Gene ZmKNL2 for inducing haploid production in maize and its application

Abstract

The present invention discloses a gene ZmKNL2 for inducing the production of corn haploids and its application, and discloses a method for preparing plant haploids. The method comprises the following steps: 1) silencing or inhibiting the expression of the ZmKNL2 gene in the genome of the target plant or knocking out the ZmKNL2 gene in the genome of the target plant to obtain a transgenic plant; 2) hybridizing the transgenic plant or its offspring obtained in step 1) as a male parent with other plants to obtain a hybrid offspring, that is, to obtain a plant haploid, and the genome sequence of the ZmKNL2 gene can be shown as Sequence 1 in the sequence table. The embodiments of the present invention prove that the ZmKNL2 mutation of corn can lead to the production of maternal haploids of corn, and provide a new idea for revealing the biological role of ZmKNL2 in the production process of maternal haploids of corn.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a gene ZmKNL2 inducing the production of corn haploids and an application thereof. Background Art

[0002] Corn is one of the most important crops in the world, and ranks first in both planting area and total output in my country. At present, almost all corn varieties used in production are hybrids, and the selection and breeding of excellent inbred lines is very important for breeding hybrids. The corn haploid breeding technology based on hybrid induction can obtain homozygous inbred lines in two generations, which greatly accelerates the breeding process and is one of the key core technologies of modern corn breeding. Obtaining haploids is the primary link in haploid breeding, and hybrid induction using haploid induction lines is the main method for corn to obtain haploids. At present, in addition to the traditional maternal haploid induction lines, the corn haploid induction lines also have haploid induction lines based on the mutation of the histone variant CENH3. By mining related genes, identifying more genes that regulate haploid induction, and further improving the induction rate, it is of great significance for breeding new induction lines with high induction rates and analyzing the genetic mechanism of haploid induction. Summary of the invention

[0003] The present invention provides a method for preparing a plant haploid, comprising the following steps:

[0004] 1) Silencing or inhibiting the expression of the ZmKNL2 gene in the genome of the target plant or knocking out the ZmKNL2 gene in the genome of the target plant to obtain a transgenic plant;

[0005] 2) using the transgenic plant obtained in step 1) or its offspring as the male parent to hybridize with other plants to obtain hybrid offspring, i.e., to obtain plant haploids,

[0006] The ZmKNL2 gene is any of the following DNA molecules:

[0007] (1) a DNA molecule whose sequence is shown in SEQ ID NO: 1 in the sequence listing;

[0008] (2) The DNA molecule comprises a first exon, a second exon, a third exon and a fourth exon, wherein the sequence of the first exon is shown in SEQ ID NOs. 98-244 of SEQ ID NOs. 1 of the sequence listing, the sequence of the second exon is shown in SEQ ID NOs. 338-494 of SEQ ID NOs. 1 of the sequence listing, the sequence of the third exon is shown in SEQ ID NOs. 672-862 of SEQ ID NOs. 1 of the sequence listing, and the sequence of the fourth exon is shown in SEQ ID NOs. 1068-2036 of SEQ ID NOs. 1 of the sequence listing;

[0009] (3) The DNA molecule comprises a first exon, a second exon, a third exon and a fourth exon, wherein the sequence of the first exon is shown in SEQ ID NOs. 98-244 of SEQ ID NOs. 1 of the sequence listing, the sequence of the second exon is shown in SEQ ID NOs. 338-452 of SEQ ID NOs. 1 of the sequence listing, the sequence of the third exon is shown in SEQ ID NOs. 672-862 of SEQ ID NOs. 1 of the sequence listing, and the sequence of the fourth exon is shown in SEQ ID NOs. 1068-2036 of SEQ ID NOs. 1 of the sequence listing;

[0010] (4) The DNA molecule comprises a first exon, a second exon, a third exon, a fourth exon, a fifth exon and a sixth exon, wherein the sequence of the first exon is shown in SEQ ID NOs. 98-244 of SEQ ID NOs. 1 in the sequence listing, the sequence of the second exon is shown in SEQ ID NOs. 338-438 of SEQ ID NOs. 1 in the sequence listing, the sequence of the third exon is shown in SEQ ID NOs. 688-862 of SEQ ID NOs. 1 in the sequence listing, the sequence of the fourth exon is shown in SEQ ID NOs. 1068-2009 of SEQ ID NOs. 1 in the sequence listing, the sequence of the fifth exon is shown in SEQ ID NOs. 2839-2902 of SEQ ID NOs. 1 in the sequence listing, and the sequence of the sixth exon is shown in SEQ ID NOs. 3083-3129 of SEQ ID NOs. 1 in the sequence listing.

[0011] Optionally, according to the above method, in step 2), the following step is also included: selecting offspring with haploid ploidy from the hybrid offspring, that is, plant haploids.

[0012] Selecting offspring with haploid ploidy from the hybrid offspring may include subjecting the hybrid offspring individual plants to haploid trait identification, flow cytometry ploidy identification and / or molecular marker identification, and selecting the offspring individual plants identified as haploid by any method as plant haploid.

