WO2020156406A1 - Wheat haploid induced gene and application thereof - Google Patents

Abstract

Provided are a wheat haploid induced gene and an application thereof. A PLA gene encoding wheat phospholipase is obtained by analyzing the homologous gene of the induced gene ZmPLA1 of maize in wheat, , which is present in the three chromosome groups of wheat A, B and D and named as PLA-A, PLA-B and PLA-D respectively. A transgenic material with PLA gene mutation is obtained through the site-directed mutagenesis technology and transgenic experiment, and using the transgenic material for inbred or hybridization with other materials, a certain proportion of haploid plants are observed in the offspring, the wheat material after PLA mutation is verified to have the function of inducing the production of wheat maternal haploid.

Description

Wheat haploid induction gene and its application Technical field

The invention relates to the field of biotechnology, in particular to wheat haploid induction genes and applications thereof.

Background technique

Wheat is the main food crop in the world. Maintaining a steady increase in wheat yield and continuous improvement of quality is still an important task for wheat breeding. As we all know, wheat is a typical self-pollinated crop, and the varieties used in production are also genetically homozygous pure line materials. Therefore, although the breeding of new wheat varieties does not require pollination every generation, it still needs to be self-purified generation by generation. After 8 generations of natural selfing or above, excellent pure line materials can be obtained. Although artificial pollination is not required, since most wheat materials can only be planted one generation per year, it still consumes a lot of time and energy. Different from the genomes of crops such as corn and rice, the genome of wheat is heterohexaploid, and the relatively complex characteristics of the genome significantly increase the difficulty of wheat breeding.

Compared with traditional breeding methods, haploid sports seeding method can significantly improve breeding efficiency, and has been widely used in corn and other crops. Therefore, it is of great significance to establish a technical system of wheat haploid seed. Predecessors explored a variety of methods to produce wheat haploids, such as gametophyte culture, distant hybridization, and apomixis. However, gametophyte culture and distant hybridization methods still require a lot of tedious operations, or use corn pollen, which is not efficient and is greatly affected by the material background, making it difficult to use on a large scale. The haploid induction method based on induced genes has been successfully applied in maize on a large scale, and its efficiency can reach more than 10%. It has become the main method for the selection of maize backbone inbred lines. Therefore, if the method of haploid induction in maize can be applied to wheat haploid seed, the efficiency of wheat haploid seed can be greatly improved.

Invention Disclosure

The technical problem to be solved by the present invention is how to improve the efficiency of wheat haplotype.

In order to solve the above technical problems, the present invention first provides a method for preparing a wheat female parent haploid induction line.

The preparation method of the wheat maternal haploid induction line provided by the present invention includes the following steps: silencing or inhibiting the expression and/or activity of the PLA gene in the target wheat genome or knocking out the PLA gene to obtain transgenic wheat, which is the wheat maternal single Ploidy induction line;

The PLA gene is the PLA-A gene in the wheat A genome and/or the PLA-B gene in the wheat B genome and/or the PLA-D gene in the wheat D genome. The genomic sequences of the PLA-A gene, the PLA-B gene and the PLA-D gene are as shown in sequence 1, sequence 4 and sequence 7 in the sequence listing, respectively.

Further, the silencing or suppressing the expression and/or activity of the PLA gene in the target wheat genome or knocking out the PLA gene is to mutate the PLA gene in the target wheat genome to reduce the PLA gene expression in the target wheat genome or to reduce the PLA gene in the target wheat genome. Gene deletion mutation or insertion mutation or base substitution.

Furthermore, the method for causing deletion mutation, insertion mutation or base substitution in the PLA gene in the target wheat genome is CRISPR/Cas9.

In a specific embodiment of the present invention, the target sequence of the CRISPR/Cas9 is 905-923 of sequence 1 and 939-957 of sequence 7.

In another specific embodiment of the present invention, the target sequence of the CRISPR/Cas9 is the 699-718th position of sequence 1.

In the method for preparing the wheat maternal haploid induction line, the silencing or suppressing the expression and/or activity of the PLA gene in the target wheat genome or knocking out the PLA gene is performed by introducing the substance that knocks out the PLA gene in the target wheat genome into the target Wheat realization.

