Parthenogenetic haploid induction gene
Provided are a parthenogenetic haploid induction gene DMP and an application thereof. The parthenogenetic haploid induction genes AtDMP8 and AtDMP9 are cloned from Arabidopsis thaliana . Experiments have shown that mutations of AtDMP8 and AtDMP9 can produce parthenogenetic haploid inducibility, to enable dicotyledonous crops to be induced to produce haploids via parthenogenetic means. The present invention was further verified in tomatoes, and it was also found in tomatoes that the mutation of SlDMP can produce parthenogenetic haploid inducibility. The invention lays an important foundation for broadening the application of haploid breeding technology on dicotyledonous plants and revealing the biological mechanism of parthenogenetic haploid production. Given the universality of the utilization of haploid breeding technology in the current breeding industry, the invention has very wide application space and market prospects.
1 . A method for preparing a plant haploid inducer line is as follows A1) or A2):
A1) knocking out the gene AtDMP8 and gene AtDMP9 in the Arabidopsis thaliana genome to obtain a transgenic Arabidopsis thaliana , namely the Arabidopsis thaliana haploid inducer line;
A2) knocking out the gene SIDMP in the tomato genome to obtain a transgenic tomato, namely the tomato haploid inducer line; wherein
the protein encoded by the gene AtDMP8 is a protein shown in SEQ ID NO: 2, or a protein having 85% or more identity with the amino acid sequence shown in SEQ ID NO. 2and having the same function, or a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in SEQ ID NO. 2;
the protein encoded by the gene AtDMP9 is a protein shown in SEQ ID NO: 4, or a protein having 85% or more identity with the amino acid sequence shown in SEQ ID NO. 4 and having the same function, or a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in SEQ ID NO. 4; and
the protein encoded by the gene SIDMP is a protein shown in SEQ ID NO: 6, or a protein having 85% or more identity with the amino acid sequence shown in SEQ ID NO. 6 and having the same function, or a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein shown in SEQ ID NO. 6.
2 . The method according to claim 1 , wherein, the gene AtDMP8 is a cDNA molecule or a genomic DNA molecule shown in SEQ ID NO. 1 or a cDNA molecule or a genomic DNA molecule having 85% or more identity to SEQ ID NO. 1, or a cDNA molecule or a genomic DNA molecule that hybridizes with SEQ ID NO. 1 under stringent conditions;
the gene AtDMP9 is a cDNA molecule or a genomic DNA molecule shown in SEQ ID NO. 3, or a cDNA molecule or a genomic DNA molecule having 85% or more identity to SEQ ID NO. 3, or a cDNA molecule or a genomic DNA molecule that hybridizes with SEQ ID NO. 3 under stringent conditions;
the gene SIDMP is a cDNA molecule or a genomic DNA molecule shown in SEQ ID NO. 5, or a cDNA molecule or a genomic DNA molecule having 85% or more identity to SEQ ID NO. 5, or a cDNA molecule or a genomic DNA molecule that hybridizes with SEQ ID NO. 5 under stringent conditions;
the genes AtDMP8 and AtDMP9 have the following functions: the Arabidopsis becomes an Arabidopsis haploid inducer line when the genes AtDMP8 and AtDMP9 are silenced or inhibited or knocked out therein; and
the gene SIDMP has the following functions: the tomato becomes a tomato haploid inducer line when the gene SIDMP is knocked out therein.
3 . The method according to claim 1 , wherein, the said knocking out gene AtDMP8 and gene AtDMP9 or said knocking out gene SIDMP is by CRISPR/Cas9.
4 . The method according to claim 3 , wherein, when selecting Arabidopsis thaliana , the target site of the CRISPR/Cas9 is located at positions 98-117 of SEQ ID NO. 1, positions 290-309 of SEQ ID NO. 3, positions 368-387 of SEQ ID NO. 3 and positions 509-528 of SEQ ID NO. 1.
5 . The method according to claim 3 , wherein, when selecting tomato, the target site of the CRISPR/Cas9 is located at positions 76-95 of SEQ ID NO. 5, positions 247-266 of SEQ ID NO. 5.