[0013] The haploid trait identification method can be carried out as follows: if the plant to be tested has the characteristics of short plant, narrow leaves, compact plant shape, male sterility, etc., then the plant is or is a candidate for being a haploid; if the plant to be tested has the characteristics of tall plant, wide and spreading leaves, normal fertility, etc., then the plant is or is a candidate for being a diploid.

[0014] The flow cytometry ploidy identification method can be performed as follows: extract the nuclei of the young leaves of the plant to be tested, and use the diploid plant leaves as a control; then use a flow cytometer to detect the signal, first detect the diploid cell nuclear signal, and set the diploid cell nuclear signal peak position as the A value (because the genetic material in the diploid cell is twice that in the haploid cell, the haploid cell nuclear signal peak position appears near half of the A value). If the cell nuclear signal peak of the plant to be tested appears near half of the A value, the plant is or can be a haploid; if the signal peak of the plant to be tested appears near the A value, the plant is or can be a diploid.

[0015] The molecular marker identification can be carried out according to the following method: PCR amplification is performed using polymorphic primers between the paternal and maternal parents, and the PCR amplification product is used to determine whether the plant to be tested is haploid or diploid: if the amplified product of the plant to be tested only has the banding pattern of the maternal parent and does not have the banding pattern of the paternal parent, then the plant is or is a candidate to be a haploid; if the amplified product of the plant to be tested has the heterozygous banding pattern of the paternal parent and the maternal parent, then the plant is or is a candidate to be a diploid.

[0016] Optionally, according to the above method, the silencing or inhibiting the expression of the ZmKNL2 gene in the genome of the target plant or knocking out the ZmKNL2 gene is to mutate the ZmKNL2 gene in the target plant, and reduce the expression level of the ZmKNL2 gene or cause functional loss. The functional loss of the ZmKNL2 gene can refer to the deletion of the 705th base C from the 5' end of sequence 1 in the sequence table, while other nucleotides remain unchanged. The deletion causes a frameshift mutation, which causes the functional loss of the ZmKNL2 gene and makes it unable to encode the ZmKNL2 protein.

[0017] Optionally, according to the above method, the mutating of the ZmKNL2 gene in the target plant is to mutate the exons of the ZmKNL2 gene in the target plant genome; the mutation is a deletion mutation and / or an insertion mutation and / or other mutations that can cause loss of gene function. For example, taking transcript T1 as an example, the first exon and / or the second exon and / or the third exon and / or the fourth exon of the ZmKNL2 gene in the target plant genome is mutated; taking transcript T2 as an example, the first exon and / or the second exon and / or the third exon and / or the fourth exon of the ZmKNL2 gene in the target plant genome is mutated; taking transcript T3 as an example, the first exon and / or the second exon and / or the third exon and / or the fourth exon and / or the fifth exon and / or the sixth exon of the ZmKNL2 gene in the target plant genome is mutated. In the embodiment of the present invention, taking transcript T3 as an example, a deletion mutation occurs in the third exon of the ZmKNL2 gene in the target plant genome, specifically, the 705th base C of the ZmKNL2 gene from the 5' end is deleted.

[0018] The above-mentioned mutation may occur on both homologous chromosomes of the above-mentioned target plant, or on only one homologous chromosome.

[0019] Optionally, according to the above method, the mutating of the exon of the ZmKNL2 gene in the target plant is achieved by using gene editing technology. The gene editing technology can use the CRISPR / Cas9 system.

[0020] The above-mentioned CRISPR / Cas9 system can be any of the following:

[0021] (b1) comprising a specific sgRNA and a Cas9 protein, wherein the target site sequence binding region of the specific sgRNA is as shown in Sequence 3 of the sequence listing;

[0022] (b2) comprising a specific DNA molecule and a Cas9 gene, wherein the specific DNA molecule is transcribed to obtain a specific sgRNA; the target site sequence binding region of the specific sgRNA is shown in Sequence 3 of the sequence listing;

[0023] (b3) comprising a plasmid having a specific DNA molecule and a plasmid having a Cas9 gene, wherein the specific DNA molecule is transcribed to obtain a specific sgRNA, and the target site sequence binding region of the specific sgRNA is shown in Sequence 3 of the sequence listing;

[0024] (b4) includes a specific recombinant plasmid, wherein the specific recombinant plasmid expresses the sgRNA and Cas9 protein, and the target site sequence binding region of the specific sgRNA is shown in Sequence 3 of the sequence listing.

[0025] The sequence of the above-mentioned specific sgRNA may be shown as Sequence 4 in the sequence table, wherein positions 1-23 in Sequence 4 are the target site sequence binding region. The above-mentioned specific recombinant plasmid contains the Cas9 gene, such as the CRISPR / Cas9 knockout vector prepared in the example.

[0026] The present invention also provides a CRISPR / Cas9 system for preparing plant haploids, wherein the CRISPR / Cas9 system is the above-mentioned CRISPR / Cas9 system.

