Maize unidirectional cross-incompatibility-related protein ZmGa2F, its encoding gene and applications

Abstract

The present invention discloses a protein ZmGa2F related to maize cross-incompatibility, its coding gene and applications. The protein provided by the present invention is as follows (a) or (b): (a) a protein consisting of the amino acid sequence shown in Sequence 1 in the sequence listing; (b) a protein derived from Sequence 1, which is obtained by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence of Sequence 1 and is related to plant cross-incompatibility. The ZmGa2F gene of the present invention can control cross-incompatibility in maize, that is, it is expressed in the maize material with ga2 compatibility, and the transgenic homozygous single plants have the ability of ga2 incompatibility. The present invention provides a new gene resource for the study of incompatibility in plants, especially maize, which can be utilized in the process of maize breeding and seed production.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and relates to a gene related to the phenomenon of unilateral cross-incompatibility in maize, its encoded protein and applications. Background Art

[0002] Maize (Zea mays) is a typical monoecious and cross-pollinated crop, with a natural outcrossing rate as high as 95%. Under natural conditions, it can set seeds through self-pollination or cross-pollination. However, there is a type of maize in nature that can set seeds through self-pollination and can also pollinate other types of maize to set seeds, but cannot be pollinated by the pollen of the vast majority of other types of maize. This phenomenon is called unilateral cross-incompatibility (UCI) in maize. Since UCI regulates the transmission of male and female gametes in maize, the UCI locus is also called Gametophyte factor (Ga). The UCI phenomenon in maize was discovered in 1902. However, due to the complexity of the genetic characteristics of the UCI locus and the particularity of the genomic background of UCI materials, little is known about its molecular mechanism. Currently, multiple UCI loci have been reported in maize, among which the unilateral cross-incompatibility of Ga1, Ga2 and Teosinte crossing barrier 1 (Tcb1) is the most complete, and thus has received extensive attention. Among them, Ga1 is the incompatibility system between dent corn, flint corn, sweet corn and popcorn, Ga2 is the incompatibility system between teosinte and maize, and Tcb1 is the incompatibility system in teosinte. Subsequent studies found that Ga1, Ga2 and Tcb1 are three independent unilateral cross-incompatibility systems, with independent incompatibility mechanisms and mutual incompatibility among them. The Ga locus has been discovered for more than 110 years, but its research progress lags far behind that of self-incompatibility. Due to the particularity of the genomic background of Ga materials and the complexity of the unilateral incompatibility mechanism, people's understanding of it has always remained at a relatively simple phenotypic description. In recent years, with the construction of the physical map of the reference genome, great progress has been made in the study of the unilateral cross-incompatibility phenomenon in maize. Currently, the pollen determinant of the Ga1 locus and the silk determinant of the Tcb1 locus have been cloned, and the molecular mechanism of their cross-incompatibility has been preliminarily revealed. However, there is still a long way to go to fully understand the molecular mechanism of unilateral cross-incompatibility in maize.

[0003] The unidirectional cross-incompatibility phenomenon controlled by the Ga2 locus is jointly regulated by two determinants, namely the pistil determinant (Female determinant) and the pollen determinant (Male determinant). Among them, the pistil determinant controls the ability of the pistil to resist the fruit set of non-homologous pollen, that is, the female function; the pollen determinant endows the pollen with the ability to break through the pistil barrier for pollination and fruit set, that is, the male function. There are three types of materials at the Ga2 locus: Ga2-S, Ga2-M, and ga2. Ga2-S plants possess both the above-mentioned female and male functions, while Ga2-M plants only possess the male function and lack the female function. The male and female functions of ga2 plants are both lost.