Further, the substance for knocking out the PLA gene in the target wheat genome may be a CRISPR/Cas9 vector. In a specific embodiment of the present invention, the CRISPR/Cas9 vector is a CRISPR/Cas9-1 vector, which is a DNA sequence encoding a sgRNA target site designed for the PLA-A gene and the PLA-D gene (sequence 1 Positions 905-923) and the coding DNA sequence of the sgRNA target site designed for PLA-D gene (positions 939-957 of sequence 7) are inserted into pBUN411 vector together. In another specific embodiment of the present invention, the CRISPR/Cas9 vector is a CRISPR/Cas9-2 vector, which is a sgRNA target site designed for PLA-A gene, PLA-B gene and PLA-D gene The vector obtained by inserting the coding DNA sequence (sequence 1 at positions 699-718) into the pBUN411 vector.

Furthermore, the wheat of interest may be wild-type wheat material CB037.

In order to solve the above technical problems, the present invention provides a method for preparing wheat female parent haploid.

The method for preparing wheat female parent haploid provided by the present invention includes the following steps: selfing the wheat female parent haploid induction line or its progeny prepared by the above method or crossing it as a male parent with other wheat materials to obtain selfed progeny Or the hybrid offspring is the haploid of the wheat female parent.

Further, the method further includes the steps of: performing haploid trait identification and/or leaf ploidy identification and/or molecular identification on the selfed progeny or the hybrid progeny single plant, and selecting at least one method for identification as The progeny of the haploid plant is the female parent haploid of wheat.

Furthermore, the method for identifying haploid traits can be carried out according to the following method: if the plant to be tested has the characteristics of short plant, narrow leaves, overshoot, compact plant type, and male sterility, then the plant is or candidate It is a haploid; if the plant to be tested has the characteristics of plant height, wide leaves, scattered, and normal fertility, then the plant is or candidate is diploid.

The leaf ploidy identification method can be carried out as follows: extract the nucleus of the young leaves of the plant to be tested, and use diploid wheat leaves as a control; then use flow cytometry to detect the signal, first detect the diploid nucleus signal, and The nuclear signal peak position of diploid cells is set to 100 (since the genetic material in diploid cells is twice that of haploid cells, the haploid nuclear signal peak position appears near 50). If the nuclear signal peak of the tested plant appears near 50, the plant is or candidate is haploid; if the signal peak of the tested plant appears near 100, which is the same as the diploid nuclear signal intensity enrichment position, then the The plant is or candidate is diploid.

The molecular marker identification can be carried out according to the following method: PCR amplification is carried out with polymorphic primers between the male parent (maternal haploid induction line) and the female parent, and the plant to be tested is judged whether it is haploid or haploid based on the PCR amplification product Diploid: If the amplified product of the plant to be tested only has the band pattern of the maternal parent, and there is no band pattern of the male parent, the plant is or candidate is haploid; if the amplified product of the tested plant has the male parent If the plant is heterozygous with the female parent, the plant is or candidate is diploid.

The wheat female parent haploid induction line or the wheat female parent haploid prepared by the above method also belong to the protection scope of the present invention.

In order to solve the above technical problems, the present invention also provides a protein.

The protein provided by the present invention is a protein shown in the following a) or b) or c) or d):

a) The amino acid sequence is the protein shown in sequence 3 or sequence 6 or sequence 9;

b) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in sequence 3 or sequence 6 or sequence 9;

c) A protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence of sequence 3 or sequence 6 or sequence 9;

d) A protein having 75% or more homology with the amino acid sequence shown in Sequence 3 or Sequence 6 or Sequence 9 and having the same function.

In order to solve the above technical problems, the present invention also provides biological materials related to the above protein.

The biological material provided by the present invention is any one of the following A1) to A12):

A1) A nucleic acid molecule encoding the above-mentioned protein;

A2) An expression cassette containing the nucleic acid molecule described in A1);

A3) A recombinant vector containing the nucleic acid molecule described in A1);

A4) A recombinant vector containing the expression cassette described in A2);

A5) A recombinant microorganism containing the nucleic acid molecule described in A1);

A6) A recombinant microorganism containing the expression cassette described in A2);

A7) A recombinant microorganism containing the recombinant vector described in A3);

A8) A recombinant microorganism containing the recombinant vector described in A4);

A9) A transgenic plant cell line containing the nucleic acid molecule described in A1);

A10) A transgenic plant cell line containing the expression cassette of A2);

A11) A transgenic plant cell line containing the recombinant vector described in A3);

A12) A transgenic plant cell line containing the recombinant vector described in A4).