[0027] The present invention also provides a ZmKNL2 mutant gene, wherein the sequence of the ZmKNL2 mutant gene is a sequence in which the 705th base C of the sequence 1 in the sequence list is deleted from the 5' end, and other nucleotides remain unchanged.

[0028] The above method, the substance for silencing or inhibiting the expression of the ZmKNL2 gene in the genome of the target plant, the substance for knocking out the ZmKNL2 gene in the genome of the target plant, the substance for silencing or inhibiting the expression of the ZmKNL2 gene in the genome of the target plant, the ZmKNL2 gene knocking out the ZmKNL2 gene in the genome of the target plant, the application of the above ZmKNL2 mutant gene or the transgenic plant containing the above ZmKNL2 mutant gene also falls within the scope of protection of the present invention. The application is any one of the following:

[0029] (1) Application in the preparation of plant haploids;

[0030] (2) Application in breeding haploid induction lines;

[0031] (3) Application in improving the efficiency of haploid plant breeding.

[0032] In the above application, the substance for knocking out the ZmKNL2 gene in the genome of the target plant can be the above CRISPR / Cas9 system.

[0033] In the above, the plant is (A1) or (A2) or (A3):

[0034] (A1) dicotyledonous plants or monocotyledonous plants;

[0035] (A2) Gramineae;

[0036] (A3)Corn.

[0037] Any of the target plants mentioned above can specifically be corn B73.

[0038] Any of the other plants mentioned above can specifically be the corn inbred line Mo17.

[0039] In an embodiment of the present invention, the substance for knocking out the ZmKNL2 gene in the genome of the target plant is a CRISPR / Cas9 knockout vector; the target site sequence of the CRISPR / Cas9 knockout vector is shown in Sequence 2 of the sequence table; the sgRNA sequence expressed by the CRISPR / Cas9 knockout vector is shown in Sequence 4 of the sequence table. The CRISPR / Cas9 knockout vector can be specifically a recombinant vector obtained by inserting the DNA molecule shown in Sequence 2 into the Esp3I site of the sgRNA-Cas9 dual expression vector.

[0040] The basic principle of the embodiment of the present invention is as follows: Taking the transcript T3 as an example, for the ZmKNL2 gene, a target site sequence is designed on its third exon, and the third exon of the gene is subjected to mutation screening by the CRISPR / Cas9 site-directed mutagenesis method to obtain a transgenic mutant with a loss of gene function. The T1 generation seeds obtained after the successful mutation are self-pollinated are planted again, and the pollen of the homozygous mutant of the T1 generation plant is used to grant the corn inbred line Mo17 to obtain offspring. The hybrid offspring are planted in the field, and the presence of maternal haploids is verified according to the growth of the offspring single plant in the field, molecular markers, and flow cytometry ploidy identification.

[0041] The present invention discloses an application of a gene ZmKNL2 for inducing the production of maize haploids. The embodiment of the present invention uses CRISPR / Cas9 technology to knock out the ZmKNL2 gene in maize to obtain a transgenic homozygous mutant plant or its offspring, which can produce a maize maternal haploid in the process of hybridization with other maize materials as a male parent. The present invention obtains a series of allelic mutations of the gene for the first time, and proves its maternal haploid induction function through hybridization. The embodiment of the present invention proves that the ZmKNL2 mutation of maize can lead to the production of maize maternal haploids, which provides a new idea for revealing the biological role of ZmKNL2 in the production process of maize maternal haploids. At the same time, the mutant individual plant obtained by the present invention has the haploid induction ability of the maize maternal parent, which is of great significance for breeding a new type of high induction rate induction line and improving the efficiency of maize haploid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of the ZmKNL2 gene structure and CRISPR / Cas9 system knockout target sites.

[0043] Figure 2 It is the mutant form of the zmknl2-1 mutant gene.

[0044] Figure 3 4 is the phenotypic identification result of the embodiment.

[0045] Figure 4 The results of flow cytometry identification of leaf ploidy in this example are shown.

[0046] Figure 5 The polymorphic molecular marker identification results of the embodiment. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0049] sgRNA-Cas9 dual expression vector: recorded in the literature: Zhu J, Song N, Sun S, Yang W, Zhao H, Song W, Lai J. Efficiency and Inheritance of targeted mutagenesis in maize using CRISPR-Cas9. J Genet Genomics. 2016; 43: 25-36.; the public can obtain it from China Agricultural University.

[0050] B73: described in the literature: Schnable, P S et al. The B73 enhances genome: complexity, diversity, and dynamics. Science. 2009; 326: 1112-1115.; available to the public from China Agricultural University.

[0051] Mo17: described in the literature: Sun S, Zhou Y, Chen J, Shi J, Zhao H, Zhao H, Song W, Zhang M, Cui Y, Dong X, Liu H, Ma X, Jiao Y, Wang B, Wei X, Stein JC, Glaubitz JC, Lu F, Yu G, Liang C, Fengler K, Li B, Rafaiski A, Schnable PS, Ware DH, Buckler ES, Lai J. Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes. Nature Genetics. 2018; 50(9): 1289-1295.; available to the public from China Agricultural University.

