Nucleic acid molecules, vectors and cells and their applications, and screening methods for plant apomixis clone seeds based on paternal imprinted genes

Abstract

The present invention belongs to the field of plant molecular biology and agricultural biotechnology, and specifically relates to nucleic acid molecules, vectors and cells and their applications, and a method for screening plant apomixis clone seeds based on paternal imprinted genes, wherein the nucleic acid molecule comprises an expression cassette E1 based on imprinted genes, and the expression cassette E1 comprises a paternal imprinted gene functional region, a first promoter, a screening marker gene and a first terminator from upstream to downstream. When the nucleic acid molecule provided by the present invention is used for screening apomixis clone seeds, the obtained positive transformed plants are self-pollinated to produce zygotic embryo seeds with fluorescence and asexual embryo seeds without fluorescence. Through photoelectric sorting, asexual embryo seeds with fixed heterosis can be efficiently sorted, thereby realizing a method for applying a hybrid apomixis system.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular biology and agricultural biotechnology, and specifically relates to a nucleic acid molecule, a recombinant vector, a transgenic cell, applications of the nucleic acid molecule, the recombinant vector or the transgenic cell, a method for cultivating transgenic plants, a method for identifying the transgenic plants, a method for screening high-purity clone seeds of plants based on paternal imprinted genes, and a method for screening apomixis based on paternal imprinted genes. Background Art

[0002] In recent years, plant biotechnology has developed rapidly. The study and improvement of crops through molecular biology and genetic engineering have achieved good application prospects in agricultural production. Recently, important progress has been made in the field of apomixis research through molecular biology and genetic engineering. At the end of 2018, the team of Professor VenkatesanSundaresan of the University of California, Davis, published a rice apomixis system; by editing the three genes PAIR1, REC8, and OSD1 (MiMe), combined with ectopic expression of BBM1 in egg cells, rice apomixis was achieved. However, the rate of cloned seeds in this apomixis system only reached 29%, which is far lower than the 96% purity requirement in production, and cloned seeds and seeds from normal pollination and fertilization cannot be sorted. As a result, the application of hybrid apomixis system has not yet been realized. Summary of the invention

[0003] The purpose of the present invention is to solve the deficiencies in the above-mentioned background technology, and to provide a nucleic acid molecule, a recombinant vector, a transgenic cell, the application of the nucleic acid molecule, the recombinant vector or the transgenic cell, a method for cultivating transgenic plants, a method for identifying the transgenic plants, a method for screening high-purity clone seeds of plants based on paternal imprinted genes, and a method for screening apomixis based on paternal imprinted genes. When the nucleic acid molecule provided by the present invention is used for screening apomixis clone seeds, the obtained positive transformed plants are self-pollinated to produce zygotic embryo seeds with fluorescence and asexual embryo seeds without fluorescence. Through photoelectric sorting, asexual embryo seeds with fixed hybrid vigor can be efficiently sorted, thereby realizing the method of applying the hybrid apomixis system.

[0004] To achieve the above objectives, in a first aspect, the present invention provides a nucleic acid molecule comprising an expression cassette E1 based on an imprinted gene, wherein the expression cassette E1 comprises, from upstream to downstream, a paternal imprinted gene functional region, a first promoter, a selection marker gene and a first terminator.

[0005] Preferably, the nucleic acid molecule further comprises an expression cassette E2, wherein the expression cassette E2 carries an autonomous embryogenesis gene.

[0006] Preferably, the nucleic acid molecule further comprises an expression cassette E3, which carries an element for producing a MiMe mutant to promote mitosis instead of meiosis.

[0007] In a second aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described above.

[0008] Preferably, the recombinant vector comprises expression cassette E1 and expression cassette E2, and the expression cassette E1 and the expression cassette E2 are inserted into the recombinant vector in the form of linked expression cassettes.

[0009] Preferably, the recombinant vector comprises a nucleic acid molecule containing the linked expression cassette and expression cassette E3.

[0010] In a third aspect, the present invention provides a transgenic cell comprising the nucleic acid molecule as described above, or the recombinant vector as described above.

[0011] In a fourth aspect, the present invention provides a use of the nucleic acid molecule as described above, or the recombinant vector as described above, or the transgenic cell as described above, wherein the use comprises at least one of the following (a1)-(a4):

[0012] (a1) Screening of parthenogenetic cloned seeds;

[0013] (a2) preparing an apomixis germ line;

[0014] (a3) One-line breeding;

[0015] (a4) Fixing plant heterosis.

[0016] In a fifth aspect, the present invention provides a method for cultivating transgenic plants, the method comprising: introducing the nucleic acid molecule as described above, or the recombinant vector as described above into a plant to obtain a transgenic plant.

[0017] Preferably, the method comprises: introducing a nucleic acid molecule containing expression cassette E1, expression cassette E2 and expression cassette E3, or a recombinant vector containing a nucleic acid molecule containing the linked expression cassette and expression cassette E3 as described above, into a plant to obtain a transgenic plant.

[0018] In a sixth aspect, the present invention provides a method for screening plant apomixis clone seeds based on paternal imprinted genes, the method comprising the following steps:

[0019] A. introducing a nucleic acid molecule containing expression cassette E1, expression cassette E2 and expression cassette E3, or a recombinant vector containing a nucleic acid molecule containing the linked expression cassette and expression cassette E3 as described above, into a plant to obtain a transgenic plant;

[0020] B. selfing the transgenic plant obtained in step A to obtain asexual embryo seeds without a screening marker and zygotic embryo seeds with a screening marker;

[0021] C. Sorting the asexual embryo seeds and zygotic embryo seeds by screening markers to obtain asexual embryo seeds without screening markers, and using them as clone seeds.

[0022] In a seventh aspect, the present invention provides a method for screening an apomixis, the method comprising:

[0023] A. introducing a nucleic acid molecule containing expression cassette E1, expression cassette E2 and expression cassette E3, or a recombinant vector containing a nucleic acid molecule containing the linked expression cassette and expression cassette E3 as described above, into a plant to obtain a transgenic plant;

[0024] B. Self-pollinating the transgenic plant obtained in step A to detect whether it meets the following conditions: (a1) having MiMe mutation, mitosis replacing meiosis; (a2) producing two types of seeds, asexual embryo seeds and zygotic embryo seeds; (a3) ​​the embryos of asexual embryo seeds do not carry the selection marker gene; (a4) the selection marker gene in the expression cassette E1 is expressed in the zygotic embryo seeds;

[0025] C. Screen the transgenic plants that meet the conditions in step B as apomixis.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The expression cassette E1 based on the paternal imprint gene provided by the present invention can make the zygotic embryo produced by pollination and fertilization produce fluorescence, and the zygotic embryo with the paternal imprint gene carries a fluorescent screening marker. When it is used for the screening of plant apomixis clone seeds, two kinds of seeds are produced in the self-pollinated offspring of the positive transformation plant, namely, zygotic embryo seeds with fluorescence in the embryo and asexual embryo seeds without fluorescence in the embryo. Through photoelectric sorting, asexual embryo seeds with fixed heterosis can be efficiently sorted out for breeding, and zygotic embryo seeds containing fluorescence can be used for commercial purposes. This apomixis method can produce and sort clone seeds with fixed heterosis without affecting seed yield, reduce the complex seed production procedure of hybrid seeds, reduce labor intensity, reduce the production cost of hybrid seeds, and meet the industrialization requirements of the one-line method for utilizing heterosis, thereby improving the benefits of the hybrid seed industry as a whole. The present invention will have broad application and market prospects in the field of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention provides a schematic structural diagram of a specific apomixis vector. DETAILED DESCRIPTION

[0029] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] In a first aspect, the present invention provides a nucleic acid molecule comprising an expression cassette E1 based on an imprinted gene, wherein the expression cassette E1 comprises, from upstream to downstream, a paternal imprinted gene functional region, a first promoter, a selection marker gene and a first terminator.

[0031] According to the present invention, the imprinted gene in the expression cassette E1 based on the paternal imprinted gene is derived from the plant father and is specifically expressed in the plant embryo, so the zygotic embryos produced by fertilization basically carry the screening marker. When it is used for the screening of plant apomixis clone seeds, the embryos of the zygotic embryo seeds basically carry the screening marker, and the embryos of the asexual embryo seeds (clone seeds) do not carry the screening marker, so the purity of the cloned seeds is significantly improved, for example, the purity can reach more than 96%, and can be 96%, 97%, 98%, 99%, 100%.