In the above biological material, the nucleic acid molecule in A1) is the gene shown in 1) or 2) or 3) as follows:

1) Its coding sequence is a cDNA molecule or genomic DNA molecule shown in sequence 1 or sequence 2 or sequence 4 or sequence 5 or sequence 7 or sequence 8;

2) A cDNA molecule or genomic DNA molecule that has 75% or more than 75% identity with the defined nucleotide sequence of 1) and encodes the above-mentioned protein;

3) A cDNA molecule or genomic DNA molecule that hybridizes to the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the above-mentioned protein.

The following 1)-6) any application also belongs to the protection scope of the present invention:

1) Application of the wheat maternal haploid induction line prepared by the above method in the preparation of wheat maternal haploid;

2) Silencing or inhibiting the expression and/or activity of the PLA gene in the target wheat genome or the application of the substance knocking out the PLA gene in the preparation of wheat maternal haploid induction lines or wheat maternal haploids;

3) Application of the wheat female parent haploid induction line prepared by the above method or the wheat female parent haploid prepared by the above method in the breeding of wheat hybrids or the application of wheat haploid species;

4) Application of the above-mentioned protein or biological material in regulating the induction rate of wheat maternal haploid induction lines;

5) Application of the above-mentioned protein or biological material in increasing the induction rate of wheat female parent haploid induction line;

6) The application of the above-mentioned protein or biological material in the cultivation of haploid wheat female parent.

In the above application, the substance for silencing or inhibiting the expression and/or activity of the PLA gene in the target wheat genome or knocking out the PLA gene may be a CRISPR/Cas9 vector for knocking out the PLA gene. In a specific embodiment of the present invention, the CRISPR/Cas9 vector is the aforementioned CRISPR/Cas9-1 vector. In another specific embodiment of the present invention, the CRISPR/Cas9 vector is the aforementioned CRISPR/Cas9-2 vector.

Description of the drawings

Figure 1 is a schematic diagram of the PLA gene structure and the setting of the target site using CRISPR/Cas9 technology.

Figure 2 shows the results of phenotypic identification. The left side is diploid; the right side is haploid.

Figure 3 shows the results of leaf ploidy identification. The left side is diploid; the right side is haploid.

Figure 4 shows the results of molecular identification. F is the male parent, CS is China Spring, F1 is the first generation of hybrids, and H is haploid.

The best way to implement the invention

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

Example 1. Obtaining wheat haploid induction genes

According to the location of maize haploid inducing gene and transgene verification, the maize haploid inducing gene ZmPLA was determined. Use the online biological information platform (www.gramene.com) to analyze and obtain the homologous genes of ZmPLA in wheat. There is a homologous gene in the A, B, and D genomes of wheat, and they are named PLA- A gene, PLA-B gene and PLA-D gene.

The genomic sequence of the PLA-A gene is shown in sequence 1, the CDS sequence is shown in sequence 2, and the amino acid sequence of the protein encoded by the PLA-A gene is shown in sequence 3.

The genomic sequence of the PLA-B gene is shown in sequence 4, the CDS sequence is shown in sequence 5, and the amino acid sequence of the protein encoded by the PLA-B gene is shown in sequence 6.

The genomic sequence of the PLA-D gene is shown in sequence 7, the CDS sequence is shown in sequence 8, and the amino acid sequence of the protein encoded by the PLA-D gene is shown in sequence 9.

Example 2. Method for inducing the haploid of wheat female parent

1. CRISPR/Cas9 system knocks out wheat PLA gene to obtain mutants

This example uses the CRISPR/Cas9 system to knock out the PLA-A gene in the wheat A genome and/or the PLA-B gene in the B genome and/or the PLA-D gene in the D genome to obtain the following PLA gene mutations and be able to induce haploids Wheat mutants: knock out the PLA-A gene in the A genome, the PLA-B gene in the B genome and the PLA-D gene in the D genome at the same time; simultaneously knock out the PLA-A gene and the D genome in the A genome Wheat mutants with PLA-D gene; wheat mutants with the PLA-A gene in the A genome knocked out alone; wheat mutants with the PLA-A gene in the D genome knocked out alone.