[0052] Example 1: Gene ZmKNL2 for inducing haploid production in maize maize parent and its application

[0053] The genomic sequence of the wild-type maize ZmKNL2 gene is shown in Sequence 1 of the sequence table. There are three transcripts in the ZmKNL2 gene. The first exon of the coding gene corresponding to transcript T1 is shown in Sequence 1 of the sequence table from the 98th to the 244th position from the 5' end, the second exon is shown in Sequence 1 of the sequence table from the 338th to the 494th position from the 5' end, the third exon is shown in Sequence 1 of the sequence table from the 672th to the 862th position from the 5' end, and the fourth exon is shown in Sequence 1 of the sequence table from the 1068th to the 2036th position from the 5' end. The first exon of the coding gene corresponding to transcript T2 is shown as positions 98-244 from the 5' end of Sequence 1 in the sequence listing, the second exon is shown as positions 338-452 from the 5' end of Sequence 1 in the sequence listing, the third exon is shown as positions 672-862 from the 5' end of Sequence 1 in the sequence listing, and the fourth exon is shown as positions 1068-2036 from the 5' end of Sequence 1 in the sequence listing. The first exon of the coding gene corresponding to transcript T3 is shown as positions 98-244 from the 5' end of Sequence 1 in the sequence listing, the second exon is shown as positions 338-438 from the 5' end of Sequence 1 in the sequence listing, the third exon is shown as positions 688-862 from the 5' end of Sequence 1 in the sequence listing, the fourth exon is shown as positions 1068-2009 from the 5' end of Sequence 1 in the sequence listing, the fifth exon is shown as positions 2839-2902 from the 5' end of Sequence 1 in the sequence listing, and the sixth exon is shown as positions 3083-3129 from the 5' end of Sequence 1 in the sequence listing.

[0054] 1. Knockout of the ZmKNL2 gene in maize using the CRISPR / Cas9 system

[0055] Taking transcript T3 as an example, the corresponding ZmKNL2 gene structure and CRISPR / Cas9 system knockout target site schematic diagram are shown in Figure 1 .

[0056] 1. Design a target site sequence on the third exon sequence of the maize ZmKNL2 gene with a length of 23 bp.

[0057] Target site sequence: 5'-AGAGAAGCATCGTCCTCAAAGGG-3' (sequence 2).

[0058] sgRNA target site sequence binding region sequence: 5'-AGAGAAGCAUCGUCCUCAAAGGG-3' (sequence 3).

[0059] 2. Preparation of CRISPR / Cas9 knockout vector

[0060] The DNA molecule shown in sequence 2 is inserted into the Esp3I site of the sgRNA-Cas9 dual expression vector, and the vector verified by sequencing is named CRISPR / Cas9 knockout vector. The CRISPR / Cas9 knockout vector is a vector in which the Esp3I site of the sgRNA-Cas9 dual expression vector is inserted into the DNA molecule shown in sequence 2, while keeping the other nucleotide sequences of the CRISPR / Cas9 knockout vector unchanged. The vector expresses the sgRNA shown in sequence 4.

[0061] 3. The CRISPR / Cas9 knockout vector prepared in step 2 was introduced into the Agrobacterium competent cell EHA105. The positive clones were identified by colony PCR and selected for sequencing. The primers for colony PCR and sequencing identification were CHE-F and CHE-R. The positive clones verified by sequencing were named recombinant bacteria EHA105 / CRISPR / Cas9.

[0062] CHE-F: 5'-AACGCAATTAATGTGAGTTAGCTCA-3';

[0063] CHE-R: 5'-TACTCAATTGTCCTTTAGACCATGT-3'.

[0064] The recombinant bacteria EHA105 / CRISPR / Cas9 were then used to transform corn B73 immature embryos using the Agrobacterium infection method (the recombinant bacteria were propagated at 28°C, and the propagated bacterial solution was used to infect corn immature embryos). After screening, differentiation and rooting, T0 generation transgenic corn plants were obtained.

[0065] 4. Collect leaves of the T0 transgenic corn plants obtained in step 3, extract genomic DNA as a template, and perform PCR amplification using a primer pair consisting of primer check-F and primer check-R to obtain amplification products of different strains.

[0066] check-F: 5'-GGAGCCCTAAAATAACTCGTCGT-3';

[0067] check-R: 5'-TCCCCCAAGATACAGTTATGTTTCCT-3'.

[0068] The PCR amplification products of different strains were sequenced, and the sequencing results were compared with the corresponding sequence of the third exon of the wild-type maize ZmKNL2 gene to identify whether the gene in different strains of T0 generation transgenic maize was mutated.

[0069] The results are as follows: Among the five T0 transgenic corn plants, one plant had a gene mutation, and the mutant gene was named zmknl2-1. The specific mutation form is as follows: Figure 2 As shown, the only difference between the ZmKNL2 mutant gene zmknl2-1 and the wild-type corn ZmKNL2 gene is that the nucleotide sequence of the ZmKNL2 mutant gene zmknl2-1 is that the 705th base C of the sequence 1 in the sequence list is deleted from the 5' end, and the other nucleotides remain unchanged. This deletion causes a frameshift mutation, resulting in the loss of function of the ZmKNL2 gene and the inability to encode the ZmKNL2 protein.