[0032] According to the present invention, the paternal imprinted gene functional region can be various paternal imprinted gene functional regions commonly used, preferably a fusion sequence of ICR (imprinting control region), DMR2′ (differentially methylated region 2′ (DMR2′) of the gene) and DMR1 (differentially methylated region 1, DMR1), and the amino acid sequence of the fusion sequence is preferably as shown in SEQ ID NO.2.

[0033] According to the present invention, in order to further improve the purity of cloned seeds, the first promoter is preferably an embryo-specific expression promoter, for example, including but not limited to OsESP1, Ole18, OsVP1P and ZmESP, preferably OsESP1 promoter, whose sequence is preferably shown in SEQ ID NO.3.

[0034] According to the present invention, the selection marker gene may include but is not limited to a fluorescent protein encoding gene, preferably a red fluorescent protein encoding gene, and further preferably a red fluorescent protein encoding gene DsRed2, and the sequence of DsRed2 is preferably as shown in SEQ ID NO.4.

[0035] According to the present invention, the first terminator may include but is not limited to Nos terminator, and the sequence of the Nos terminator is preferably as shown in SEQ ID NO.5.

[0036] According to the present invention, preferably, the expression cassette E1 comprises the nucleotide sequence shown as SEQ ID NO.1.

[0037] According to the present invention, when the nucleic acid molecule is used to screen plant apomixis clonal seeds, those skilled in the art can make the plant produce clonal seeds, that is, asexual embryo seeds (for example, ectopic expression of BBM1 in egg cells is sufficient to achieve parthenogenesis) according to conventional technical means in the art, and distinguish the embryoless seeds with screening markers produced by fertilization from the asexual embryo seeds without screening markers produced by apomixis by screening markers and the like according to conventional technical means in the art, thereby screening out high-purity clonal seeds.

[0038] According to a preferred embodiment of the present invention, the nucleic acid molecule further comprises an expression cassette E2, wherein the expression cassette E2 carries an autonomous embryogenesis gene.

[0039] More preferably, the expression cassette E2 comprises from upstream to downstream: a second promoter, an embryo autonomous gene and a second terminator. More preferably, in order to further improve the purity of cloned seeds, the expression cassette E2 comprises an egg cell-specific promoter, an embryo autonomous gene and a second terminator.

[0040] According to the present invention, the egg cell-specific promoter can be any conventional promoter that can specifically promote the expression of embryonic autonomous genes, preferably selected from but not limited to AtDD45, Os03g0296600pro, ECA1-like1 pro, DCL2, AT1G74480.1 and ZmEAl promoter; more preferably AtDD45, the sequence of AtDD45 is preferably as shown in SEQ ID NO.7.

[0041] According to the present invention, the autonomous embryogenesis gene can be a conventional gene capable of achieving parthenogenesis to obtain asexual embryos, preferably, selected from but not limited to BBM1, WUS, LEC, CLAVATA and MYB115, preferably BBM1, preferably as shown in SEQ ID NO.8.

[0042] According to the present invention, the second terminator may be any conventional terminator capable of expressing a seed gene, for example, it may be but is not limited to a Nos terminator, and the sequence of the Nos terminator is preferably as shown in SEQ ID NO.5.

[0043] According to the present invention, preferably, the expression cassette E2 comprises the nucleotide sequence shown as SEQ ID NO.6.

[0044] According to the present invention, the expression of the expression cassette E2 causes the oocyte to undergo parthenogenesis under the action of the oocyte-specific promoter driving the embryo to autonomously generate gene expression, thereby obtaining an asexual embryo, which contains only the maternal genome but not the paternal genome, and therefore does not carry a screening marker, thereby enabling the zygotic embryo to be screened by the screening marker.

[0045] According to the present invention, the nucleic acid molecule further comprises an expression cassette E3, which carries elements for producing MiMe mutants to force mitosis instead of meiosis.

[0046] Preferably, the MiMe mutant includes mutations of PAIR1, REC8 and OSD1, and the elements for producing the MiMe mutant include: a target sequence for constructing a PAIR1 mutation as shown in SEQ ID NO.9 and SEQ ID NO.10, a target sequence for constructing a REC8 mutation as shown in SEQ ID NO.11 and SEQ ID NO.12, and a target sequence for constructing an OSD1 mutation as shown in SEQ ID NO.13 and SEQ ID NO.14.

[0047] Wherein, the MiMe mutant can be obtained by gene editing, and the vector can be a Cas9 vector.

[0048] According to the present invention, MiMe mutation can be used to induce mitosis to replace meiosis, thereby blocking meiosis, so that under the condition that the expression cassette E3 produces a MiMe mutant, the asexual embryo can further develop into a clonal seed.

[0049] It is well known in the art that among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are mostly more than one codon to determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the composition of the amino acid, so the nucleotide sequence of the gene encoding the same protein can be different. According to the known codon table, those skilled in the art can completely deduce other nucleotide sequences that can encode the same protein molecule from the nucleotide sequence disclosed in the present invention, and obtain the nucleotide sequence by biological methods (such as PCR method, mutation method) or chemical synthesis method, so this part of the nucleotide sequence should be included in the scope of the present invention.

[0050] According to a preferred embodiment of the present invention, the present invention provides a nucleic acid molecule, which comprises the expression cassette E1 and expression cassette E2 as described above, wherein the expression cassette E1 and expression cassette E2 preferably form a linked expression cassette.

[0051] According to a preferred embodiment of the present invention, the present invention provides a nucleic acid molecule, which comprises the expression cassette E1, expression cassette E2 and expression cassette E3 as described above.

[0052] When expression cassette E2 and expression cassette E3 are used in combination, the expression of expression cassette E2 causes the egg cell to undergo parthenogenesis to obtain asexual embryos under the action of the egg cell-specific promoter driving the embryo's autonomous gene expression, and under the condition that the expression cassette E3 produces MiMe mutants, the asexual embryo can further develop into cloned seeds with exactly the same genotype as the maternal seed.

[0053] In a second aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described above.

[0054] According to the present invention, the recombinant vector can be constructed according to conventional technical means in the art. For example, when the recombinant vector also includes at least one of expression cassette E2 and expression cassette E3, different expression cassettes can be constructed in the same vector or in different vectors.

[0055] The vector may be any conventional vector in the art, for example, may be, but is not limited to, a Cas9 vector.

[0056] According to the present invention, the recombinant vector of the present invention can be constructed by conventional technical means in the art. For example, various nucleases that can have a cleavage site in the vector multiple cloning site can be used to perform enzymatic digestion to obtain a linear plasmid, which can be connected to the target gene fragment cut by the same nuclease to obtain the recombinant vector of the present invention.

[0057] According to a preferred embodiment of the present invention, the recombinant vector comprises a nucleic acid molecule containing a linked expression cassette, and the linked expression cassette comprises the expression cassette E1 and the expression cassette E2.

[0058] According to another preferred embodiment of the present invention, the recombinant vector comprises a nucleic acid molecule containing the linked expression cassette and expression cassette E3.

[0059] According to a preferred embodiment of the present invention, the vector used to construct the recombinant vector is a Cas9 vector, and the recombinant vector is obtained by introducing the MiMe mutation into the gene editing vector Cas9 vector by the Gate-Way method, and then introducing the linkage expression cassette as described above by subcloning using an enzyme cutting and ligation method.

[0060] In a third aspect, the present invention provides a transgenic cell, which comprises the nucleic acid molecule as described above, or the recombinant vector as described above.

[0061] The transgenic cell may be a bacterial cell, for example, an Escherichia coli cell (which may be Escherichia coli DH5a) or an Agrobacterium cell (which may be Agrobacterium EHA105).

[0062] The nucleic acid molecules described above can be introduced into the host cells by conventional techniques in the art, such as calcium chloride chemical transformation, heat shock transformation, electroporation transformation, and the like.