The specific preparation method of the mutant is as follows:

1. Selection of sgRNA target site sequence

Figure 1 is a schematic diagram of gene structure and target sites.

The genomic sequences of wheat PLA-A gene, PLA-B gene and PLA-D gene are shown in sequence 1, sequence 4, and sequence 7 in the sequence table, respectively. For the three target genes, two target site designs were carried out, as follows:

1) Design two target sites for PLA-A gene and PLA-D gene for the first time

a. The target site sequence designed for the PLA-A gene and the PLA-D gene to knock out the two genes at the same time is CCAGGGACGTCAACCGCTT (the target site sequence is located at positions 905-923 of sequence 1 or 905-923 of sequence 7) . The sgRNA target site sequence designed for the target site sequence is CCAGGGACGUCAACCGCUU, and the coding DNA sequence of the sgRNA target site is CCAGGGACGTCAACCGCTT.

b. The target site sequence for the PLA-D gene is CCCCTACATCTTCCCGCAA (the target site sequence is located at positions 939-957 in sequence 7). The sgRNA target site sequence designed for the target site sequence is CCCCUACAUCUUCCCGCAA, and the coding DNA sequence of the sgRNA target site is CCCCTACATCTTCCCGCAA.

2) Design a target site for the PLA-A gene, PLA-B gene and PLA-D gene for the second time

c. The target site sequence designed for the PLA-A gene, PLA-B gene and PLA-D gene to knock out the three genes at the same time is GACGGTGCTGACCATCGACG (the target site sequence is located at the 699-718th of the sequence 1 or the sequence 4 702-721 bits or 699-718 bits in sequence 7). The sgRNA target site sequence designed for the target site sequence is GACGGUGCUGACCAUCGACG, and the coding DNA sequence of the sgRNA target site is GACGGTGCTGACCATCGACG.

2. Construction of CRISPR/Cas9 vector

The CRISPR/Cas9-1 vector is the coding DNA sequence of the sgRNA target site designed for the PLA-A gene and PLA-D gene in step 1 a and the sgRNA target site designed for the PLA-D gene in step 1 b The coding DNA sequences of the two are inserted into the pBUN411 vector (pBUN411 is recorded in the following documents: Xing H L, Dong L, Wang Z P, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants[J]. BMC plant biology, 2014 ,14(1):1.).

The CRISPR/Cas9-2 vector inserts the coding DNA sequences of the sgRNA target sites designed for the PLA-A gene, PLA-B gene and PLA-D gene in step 1 c into the pBUN411 vector (pBUN411 is described in the following document: Xing H L, Dong L, Wang Z P, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants[J]. BMC plant biology, 2014, 14(1):1.).

3. Obtaining genetically modified wheat

Firstly, CRISPR/Cas9-1 vector and CRISPR/Cas9-2 vector were transformed into Agrobacterium competent cell EHA105 (purchased from Huayueyang Biotechnology Co., Ltd., publicly available through purchase) through heat shock transformation, respectively, and recombinant bacteria were obtained. EHA105/CRISPR/Cas9-1 and recombinant strain EHA105/CRISPR/Cas9-2.

Then, the recombinant bacteria EHA105/CRISPR/Cas9-1 and the recombinant bacteria EHA105/CRISPR/Cas9-2 were respectively infected with Agrobacterium (recombinant Agrobacterium was expanded at 28℃, and the expanded bacterial solution was used to infect wheat ) Transformation of wheat receptor material CB037 (CB037 is recorded in the following documents: Ye Xingguo, Chen Ming, Du Lipu, & Xu Huijun. (2011). Wheat transgenic method and its review (Doctoral dissertation)) immature embryos.

Finally, the T0 generation transgenic wheat plants were obtained after screening, differentiation and rooting according to conventional methods.

4. Identification of transgenic wheat with mutations in PLA gene

The leaves of T0 generation transgenic wheat plants were collected, and genomic DNA was extracted as a template, and PCR amplification was performed with the following primers to obtain PCR amplification products of different strains.