[0070] Plants with homozygous mutations in the gene were recorded as positive T0 transgenic corn, that is, the ZmKNL2 genes in the two homologous chromosomes in the genome of the T0 transgenic corn were both ZmKNL2 mutant genes zmknl2-1.

[0071] 5. After harvesting the seeds of the positive T0 transgenic corn obtained in step 4, sow and self-pollinate to obtain T1 transgenic corn. The specific method for identifying whether the gene of the T1 transgenic corn is a mutant genotype is as follows: using the genomic DNA of the T1 transgenic corn as a template, using a primer pair consisting of primer check-F and primer check-R to perform PCR amplification, sequencing the PCR product, and classifying the genotype of the T1 transgenic corn according to the sequencing result.

[0072] In the sequencing results, (1) the sequence with a double peak characteristic from the target site sequence is a heterozygous genotype, which is a heterozygous gene mutation in the T1 generation transgenic corn (the gene in one homologous chromosome is mutated, and the gene in the other homologous chromosome is not mutated); (2) the sequence with a specific single peak characteristic from the target site sequence is compared with the third exon sequence of the wild-type corn ZmKNL2 gene. If they are the same, it is wild type and no mutation has occurred, which is not considered in the following analysis; if there is a mutation, it is a homozygous mutation obtained after self-pollination of the T0 generation plants, which is a homozygous gene mutation in the T1 generation transgenic corn (the ZmKNL2 genes in both homologous chromosomes are mutated).

[0073] After analysis, the T1 generation transgenic corn homozygous gene mutation strain includes zmknl2-1, and its mutation type is as follows: the two homologous chromosomes of the T1 generation transgenic corn gene mutation homozygous strain zmknl2-1 contain the mutant gene zmknl2-1 (the only difference between the nucleotide sequence of this mutant gene and the wild-type corn ZmKNL2 gene is that the nucleotide sequence of the ZmKNL2 mutant gene zmknl2-1 is that the 705th base C from the 5' end of sequence 1 in the sequence list is deleted, and the other nucleotides remain unchanged).

[0074] II. Identification of the haploid-inducing ability of mutants obtained by knocking out the maize ZmKNL2 gene using the CRISPR / Cas9 system

[0075] The pollen of the T1 generation transgenic corn gene homozygous mutant strain zmknl2-1 prepared above was inoculated to the inbred line Mo17 to obtain hybrid offspring, and determine whether the hybrid offspring contained the maize maternal haploid.

[0076] The criteria for determining the haploidy of the maize maternal parent are as follows: in the hybrid offspring, if the plant is identified as haploid according to any of the following three methods (polymorphic molecular markers, flow cytometry identification, and phenotypic identification), then the plant is the maize maternal parent haploid; if the identification results of none of the following three methods are haploid, then the plant is not the maize maternal parent haploid.

[0077] (1) Identification of polymorphic molecular markers

[0078] Ten pairs of molecular markers were randomly designed on the maize B73 reference genome. The genomic DNA of inbred lines B73 and Mo17 were used as templates for amplification and polymorphic molecular marker screening, and finally one pair of molecular markers was obtained.

[0079] Molecular marker: CHR-10-53.5-F: 5′-GGTGTTTCCTCGCTGTCGTTT-3′;

[0080] CHR-10-53.5-R: 5'-GGTCCAGATCAGATCCGCATTA-3'.

[0081] The length of the PCR product of the genomic DNA of corn B73 amplified by the molecular marker is 400bp, and the length of the PCR product of the genomic DNA of corn Mo17 amplified by the molecular marker is 300bp, which has a large difference and can be distinguished by agarose gel electrophoresis. The PCR product of B73 is larger and the electrophoresis speed is slow, while the product fragment of Mo17 is smaller and the electrophoresis speed is fast. Therefore, the band of B73 is located above the band of Mo17 (such as Figure 5 As shown in the figure, the second lane is the Mo17 band type, and the third lane is the B73 band type). If the tested individual strain has only the Mo17 band (such as Figure 5 If the hybrid offspring has both B73 and Mo17 bands (such as the 5th or 6th lane shown in Figure 1), the individual plant is considered to have no band pattern of the paternal material and is therefore a maternal haploid. Figure 5 If the single plant is not hybridized, it is considered to be a diploid offspring of normal hybridization.

[0082] The hybrid offspring obtained above were sown in a greenhouse, and genomic DNA was extracted from leaves of 14-day-old seedlings. The molecular markers were amplified by PCR and the band patterns were detected by agarose gel electrophoresis. The molecular marker identification results were as follows:

[0083] Among the 1377 offspring obtained by crossing the T1 generation transgenic corn homozygous gene mutant strain zmknl2-1 with the inbred line Mo17, two plants with only the Mo17 band were obtained and named as pseudohaploid plants.