[0063] In a fourth aspect, the present invention provides a use of the nucleic acid molecule as described above, or the recombinant vector as described above, or the transgenic cell as described above, wherein the use comprises at least one of the following (a1)-(a4):

[0064] (a1) Screening of parthenogenetic cloned seeds;

[0065] (a2) preparing an apomixis germ line;

[0066] (a3) One-line breeding;

[0067] (a4) Fixing plant heterosis.

[0068] According to a specific application of the present invention, the nucleic acid molecule contains an expression cassette E1. As described above, the expression cassette E1 can make the zygotic embryo produce fluorescence. The zygotic embryo with the paternal imprint gene carries a fluorescent screening marker and can be easily sorted out.

[0069] When used for the above applications, those skilled in the art can achieve the applications by combining other operations according to conventional technical means in the art. For example, when used for parthenogenesis to prepare cloned seeds, those skilled in the art can achieve the acquisition of asexual embryos and asexual embryo seeds according to conventional technical means in the art.

[0070] According to a specific application of the present invention, the nucleic acid molecule contains expression cassette E1, expression cassette E2 and expression cassette E3, and expression cassette E1 and expression cassette E2 exist in the nucleic acid molecule in the form of linked expression cassettes.

[0071] In a fifth aspect, the present invention also provides a method for cultivating transgenic plants, the method comprising: introducing the nucleic acid molecule as described above, or the recombinant vector as described above into a plant to obtain a transgenic plant.

[0072] Preferably, the method comprises: introducing a nucleic acid molecule containing expression cassette E1, expression cassette E2 and expression cassette E3, or a nucleic acid molecule containing the linked expression cassette and expression cassette E3 into a plant to obtain a transgenic plant.

[0073] According to the present invention, the method for introducing the nucleic acid molecule as described above into a plant may include: constructing an expression vector, and introducing the expression vector into Escherichia coli to verify its correctness, and then transforming the verified correct expression vector into Agrobacterium; and finally infecting the plant callus with the Agrobacterium to introduce it into the plant.

[0074] Preferably, the plant includes monocotyledonous plants or dicotyledonous plants, and the monocotyledonous plants or dicotyledonous plants are rice, corn, rapeseed, millet, wheat, pepper; preferably monocotyledonous plants.

[0075] Preferably, the monocotyledonous plant is rice, more preferably hybrid rice.

[0076] According to the present invention, the above nucleic acid or vector can be introduced into any part of the target plant as long as it can be expressed and achieve the purpose of the present invention, for example, it can be introduced into the nucleus, chloroplast, mitochondria and / or plastid of the cell.

[0077] The present invention also provides a method for identifying the transgenic plant as described above, the method comprising at least one of the following methods:

[0078] (1) using specific primers to specifically amplify the transgenic plant or part thereof;

[0079] (2) by observing the screening marker;

[0080] (3) Sorting by fluorescence quantitative analysis;

[0081] (4) Detection by probe hybridization.

[0082] According to a preferred embodiment of the present invention, the specific primers can specifically amplify the full length or any fragment of the expression cassette E1, and the screening marker gene is the red fluorescent protein gene DsRed2 as shown in SEQ ID NO.4, and the primers include an upstream primer as shown in SEQ ID NO.15 and a downstream primer as shown in SEQ ID NO.16.

[0083] In a sixth aspect, the present invention provides a method for screening plant apomixis clone seeds based on paternal imprinted genes, the method comprising the following steps:

[0084] A. introducing a nucleic acid molecule containing expression cassette E1, expression cassette E2 and expression cassette E3, or a recombinant vector containing a nucleic acid molecule containing the linked expression cassette and expression cassette E3 as described above, into a plant to obtain a transgenic plant;

[0085] B. selfing the transgenic plant obtained in step A to obtain asexual embryo seeds without a screening marker and zygotic embryo seeds with a screening marker;

[0086] C. Sorting the asexual embryo seeds and zygotic embryo seeds by screening markers to obtain asexual embryo seeds without screening markers, and using them as clone seeds.

[0087] The expression cassette E1 based on the paternal imprint gene provided by the present invention can make the zygotic embryo produced by pollination and fertilization produce fluorescence, and the zygotic embryo with the paternal imprint gene has a fluorescent screening marker. The expression of the expression cassette E2 allows the egg cell to undergo parthenogenesis under the action of the egg cell-specific promoter driving the embryo to autonomously generate gene expression to obtain an asexual embryo without a screening marker, and under the condition that the expression cassette E3 produces a MiMe mutant, the asexual embryo can further develop into a clone seed with the same genotype as the maternal parent, containing only the maternal genome and not the paternal genome. Therefore, the zygotic embryo seeds and the asexual embryo seeds can be efficiently screened.

[0088] In a seventh aspect, the present invention provides a method for screening an apomixis, the method comprising:

[0089] A. introducing a nucleic acid molecule containing expression cassette E1, expression cassette E2 and expression cassette E3, or a recombinant vector containing a nucleic acid molecule containing the linked expression cassette and expression cassette E3 as described above, into a plant to obtain a transgenic plant;

[0090] B. Self-pollinating the transgenic plant obtained in step A to detect whether it meets the following conditions: (a1) having MiMe mutation, mitosis replacing meiosis; (a2) producing two types of seeds, asexual embryo seeds and zygotic embryo seeds; (a3) ​​the embryos of asexual embryo seeds do not carry the selection marker gene; (a4) the selection marker gene in the expression cassette E1 is expressed in the zygotic embryo seeds;

[0091] C. Screen the transgenic plants that meet the conditions in step B as apomixis.

[0092] The present invention will be described in detail below by way of examples. In the following examples:

[0093] Example 1

[0094] This example is used to illustrate the acquisition of transgenic plants.

[0095] 1. Construction of the apomixis vector p76C

[0096] 1. Synthetic expression cassettes E1 and E2

[0097] The synthetic expression cassette E1, the sequence of which is shown in SEQ ID NO.1: includes the fusion sequence of the paternal imprinted gene functional region ICR, DMR2′, and DMR1 (SEQ ID NO.2), the embryo-specific expression promoter OsESP1 (SEQ ID NO.3), the red fluorescent protein gene DsRed2 (SEQ ID NO.4), and the NOS terminator (SEQ ID NO.5).

[0098] The synthetic expression cassette E2, the sequence of which is shown in SEQ ID NO.6, comprises an oocyte-specific promoter AtDD45 (SEQ ID NO.7), an embryo autonomous gene selected from BBM1 (SEQ ID NO.8), and a NOS terminator (SEQ ID NO.5).

[0099] E1 and E2 were constructed into a linked expression cassette and named 76E.

[0100] 2. Construction of expression vector p76C

[0101] Two targets were designed in the coding regions of PAIR1, REC8 and OSD1 genes, respectively (PAIR1: SEQ ID NO.9 and SEQ ID NO.10; REC8: SEQ ID NO.11 and SEQ ID NO.12; OSD1: SEQ ID NO.13 and SEQ ID NO.14), and constructed into the gene editing vector Cas9 vector through the Gate-Way method to construct the MiMe mutant, which was named p56DM.

[0102] 76E was subcloned into the gene editing vector p56DM by restriction ligation, and the vector was renamed p76C. The complete vector map of the expression vector p76C is attached. Figure 1 .

[0103] 3. Transformation of Recombinant Plasmid

[0104] (1) Take a tube of 200 μL E. coli competent cells DH5a and mix with 5 μL ligation product and incubate on ice for 30 min;

[0105] (2) Rapidly place in a 42°C constant temperature water bath, heat shock for 90 seconds, and ice bath for 2 minutes;

[0106] (3) Add 500 μL LB liquid medium and mix well;

[0107] (4) Cultivate at 37°C and 200 rpm for 45 min to allow the cells to return to normal growth;

[0108] (5) Spread the bacterial solution evenly on the LB solid medium plate;

[0109] (6) After 30 minutes, place the cells in a 37°C incubator for overnight culture.

[0110] (7) Select the correct monoclonal inoculum, extract the plasmid, and perform enzyme digestion verification.

