruments typically provide recommended parameters. Gently pipette the mixture of competent cells and DNA into an electroporation vessel, tapping lightly to distribute the mixture evenly at the bottom. Cover the vessel and place it in the electroporation tank, close the safety cap, and press the red electroporation button. After electroporation is complete, place the preheated SOC (Sodium Oxygenate) at 37 ℃ into the electroporation vessel, rotate the mixture, and transfer it to a shaker tube using a pipette tip. Incubate at 28 ℃ for 2 hours using a shaker at 180 rpm. Spread 50 μL of the bacterial culture onto LB solid medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL), and incubate in the dark at 28 ℃ for 2 days. Transform single colonies of Agrobacterium and inoculate them into LB liquid medium containing the same antibiotics. Incubate at 28 ℃ with shaking for 2 days. Mix an appropriate amount of the bacterial culture with an equal volume of 50% sterile glycerol and store at -80 ℃ for later use.

[0176] Example 2: Obtaining transgenic plants

[0177] Single colonies of Agrobacterium expressing the rice vector p29C were selected and inoculated onto LB medium containing 50 mg / L kanamycin. After incubation in the dark at 26°C for 2 days, the Agrobacterium cells were washed off with NB-AS liquid medium and cultured at 28°C with shaking at 180 rpm for 90-120 min. The colony concentration was adjusted to an OD600 of 0.8-1.0, and the colonies were then transformed into hybrid rice. The transformation process is as follows:

[0178] For hybrid rice seed disinfection, select plump seeds, soak them in 75% alcohol for 30 seconds, discard the alcohol, rinse once with sterile water, disinfect with HgCl2 for 8 minutes, rinse twice with sterile water, soaking for 1 minute each time, and soak in sterile water for 1 hour. After disinfection, inoculate the seeds onto induction medium and grow under light for 7 days.

[0179] Collect the sterile callus together. Immerse it in Agrobacterium suspension for 5-10 minutes, remove it, and air dry it with filter paper. Inoculate it onto co-culture medium and co-culture for 2 days. Wash the co-cultured callus 6 times, air dry it with filter paper, and then inoculate it onto hygromycin-resistant selection medium for 45 days.

[0180] The resistant callus was transferred to differentiation medium. After two weeks of culture, the callus began to turn green, and after three weeks, shoots emerged, followed by root growth. The seedlings were then transferred to small Erlenmeyer flasks containing rooting medium, one seedling per flask, and cultured under light. When the plantlets reached approximately 7-10 cm in height, they were hardened off indoors for 3-4 days, and then transplanted into soil. Transformed plants were selected by observing green fluorescence.

[0181] Example 3: Molecular detection of transgenic plants

[0182] DNA extraction: Take 1.0 g of leaf tissue, grind it into powder using liquid nitrogen, transfer it to a 2 mL EP tube, and add 700 μL of preheated CTAB solution. Incubate at 65 ℃ for 30-60 min, gently mixing during incubation. After cooling, add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, centrifuge at 12000 rpm for 10 min, transfer the supernatant to a new centrifuge tube, add 500 μL of isopropanol, and incubate at -20 ℃ for 30-60 min. Collect the precipitate at 4 ℃, 12000 rpm for 10 min, and discard the supernatant. Wash the precipitate twice with 70% ethanol, dry off any residual alcohol, and dissolve the precipitate in 50-100 μL of ddH2O for later use.

[0183] PCR analysis: Primers were designed using the Cas9 gene as a template, and the amplified product fragment was 439 bp.

[0184] Forward primer:

[0185] F: 5'-GAACGGTCGTAAGAGGATGCTG-3' (SEQ ID NO. 6).

[0186] Reverse primer:

[0187] R: 5'-GGTGATGGACTGGTGGATGAGA-3' (SEQ ID NO. 7).

[0188] PCR reaction system: DNA 30-90 ng, 10×Buffer 2.0 μL, 1 mM dNTP 1.8 μL, 25 mM MgCl2 1.5 μL, 10 μM primers (0.5 μL each), Tag enzyme 1.5 U, and ddH2O added to a reaction volume of 20 μL. The PCR reaction program was: 95 ℃ for 5 min; 95 ℃ for 30 s, 62 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles; 72 ℃ for 10 min, then cooled to 10 ℃. The results were detected by 1.5% agarose gel electrophoresis and photographed.