[0084] The electrophoresis results of PCR products of two pseudohaploid plants are as follows Figure 5 As shown, M is a marker, Mo17 is the electrophoresis result of the PCR product of maize Mo17, B73 is the electrophoresis result of the PCR product of maize B73, D is the electrophoresis result of the PCR product of the heterozygous diploid in the hybrid offspring, and H1 and H2 are the electrophoresis results of the PCR product of the pseudohaploid plants in the hybrid offspring.

[0085] (2) Flow cytometry to identify leaf ploidy

[0086] A total of 2 pseudo-haploid plants showing haploid banding patterns obtained by (1) in the above hybrid offspring were subjected to flow cytometry identification, and the method is as follows: extract the nuclei of the young leaves of the test plant, and use diploid corn leaves as a control; then use a flow cytometer to detect the signal. First, the diploid cell nuclear signal is detected, and the diploid cell nuclear signal peak is set to the A value (because the genetic material in the diploid cell is twice that in the haploid cell, the haploid cell nuclear signal peak appears near half of the A value). If the signal peak of the test plant appears near the A value, it is considered to be enriched with the diploid cell nuclear signal intensity and at the same position, and the test plant is diploid. If the cell nuclear signal peak of the test plant appears near half 2 of the A value, the test plant is considered to be a haploid plant.

[0087] The results are as follows Figure 4 As shown, the left figure is the flow cytometry test result of B73 wild-type corn, and the right figure is the flow cytometry test result of one of the pseudohaploid plants in the T1 generation of transgenic corn hybrid offspring. Figure 4 The middle right figure is the same. Compared with the B73 wild-type corn, where the nuclear signal peak appears near 100, the nuclear signal peak of the pseudohaploid plant appears near 50.

[0088] The results showed that the ploidy of two pseudohaploids identified by polymorphic molecular markers in the hybrid offspring of zmknl2-1 and inbred line Mo17 were both haploid after detection by flow cytometry.

[0089] (3) Phenotypic identification

[0090] The phenotypes of the two pseudohaploid plants that emerged from the hybrid offspring of the T1 generation homozygous gene mutant strain zmknl2-1 and the inbred line Mo17 were continuously observed. The haploids have the characteristics of short plants, narrow leaves, compact plant shape, and male sterility, while the diploids have tall plants, wide and spreading leaves, and normal fertility.

[0091] Observation results such as Figure 3 As shown, the upper left and lower left pictures are heterozygous diploid plants and their anthers in the hybrid offspring of the T1 generation homozygous gene mutation strain zmknl2-1 and the inbred line Mo17; the upper right and lower right pictures are plants and their anthers of one of the pseudohaploids in the hybrid offspring, and the plants and anthers of the above two pseudohaploid plants are similar in morphology to the upper right and lower right pictures. Compared with the diploid plants and anthers of the control, the pseudohaploid plants are short, compact, have smaller anthers, and are male sterile.

[0092] The results showed that the pseudohaploid offspring identified by polymorphic molecular markers and flow cytometry in the hybrid offspring of zmknl2-1 and Mo17 were all haploid plants of the maternal parent after phenotypic identification.

[0093] According to the formula, induction rate (%) = (number of haploid plants / total number of plants tested) * 100%, in this embodiment, the induction rate of the T1 generation transgenic corn gene homozygous mutant strain zmknl2-1 is 0.15% ((2 / 1377) * 100%), indicating that when corn plants with ZmKNL2 gene mutation are hybridized with other materials, corn maternal haploids can be obtained in the offspring.