[0111] 4. Obtaining recombinant Agrobacterium

[0112] The correctly constructed plant expression vector was transformed into Agrobacterium EHA105 using the electroporation method. The electroporation transformation method was adopted, mainly referring to the instruction manual of the electroporator produced by Bio-Rad. The specific steps are as follows:

[0113] Take out the EHA105 competent cells stored at -80℃ and freeze and thaw them on ice. Precool the 1mm electroporation cup on ice and thaw the frozen SOC at 37℃. Precool a clean EP centrifuge tube on ice. Generally, there are two more EP centrifuge tubes than the samples to be transformed, one for negative control (no DNA added) and the other for positive control (add 1μl of 10ng / μl pUC19). Pipette 1μl of the DNA sample to be transformed into the precooled EP centrifuge tube, then gently take out 20μl of the melted EHA105 competent cells and put them at the bottom of the precooled centrifuge tube. Gently mix the two, try not to produce bubbles, do not touch the bottom of the centrifuge tube with your hands, avoid temperature changes that affect the efficiency of competent transformation, and operate as quickly as possible. Set the transformation parameters, resistance 200Ω, capacitance 25μF, voltage 1800V, electroporation cup size 1mm, BioRad electroporation instrument generally has recommended parameters. Gently aspirate the competent state and DNA mixture, put it into the electroporation cup, and tap it to evenly distribute the mixture on the bottom of the cup. Cover the lid and put it into the electroporation tank, close the safety cover, press the red electroporation button, and after the electroporation is completed, put the SOC preheated at 37℃ into the electroporation cup, spin up the mixture, and use a pipette to transfer the mixture into the shaking tube, and shake it at 28℃, 180rpm, 2h. Take 50μl of bacterial solution and spread it on LB solid culture medium containing kanamycin (50μg / mL) and rifampicin (25μg / mL), culture it in the dark at 28℃ for 2d, pick a single colony of Agrobacterium transformant, inoculate it into LB liquid culture medium with the same antibiotics, and culture it at 28℃ for 2d. Take an appropriate amount of bacterial solution and add an equal volume of 50% sterile glycerol, mix it, and store it at -80℃ for use.

[0114] 2. Obtaining transgenic plants

[0115] A single colony of Agrobacterium tumefaciens expressing rice vector p76C was selected and inoculated on LB medium containing 50 mg / L kanamycin and cultured in the dark at 26°C for 2 days. The Agrobacterium tumefaciens was washed with NB-AS liquid medium and cultured with liquid shaking at 28°C and 180 rpm for 90-120 min. The colony concentration was adjusted to OD600 of 0.8-1.0, and hybrid rice was transformed. Hybrid rice seeds were disinfected, and seeds with full grains were selected and soaked in 75% alcohol for 30 seconds. The alcohol was poured out, and the seeds were rinsed with sterile water once, disinfected with HgCl2 for 8 minutes, washed with sterile water twice, soaked for 1 minute each time, and soaked in sterile water for 1 hour. The disinfected seeds were inoculated on the induction medium and grown under light for 7 days to obtain sterile callus tissue.

[0116] The sterile callus tissues were collected together, placed in the Agrobacterium suspension, soaked for 5-10 minutes, taken out, and dried with filter paper. Inoculated into the co-cultivation medium, co-cultivated for 2 days, the co-cultivated callus was washed 6 times, dried with filter paper, and inoculated into the selection medium with hygromycin resistance for 45 days.

[0117] The resistant callus is transferred to the differentiation medium. After 2 weeks of culture, the callus begins to turn green. After 3 weeks, young shoots will grow, and then roots will grow. The seedlings are transferred to small triangular bottles containing rooting medium, one plant per bottle, and continue to be cultured under light. When the plantlets grow to about 7-10 cm, they are hardened indoors and transplanted to the soil after 3 to 4 days. The transformed plants are selected by observing the green fluorescence (which comes with the carrier).

[0118] Example 2

[0119] This example is used to illustrate the acquisition of transgenic plants.

[0120] The transgenic plants were prepared according to the method of Example 1, except that the paternal imprinted gene functional region in the expression cassette E1 did not include ICR, DMR2′, and DMR1.

[0121] Comparative Example 1

[0122] This comparative example is used to illustrate the acquisition of the reference transgenic plants.

[0123] The transgenic plants were prepared according to the method of Example 1, except that the paternal imprinted gene functional region was not provided in the expression cassette E1.

[0124] Test Example 1

[0125] This test case is used to illustrate the molecular detection of transgenic plants

[0126] DNA extraction: Take 1.0g of leaves from Example 1, Example 2 and Comparative Example 1, grind them into powder with liquid nitrogen, transfer them into a 2ml EP tube, and add 700μl preheated CTAB solution. Place in a 65℃ water bath for 30-60min, gently mix during the period, add an equal volume of chloroform: isoamyl alcohol (24:1) after cooling, mix, centrifuge at 12000rpm for 10min, take the supernatant into a new centrifuge tube, add 500μl isopropanol, and let stand at -20℃ for 30-60min. Collect the precipitate at 4℃, 12000rpm, 10min, and discard the supernatant. Wash the precipitate twice with 70% ethanol, blow dry the alcohol residue, dissolve the precipitate with 50-100μl ddH2O, and set aside.

[0127] PCR analysis: Primers were designed using the DsRed2 gene as a template, and the amplified product fragment was 431 bp.

[0128] Forward Primer:

[0129] F: 5'-CCCAGTTCCAGTACGGCTCCAAG-3' (SEQ ID NO. 15);

[0130] Reverse Primer:

[0131] R: 5'-CTCGTTGTGGGAGGTGATGTCCAG-3' (SEQ ID NO. 16).

[0132] PCR reaction system: DNA 30-90ng, 10×Buffer 2.0μl, 1mM dNTP 1.8μl, 25mM MgCl21.5μl, 10uM primer 0.5μl each, Tag enzyme 1.5U, add ddH2O to 20μl reaction volume. PCR cycle conditions 94℃, 3min; 94℃, 1min, 64℃, 1.5min, 72℃ 30s, 40 cycles; 72℃, extension 5min. Detection by 1.4% agarose gel electrophoresis, photographic recording of electrophoresis results, the results show that the amplified fragments of the transgenic plants obtained using Example 1, Example 2 and Comparative Example 1 are consistent with the expected size.

[0133] Fluorescence quantitative analysis: RNA was extracted from T0 generation transgenic hybrid rice and wild-type hybrid rice controls using a plant total RNA extraction kit (Tiangen DP432). The method was referred to the kit instructions. The total RNA was used as a template for reverse transcription to synthesize the corresponding cDNA. Actin was used as an internal reference gene and the wild-type plant was used as a control. The RFP (red fluorescent protein) gene was detected by quantitative PCR using the RT-PCR method. It was shown that in the transgenic plants obtained using Example 1, Example 2 and Comparative Example 1, the transcription level of RFP detected by quantitative PCR should correspond to the translation expression level observed by fluorescence microscopy.

[0134] Southern blot analysis: Take the total DNA of rice, digest it with EcoRI, electrophoresed it, and transferred it to nitrocellulose membrane Hybond-N. Use the random primer labeling kit (Promega) and [α-32P]dATP (Beijing Yahui Company) to prepare the α-32P labeled RFP gene fragment by random primer method, as a molecular probe, and perform Southern hybridization analysis of transformed plants according to the molecular cloning method of Sambrook et al. Southern hybridization analysis can show the copy number of transgenics. The copy number of transgenics transformed by Agrobacterium-mediated transformation is mostly 1-3 copies. However, since the MiMe mutant is used in the embodiment of the present invention, meiosis is replaced by mitosis, and there is no free combination and separation of genes in the offspring. Therefore, the number of transgenic copies in the transgenic plants obtained using Example 1, Example 2 and Comparative Example 1 is not restricted.

[0135] Test Example 2

[0136] This test case is used to illustrate the screening of apomixis

[0137] The transgenic plants that had passed molecular identification were self-pollinated, and their agronomic traits such as plant height, number of tillers, flag leaf length, fruit set rate, and 1000-grain weight were examined, which were no different from those of the wild type. The harvested seeds were observed for size differences, which were no different from those of the wild type. Under a handheld fluorescent lamp, it was found that some seeds had embryos showing red fluorescence while others did not. The ratio of fluorescent seeds to non-fluorescent seeds was counted.

[0138] The purity of cloned seeds in the transgenic plants obtained in Example 1 was 98%, the purity of cloned seeds in the transgenic plants obtained in Example 2 was 96%, and the purity of cloned seeds in the transgenic plants obtained in Comparative Example 1 was 29%.