PLA mutation sequence detection primer:

PLA-A: 4AF: GTCAAGATCTCCAGCCGAGAC;

4AR: GGTACTTGCCGCTGTACCT;

PLA-B: 4BF: AACTCAACATGGGGCGTCCTC;

4BR: ACGTCGTATGTGGAGAAGATGATG;

PLA-D: 4DF: TTCGGGTCCGGATTCTATTGTG;

4DR: GCAGGTACTTGCCGTTGTACC.

The PCR products of different strains were sequenced by Sanger and compared with the wild-type wheat PLA gene according to the sequencing results to identify whether the PLA-A gene, PLA-B gene and PLA-D gene in different strains of T0 generation transgenic wheat Mutation occurred. Plants with mutations in the PLA gene were recorded as positive T0 generation transgenic wheat.

5. Genotype identification of transgenic wheat with mutations in PLA gene in T1 generation

The positive T0 generation transgenic wheat obtained in the above step 4 is harvested and then sown to obtain T1 generation transgenic wheat. To identify the genotype of the PLA gene of the T1 generation transgenic wheat, the specifics are as follows: using the genomic DNA of the T1 generation transgenic wheat as a template, using the PLA mutation sequence detection primers for amplification, the PCR product is subjected to Sanger sequencing, and the T1 generation transgenic according to the sequencing results The mutation of PLA gene in wheat is described.

1) T1 generation transgenic wheat PLA gene mutant lines with double and single mutations

In the selfed progeny of T0 transgenic wheat plants obtained by the recombinant strain EHA105/CRISPR/Cas9-1 infecting the wheat receptor material CB037, the PLA gene of T1 transgenic wheat with both PLA-A and PLA-D gene mutations was obtained Mutant line Da14-1, PLA-A gene mutation T1 generation transgenic wheat PLA gene mutant line Da14-1-A and PLA-D gene mutation alone T1 generation transgenic wheat PLA gene mutant line Da14-1 -D.

The PLA-A genes on the two homologous chromosomes in the A genome of the T1 transgenic wheat PLA gene mutant strain Da14-1 are both PLA-A-1 mutant genes, and the PLA on the two homologous chromosomes in the D genome -D genes are all PLA-D-1 mutant genes. The PLA-A-1 mutant gene is the gene sequence obtained by deleting the 909th base G of the PLA-A gene shown in sequence 1; the PLA-D-1 mutant gene is the PLA-D gene shown in sequence 7 The gene sequence obtained after deletion of GG at positions 908-909.

The PLA-A genes on the two homologous chromosomes in the A genome of the T1 transgenic wheat PLA gene mutant strain Da14-1-A are both PLA-A-1 mutant genes. The PLA-A-1 mutant gene is a gene sequence obtained by deleting the 909th base G of the PLA-A gene shown in SEQ ID NO:1.

The PLA-D genes on the two homologous chromosomes in the D genome of the T1 generation transgenic wheat PLA gene mutant strain Da14-1-D are both PLA-D-1 mutant genes. The PLA-D-1 mutant gene is a gene sequence obtained by deleting the GG at positions 908-909 of the PLA-D gene shown in SEQ ID NO: 7.

2) The PLA gene mutant line of T1 generation transgenic wheat with three mutations

In the selfed progeny of T0 transgenic wheat plants obtained by the recombinant strain EHA105/CRISPR/Cas9-2 infecting the wheat receptor material CB037, the T1 generation with simultaneous mutations of PLA-A, PLA-B and PLA-D genes was obtained Transgenic wheat PLA gene mutant line Ne147-1.

The PLA-A gene on the two homologous chromosomes in the A genome of the T1 transgenic wheat PLA gene mutant line Ne147-1 are both PLA-A-2 mutant genes, and the PLA on the two homologous chromosomes in the B genome -B genes are both PLA-B-1 mutant genes, and the PLA-D genes on the two homologous chromosomes in the D genome are both PLA-D-2 mutant genes. The PLA-A-2 mutant gene is the gene sequence obtained by deleting the TCGA at bases 713-716 of the PLA-A gene shown in sequence 1; the PLA-B-1 mutant gene is the PLA- shown in sequence 4. The gene sequence obtained by inserting 1 base C between the 718th and 719th positions of the B gene; the PLA-D-2 mutant gene is the 716th and 717th positions of the PLA-D gene shown in sequence 7 The gene sequence obtained by inserting a base G between.