[0004] Maize is the crop with the largest planting area in China. With the transformation and application of maize from food use to fields such as feed, energy, chemical industry, and medicine, the global demand for maize continues to grow. The increase in maize yield mainly lies in the breeding and promotion of hybrid varieties. However, in order to prevent the mixing of hybrid varieties, different types of maize need to be isolated in maize seed production fields, which is a very difficult challenge. Currently, time isolation and space isolation are commonly used isolation methods in production. However, due to the characteristics of maize, such as a large amount of pollen, a relatively long pollen survival time, and the ability of pollen to be transmitted by wind over a long distance, it is not easy to implement time isolation and space isolation in practice, and the isolation effect is difficult to guarantee, easily resulting in the mixing of varieties. The application of the Ga2 locus provides a new biological solution for the isolation between maize varieties and has broad application prospects in maize hybrid seed production and the production of special maize. In addition, the biological isolation between transgenic maize and conventional maize is also an important issue faced by global maize production. In the practice of transgenic maize cultivation in the United States, usually about 20% of non-transgenic maize is planted together with transgenic maize to avoid the rapid evolution of pathogens and pests due to strong artificial selection pressure. This strategy of non-isolated planting and mixed harvesting will inevitably lead to all maize foods containing transgenic components. Currently, some transgenic maize in China has obtained safety certificates, and the commercial cultivation of transgenic maize may not be far away. How to learn from the experience and lessons in the United States to avoid the contamination of transgenic maize is a potential challenge for the Chinese maize industry. The application of the Ga2 locus will provide an important way for the biological isolation between transgenic maize and conventional maize in China. Summary of the Invention

[0005] The object of the present invention is to provide a protein related to the unidirectional cross-incompatibility phenomenon of maize, its coding gene, and applications.

[0006] The protein provided by the present invention, named ZmGa2F, is derived from Zea mays L. of the genus Zea mays and is as follows (a) or (b):

[0007] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing;

[0008] (b) A protein derived from SEQ ID NO: 1, which is obtained by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 and is related to plant hybridization incompatibility.

[0009] SEQ ID NO: 1 in the Sequence Listing is the amino acid sequence of ZmGa2F, which consists of 379 amino acids. In this protein sequence, there are 172 hydrophobic amino acids, 118 hydrophilic amino acids, 47 basic amino acids, and 69 acidic amino acids. The molecular weight of this protein is 41.29 KD, and the isoelectric point is 7.59.

[0010] For the purpose of facilitating the purification of the protein shown in (a) above, a tag shown in the following table can be linked to the amino terminus or carboxyl terminus of the protein consisting of the amino acid residue sequence of SEQ ID NO: 1 in the Sequence Listing.

[0011] Table: Sequences of tags

[0012] Label Residue Sequence Poly-Arg 5 - 6 (usually 5) RRRRR Poly-His 2 - 10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0013] The protein in (b) above can be artificially synthesized, or its coding gene can be synthesized first and then expressed biologically. The coding gene of the protein in (b) above can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2 in the Sequence Listing and / or performing a missense mutation of one or several base pairs.

[0014] The nucleic acid molecule encoding the said protein also belongs to the scope of protection of the present invention.

[0015] The said nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the said nucleic acid molecule can also be RNA, such as mRNA, hnRNA or tRNA, etc.

[0016] In an embodiment of the present invention, the said nucleic acid molecule is specifically the gene encoding the said protein (named ZmGa2F), and the said gene can specifically be any one of the following 1)-4) DNA molecules:

[0017] The said gene is any one of the following 1)-4) DNA molecules:

[0018] 1) The DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing;

[0019] 2) The DNA molecule shown in SEQ ID NO: 3 in the Sequence Listing;

[0020] 3) The DNA molecule shown in SEQ ID NO: 4 in the Sequence Listing;

[0021] 4) A DNA molecule that hybridizes under stringent conditions with any of the DNA molecules defined in 1)-3) and encodes a protein derived from SEQ ID NO: 1 that is related to the phenomenon of unilateral cross-incompatibility in maize;

[0022] 5) A DNA molecule having more than 90% identity with any of the DNA sequences defined in 1)-4) and encoding a protein derived from SEQ ID NO: 1 that is related to the phenomenon of unilateral cross-incompatibility in maize.

[0023] Among them, SEQ ID NO: 2 is the cDNA sequence of the ZmGa2F gene, SEQ ID NO: 3 is the sequence of the ZmGa2F gene in the maize genome, and SEQ ID NO: 4 contains a 2052-bp promoter sequence upstream of the ZmGa2F gene, a 1288-bp coding sequence, and a 1092-bp terminator sequence.