[0189] MiMe mutation detection: Specific primers were designed based on the sequence information of the target genes OSD1, PAIR1, and REC8.

[0190] The primers for OSD1 are:

[0191] F: 5'-GGGATTCGTTGGTTCGTGTT-3' (SEQ ID NO. 8);

[0192] R: 5'-TTGCGATAAGCAGAAAGAAATG-3' (SEQ ID NO. 9).

[0193] The primers for PAIR1 are:

[0194] F: 5'-CGAAGGAGAAGGCTACGGC-3' (SEQ ID NO. 10);

[0195] R: 5'-CAGGGACAGGAGTGAGTGGAA-3' (SEQ ID NO. 11).

[0196] The primers for REC8 are:

[0197] F: 5'-GCGACGCTTCACTCGAAGATCA-3' (SEQ ID NO. 12);

[0198] R: 5'-CGCCATGCCTCGTTGATCTCAA-3' (SEQ ID NO. 13).

[0199] The primers were designed to avoid the target sites and cover both ends of the three genes, so both mutated and non-mutated genes could be amplified.

[0200] Using a transgenic recipient rice variety as a negative control, PCR amplification was performed on the mutated or non-mutated target genes OSD1, PAIR1, and REC8 in T0 generation Cas9-positive transgenic plants. The PCR reaction system was as follows: DNA (mutated or non-mutated target genes in the transgenic plants) 30-90 ng, 2×Taq Master Mix 10 μL, 10 mmol / L F 1 μL, 10 mmol / L R 1 μL, and ddH2O added to a final volume of 20 μL. The reaction program was: 95 ℃ denaturation for 5 min; 35 cycles of 95 ℃ denaturation for 30 s, 63 ℃ annealing for 30 s, 72 ℃ extension for 75 s, and 72 ℃ extension for 10 min. The reaction products were detected by 1% agarose gel electrophoresis. The amplified products were sequenced, and the coding sequence of the negative control was used as a reference sequence to analyze the mutation status of target genes OSD1, PAIR1 and REC8 in positive transformed plants. MiMe mutants in which all three genes were mutated were selected.

[0201] Example 4: Screening of apomixis

[0202] MiMe mutant plants that were identified as positive by molecular identification were harvested at maturity to examine their agronomic traits such as seed setting rate, thousand-seed weight, and seed morphology.

[0203] T1 generation seeds were sampled during germination at the seedling stage, and their chromosome ploidy was determined by flow cytometry using the transformed plant recipient as a control. Genotyping was performed using whole-genome targeted sequencing, and heterozygosity was analyzed using the transformed plant recipient as a control. Transformed plants exhibiting the following two characteristics were selected as apomixis: ① T1 generation flow cytometry showed diploidity; ② Targeted sequencing results showed that the genotype at the heterozygous locus was consistent with the wild-type control.

[0204] Example 5: Detection of genetic stability of hybrid rice apomixis

[0205] Apomixis were selected and propagated to the T2 generation. Agronomic traits such as plant height, number of tillers, flag leaf length, seed setting rate, and thousand-grain weight were examined in each generation. Wild-type hybrid rice was used as a control, and its chromosome number was determined by flow cytometry. Heterozygosity was determined by whole-genome targeted sequencing, again using wild-type hybrid rice as a control.

[0206] The above embodiments are merely illustrative of the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the scope of protection of the present invention.

[0207] sequence list

[0208] <110> Guizhou Provincial Rice Research Institute

[0209] <120> A method for obtaining plant apomixis lines with high efficiency

[0210] <160> 13

[0211] <210> 1

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[0213] <212> DNA

[0214] <213> Rice (Oryza sativa)

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[0216]

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[0222]

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[0226] <213> Rice (Oryza sativa)

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[0228] GCGCTCGCCGACCCCTCGGGTGG

[0229] <210> 4

[0230] <211> twenty three

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[0232] <213> Rice (Oryza sativa)

[0233] <400> 4

[0234] CACCGCCACCGCCACGGAACCGG

[0235] <210> 5

[0236] <211> twenty three

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[0238] <213> Rice (Oryza sativa)

[0239] <400> 5

[0240] GTGTGGCGATCGTGTACGAGAGG

[0241] <210> 6

[0242] <211> twenty two

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[0244] <213> Artificial sequence