[0139] Purity detection: Since the plant of the present invention contains the E3 expression cassette, the offspring will not undergo trait segregation. If two different seeds (fluorescent / non-fluorescent) appear, it indicates that the paternal imprint is successfully expressed, so the zygotic embryos and asexual embryos can be screened; if the paternal imprint is not successfully expressed, the heterozygosity of the seeds can be determined by gene chip, so that the zygotic embryos and asexual embryos can also be screened.

[0140] Seeds with embryos without red fluorescence were germinated and their chromosome ploidy was detected by flow cytometry. The parent was used as a control and its heterozygosity was detected by whole genome sequencing. Transformed plants with the following characteristics were selected as apomixis: (1) self-pollination produced embryos with red fluorescence and embryos without fluorescence; (2) embryos without red fluorescence were diploid and retained maternal heterozygosity; (3) had MiMe mutation.

[0141] Test example

[0142] This experiment is used to illustrate the apomixis reproduction of hybrid rice.

[0143] The seeds without red fluorescence in Example 1 were propagated, and their agronomic traits such as plant height, tiller number, sword leaf length, fruit set rate, and 1000-grain weight were examined, which were no different from the wild type. The harvested seeds were observed for size differences, which were no different from the wild type, and their embryos were observed for red fluorescence under a handheld fluorescent lamp, and the ratio of fluorescent seeds to non-fluorescent seeds was counted.

[0144] Embryos without red fluorescence were germinated and their chromosome ploidy was detected by flow cytometry. The parent was used as a control and its heterozygosity was detected by whole genome sequencing. Repeat for multiple generations and select stable strains for application in one-line breeding.