[0024] Recombinant vectors, expression cassettes, transgenic cell lines or recombinant microorganisms containing the above nucleic acid molecules also fall within the scope of protection of the present invention. The recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

[0025] The recombinant expression vector can be constructed using existing plant expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vectors. The plant expression vector may also contain the 3' untranslated region of the foreign gene, i.e., containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. When using the gene to construct the recombinant expression vector, any one of the enhancer-type, constitutive, tissue-specific or inducible promoters can be added before the transcription start nucleotide, such as the cauliflower mosaic virus CaMV35S promoter, the ubiquitin gene Ubiquitin promoter (pUbi), the stress-inducible promoter rd29A, etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct the recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vector used can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants, an antibiotic marker with resistance, or an anti-chemical reagent marker gene, etc. It is also possible not to add any selectable marker gene and directly screen the transformed plants under stress conditions.

[0026] In the present invention, the recombinant expression vector is a recombinant plasmid obtained by inserting the ZmGa2F gene into the multiple cloning sites (such as EcoR I and Hind III) of the pCAMBIA3300 vector. More specifically, it is a recombinant plasmid (named pCAMBIA3300-ZmGa2F) obtained by replacing the small fragment between the restriction enzyme sites EcoR I and Hind III of the pCAMBIA3300 vector with the DNA fragment shown in Sequence 4 of the sequence listing.

[0027] The expression cassette consists of a promoter capable of initiating the expression of the gene, the gene, and a transcription termination sequence.

[0028] The transgenic cell line is non-reproductive material into which the gene has been transferred.

[0029] The use of the protein, nucleic acid molecule, recombinant vector, expression cassette, transgenic cell line or recombinant microorganism in any of the following also falls within the scope of protection of the present invention:

[0030] (a) Plant breeding and / or seed production;

[0031] (b) Regulating unilateral cross-incompatibility in plants.

[0032] The present invention also provides a method for cultivating transgenic plants.

[0033] The method for cultivating transgenic plants provided by the present invention may be as follows:

[0034] Here, the inability to set seeds due to the rejection of pollination by maize materials of the ga2 / ga2 type is called ga2 incompatibility; the ability to set seeds due to the acceptance of pollination by maize materials of the ga2 / ga2 type is called ga2 compatibility.

[0035] To cultivate a transgenic plant with ga2 incompatibility, the following steps are included:

[0036] (1) Introduce the coding gene of the ZmGa2F protein into a recipient plant (the recipient plant is ga2 compatible), and obtain a transgenic plant expressing the coding gene; the ga2-incompatible trait of the homozygous transgenic recipient plant is caused by the expression of the functional ZmGa2F protein in the recipient plant;

[0037] (2) Obtain a transgenic plant from the transgenic plant obtained in step (1) that cannot accept maize pollen of the ga2 / ga2 genotype.

[0038] In step (1) of the method, the coding gene can be introduced into the recipient plant through the above recombinant expression vector pCAMBIA3300-ubi-ZmGa2F;

[0039] In the present invention, the plant can be either a monocotyledon or a dicotyledon. Among them, the monocotyledon is such as a gramineous plant, specifically maize.

[0040] When cultivating a transgenic plant with ga2 incompatibility, any ga2-compatible maize material can be used as the recipient plant to obtain the corresponding ga2-incompatible transgenic maize.

[0041] In one embodiment of the present invention, the recipient plant used when cultivating a transgenic plant with ga2 incompatibility is specifically the maize variety B104.

[0042] The present invention adopts the strategy of map-based cloning. A BC1F1 population is developed by crossing the maize inbred line 511L with the genotype Ga2-S / Ga2-S and the maize inbred line W22 with the genotype ga2 / ga2. The gene controlling this trait is mapped between two markers M3 and M8 on chromosome 5 of maize. Referring to the B73V4 genome, the physical distance is 1.7 Mb, which includes a total of eight genes. Using the filament transcriptome data of the maize inbred line 511L with the genotype Ga2-S / Ga2-S and the maize inbred line B73 with the genotype ga2 / ga2 for de novo assembly, a gene specifically expressed in the filaments of the Ga2-S / Ga2-S genotype material is obtained, and this gene is named ZmGa2F. The ZmGa2F gene is introduced into ga2-compatible maize materials by transgenic technology. Homozygous ZmGa2F transgenic receptor plants do not accept the pollen of maize with the ga2 / ga2 genotype, resulting in infertility.