2. Identification of haploid inducibility of mutants obtained by knocking out wheat PLA gene by CRISPR/Cas9 system

1. Field phenotype identification

T1 generation transgenic wheat PLA gene mutant lines (T1 generation transgenic wheat PLA gene mutant line Da14-1, T1 generation transgenic wheat PLA gene mutant line Da14-1-A, T1 generation transgenic wheat PLA gene mutant line Da14- 1-D, T1 generation transgenic wheat PLA gene mutant line Ne147-1) materials were selfed to obtain selfed progeny.

T1 generation transgenic wheat PLA gene mutant lines (T1 generation transgenic wheat PLA gene mutant line Da14-1, T1 generation transgenic wheat PLA gene mutant line Da14-1-A, T1 generation transgenic wheat PLA gene mutant line Da14- The pollen of the 1-D and T1 transgenic wheat PLA gene mutant strain Ne147-1) was granted to wild-type wheat material, China Spring, to be crossed to obtain hybrid offspring. At the same time, the pollen of wild-type wheat CB037 (with no mutation in the PLA gene) was granted to the offspring of wild-type wheat China Spring as a control.

The progeny obtained above were sown in the field, and the phenotype of the individual plants of the progeny was observed. The haploid has the characteristics of short plants, narrow leaves, and upswing, compact plant type, and male sterility. The diploid is characterized by tall plants and leaves. It is wide, loose and fertile (Figure 2).

The system calculation results for each strain are shown in Table 1.

T1 generation transgenic wheat PLA gene mutant line Da14-1 selfed 154 progeny, 17 out of 154 progeny showing haploid traits single plant, proposed to be haploid plants;

Two of the 248 progeny of the T1 transgenic wheat PLA gene mutant line Da14-1-A selfed showed haploid traits, which were planned as haploid plants.

One of the 225 progenies of the T1 generation transgenic wheat PLA gene mutant line Da14-1-D self-bred was obtained as a single plant with haploid traits, which was proposed as a haploid plant.

Among the 179 progeny of the T1 transgenic wheat PLA gene mutant line Ne147-1 selfed, 31 single plants with haploid traits were obtained, which were planned to be haploid plants.

Fifteen of the 140 offspring of the T1 generation transgenic wheat PLA gene mutant line Da14-1 crossed with wild-type Chinese spring were single plants with haploid traits, which were planned as haploid plants;

One of the 215 progeny of the T1 generation transgenic wheat PLA gene mutant line Da14-1-A crossed with wild-type Chinese spring was a single plant with haploid traits, and it was planned to be a haploid plant.

Two of the 210 offsprings of the T1 generation transgenic wheat PLA gene mutant line Da14-1-D crossed with wild-type Chinese spring showed haploid traits, which were planned to be haploid plants.

Among the 137 progenies of the T1 generation transgenic wheat PLA gene mutant line Ne147-1 crossed with wild-type Chinese spring, 24 single plants with haploid traits were obtained, which were planned to be haploid plants.

2. Flow cytometry to detect leaf ploidy

Perform flow cytometric detection of the plants with haploid traits identified in step 1 above. The method is as follows:

Extract the nucleus of the young leaves of the plant to be tested, and use diploid wheat leaves as a control; then use flow cytometry to detect the signal, first detect the diploid nuclear signal, and set the diploid nuclear signal peak position to 100 (due to The genetic material in diploid cells is twice that in haploid cells. Therefore, the haploid cell nuclear signal peak position theoretically appears near 50); if the signal peak of the tested plant appears near 100, then It is considered that it is the same as the enrichment position of diploid cell nuclear signal intensity, and the plant to be tested is diploid. If the nuclear signal peak of the plant to be tested appears near 50, the plant to be tested is considered to be a haploid plant (Figure 3). The error of flow cytometry measurement is small, so the tested plants can be accurately divided into two groups of haploid and diploid after the measurement.

