Cloning and application of maize nucleus male fertility gene IPE2
阅读说明：本技术 一个玉米细胞核雄性育性基因ipe2的克隆与应用 (Cloning and application of maize nucleus male fertility gene IPE2 ) 是由 刘娟 臧杰 陈化榜 张怀仁 于 2020-07-13 设计创作，主要内容包括：本发明公开了一种与植物雄性育性相关的蛋白及其编码基因与应用。本发明所提供的蛋白是如下(a)或(b)：(a)由序列表中序列1所示的氨基酸序列组成的蛋白质；(b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加,且与植物雄性育性相关的由序列1衍生的蛋白质。本发明的IPE2基因在玉米中能够控制雄性育性。本发明为植物特别是玉米的雄性育性研究提供了新的基因资源,其在玉米制种和育种领域的应用中将发挥重要作用。(The invention discloses a protein related to plant male fertility and a coding gene and application thereof. The protein provided by the invention is (a) or (b) as follows: (a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table; (b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1, is related to the male fertility of plants and is derived from the sequence 1. The IPE2 gene of the present invention is capable of controlling male fertility in maize. The invention provides a new gene resource for the male fertility research of plants, particularly corn, and the gene resource plays an important role in the application of the corn seed production and breeding fields.)
1. A protein which is (a) or (b) below:
(a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;
(b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1, is related to the male fertility of plants and is derived from the sequence 1.
2. A nucleic acid molecule encoding the protein of claim 1.
3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a gene encoding the protein of claim 1, and the gene is a DNA molecule selected from the group consisting of 1) to 5) below:
1) DNA molecule shown in sequence 2 in the sequence table;
2) DNA molecule shown in sequence 3 in the sequence table;
3) DNA molecule shown in sequence 4 in the sequence table;
4) a DNA molecule which hybridizes with the DNA molecule defined in any one of 1) to 3) under strict conditions and codes for a protein which is related to the male fertility of plants and is derived from the sequence 1;
5) a DNA molecule which has more than 90% of identity with the DNA sequence defined in any one of 1) to 4) and codes a protein which is related to the male fertility of plants and is derived from the sequence 1.
4. A recombinant vector, expression cassette, transgenic cell line or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.
5. The recombinant vector according to claim 4, wherein: the recombinant vector is a recombinant expression vector or a recombinant cloning vector.
6. Use of a protein according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a recombinant vector, expression cassette, transgenic cell line or recombinant microorganism according to claim 4 or 5 for plant breeding and/or seed production.
7. The method for cultivating the transgenic plant comprises the following steps:
the method for cultivating the transgenic plant provided by the invention can be as follows:
(1) introducing an IPE2 protein coding gene into an IPE2/IPE2 receptor plant to obtain a transgenic plant expressing the coding gene; the male sterility trait of said recipient plant is due to the loss or reduced ability of said recipient plant to express functional said IPE2 protein;
(2) obtaining a male-fertile transgenic plant from the transgenic plant obtained in the above step; the coding gene can be introduced into the recipient plant by the above recombinant expression vector p3300-IPE 2.
8. The method of claim 7, wherein: in step 1), the encoding gene is introduced into the recipient plant through the recombinant expression vector of claim 5.
9. Use or method according to claim 7 or 8, characterized in that: the plant is a monocotyledon or a dicotyledon.
10. Use or method according to any of claims 7-9, wherein: the monocotyledon is a gramineous plant;
the Gramineae plant is corn.
Technical Field
The invention belongs to the field of plant genetic engineering, and relates to cloning and application of a corn nucleus male fertility gene IPE 2.
Background
Plant male sterility refers broadly to the fact that dysplasia of the male organs of plants ultimately leads to male sterility in the mature stage (Chen and Liu, 2014) and can stabilize remainsA phenomenon that is passed on to offspring, but the female reproductive system develops normally. The male sterility of the plant is mainly expressed by the developmental deformity of a male reproductive system and the disappearance of male flower atrophy; normal microspore generating tissues cannot be formed, and microspores are abnormal; pollen grain malformation, immaturity, inevitability, and anther dehiscence, etc. 1763 Joseph Gottlieb in GermanyProfessor finds the phenomenon of plant male sterility for the first time (Mayr, 1986); in 1876, Coleman introduced the concept of "male sterility of plants" for the first time. In 1908, Bateson et al found a nuclear male sterility controlled by a recessive gene in sweet pea. Male sterility has been reported to occur in 617 plant species, including 43 families, 162 genera and 320 species (Kaul, 1988).
The entire complex developmental process of plant gametophytes (gammophytes) is finely regulated by the cooperation of their own sporophyte genes and Gametophyte genes (Boavida et al, 2005). When plants are transformed from vegetative to reproductive growth, there is differentiation from sub-epidermal lamellar cells to form flower primordia in the flower (Ma, 2005; Ma et al, 2008), but only a few of them differentiate to form germ cells (Kelliher and Walbot, 2011), and anther primordia undergo a complex differentiation process to form sporocytes, which undergo one meiosis and two mitoses to form microspores (McCormick, 1993; Scott et al, 2004). Male sterility of plants may result if any gene involved in male gamete development is mutated.