[0043] The ZmGa2F gene of the present invention can control cross-incompatibility in maize, that is, it is expressed in ga2-compatible maize materials, and transgenic homozygous single plants have the ability of ga2-incompatibility.

[0044] The present invention provides a new gene resource for the study of unilateral cross-incompatibility in maize, which can play an important role in the application of maize breeding and seed production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a map-based cloning map of the ZmGa2F gene.

[0046] Figure 2 It is an expression map of the annotated genes in the mapped interval in the pollen of maize inbred lines 511L and B73.

[0047] Figure 3 It is a tissue-specific expression map of XLOC_039725 in maize inbred lines 511L and B73.

[0048] Figure 4 It is an expression map of XLOC_039725 in the filaments of different types of maize inbred lines.

[0049] Figure 5This is a figure for transgenic function verification. (A) Hybrid compatibility analysis. The fruiting situation of 511L (Ga2-S / Ga2-S), B104 (ga2 / ga2), and T1 transgenic lines (CS1, CS2, and CS3) pollinated with purple kernel ZYM1 (ga2 / ga2) on the first day and yellow kernel Mo17 (Ga2-M) on the second day; (B) Hybrid compatibility analysis. The fruiting situation of 511L (Ga2-S / Ga2-S), B104 (ga2 / ga2), and T1F2 transgenic lines (CS1, CS2, and CS3) pollinated with purple kernel ZYM1 (ga2 / ga2) and yellow kernel Mo17 (Ga2-M). Detailed implementation mode

[0050] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0051] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0052] The maize inbred line 511L of Ga2-S / Ga2-S type was provided by MaizeGDB Stock Center( https: / / www.maizegdb.org / data_center / stock?id=9020818 ). The maize inbred lines GSW1 of Ga2-S / Ga2-S type, DM-1 and HN160 of Ga2-M / Ga2-M type, and B73 and W22 of ga2 / ga2 type were collected and preserved in this laboratory. The maize genome sequencing information refers to the MaizeGDB database, and the link to this database is as follows: http: / / www.maizegdb.org / .

[0053] Example 1: Map-based cloning of the maize unilateral cross-incompatibility gene ZmGa2F

[0054] I. Construction of the genetic mapping population

[0055] Using 511L (Ga2-S / Ga2-S) as the male parent and common maize W22 (ga2 / ga2) as the female parent, F1 (Ga2-S / ga2) was crossed. Pollinating F1 with 511L pollen, a BC1F1 segregating population was obtained. Taking the pollen of individual offspring plants to pollinate 511L one by one to identify their phenotypes, it was found that the ratio of fruiting to non-fruiting was in line with 1:1 (104:116, χ 2 = 0.0233, P > 0.05), proving that this trait is controlled by a recessive single gene, and the BC1F1 population was used as the genetic mapping population.

[0056] II. Mapping of the ZmGa2F gene

[0057] Using 511L (Ga2-S / Ga2-S) as the male parent and common maize W22 (ga2 / ga2) as the female parent, F1 (Ga2-S / ga2) was crossed. Then, 511L pollen was used to pollinate F1 to obtain the BC1F1 segregating population. The BC1F1 segregating population was planted, and mature individual plants were pollinated one by one with W22. The genotypes of the plants in the segregating population were determined based on the fruiting situation. Then, 10 pollinated individual plants with fruiting and non-fruiting were selected from the BC1F1 population respectively to verify whether the polymorphic primers were linked to the trait of unilateral cross-incompatibility. Linked primers were screened out and used to determine the genotypes of 2,600 individual plants in the population. By combining the ear fruiting phenotypes, the individual plants with inconsistent genotypes and phenotypes were selected as crossover individuals. According to the different numbers of crossover individuals screened by different primers, the mapping interval was determined according to the decreasing trend. Thus, the ZmGa2F gene was mapped between the primer markers M3 and M8 on chromosome 5 of maize. Referring to the published genome sequencing results of maize inbred line B73, the physical distance was approximately 1.7 Mb( Figure 1 ). Among them, the primer sequences of the molecular markers used for gene mapping are shown in Table 1.