The results showed that the haploid plants identified by the phenotype in the selfed progeny of the T1 generation transgenic wheat PLA gene mutant line or the progeny of crosses with China Spring were all haploid plants after flow cytometry.

3. Molecular marker identification

The haploid plants in the hybrid offspring of the mutant material in the above step 1 and the Chinese spring were used for genotype identification using polymorphic molecular markers (Xbarc284 and xgwm124-1B). The results are shown in Figure 4, and the results show that the haploid plants only have the band type of Chinese Spring (mother parent). It is further proved that the haploid obtained by this method is parthenogenetic maternal haploid. The primer sequence of the polymorphism marker is as follows:

Xbarc284-L: GCGTCAGAAATGCAAGAAAAATAGG;

Xbarc284-R: GCGGAAGAAAAGGACGAAGACAAG;

Xgwm124-1B-F: GCCATGGCTATCACCCAG;

Xgwm124-1B-R: ACTGTTCGGTGCAATTTGAG.

Therefore, if the T1 generation transgenic wheat PLA gene mutant line is selfed or crossed with hybrids, if it is identified as haploid according to any of the above three methods, the plant is or candidate It is a wheat female parent haploid; if none of the above three methods is a haploid, the plant is not or the candidate is not a wheat female parent haploid.

The haploid induction rate was calculated based on the number of haploids obtained by each strain, and the haploid induction rate (%)=(number of haploids/total number of plants tested)×100%. The statistical results are shown in Table 1: It can be seen that the T1 generation transgenic wheat PLA gene mutant line Ne147-1 has the highest haploid induction rate, and the T1 generation transgenic wheat PLA gene mutant strain It is followed by Da14-1.

In summary, the PLA gene mutant plants obtained by mutating the PLA-A gene in the wheat A genome and/or the PLA-B gene in the wheat B genome and/or the PLA-D gene in the wheat D genome can be used as wheat A female parent haploid induction line, which is selfed or crossed with other wheat materials to obtain a wheat female parent haploid in the offspring.

Table 1. T1 generation transgenic wheat PLA gene mutant lines selfed and haploid plants in the offspring of crosses with Chinese spring

Conversion event	Mother parent	Number of offspring	Number of haploids	Induction rate
Da14-1	Chinese Spring	140	15	10.71%

Da14-1-A	Chinese Spring	215	1	0.47%
Da14-1-D	Chinese Spring	210	2	0.95%
Ne147-1	Chinese Spring	137	twenty four	17.52%
Da14-1	Selfing	154	17	11.03%
Da14-1-A	Selfing	248	2	0.81%
Da14-1-D	Selfing	225	1	0.44%
Ne147-1	Selfing	179	31	17.32%
Wild type control	Wild type CB037	485	0	0

Industrial application

The present invention obtains the homologous gene PLA encoding wheat phospholipase by analyzing the homologous genes of the induced gene ZmPLA1 in maize in wheat. The gene exists in the three chromosomes A, B, and D of wheat, respectively named PLA -A gene, PLA-B gene and PLA-D gene, through CRISPR/Cas9 site-directed mutagenesis technology and transgenic experiments, successfully obtained PLA gene mutation transgenic materials, using transgenic materials for selfing or hybridization with other materials, in the offspring A certain proportion of haploid plants was observed, which verified that the wheat material after PLA mutation has the function of inducing the haploid of the female parent of wheat. The present invention not only lays an important foundation for revealing the genetics and biological mechanism of wheat female parent haploid production, but also is important for breeding new induction lines, further increasing the induction rate, and improving the efficiency of wheat haploid species. Meaning.

Claims (13)

1. A method for preparing a wheat maternal haploid induction line, comprising the steps of: silencing or inhibiting the expression and/or activity of PLA gene in the target wheat genome or knocking out the PLA gene to obtain transgenic wheat, which is a wheat maternal haploid Body induction line