[0145] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention. SEQUENCE LISTING <110> Hunan Hybrid Rice Research Center <120> Nucleic acid molecules, vectors and cells and their applications and plant apomixis cloning based on paternal imprinted genes Seed screening method <130> YXI66057HHRRC <160> 16 <170> PatentIn version 3.3 <210> 1 <211> 4219 <212> DNA <213> Expression cassette E1 <400> 1 caacccttac tccctttctt tcttctcata gagtgaacct aaatgcaaga tattagtgct 60 aaattgcttt aaaaataata atttaaattc aattggattt tataccataa atggttttca 120 tggttcatca ttgtgggata gatgcatgtt ttggcctgga cttgaaaatt tgtagcatta 180 caaaatattc tctccatttc tagatgaaag attttgggtt gacttcccta aacaccaaat 240 atagtgttta ttgtttatac ttactttttt gtggtatgga tatctttgaa gcattaatat 300 ataatttatt atcagaatta acttgattta cagattgtat tagattaggg atttgatatt 360 tcagagattt attaggttgt tatgtattct tttaaaaaca aaaaagaaaa gaaaagaaaa 420 gtaaatctca tcagttttag ttttaatttt aagaaataca agccgaaatg gaaatataag 480 aaaggagaga tatatccaat tgagattgaa gggataactt ttgactagaa atttatttaa 540 aataactact ttcaagtttt atttactcag tggcaaaaag ccgaaattag taaaaagcaa 600 tatcttctat gtacgcacat cggtatatgt tacattattt ttggtagatt gtacgagatt 660 tttctcctac attttattta attack atccctaata tctaactata attack 780. aaccaacgcc gaatccatct ccttctaat attttctccc aagctacaac aagccattaa tacacagccc acaattccgc taataggctg agcaaacacc aatattaaaa 900. acaaaactac ctaaccacgt cccatactca tcacggatca cacctcccgc tgttgccttc cccgggtttc catgcgaggc tccgtcggta ttgattgtaa cccatccctc acttggtttc 960 ccaaacagtc ggtttcaagg tttaagttc gagaggat taggtgaaac gatagaggtt aatttgattg attttatgag ttttcacata tattaaaaaa attack attack aaatattatt tttggtatta atttgtttct ttattacttt aaaccaatag taattttata 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200 aaaaaaggca tggtaaagca aaaaataat ctcttaaaga acaaggaaaa agacatcgaa attatttggt tgacttcact agacccaggc tactttcaag tcaataagag acatttgtta gttataagac atatctctta acataaga tgtgttaaga gaccatcaat aaggatgctc taataatctt cctttcactg cgttacaaac gtttgctatg gatttgttct cttatcgcaa 1440 aaacaataca cgatttgtgc caaggccaca tttatgtaa taattttggg tgccacacta 1500 atcaataatt ttctgttagt tggtcaaatt gtcattctaa aagtcattt tacaaaggct 1560 ttaaccctaa aatttgtaac atttattatt catttttgat ttgttgtgtc ttagtctcat 1620 atatttgttg ggcttttgtt tttgtttatt tagaatatat aatcaatcac ctatgaaaca 1680 taattaaatc tttttgccta agtacaaaca ctatttgggg tccctgtttt ggtgtagttt 1740 agggaagtca acccaaaata tttttgtcta caaatggaga gagtatttac tctttaagta 1800 ctgctattaa atttcaattc aacatatgga attacaagga cagctgagca aagaatgcac 1860 accctagttt atgcttaaag atcagttcag atttaggtga tgctgcacta tcaaaattac 1920 caggcctaac agcttagtgt gtatttacca aaactactat caaacaccag atttcccatg 1980 ttcttttatt tatatcagat aaacatttt tttctacgag tatgccacaa ttgccaccaa 2040 gtcagcaatt gtcgtttctc ttgttttaac gcatttcttt caattttagc atggaaaact 2100 tcgcattcga gtgggatgga acatagcata taattcgata atgcatgttt tgcatctctg 2160 aagattgtgg aaatttgcat tagatgcata gattggttgc tttggtgtga ttttttccc 2220 aatgctttct cgtgctagta attaccatc atgtgttatt cattcatgat atgctccatt 2280 ttaaayaag ctgctaatgt tgggccatga actaaaattc tgaccaggta aaattgttag 2340 gccagtttct cactatatca agtcaatggt acatgccatg tgggtttt tggtgcaaa 2400 tgtctcagaa agaagaaag aattgatctg cctaccagt aaaaaatgg tccgcttgtg 2460 ccttttcctg ccttatagtg attggaatga atgcctaag atgctaaat tggccaatta 2520 ttctcccaa tagtgacaaa tcctgtta acaccatata ttgttctct tgcggtttt 2580 cctattccag aggaatccc tccttattc agtatgcatg tactttgt cgttttattc 2640 atgacattat cagcatgcca gggatgaaag taaatcactg gtcatagcaa gtccacgttg 2700 atcaaaaaaa tagctttcca gccgaatggt gtcattccc taacttggaa agtgagta 2760 taattgatga ggaaaaga tgaaaaagtc atcagtttta gctatatata gcgctattga 2820 gataaagatg tttggccagt ttggtgtcat accaacagta catgactgct aggtagttga 2880 ctgtcctcat cagttttggt gcaataaatt gaaagatctc actggcaaac acattattct 2940 tcctgtttct aaacaaatct aattaccaca gtgaaaactg caaaagcgga aaggttacga 3000 tggaaaccag agcaccatgg atattcttca atctttactc aaggtatcca tccttattgc 3060 gattttgttt gttcagtatg ttcatattaa tcatttcctt atgttcacct gtctattctt 3120 ctggggcagt gcttgaagat aagaaattga actgcaagca ttctatgctc atattaatca 3180 tttccttatg tatatctgtc ttttcttcag ggatagtgct tgaagataag aaatgaacca 3240 caagcattct ttctctgccc tctatggcct cctccgagaa cgtcatcacc gagttcatgc 3300 gcttcaaggt gcgcatggag ggcaccgtga acggccacga gttcgagatc gagggcgagg 3360 gcgagggccg cccctacgag ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc 3420 ccctgccctt cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg 3480 tgaagcaccc cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt 3540 gggagcgcgt gatgaacttc gaggacggcg gcgtggcgac cgtgacccag gactcctccc 3600 tgcaggacgg ctgcttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg 3660 gccccgtgat gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc 3720 gcgacggcgt gctgaagggc gagacccaca aggccctgaa gctgaaggac ggcggccact 3780 acctggtgga gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact 3840 actacgtgga cgccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc 3900 agtacgagcg caccgagggc cgccaccacc tgttcctgta gggtgaccag ctcgaatttc 3960 cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 4020 gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 4080 tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 4140 tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 4200 tctatgttac tagatcggg 4219 <210> 2 <211> 1824 <212> DNA <213> ICR+DMR2'+DMR1) <400> 2 caacccttac tccctttctt tcttctcata gagtgaacct aaatgcaaga tattagtgct 60 aaattgcttt aaaaataata atttaaattc aattggattt tataccataa atggttttca 120 tggttcatca ttgtgggata gatgcatgtt ttggcctgga cttgaaaatt tgtagcatta 180 caaaatattc tctccattc tagatgaaag atttgggtt gacttcccta aacaccaaat 240 atagtgttta ttgtttatac ttacttttttt gtggtatgga tatctttgaa gcattaatat 300 ataatttatt atcagaatta acttgattta cagattgtat tagattaggg atttgatatt 360 tcagagattt attaggttgt tatgtattct tttaaaaaca aaaaagaaaa gaaaagaaaa 420 gtaaatctca tcagttttag ttttaatttt aagaaataca agccgaaatg gaaatataag 480 aaaggagaga tatatccaat tgagattgaa gggataactt ttgactagaa attatttaa 540 aataactact ttcaagtttt atttactcag tggcaaaaag ccgaaattag taaaaagcaa 600 tatcttctat gtacgcacat cggtatatgt tacattattt ttggtagatt gtacgagatt 660 tttctcctac attttattta attack atccctaata tctaactata attack 780. aaccaacgcc gaatccatct ccttctaat attttctccc aagctacaac aagccattaa tacacagccc acaattccgc taataggctg agcaaacacc aatattaaaa 900. acaaaactac ctaaccacgt cccatactca tcacggatca cacctcccgc tgttgccttc cccgggtttc catgcgaggc tccgtcggta ttgattgtaa cccatccctc acttggtttc 960 ccaaacagtc ggtttcaagg tttaagttc gagaggat taggtgaaac gatagaggtt aatttgattg attttatgag ttttcacata tattaaaaaa attack attack aaatattatt tttggtatta atttgtttct ttattacttt aaaccaatag taattttata 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200 aaaaaaggca tggtaaagca aaaaataat ctcttaaaga acaaggaaaa agacatcgaa attatttggt tgacttcact agacccaggc tactttcaag tcaataagag acatttgtta gttataagac atatctctta acataaga tgtgttaaga gaccatcaat aaggatgctc tataatctt cctttcactg cgttacaaac gtttgctatg gatttgttct cttatcgcaa 1440 aaacaataca cgatttgtgc caaggccaca ttttatgtaa taattttggg tgccacacta 1500 atcaataatt ttctgttagt tggtcaaatt gtcattctaa aagtcatttt tacaaaggct 1560 ttaaccctaa aatttgtaac atttattatt catttttgat ttgttgtgtc ttagtctcat 1620 atatttgttg ggctttgtt tttgtttatt tagaatatat aatcaatcac ctatgaaaca 1680 taattaaatc ttttgccta agtacaaaca ctatttgggg tccctgtttt ggtgtagtttt 1740 agggaagtca acccaaaata ttttgtcta caaatggaga gagtatttac tctttaagta 1800 ctgctattaa atttcaattc aaca 1824 <210> 3 <211> 1439 <212> DNA <213> The OsESP1 <400> 3 tatggaatta caaggacagc tgagcaaaga atgcacaccc tagtttatgc ttaaagatca 60 gttcagattt aggtgatgct gcactatcaa aattaccagg cctaacagct tagtgtgtat 120 ttaccaaaac tactatcaaa caccagattt cccatgttct tttatttata tcagataaaaa 180 catttttttc tacgagtatg ccacaattgc caccaagtca gcaattgtcg tttctcttgt 240 tttaacgcat ttctttcaat tttagcatgg aaaacttcgc attcgagtgg gagggaacat 300 agkatataat tcgataatgc atgttttgca tctctgaaga ttgtggaaat ttgcattaga 360 tgcatagatt ggttgctttg gtgtgatttt tttcccaatg ctttctcgtg ctagtaattt 420 accatcatgt gttatcatt catgatatgc tccattttaa taaaagctgc taatgttggg 480 ccatgaacta aaattctgac caggtaaaat tgttaggcca gtttctcact atatcaagtc 540 aatggtacat gccatgttgg gctttttgga tgcaaatgtc tcagaaagaa agaaagaatt 600 gatctgccta tccagtaaaa aaatggtccg cttgtgcctt ttcctgcctt atagtgattg 660 gaatgaaatg cctaagatgc taaaattggc caattattct tcccaatagt gacaaatccc 720 ttgttaacac catatattgt tcctctttgc ggttttccta ttccagagga aatccctcct 780 tatttcagta tgcatgtact ttttgtcgtt ttattcatga cattatcagc atgccaggga 840 tgaaagtaaa tcactggtca tagcaagtcc acgttgatca aaacaatagc tttccagccg 900 aatggtgtca tttccctaac ttggaaagtg atagtataat tgatgaggag aaaagatgaa 960 aaagtcatca gttttagcta tatatagcgc tattgagata aagatgtttg gccagtttgg 1020 tgtcatacca acagtacatg actgctaggt agttgactgt cctcatcagt tttggtgcaa 1080 taaattgaaa gatctcactg gcaaacacat tattcttcct gtttctaaac aaatctaatt 1140 accacagtga aaactgcaaa agcggaaagg ttacgatgga aaccagagca ccatggatat 1200 tcttcaatct ttactcaagg tatccatcct tattgcgatt ttgtttgttc agtatgttca 1260 tattaatcat ttccttatgt tcacctgtct attcttctgg ggcagtgctt gaagataaga 1320 aattgaactg caagcattct atgctcatat taatcatttc