[0058] Table 1 Primer sequences of molecular markers used for gene mapping

[0059]

[0060] III. Cloning of the ZmGa2F gene

[0061] Based on the speculation that the filament determinant controls the fruiting of filaments that hinder non-homotypic pollen pollination, it is speculated that the filament determinant is specifically expressed in the filament tissue. Referring to the B73V4 genome sequence information, primers were designed for 8 annotated genes in the filament determinant region, and the expression levels of these 8 genes in the filament tissues of 511L and B73 materials were detected. The results of fluorescence quantitative PCR showed that only the Zm00001d016248 gene had trace expression, but there was no significant difference between the two materials, and the other 7 genes were not expressed( Figure 2 ). This result implies that the 8 annotated genes in the filament determinant region are not candidate genes for the filament determinant.

[0062] Since none of the 8 annotated genes in the filament determinant region are candidate genes for the filament determinant, it is speculated that the candidate gene is not expressed or absent in the filament tissue of B73, resulting in its non-annotation in the B73 reference genome. Therefore, through transcriptome sequencing, we aimed to find the candidate gene for the filament determinant at the Ga2 locus.

[0063] We selected the unpollinated filament tissues of 511L and B73 for transcriptome sequencing. Through de novo assembly, the transcripts were re-aligned to the B73 V4 reference genome. The results showed that there were 8 transcripts within the 1.7 Mb filament determinant locus region, and only two transcripts were annotated, namely Zm00001d016245 and Zm00001d016248. Zm00001d016245 was hardly expressed in both materials, while the Zm00001d016248 transcript was slightly expressed in both materials, but there was no significant difference between them. Only the transcript XLOC_039725 was highly expressed in the filaments of 511L and hardly expressed in the filaments of B73 (Table 2).

[0064] Table 2 Transcripts obtained by de novo assembly within the mapped interval

[0065]

[0066] The expression levels of the transcript XLOC_039725 were detected in the pollen tissues of 511L and B73. The results showed that XLOC_039725 was highly expressed in the pollen of 511L ( Figure 2 ), and hardly expressed in B73. The expression levels of XLOC_039725 were analyzed in the roots, stems, leaves, pollen, and filaments of 511L and B73. The results showed that this gene was specifically expressed in the pollen of 511L ( Figure 3 ). The filament RNA of the Ga2-S / Ga2-S inbred line 511L and GSW1, the Ga2-M / Ga2-M inbred lines DM-1 and HN160, and the ga2 / ga2 inbred lines B73 and W22 was extracted, reverse transcribed into cDNA, and the XLOC_039725 gene was detected by fluorescence quantitative PCR with the primer pair F1 / R1, and the GAPDH gene was amplified with the primer pair F2 / R2 as an internal reference. The results showed that XLOC_039725 was expressed in the filaments of Ga2-S materials and not expressed in the filaments of Ga2-M materials and ga2 materials ( Figure 4) Therefore, it is speculated that the candidate gene XLOC_039725 is the target gene ZmGa2F. Using the cDNA of maize inbred line 511L as a template, PCR amplification was carried out with the primer pair F3 / R3, and the sequence of the obtained PCR product is Sequence 2 in the sequence listing, and Sequence 2 is the cDNA sequence of the ZmGa2F gene. Using the genomic DNA of maize inbred line 511L as a template, Sequence 3 was amplified by the primer pair F3 / R3, and Sequence 3 is the coding sequence of the ZmGa2F gene in the maize genome. Using the genomic DNA of maize inbred line 511L as a template, Sequence 4 was amplified by the primer pair F4 / R4, and Sequence 4 contains a 2052-bp promoter sequence upstream of the ZmGa2F gene, a 1288-bp coding sequence, and a 1092-bp terminator sequence.

[0067] F1: 5′-ACCGTGAATGTTGTAGCCGAC-3′;

[0068] R1: 5′-TCACCACGTTGTGCTTGCC-3′.

[0069] F2: 5′-CTGGTTTCTACCGACTTCCTTG-3′;

[0070] R2: 5′-CGGCATACACAAGCAGCAAC-3′.