[0094] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. Sequence Listing <110> China Agricultural University <120> Gene ZmKNL2 for inducing haploid production in maize and its application <130> 211825 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3456 <212> DNA <213> Zea mays <400> 1 ctaaaataac tcgtcgttct cgtaggcagc gcgccagcgc cgccgtctac tctcctttct 60 tgccgcgcgg cgccgccgtc tactctcgct caccggaatg ccgtcccagc ctcagccgag 120 ctccatcggc ggaacctccc agaccccggc gccgttccag accgcgcccg tcttcaccgg 180 cggaagcccc cggaccctgg cgacgctctc cgctcagtcc gtcccccgcg tcacgcggag 240 aagcgtacgc gcctctcacc cgtcctgtct ctcatttttc gtctttgcgc gctgtgcggt 300 cttatcgact gatgtgttcc ttcgctgctt cttttagatt gcgctcgtcg attggtgttt 360 ggagagggtg gagggcgagg agggcaagat ccgcgtcgct ggaaccacct acaccccgca 420 aacgagcccg caaacgaggc atctcctccg cggttccatt tatcgatttc gttttcccaa 480 ttaccctccc aaatgtgctt tagttgtaca tcctgtttgg aaaattttga tagctttctt 540 ttcctcaatt aagaaattta gtagatcggt aaatatgata ttttctttgg caatttgaga 600 aattcaatgg tagcaaggga atgccaaaag atttccatcg gtactccaca attaatctca 660 cggtgctgta gcctactatg caaacaggag agaagcatcg tcctcaaagg caaacagga 720 agtggcaggc cgagtgttca gatcatctgc tattgttagg cgccatgatc acttcagaat tatgtccgag gatggttacc tcatacggat aggctgtctc ttgaatattc ccaaaacacg taacaatgga ttttctgaag aggtaattgc tatgattggt ctccagggac tggaattttg gaggcgcat tgctagtaaa ataatggaca atcctgtgca ccaattcatt atattctttt tcattaatcc tttagatacc cagctgatcc taggtaattt aatttctagt gccttagaag atgaccagca aattctcatc tgtatatata tccatgactt tttgtaggtg tgtgaatgct ttgagtttgg ctttccaatt caatggcaca ggcttgttaa tccaaagatg gtgcccgata ataacatgc actgtcacca tcagaaacaa ctgctagtgc accaagtcat tcagttggat attacatgga gaagttctta agtgacagtt tttccaattc taacagatat tctttcacag aatgattc ttacacatct gtagtgtgta ctggcagcag aaatggactt actacccaaa ccctttcaaa cttaccagat gataatgcag gaaacataac tgtatcttgg ggattatatg gcctcgggat ggacatgtct gagaaacctt ggacaccacc tgcagaagcg tgtaataata 1440 gacaagaaag tgatcaacat gaaagtatgc aaatagatgc atgtaaacaa gagttagtta 1500 atcgctccat gagttctgtt tcagttaagc aaagtataag ttccatcagc cccaactcaa 1560 aagtagacgg caacagaatt gtaccatcca aaattatgtc agttgtgaat gaaagttaca 1620 ggagcacagt aggttgtggt caggctgagg aagacgttgg tattcagcaa gagaaaaagc 1680 acagctgttc aagtgagcat ggaatggtta ctctttctat taactgtaca tcctctcaac 1740 taggtgcacc aggaattcca aaatttggga aagattcagt gaacctttgg actacagatg 1800 cattagaact atcaactgaa gggatgactc caaagattgg agctgacagt actgacagga 1860 gattacgaag tggcaaaatt cttggaatgc caagtggtgg actaatgaat agaggccaca 1920 agctgaaaaa gatcaagcag gaagcatcca gtaaacagat ggttaatcaa ggggctacat 1980 gcactgtgga tctgacttct catgaaaatg taggtttaat tttacacctt gaataaatca 2040 gttaacaaca gttattctga tttaattaat attcttttga taccctgata ctctttttgg 2100 cctgtggttc acccttcact ctgatcacac tacttttat tatcttat tatgtgcata 2160 ttgccaattg ccattcattg ttgtggtttt acacaatgca taatatcctg ttctacacc 2220 tttagttccc tgaatggta tctgtaaga tgagttggtt ggttaactga tgtctaaggg 2280 taatgtatta ttcctacaag tattaactt tgtaagcct ttgaagaga ctaatggtga 2340 atgaaccata attctcatg ttaataaatc attgaagaat tattttttca tgtggttatt 2400 tttatataaa acaatcag gggctctttc tgttatgttt tgtaatctta cttcctataa 2460 aactttagca gatagctga gcagcacgggg aagattgtga aaaagcaac ttttttaac 2520 tatacacctt atatatgctt agaccaaact tctgaagt tcctgcttac ccagccacca 2580 tgtgcagtaa aagtctgttt cttacatgtt atgtttgttg ttgtactttg tttccattat 2640 gabaaatatt ttatacaca tagttgaagg gaacagtggt atgcctgttc atgttctgtt 2700 aacattgtat tgatcatctc cagtcatatg aagctttgct ggaatgctgt caagcaatca 2760 tgtgacttgt tgaatcgtta ctgggcaaag acctgtacgg cctatttggt aggtctcatt 2820 agatgtcaat aattgcagga tttttcagca gctgagattg tcgttgaaga aaagctggaa 2880 tcacatagtt catgccttaa aggtcagttt gataatctgc agagtttgca cttattcttg 2940 agcactactc ctgtctataa tatcactaag gttggtttag gagctaaatg ccactaactc 3000 tgttttggta tgttgttttg aactatgaaa aagaagacta tgtataacct ccccccaagt 3060 actaatcaca aaatattgac aggacgagga ggaccagcga aggggaagag gaagagggag 3120 agatggtagg attcagcgtg tctgttttca agccggatgt gaaccgtgtt tcagtttgtt 3180 tctcggcttg agtagctaac ttatgtagat gggtcgtgtg tgccagttcc atgagcagat 3240 ggtgccacta agggtgtgtt tagtttgagg tcaaagtagg atgagtcagg ggcgacatcg 3300 ttccctcgac tttttgggat cacgtcgcca caaaaaacta ctccggggtt tttctcgctc 3360 atgcgtgatt ctcatccaaa cactgttgaa actgtggtca ctctatttca ttcctccatt 3420 tacatccaac caaatacact ctaatttaca cgcctc 3456 <210> 2 <211> 23 <212> DNA <213> Zea mays <400> 2 agagaagcat cgtcctcaaa ggg 23 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <400> 3 agagaagcau cguccucaaa ggg 23 <210> 4 <211> 99 <212> RNA <213> Artificial Sequence <400> 4 agagaagcau cguccucaaa gggguuuuag agcuagaaau agcaaguuaa aauaaggcua 60 guccguuauc aacuugaaaa aguggcaccg agucggugc 99