cttatgtata tctgtctttt 1380 cttcagggat agtgcttgaa gataagaaat gaaccacaag cattctttct ctgccctct 1439 <210> 4 <211> 678 <212> DNA <213> Red fluorescent protein gene DsRed2 <400> 4 atggcctcct ccgagaacgt catcaccgag ttcatgcgct tcaaggtgcg catggagggc 60 accgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120 cacaacaccg tgaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180 ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 240 gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300 gacggcggcg tggcgaccgt gacccaggac tcctccctgc aggacggctg cttcatctac 360 aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtgatgca gaagaagacc 420 atgggctggg aggcctccac cgagcgcctg taccccgcg acggcgtgct gaagggcgag 480 acccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 540 tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacgc caagctggac 600 atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcac cgagggccgc 660 caccacctgt tcctgtag 678 <210> 5 <211> 278 <212> DNA <213> Our <400> 5 ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg caataaagtt tcttaagatt 60 gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca 120 tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt 180 cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa 240 attatcgcgc gcggtgtcat ctatgttact agatcggg 278 <210> 6 <211> 2960 <212> DNA <213> Expression cassette E2 <400> 6 aaatgttcct cgctgacgta agaagacatt agtaatggtt ataatatata gctttctatg 60 aatgtatggt gagaaaatgt ctgttcactg attttgagtt tggaataaaa gcatttgcgt 120 ttggtttatc attgcgttta tacaaggaca gagatccact gagctggaat agcttaaaac 180 cattatcaga acaaaataaa ccattttttg ttaagaatca gagcatagta aacaacagaa 240 acaacctaag agaggtaact tgtccaagaa gatagctaat tatatctatt ttataaaagt 300 tatcatagtt tgtaagtcac aaaagatgca aataacagag aaactaggag acttgagaat 360 atacattctt gtatatttgt attcgagatt gtgaaaattt gaccataagt ttaaattctt 420 aaaaagatat atctgatcta gatgatggtt atagactgta attttaccac atgtttaatg 480 atggatagtg acacacatga cacatcgaca acactatagc atcttatta gattacaaca 540 tgaaattttt ctgtaataca tgtctttgta cataatttaa aagtaattcc taagaaatat 600 atttatacaa ggagtttaaa gaaaacatag cataaaagttc aatgagtagt aaaaaccata 660 tacagtatat agcataaagt tcaatgagtt tattacaaaa gcattggttc actttctgta 720 acacgacgtt aaaccttcgt ctccaatagg agcgctactg attcaacatg ccaatatata 780 ctaaatacgt ttctacagtc aaatgcttta acgtttcatg attaagtgac tatttaccgt 840 caatcctttc ccattcctcc cactaatcca acttttaat tactcttaaa tcaccactaa 900 gcttcgaatc catccaaaac cacaataataa aaacagaact ctcgtaactc aatcatcgca 960 aaacaaaaca aaacaaaaca aaaaccccaa aaagaaagaa taatggcctc catcaccaac 1020 tggctcggct tctcctcctc ctccttctcc ggcgccggcg ccgaccccgt cctgccccac 1080 ccgccgctgc aagagtgggg gagcgcttat gagggcggcg gcacggtggc ggccgccggc 1140 ggggaggaga cggcggcgcc gaagctggag gacttcctcg gcatgcaggt gcagcaggag 1200 acggccgccg cggcggcggg gcacggccgt ggaggcagct cgtcggtcgt tgggctgtcc 1260 atgatcaaga actggctacg cagccagccg ccgcccgcgg tggttggggg agaagacgct 1320 atgatggcgc tcgcggtgtc gacgtcggcg tcgccgccgg tggacgcgac ggtgccggcc 1380 tgcatttcgc cggatgggat ggggtcgaag gcggccgacg gcggcggcgc ggccgaggcg 1440 gcggcggcgg cggcggcgca gaggatgaag gcggccatgg acacgttcgg gcagcggacg 1500 tccatctacc ggggtgtcac caagcacagg tggacaggaa ggtatgaagc ccatctttgg 1560 gataacagct gcagaagaga aggtcagact cgcaaaggca gacaagtcaa tgcaggagga 1620 tatgataagg aagaaaaagc tgctagggct tatgatttgg ctgcccttaa atactggggc 1680 actacaacga cgacgaattt tccggtaagc aactacgaaa aagagttgga tgaaatgaag 1740 cacatgaata ggcaggaatt tgttgcatcc cttagaagaa aaagcagtgg attttcacgt 1800 ggtgcttcca tatatcgtgg tgttacaaga caccatcagc atggaaggtg gcaagcaagg 1860 ataggacggg tggcaggaaa caaggatctg tatttgggca catttggcac ccaaggaa 1920 gctgcagagg catatgatat cgctgcaatc aaattccgtg gtctcaatgc tgtgacaaac 1980 tttgacatga gccggtacga tgtcaagagc atcattgaaa gcagcaatct cccaattggt 2040 actggaacca cccggcgatt gaaggactcc tctgatcaca ctgataatgt catggacatc 2100 aatgtcaata ccgaacccaa taatgtggta tcatcccact tcaccaatgg ggttggcaac 2160 tatggttcgc agcattatgg ttaacaatgga tggtcgccaa ttagcatgca gccgatcccc 2220 tcgcagtacg ccaacggcca gcccagggca tggttgaaac aagagcagga cagctctgtg 2280 gttacagcgg cgcagaacct gcacaatcta catcatttta gttccttggg ctacacccac 2340 aacttcttcc agcaatctga tgttccagac gtcacaggtt tcgttgatgc gccttgagg 2400 tccagtgact catactcctt caggtacaat ggaacaaatg gctttcatgg tctcccgggt 2460 ggaatcagct atgctatgcc ggttgcgaca gcggtggacc aaggtcaggg catccatggc 2520 tatggagaag atggtgtggc aggcattgac accacacatg acctgtatgg cagccgtaat 2580 gtgtactacc tttccgaggg ttcgcttctt gccgatgtcg aaaaagaagg cgactatggc 2640 caatctgtgg ggggcaacag ctgggttttg ccgacaccgt agggtgacca gctcgaattt 2700 ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 2760 tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 2820 atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 2880 atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 2940 atctatgtta ctagatcggg 2960 <210> 7 <211> 1002 <212> DNA <213> Oocyte-specific promoter AtDD45 <400> 7 aaatgttcct cgctgacgta agaagacatt agtaatggtt ataatatata gctttctatg 60 aatgtatggt gagaaaatgt ctgttcactg attttgagtt tggaataaaa gcatttgcgt 120 ttggtttatc attgcgttta tacaaggaca gagatccact gagctggaat agcttaaaac 180 cattatcaga acaaaataaa ccattttttg ttaagaatca gagcatagta aacaacagaa 240 acaacctaag agaggtaact tgtccaagaa gatagctaat tatatctatt ttataaaagt 300 tatcatagtt tgtaagtcac aaaagatgca aataacagag aaactaggag acttgagaat 360 atacattctt gtatatttgt attcgagatt gtgaaaattt gaccataagt ttaaattctt 420 aaaaagatat atctgatcta gatgatggtt atagactgta attttaccac atgtttaatg 480 atggatagtg acacacatga cacatcgaca acactatagc atcttatta gattacaaca 540 tgaaattttt ctgtaataca tgtctttgta cataatttaa aagtaattcc taagaaatat 600 atttatacaa ggagtttaaa gaaaacatag cataaaagttc aatgagtagt aaaaaccata 660 tacagtatat agcataaagt tcaatgagtt tattacaaaa gcattggttc actttctgta 720 acacgacgtt aaaccttcgt ctccaatagg agcgctactg attcaacatg ccaatatata 780 ctaaatacgt ttctacagtc aaatgcttta acgtttcatg attaagtgac tatttaccgt 840 caatcctttc ccattcctcc cactaatcca acttttaat tactcttaaa tcaccactaa 900 gcttcgaatc catccaaaac cacaatataa aaacagaact ctcgtaactc aatcatcgca aaacaaaaca aaaacaaaca aaaaccccca aaaagaaga ta <210> 8 <211> 1680 <212> DNA <213> Install the license plate for BBM1 <400> 8 atggcctcca tcaccaactg gctcggcttc tcctcctcct ccttctccgg cgccggcgcc gacccgtcc tgccccaccc gccgctgcaa gagtggggga gcgcttatga gggcggcggc 120 acggtggcgg ccgccggcgg ggaggagacg gcggcgccga ctggaga cttcctcggc 180 atgcaggtgc agcaggagac ggccgccgcg gcggcggggc acggccgtgg aggcagctcg 240 tcggtcgttg ggctgtccat gatcaagaac tggctacgca gccagccgcc gcccgcggtg 300 gttggggggag aagacgctat gatggcgctc gcggtgtcga cgtcggcgtc gccgccggtg 360 gacgcgacgg tgccggcctg catttcgccg gatgggatgg ggtcgaaggc ggccgacggc 420 ggcggcgcgg ccgaggcggc ggcggcggcg gcggcgcaga ggatgaaggc ggccatggac 480 acgttcgggc agcggacgtc catctaccgg ggtgtcacca agcacaggtg gacaggaagg 540 tatgaagcccc atctttggga taacagctgc agagagaag gtcagactcg aaggcaga 600 caagtcaatg caggagata tgataggaa gaaaagctg ctaggctta tgatttggct 660 gcccttaaat actggggcac tacaacgacg acgaatttc cggtaagcaa ctacgaaaaa 720 gagttggatg aaatgaagca catgaatagg caggaatttg ttgcatccct tagagaaaa 780 agcagtggat ttcacgtgg tgcttccata tatcgtggtg ttacaagaca ccatcagcat 840 ggaaggtggc aagcaggat aggacgggtg gcaggaaaca aggatctgta ttggggcaca 900 ttggcacccc agaggaagc tgcagaggca tatgatatcg ctgcaatcaa attccgtggt 960 ctcaatgctg tgacaactt tgacatgagc cggtacgatg tcagagcat cattgaagc 1020 agcaatctcc caatgtac tggaaccacc cggcgattga agcactcctc tgatcacact 1080 gataatgtca tggacatca tgtcaatacc gaacccaata atgtggtatc atcccactc 1140 accaatgggg ttggcaacta tggttcgcag cattatggtt acaatggattg gtcgccaatt 1200 agcatgcagc cgatccccctc gcagtacgcc aacggccagc ccagggcatg gttgaaacaa 1260 gagcaggaca gctctgtggt tacagcggcg cagaacctgc acaatctaca tcattttagt 1320 tccttgggct acacccacaa cttcttccag caatctgatg ttccagacgt cacaggtttc 1380 gttgatgcgc cttcgaggtc cagtgactca tactccttca ggtacaatgg aacaaatggc 1440 tttcatggtc tcccgggtgg aatcagctat gctatgccgg ttgcgacagc ggtggaccaa 1500 ggtcagggca tccatggcta tggagaagat ggtgtggcag gcattgacac cacacatgac 1560 ctgtatggca gccgtaatgt gtactacctt tccgagggtt cgcttcttgc cgatgtcgaa 1620 aaagaaggcg actatggcca atctgtgggg ggcaacagct gggttttgcc gacaccgtag 1680 <210> 9 <211> twenty three <212> DNA <213> Target sequence R1 of PAIR1 mutation <400> 9 ggtgaggagg ttgtcgtcga ggg 23 <210> 10 <211> twenty two <212> DNA <213> Target sequence R2 of PAIR1 mutation <400> 10 aagcaaccca gtgcaccgct gg 22 <210> 11 <211> twenty three <212> DNA <213> REC8 mutated target sequence C1 <400> 11 gtgtggcgat cgtgtacgag agg 23 <210> 12 <211> twenty two <212> DNA <213> REC8 mutated target sequence C2 <400> 12 cccatggcac taaggctctc cg 22 <210> 13 <211> twenty three <212> DNA <213> Target sequence D1 for OSD1 mutation <400> 13 gcgctcgccg acccctcggg tgg 23 <210> 14 <211> twenty two <212> DNA <213> Target sequence R1 for OSD1 mutation <400> 14 ctgccgccga cgagcaacaa gg 22 <210> 15 <211> twenty three <212> DNA <213> DsRed2 gene amplification forward primer <400> 15 cccagttcca gtacggctcc aag 23 <210> 16 <211> twenty four <212> DNA <213> DsRed2 gene amplification reverse primer <400> 16 ctcgttgtgg gaggtgatgt ccag 24

