Gene ZmPLD3 for inducing maize maternal haploid production and its application thereof
A gene ZmPLD3 for inducing maize maternal haploid production and its application thereof. Transgenic homozygous mutant plants or their progeny can be obtained by knocking out the ZmPLD3 gene in maize, and maize maternal haploids can be produced by hybridizing them as paternal materials with other maize materials. A series of allelic mutations of the gene having maternal haploid induction function through hybridization were obtained. The experiments showed that the mutation of maize phospholipase PLD3 could lead to the production of maize maternal haploid, which provides new thoughts for revealing the biological role of phospholipase in maize maternal haploid induction process. At the same time, the mutant individuals have the maternal haploid induction ability in maize, which is of great significance for breeding new types of haploid induction lines with high haploid induction rate and improving the efficiency of maize haploid breeding.
1 . A method for inducing maternal haploids, comprising the following steps:
(1) silencing or knocking out a ZmPLD3 gene in a target plant genome to obtain a transgenic plant;
(2) crossing the transgenic plant obtained in step (1) as a male parent with female plants to obtain hybrid offspring which are maternal haploids;
wherein the ZmPLD3 gene according to step (1) is any one of the following DNA molecules:
(1) a DNA molecule whose genome sequence is as shown in SEQ ID NO: 1 of the sequence listing; and
(2) a DNA molecule that originated from maize, whose sequence is at least 90%, identical to the DNA sequence defined in (1) and with the same function, wherein the plant is maize.
2 . The method according to claim 1 , wherein silencing or knocking out the ZmPLD3 gene in the target plant genome, comprises mutating the ZmPLD3 gene in the target plant genome to reduce an expression of the ZmPLD3 gene in the target plant genome or cause the ZmPLD3 gene in the target plant genome to lose function.
3 . The method according to claim 2 , wherein reducing the expression of the ZmPLD3 gene in the target plant genome or causing the ZmPLD3 gene in the target plant genome to lose function comprises mutating a first exon and/or a second exon and/or a third exon of the ZmPLD3 gene in the target plant genome; wherein the mutation is at least one of a deletion mutation, an insertion mutation, and a substitution mutation.
4 . The method according to claim 3 , wherein the mutation comprises inserting the base T between the 2205 th base and the 2206 th base of the 5′ end of the ZmPLD3 gene in the target plant genome.
5 . The method according to claim 3 , wherein the mutation is achieved by CRISPR/Cas9 gene editing technology; and wherein a target sequence of the CRISPR/Cas9 is shown in SEQ ID NO: 2 of the sequence listing.
6 . The method according to claim 1 , wherein step (2) further comprises the following steps of:
screening individual plants of the hybrid progeny by at least one method selected from the group consisting of haploid phenotypic identification, leaf ploidy identification and molecular marker identification to identify haploid plants; and
selecting the individual plants of the hybrid progeny identified as haploid as maternal haploids.
7 . A method for preparing a maternal haploid induction line comprising silencing or knocking out a ZmPLD3 gene in the target plant genome to obtain a transgenic plant comprising a plant maternal haploid induction line;
the ZmPLD3 gene is any one of the following DNA molecules:
(1) a DNA molecule whose genome sequence is as shown in SEQ ID NO: 1 of the sequence listing; and
(2) a DNA molecule that originated from maize, whose sequence is at least 90%, identical to the DNA sequence defined in (1) and with the same function, and wherein the plant is maize.
8 . The method according to claim 7 , wherein silencing or knocking out the ZmPLD3 gene comprises:
mutating the ZmPLD3 gene in the target plant genome to reduce an expression of the ZmPLD3 gene in the target plant genome or cause the ZmPLD3 gene in the target plant genome to lose function.
9 . The method according to claim 8 , wherein reducing the expression of the ZmPLD3 gene in the target plant genome or causing the ZmPLD3 gene in the target plant genome to lose function comprises:
mutating at least one of a first exon and/or a second exon and/or a third exon of the ZmPLD3 gene in the target plant genome, wherein the mutation is at least one of a deletion mutation, an insertion mutation, and a substitution mutation.
10 . The method according to claim 9 , wherein the mutation comprises inserting the base T between the 2205 th base and the 2206 th base of the 5′ end of the ZmPLD3 gene in the target plant genome.
11 . The method according to claim 10 , wherein the mutation is achieved by CRISPR/Cas9 gene editing technology; and wherein a target sequence of the CRISPR/Cas9 is shown in SEQ ID NO: 2 of the sequence listing.