Claims

1. A method for preparing plant haploids, characterized in that: The steps include: 1) Knockout in the target plant genome ZlUT Gene, to obtain transgenic plants; 2) hybridizing the transgenic plant or its offspring obtained in step 1) as the male parent with other plants to obtain hybrid offspring, that is, to obtain plant haploids, Said ZlUT A gene is a DNA molecule that is any of the following: (1) A DNA molecule having a sequence as shown in SEQ ID NO: 1 of the sequence listing; (2) The DNA molecule comprises a first exon, a second exon, a third exon and a fourth exon, wherein the sequence of the first exon is shown in positions 98 to 244 of SEQ ID NO. 1 of the sequence listing, the sequence of the second exon is shown in positions 338 to 494 of SEQ ID NO. 1 of the sequence listing, the sequence of the third exon is shown in positions 672 to 862 of SEQ ID NO. 1 of the sequence listing, and the sequence of the fourth exon is shown in positions 1068 to 2036 of SEQ ID NO. 1 of the sequence listing; (3) The DNA molecule comprises a first exon, a second exon, a third exon and a fourth exon, wherein the sequence of the first exon is shown in SEQ ID NOs. 98-244 of SEQ ID NOs. 1 of the sequence listing, the sequence of the second exon is shown in SEQ ID NOs. 338-452 of SEQ ID NOs. 1 of the sequence listing, the sequence of the third exon is shown in SEQ ID NOs. 672-862 of SEQ ID NOs. 1 of the sequence listing, and the sequence of the fourth exon is shown in SEQ ID NOs. 1068-2036 of SEQ ID NOs. 1 of the sequence listing; (4) The DNA molecule comprises a first exon, a second exon, a third exon, a fourth exon, a fifth exon and a sixth exon, the sequence of the first exon is shown as positions 98-244 of SEQ ID NO. 1 of the sequence listing, the sequence of the second exon is shown as positions 338-438 of SEQ ID NO. 1 of the sequence listing, the sequence of the third exon is shown as positions 688-862 of SEQ ID NO. 1 of the sequence listing, the sequence of the fourth exon is shown as positions 1068-2009 of SEQ ID NO. 1 of the sequence listing, the sequence of the fifth exon is shown as positions 2839-2902 of SEQ ID NO. 1 of the sequence listing, and the sequence of the sixth exon is shown as positions 3083-3129 of SEQ ID NO. 1 of the sequence listing; The knockout ZlUT Genes are achieved through gene editing technology; The gene editing technology uses the CRISPR / Cas9 system; The CRISPR / Cas9 system is any of the following: (b1) comprising a specific sgRNA, wherein the target site sequence binding region of the specific sgRNA is shown in sequence 3 of the sequence listing; (b2) includes a specific DNA molecule, wherein the specific DNA molecule is transcribed to obtain the specific sgRNA described in (b1); (b3) comprising a plasmid having the specific DNA molecule described in (b2); (b4) comprising a specific recombinant plasmid, wherein the specific recombinant plasmid expresses a specific sgRNA, and the target site sequence binding region of the specific sgRNA is shown in Sequence 3 of the sequence listing; The plant is corn.

2. The method according to claim 1, characterized in that: In step 2), the following step is also included: selecting offspring with haploid ploidy from the hybrid offspring, that is, plant haploids.

3. A CRISPR / Cas9 system for preparing plant haploids, characterized in that: The CRISPR / Cas9 system is the CRISPR / Cas9 system described in claim 1.

4. ZlUT A mutant gene characterized by: The nucleotide sequence is a sequence in which the 705th base C of the sequence 1 in the sequence list is deleted from the 5' end, and the other nucleotides remain unchanged.

5. Knockout in the target plant genome ZlUT Genetic material or the one described in claim 4 ZlUT The application of mutant genes is characterized by: The application is any of the following: (1) Application in the preparation of plant haploids; (2) Application in breeding haploid induction lines; (3) Application in improving the efficiency of haploid plant breeding; The knockout target plant genome ZlUT The genetic material is the CRISPR / Cas9 system; The CRISPR / Cas9 system is any of the following: (b1) comprising a specific sgRNA, wherein the target site sequence binding region of the specific sgRNA is shown in sequence 3 of the sequence listing; (b2) includes a specific DNA molecule, wherein the specific DNA molecule is transcribed to obtain the specific sgRNA described in (b1); (b3) comprising a plasmid having the specific DNA molecule described in (b2); (b4) comprising a specific recombinant plasmid, wherein the specific recombinant plasmid expresses a specific sgRNA, and the target site sequence binding region of the specific sgRNA is shown in Sequence 3 of the sequence listing; The plant is corn.

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