[0071] F3: 5′-ATGGCAGCAGTTGGCAGAT-3′;

[0072] R3: 5′-GCCGGCTAGTTGACGATGAT-3′.

[0073] F4: 5′-GCCGTCGCCTACTCACAAG-3′;

[0074] R4: 5′-CGGAGTGGTAGGGGAGGGTA-3′.

[0075] Example 2. Functional verification of the maize unilateral cross-incompatibility gene ZmGa2F

[0076] I. Construction of an expression vector driven by the self-promoter of the ZmGa2F gene

[0077] Specific primers were designed for the gDNA sequence of the ZmGa2F gene. The specific sequences are as follows:

[0078] P1-F: 5′-aacagctatgacatgattacgaattc GCCGTCGCCTACTCACAAG-3′;

[0079] P1-R: 5'-gtaaaacgacggccagtgccaagctt CGGAGTGGTAGGGGAGGGTA-3'.

[0080] Among them, the lowercase letters indicate the homologous recombination arms required for ligation with the vector. The uppercase letters of P1-F are the first 21 bases shown in Sequence 4, and the uppercase letters of P1-R are the reverse complementary sequence of the last 22 bases shown in Sequence 2. Using the gDNA of maize inbred line 511L as a template, PCR amplification was carried out with the primer pair P1-F / P1-R. The obtained PCR product was detected by 1.0% agarose gel electrophoresis and then recovered for sequencing, showing that its sequence was as shown in Sequence 4 in the sequence listing. The Sequence 4 was ligated with the pCAMBIA3300 vector after digestion with EcoR I and Hind III and recovery. After digestion and sequencing identification, the pCAMBIA3300-ZmGa2F expression vector driven by the gene's own promoter was constructed.

[0081] Structural description of the recombinant pCAMBIA3300-ZmGa2F expression vector: A recombinant plasmid obtained by replacing the small fragment between the EcoR I and Hind III restriction sites of the pCAMBIA3300 vector with the DNA fragment shown in Sequence 4 in the sequence listing.

[0082] II. Maize genetic transformation experiment

[0083] Tianjin Genovo Biotechnology Co., Ltd. completed the genetic transformation of the recombinant pCAMBIA3300-ZmGa2F expression vector into maize inbred line B104. The specific transformation method was the conventional Agrobacterium-mediated genetic transformation of maize immature embryos.

[0084] III. Functional verification of transgenic offspring

[0085] Since the pCAMBIA3300 transformation vector contains the basta resistance gene, the method for determining whether it is a positive transgenic plant is as follows: At the seedling stage, the leaves of individual plants in the transgenic offspring population were smeared or sprayed with 5% basta solvent (10% glufosinate-ammonium, Beijing Coolaber Technology Co., Ltd.). After 2-3 days, if the leaf color did not change, it was a transgenic positive, and if the leaf turned yellow and slightly withered, it was a transgenic negative.