Male sterile materials have been used in hybrid seed production of various crops such as corn (Wan et al, 2019), rice (Li et al, 2007), sorghum (stephenland Holland, 1954) (Bohra et al, 2016). The corn is the crop with the widest planting range, the most use and the highest total yield in China, and is the crop which has the earlier and the most thorough initial time for utilizing the heterosis in the three food crops. Corn is a hermaphrodite, cross-pollinated crop and is a basic research model plant with great advantages (Schnable et al, 2012). The research on the plant male sterile mutant is beneficial to deeply understanding the regulation and control mechanism of the related genes participating in the development of male organs and the life process of the plant generation alternation, and the sterile material provides valuable resources for the practical production of seed production.
Disclosure of Invention
The invention aims to provide a protein related to plant male fertility and a coding gene and application thereof.
The protein provided by the invention is named IPE2, is derived from corn (Zea mays L.) of Zea, and is (a) or (b) as follows:
(a) a protein consisting of an amino acid sequence shown in a
(b) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the
In order to facilitate the purification of the protein shown in the above (a), a tag shown in the following table may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid residue sequence of
Table: sequence of tags
Label (R)
Residue of
Sequence of
Poly-Arg
5-6 (typically 5)
RRRRR
Poly-His
2-10 (generally 6)
HHHHHH
8
DYKDDDDK
Strep-tag II
8
WSHPQFEK
c-
10
EQKLISEEDL
The protein of (b) may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression. The gene encoding the protein of (b) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in
Nucleic acid molecules encoding such proteins are also within the scope of the invention.
The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA, or tRNA, and the like.
In one embodiment of the invention, the nucleic acid molecule is in particular a gene (named IPE2) encoding the protein, which gene may in particular be a DNA molecule according to any one of the following 1) to 5):
1) DNA molecule shown in
2) DNA molecule shown in
3) DNA molecule shown in
4) a DNA molecule which hybridizes with the DNA molecule defined in any one of 1) to 3) under strict conditions and codes for a protein which is related to male fertility of plants and is derived from the
5) a DNA molecule which has more than 90 percent of identity with the DNA sequence defined in any one of 1) to 4) and codes the protein which is related to the plant male fertility and is derived from the
Wherein, the
Recombinant vectors, expression cassettes, transgenic cell lines or recombinant microorganisms containing the above-described nucleic acid molecules are also within the scope of the present invention.
The recombinant vector can be a recombinant expression vector and can also be a recombinant cloning vector.
The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UBIN or other derived plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CaMV)35S promoter, Ubiquitin gene Ubiquitin promoter (pUbi), stress-inducible promoter rd29A and the like can be added before the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. The transformed plants can also be screened directly in a stress environment without adding any selective marker gene.
The expression cassette consists of a promoter capable of driving expression of the gene, a reporter gene, and a transcription termination sequence.
The transgenic cell line is a non-propagating material into which the gene is transferred.
The invention also provides a method for cultivating the transgenic plant.
The application of the protein or the nucleic acid molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant microorganism in plant breeding and/or seed production also belongs to the protection scope of the invention.
The invention also provides a method for cultivating the transgenic plant.
The method for cultivating the transgenic plant provided by the invention can be as follows:
(1) introducing an IPE2 protein coding gene into an IPE2/IPE2 receptor plant to obtain a transgenic plant expressing the coding gene; the male sterility trait of said recipient plant is due to the loss or reduced ability of said recipient plant to express functional said IPE2 protein;
(2) obtaining a male-fertile transgenic plant from the transgenic plant obtained in the above step; the coding gene can be introduced into the recipient plant by the above recombinant expression vector p3300-IPE 2.
In the invention, the male sterility of the corn is that the anther is not externally hung in the full-bloom stage and/or 1 percent I is not added in the anther2The KI staining was identified as havingViable pollen ".
In the present invention, the plant may be either a monocotyledon or a dicotyledon. Wherein the monocotyledonous plant is selected from Gramineae, specifically semen Maydis.
The invention uses the map-based cloning strategy to combine BC by using a corn male sterile mutant ipe2 and a corn inbred line B731F1The population, which maps the gene controlling this mutation trait to maize chromosome five 131.799Mb to 134.582Mb, has a reference physical distance of about 2.78Mb based on published B73 genome sequencing results, and comprises twenty-seven genes in total. Wherein the sequence with the gene number Zm00001d015960 has difference between the mutant and the wild type, the mutant has deletion of 12bp and insertion of 314bp, and the wild type does not have the deletion and insertion. The IPE2 gene is complementarily expressed in the maize male sterile mutant by using a transgenic technology, and the male fertility can be restored. Therefore, Zm00001d015960 gene is named IPE2 as the target gene.
The IPE2 gene of the invention can control male fertility in maize, namely, homozygous mutation or deletion of the gene can make maize male sterile, and normal expression of the IPE2 gene in the material with mutation or deletion of the gene can restore male fertility.
The invention provides a new gene resource for the research of male fertility of corn.
Drawings
FIG. 1 is a graph comparing the phenotype of maize mutant ipe2 with that of the wild type. Wherein A, C, E, G, I is wild type, B, D, F, H, J is ipe2 mutant. A and B, plant morphology; c and D, the condition of externally hanging tassel anthers; e and F, spikelet morphology; g and H, anther morphology; i and J,
FIG. 2 is a map of the ipe2 gene.
FIG. 3 is a schematic diagram of the insertion site of IPE2 in
FIG. 4 shows the verification of genetic complementation of IPE2 gene. Wherein A is IPE2 genetic complementation flow chart; b is the PCR assay of the transgenic line (
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The maize mutant ipe2 was collected and stored for this laboratory. The maize genome sequencing information is referenced to the MaizeGDB database linked as follows: http: // www.maizegdb.org/.