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[0246] GAACGGTCGTAAGAGGATGCTG

[0247] <210> 7

[0248] <211> twenty two

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[0250] <213> Artificial sequence

[0251] <400> 7

[0252] GGTGATGGACTGGTGGATGAGA

[0253] <210> 8

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[0255] <212> DNA

[0256] <213> Artificial sequence

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[0258] GGGATTCGTTGGTTCGTGTT

[0259] <210> 9

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[0264] TTGCGATAAGCAGAAAGAAATG

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[0270] CGAAGGAGAAGGCTACGGC

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[0276] CAGGGACAGGAGTGAGTGGAA

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[0280] <213> Artificial sequence

[0281] <400> 12

[0282] GCGACGCTTCACTCGAAGATCA

[0283] <210> 13

[0284] <211> twenty two

[0285] <212> DNA

[0286] <213> Artificial sequence

[0287] <400> 13

[0288] CGCCATGCCTCGTTGATCTCAA.

Claims

1. A method for efficiently obtaining apomixis lines in plants, characterized in that, Includes the following steps: A. Constructing the gene expression cassette E1, driven by a first oocyte-specific promoter for autonomous embryogenesis; B. Constructing a second oocyte-specific promoter to drive the expression cassette E2 of paternally derived transcription factor genes; C. Constructing the gene editing vector expression cassette E3 for the MiMe mutant; D. Introduce the above E1, E2, and E3 linkage expression cassettes into plants to obtain transgenic plants; The expression cassette E1 can induce parthenogenesis in oocytes to form cloned embryos, maintain plant fertility stability, and ensure the seed setting rate of cloned plants; the expression cassette E2 combines a highly efficient parthenogenesis enhancement factor with a gene for autonomous development of the second embryo to improve the cloned embryo induction rate; the expression cassette E3 is used to obtain the MiMe mutant, which achieves the substitution of meiosis with mitosis. The expression cassette E1 in step A comprises the following elements from upstream to downstream: a first oocyte-specific promoter, a coding sequence of the first autogenic embryogenesis gene, and a terminator; the first oocyte-specific promoter is selected from AtDD45, Os03g0296600pro, DCL2, AT1G74480.1, or ZmEAl pro; the first autogenic embryogenesis gene is BBM4; the terminator is selected from NOS, OCS, or PINII; the sequence of expression cassette E1 is shown in SEQ ID NO.1; In step B, the expression cassette E2 comprises the following elements from upstream to downstream: a second oocyte promoter, a parthenogenetic enhancement factor, a coding sequence for a second embryo autonomous development gene, and a terminator; the second oocyte-specific promoter is selected from AtEC1.1, AtEC1.2, AtEC1.3, AtEC1.4, or AtEC1.5; the parthenogenetic enhancement factor is selected from OsWOX9A, OsWOX9B, OsWOX9C, or AtWOX9; the coding sequence for the second embryo autonomous development gene is selected from BBM1, WUS, SERK, LEC, CLAVATA, or MYB115; the terminator is selected from NOS, OCS, or PINII; the sequence of expression cassette E2 is shown in SEQ ID NO.2; In step C, the expression cassette E3 includes the MiMe mutant gene. The gene edited by the MiMe mutant gene is a key gene for meiosis, which enables mitosis to replace meiosis. The gene edited by the MiMe mutant gene includes OSD1, PAIR1, and REC8. The sequences of OSD1, PAIR1, and REC8 are shown in SEQ ID NO.3-SEQ ID NO.5, respectively.

2. A nucleic acid molecule, characterized in that, Nucleic acid sequences comprising the expression cassettes E1, E2, and E3 as described in claim 1.

3. An application of the nucleic acid molecule as described in claim 2 in the cultivation of transgenic plants, comprising the following steps: introducing the nucleic acid molecule into a plant to obtain a transgenic plant.

4. The application according to claim 3, characterized in that: The plants include monocotyledons and dicotyledons.

5. A recombinant vector, characterized in that: It includes the nucleic acid molecule as described in claim 2.

6. An application of the recombinant vector as described in claim 5, characterized in that: 1) Preparation of apomixis; 2) Obtaining parthenogenetic clone seeds; 3) Fixation of hybrid vigor.

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