[0086] A total of 8 T0 transgenic positive plants were obtained through genetic transformation and basta screening. The T0 transgenic positive plants were self-crossed to obtain T1 transgenic plants. At the seedling stage, 5% basta solution was used to screen out positive transgenic plants. The genotype with the ZmGa2F candidate gene vector was defined as A, and the genotype without the transgenic fragment was defined as a. Then there are three genotypes AA, Aa, and aa in the transgenic T1 single plants. In theory, AA self-crossing does not produce seeds because there is no pollen determinant. However, there are many factors that cause pollination failure, such as pollination method, pollen inactivation, weather temperature and humidity. Therefore, we adopted the following pollination verification strategy: At the seedling stage, 5% basta herbicide was sprayed on the T1 plants. Since the genotype aa plants do not have basta resistance and cannot grow, only the single plants with genotypes AA and Aa remain. On the first day of the full-bloom stage, the purple-seeded maize (ZYM1) of the ga2 material was used to pollinate the T1 transgenic lines. On the second day, the yellow-seeded maize Mo17 of the Ga2-M material was used to cover and pollinate the transgenic lines. Only the homozygous plants have the ability to prevent pollen from pollinating the filaments. Therefore, the seeds of the homozygous transgenic lines with genotype AA are all yellow. It cannot accept the pollen of the purple maize of the ga2 type and only accepts the yellow-seeded pollen of the Ga2-M; while the seeds of the plants with genotype Aa are mostly purple and a small part are yellow. The results showed that among the 8 transgenic lines, the seeds of the homozygous genotype AA single plants in 3 transgenic lines (CS1, CS2, and CS3) were all yellow( Figure 5 A). To further confirm this result, we planted the yellow seeds (T2) produced by the AA plants of the T1 generation in the greenhouse and sprayed 5% basta herbicide on the T2 plants. The results showed that all the T2 plants grew normally and were positive, proving that the genotype of the T1 plants was AA. Subsequently, the T2 plants were self-crossed to obtain T3 plants. The T3 plants obtained by self-crossing were planted in the greenhouse, and 5% basta herbicide was sprayed to remove the non-positive plants. The following pollination verification strategy was adopted for the remaining positive plants: On the first day, the pollen of the purple-seeded maize (ZYM1) of the ga2 material was used to pollinate the T3 transgenic lines. On the second day, the T3 transgenic lines were self-crossed. We defined the pollen determinant genotype as B. Among the T1 plants, the genotype of the yellow seeds (T2) produced by the AA plants was AaBb. After screening with basta herbicide, the positive T3 plants would have genotypes AAB_, AAbb, and Aa__. Pollination experiments were carried out on the T3 positive plants according to the above strategy. The results showed that the seeds of the plants with genotype AAB_ in the three transgenic events were all yellow, that is, they could not accept the pollen of the ga2 male gametes; most of the seeds of the plants with genotype Aa__ were purple( Figure 5B). Based on the above results, we have demonstrated that homozygous ZmGa2F can endow the silks of the ga2 inbred line B104 with the ability to hinder the pollination of ga2 pollen. The ZmGa2F gene is the silk determinant at the Ga2 locus and is a gene related to the phenomenon of unilateral cross-incompatibility in maize.

[0087] Based on the research results of the above embodiments, it can be seen that: through map-based cloning and transgenic function verification, the ZmGa2F gene cloned in the present invention is a gene related to the phenomenon of unilateral cross-incompatibility in maize. The protein encoded by this gene can endow the ga2 / ga2 genotype material with cross-incompatibility barriers, thereby preventing the ga2 / ga2 genotype material from setting seeds and can be utilized in the process of maize breeding and seed production.

Claims

1. A protein, as follows: A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.

2. A nucleic acid molecule encoding the protein according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein: The nucleic acid molecule is a gene encoding the protein according to claim 1, and the gene is a DNA molecule of any one of the following 1)-2): 1) The DNA molecule shown in SEQ ID NO: 2 in the Sequence Listing; 2) The DNA molecule shown in SEQ ID NO: 3 in the Sequence Listing.

4. A recombinant vector containing the nucleic acid molecule according to claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is a recombinant expression vector or a recombinant cloning vector.

6. An expression cassette containing the nucleic acid molecule according to claim 2 or 3.

7. A recombinant microorganism containing the nucleic acid molecule according to claim 2 or 3.

8. Use of the protein according to claim 1 or the nucleic acid molecule according to claim 2 or 3 or the recombinant vector according to claim 4 or 5 or the expression cassette according to claim 6 or the recombinant microorganism according to claim 7 in any of the following: (a) Maize breeding and / or seed production; (b) Regulating maize cross-incompatibility.

9. A method for cultivating a transgenic plant, as follows: Here, the inability to set seeds due to the pollination of maize materials of the ga2 / ga2 type is called ga2 incompatibility; the ability to set seeds due to the pollination of maize materials of the ga2 / ga2 type is called ga2 compatibility; Cultivating a transgenic plant to make it ga2 incompatible, including the following steps: (1) Introducing the encoding gene of the ZmGa2F protein into a recipient plant, the recipient plant being ga2 compatible, to obtain a transgenic plant expressing the encoding gene; the ga2-incompatible trait of the homozygous transgenic recipient plant is caused by the expression of the functional ZmGa2F protein in the recipient plant; (2) Obtaining a transgenic plant from the transgenic plant obtained in step (1) that cannot accept maize pollen of the ga2 / ga2 genotype; The ZmGa2F protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing; The plant is maize.

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