6 . The method according to claim 1 , wherein, the plant haploid inducer line specifically comprises an Arabidopsis thaliana mutant line T1-34, an Arabidopsis thaliana mutant line T1-6, an Arabidopsis thaliana mutant line T1-11, an Arabidopsis thaliana mutant line T1-19, an Arabidopsis thaliana mutant line T1-24, an Arabidopsis thaliana mutant line T1-25, an Arabidopsis thaliana mutant line T1-28, or an Arabidopsis thaliana mutant line T1-32;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-34 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID NO. 1, and a base (T) insertion occurs in the other chromosome at positions 114-115 of SEQ ID NO. 1;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-6 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID NO. 1, and a fragment deletion occurs in the other chromosome at positions 113-114 of SEQ ID NO. 1; and in a gene encoding protein AtDMP9, a fragment insertion occurs in both chromosomes at positions 160-161 of SEQ ID NO. 3, with the nucleotide sequence of the inserted fragment shown as SEQ ID NO. 13;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-11 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) insertion occurs at positions 114-115 of SEQ ID NO. 1, and in a gene encoding protein AtDMP9, a base (T) insertion occurs in one chromosome at positions 160-161 of SEQ ID NO. 3, and a fragment insertion occurs in the other chromosome at positions 160-161 of SEQ ID NO. 3, with the nucleotide sequence of the inserted fragment shown as SEQ ID NO. 13;
the difference between the genome of the Arabidopsis thaliana mutant line T1-19 and the genome of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) deletion occurs in one chromosome at position 114 of SEQ ID NO. 1, and a fragment deletion occurs in the other chromosome at positions 115-511 of SEQ ID NO. 1, and in a gene encoding protein AtDMP9, a fragment deletion occurs in one chromosome at positions 161-560 of SEQ ID NO. 3, and a fragment deletion occurs in the other chromosome at positions 161-564 of SEQ ID NO. 3;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-24 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID NO. 1, and a base (T) insertion occurs in the other chromosome at positions 114-115 of SEQ ID NO. 1, and in a gene encoding protein AtDMP9, a fragment deletion occurs in one chromosome at positions 161-560 of SEQ ID NO. 3, and a fragment deletion occurs in the other chromosome at positions 159-160 of SEQ ID NO. 3;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-25 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a base (T) deletion occurs in one chromosome at position 114 of SEQ ID NO. 1, and a fragment CGT insertion occurs in the other chromosome at positions 114-115 of SEQ ID NO. 1; and in a gene encoding protein AtDMP9, a fragment deletion occurs in one chromosome at positions 161-162 of SEQ ID NO. 3, and a base (A) insertion occurs in the other chromosome at positions 160-161 of SEQ ID NO. 3;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-28 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-512 of SEQ ID NO. 1, and a base (T) deletion occurs in the other chromosome at position 114 of SEQ ID NO. 1, and in a gene encoding protein AtDMP9, and a base (A) insertion occurs in both chromosomes at positions 160-161 of SEQ ID NO. 3; and
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T1-32 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in one chromosome at positions 115-511 of SEQ ID NO. 1, and a base (T) insertion occurs in the other chromosome at positions 114-115 of SEQ ID NO. 1, and in a gene encoding protein AtDMP9, and a base (C) deletion occurs in both chromosomes at position 161 of SEQ ID NO. 3.
7 . The method according to claim 1 , wherein the method further comprising the step of producing the offspring of the plant haploid inducer line: selfing the plant haploid inducer line.
8 . The method according to claim 7 , wherein, the plant haploid inducer line is an Arabidopsis thaliana mutant line T2-33, an Arabidopsis thaliana mutant line T2-38, a tomato mutant line sldmp-1 or a tomato mutant line sldmp-2;
the difference of the genomic DNA of the Arabidopsis thaliana mutant line T2-33 and the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP9, a base (T) insertion occurs in both chromosomes at positions 160-161 of SEQ ID NO. 3, and a fragment insertion occurs in both chromosomes at positions 561-562 of SEQ ID NO. 3, with the nucleotide sequence of the inserted fragment shown as SEQ ID NO. 14;
the difference between the genomic DNA of the Arabidopsis thaliana mutant line T2-38 and the genomic DNA of the wild-type Arabidopsis thaliana Col-0 only lies in that in a gene encoding protein AtDMP8, a fragment deletion occurs in both chromosomes at positions 115-127 of SEQ ID NO. 1, and in a gene encoding protein AtDMP9, a base (T) and a base (G) insertion occurs in both chromosomes at positions 160-161 of SEQ ID NO. 3 and positions 562-563 of SEQ ID NO. 3, respectively;
the difference between the genome DNA of the tomato mutant line sldmp-1 and the genome DNA of wild-type tomato AC only lies in that in a gene encoding SIDMP protein, and a base (C) insertion occurs in both chromosomes at positions 92-93 of SEQ ID NO. 5; and
the difference between the genome DNA of the tomato mutant line sldmp-2 and the genome DNA of wild-type tomato AC only lies in that in a gene encoding protein SIDMP, a fragment deletion occurs in both chromosomes at positions 93-249 of SEQ ID NO. 5.