   The PLA gene is the PLA-A gene in the wheat A genome and/or the PLA-B gene in the wheat B genome and/or the PLA-D gene in the wheat D genome.
2. The method according to claim 1, wherein the silencing or suppressing the expression and/or activity of the PLA gene in the target wheat genome or knocking out the PLA gene is to mutate the PLA gene in the target wheat genome to make the PLA gene in the target wheat genome The decrease in expression level may cause deletion mutation or insertion mutation or base substitution in the PLA gene in the target wheat genome.
3. The method according to claim 2, wherein the method for causing the PLA gene in the target wheat genome to undergo deletion mutation, insertion mutation or base substitution is CRISPR/Cas9.
4. The method according to claim 3, wherein the target sequence of the CRISPR/Cas9 is the 905-923th sequence 1 and the 939-957 sequence 7th.
5. The method according to claim 3, wherein the target sequence of the CRISPR/Cas9 is sequence 1 699-718.
6. A method for preparing a wheat female parent haploid, comprising the steps of: selfing the wheat female parent haploid induction line or its progeny prepared by the method of any one of claims 1-5 or as a male parent with other Crossing wheat materials to obtain selfed progeny or hybrid progeny is the haploid of the wheat female parent.
7. The method according to claim 6, characterized in that: the method further comprises the steps of: performing haploid character identification and/or leaf ploidy identification and/or the selfed progeny or the hybrid progeny single plant Molecular identification, selecting at least one method to identify the progeny individual plants as haploids are wheat maternal haploids.
8. A wheat maternal haploid induction line prepared by the method of any one of claims 1-5.
9. A wheat female parent haploid prepared by the method of claim 6 or 7.
10. Protein is the protein shown in a) or b) or c) or d) as follows:

    a) The amino acid sequence is the protein shown in sequence 3 or sequence 6 or sequence 9;

    b) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in sequence 3 or sequence 6 or sequence 9;

    c) A protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence of sequence 3 or sequence 6 or sequence 9;

    d) A protein having 75% or more homology with the amino acid sequence shown in Sequence 3 or Sequence 6 or Sequence 9 and having the same function.
11. The biological material related to the protein of claim 10 is any one of the following A1) to A12):

    A1) A nucleic acid molecule encoding the protein of claim 10;

    A2) An expression cassette containing the nucleic acid molecule described in A1);

    A3) A recombinant vector containing the nucleic acid molecule described in A1);

    A4) A recombinant vector containing the expression cassette described in A2);

    A5) A recombinant microorganism containing the nucleic acid molecule described in A1);

    A6) A recombinant microorganism containing the expression cassette described in A2);

    A7) A recombinant microorganism containing the recombinant vector described in A3);

    A8) A recombinant microorganism containing the recombinant vector described in A4);

    A9) A transgenic plant cell line containing the nucleic acid molecule described in A1);

    A10) A transgenic plant cell line containing the expression cassette of A2);

    A11) A transgenic plant cell line containing the recombinant vector described in A3);

    A12) A transgenic plant cell line containing the recombinant vector described in A4).
12. The biological material according to claim 11, characterized in that: A1) the nucleic acid molecule is the gene shown in 1) or 2) or 3):

    1) Its coding sequence is a cDNA molecule or genomic DNA molecule shown in sequence 1 or sequence 2 or sequence 4 or sequence 5 or sequence 7 or sequence 8;

    2) A cDNA molecule or genomic DNA molecule that has 75% or more than 75% identity with the defined nucleotide sequence of 1) and encodes the protein of claim 10;

    3) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein of claim 10.
13. The following 1)-6) any application:

    1) Application of the wheat maternal haploid induction line prepared by the method of any one of claims 1-5 in the preparation of wheat maternal haploid;

    2) Silencing or suppressing the expression and/or activity of the PLA gene in the target wheat genome or the application of the substance knocking out the PLA gene in the preparation of wheat maternal haploid induction lines or wheat maternal haploids;

    3) The wheat maternal haploid induction line prepared by the method of any one of claims 1-5 or the wheat maternal haploid prepared by the method of claim 6 or 7 is selected in wheat hybrids or Application of wheat haploid sports species;

    4) Use of the protein of claim 10 or the biological material of claim 11 or 12 in regulating the induction rate of wheat maternal haploid induction lines;

    5) Use of the protein of claim 10 or the biological material of claim 11 or 12 in increasing the induction rate of wheat maternal haploid induction lines;

    6) Use of the protein of claim 10 or the biological material of claim 11 or 12 in the cultivation of wheat female parent haploid.

Images