Claims

1. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises expression cassettes E1, E2 and E3 based on imprinted genes, wherein the expression cassette E1 comprises, from upstream to downstream, a paternal imprinted gene functional region, a first promoter, a selection marker gene and a first terminator; The expression cassettes E1 and E2 constitute a linked expression cassette; The nucleic acid molecule is used to cultivate transgenic plants, and the plant is rice; The expression cassette E2 includes, from upstream to downstream: a second promoter, an embryo autonomous gene and a second terminator; the second promoter is an egg cell specific promoter; The expression cassette E3 carries elements for generating MiMe mutants to promote mitosis instead of meiosis; the MiMe mutants include mutations in PAIR1, REC8 and OSD1, and the elements for generating MiMe mutants include: target sequences for constructing PAIR1 mutations as shown in SEQ ID NO: 9 and SEQ ID NO: 10, target sequences for constructing REC8 mutations as shown in SEQ ID NO: 11 and SEQ ID NO: 12, and target sequences for constructing OSD1 mutations as shown in SEQ ID NO: 13 and SEQ ID NO: 14; The first promoter is an embryo-specific expression promoter; the selection marker gene is a fluorescent protein encoding gene; The autonomous embryogenesis gene is a gene that can achieve parthenogenesis to obtain asexual embryos.

2. The nucleic acid molecule according to claim 1, wherein The paternal imprinted gene functional region includes the fusion sequence of ICR, DMR2′ and DMR1.

3. The nucleic acid molecule according to claim 2, wherein The fusion sequence is shown in SEQ ID NO:

2.

4. The nucleic acid molecule according to claim 1, wherein The embryo-specific expression promoter is selected from OsESP1, Ole18, OsVP1P and ZmESP.

5. The nucleic acid molecule according to claim 4, wherein The embryo-specific expression promoter is OsESP1 promoter.

6. The nucleic acid molecule according to claim 5, wherein The sequence of the OsESP1 promoter is shown in SEQ ID NO:

3.

7. The nucleic acid molecule according to claim 1, wherein The screening marker gene is a red fluorescent protein encoding gene.

8. The nucleic acid molecule according to claim 7, wherein The screening marker gene is the red fluorescent protein gene DsRed2.

9. The nucleic acid molecule according to claim 8, wherein The sequence of DsRed2 is shown in SEQ ID NO:

4.

10. The nucleic acid molecule according to claim 1, wherein The first terminator is the Nos terminator.

11. The nucleic acid molecule according to claim 10, wherein The sequence of the Nos terminator is shown in SEQ ID NO:

5.

12. The nucleic acid molecule according to claim 1, wherein The imprinted gene-based expression cassette E1 comprises the nucleotide sequence shown in SEQ ID NO:

1.

13. The nucleic acid molecule according to claim 1, wherein The second promoter is selected from AtDD45, Os03g0296600pro, ECA1-like1 pro, DCL2, AT1G74480.1 and ZmEAlpromoter; and / or The embryo autonomous genes are selected from BBM1, WUS, LEC, CLAVATA and MYB115; and / or The second terminator is the Nos terminator.

14. The nucleic acid molecule according to claim 13, wherein The second promoter is AtDD45.

15. The nucleic acid molecule according to claim 14, wherein The sequence of AtDD45 is shown in SEQ ID NO:

7.

16. The nucleic acid molecule according to claim 13, wherein The embryo autonomous gene is BBM1.

17. The nucleic acid molecule according to claim 16, wherein The sequence of BBM1 is shown in SEQ ID NO:

8.

18. The nucleic acid molecule according to claim 13, wherein The sequence of the Nos terminator is shown in SEQ ID NO:

5.

19. The nucleic acid molecule according to claim 1, wherein The expression cassette E2 comprises the nucleotide sequence shown in SEQ ID NO:

6.

20. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to any one of claims 1 to 19.

21. A transgenic cell, characterized in that: The transgenic cell contains the nucleic acid molecule according to any one of claims 1 to 19, or the recombinant vector according to claim 20; wherein the cell is a bacterial cell.

22. Use of the nucleic acid molecule according to any one of claims 1 to 19, or the recombinant vector according to claim 20, or the transgenic cell according to claim 21, wherein: The application is to fix plant heterosis, and the plant is rice.

23. The use according to claim 22, wherein: The fixing of plant heterosis includes one-line breeding.

24. The use according to claim 23, wherein: The one-line breeding method includes preparing an apomictic line.

25. The use according to claim 24, wherein: The preparation of the apomictic line includes the screening of parthenogenetic clone seeds.

26. A method for cultivating transgenic plants, characterized in that: The method comprises: introducing the nucleic acid molecule described in any one of claims 1 to 19 or the recombinant vector described in claim 20 into a plant to obtain a transgenic plant; wherein the plant is rice.

27. A method for screening plant apomixis clone seeds based on paternal imprinted genes, characterized in that: The method comprises the following steps: A. introducing a nucleic acid molecule containing the expression cassette E1, expression cassette E2 and expression cassette E3 according to any one of claims 1 to 19, or the recombinant vector according to claim 20 into a plant to obtain a transgenic plant; B. selfing the transgenic plant obtained in step A to obtain asexual embryo seeds without a screening marker and zygotic embryo seeds with a screening marker; C. sorting the asexual embryo seeds and zygotic embryo seeds by screening markers to obtain asexual embryo seeds without screening markers, and using them as clone seeds; Wherein, the plant is rice.

28. A method for screening apomixis based on paternal imprinted genes, characterized in that: The method includes: A. introducing a nucleic acid molecule containing the expression cassette E1, expression cassette E2 and expression cassette E3 according to any one of claims 1 to 19, or the recombinant vector according to claim 20 into a plant to obtain a transgenic plant; B. Self-pollinating the transgenic plant obtained in step A to detect whether it meets the following conditions: (a1) having MiMe mutation, mitosis replacing meiosis; (a2) producing two types of seeds, asexual embryo seeds and zygotic embryo seeds; (a3) ​​the embryos of asexual embryo seeds do not carry the selection marker gene; (a4) the selection marker gene in the expression cassette E1 is expressed in the zygotic embryo seeds; C. Screening transgenic plants that simultaneously meet the conditions in step B as apomixis; Wherein, the plant is rice.

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