Gene zmfie2 for regulating maize kernel development, encoded protein thereof, indel molecular marker, and use thereof

Abstract

Provided is a gene ZmFIE2 for regulating maize kernel development, an encoded protein thereof, an InDel molecular marker, and a use thereof, aiming to solve the current technical problems of low maize haploid induction rate, long breeding cycle, and low maize yield. An InDel molecular marker for detecting ZmFIE2 mutants was screened out, and a primer for InDel molecular marker was designed, the InDel molecular marker and the primer thereof can be applied to the breeding of maize kernel size varieties and maize haploid or diploid lines. From the epigenetic level, it was found that ZmFIE2 regulates the development of maize embryo and endosperm, affects the accumulation of kernel storage materials, and affects the cell cycle process of endosperm cells, which has great value in improving maize yield. By using multiple created ZmFIE2 allele mutants, research has found that the loss of ZmFIE2 function can directly determine the division of endosperm cells and apomixis, which will provide new gene resources and new ways to achieve high-frequency haploid induction and heterosis fixation in maize.

Description

Maize kernel development regulatory gene ZmFIE2, its encoding protein, Indel molecular markers and their applications Technical Field

[0001] The present invention relates to the field of bioengineering technology, and specifically to a corn kernel development regulating gene ZmFIE2, its encoded protein, Indel molecular markers and applications thereof. Background Art

[0002] Maize (Zea mays L.) is one of the three major grain crops worldwide and in my country. The kernel, as its primary harvested organ, directly determines its yield and quality. Currently, human society is facing challenges such as population growth, shrinking arable land, and environmental degradation, all of which are closely linked to the continued increase in staple crop yields. Maize kernel development is a complex and delicate process, regulated not only by genetic factors but also by epigenetic factors such as DNA methylation, histone modification, and chromatin remodeling.

[0003] In plants, PRC2 catalyzes H3K27me3 modification, triggering epigenetic silencing of key regulatory genes. To perform diverse biological functions during development, plants have evolved functionally differentiated PRC2 complexes. FIS-PRC2 primarily functions in gametophyte and seed development. The FIS-PRC2 complex comprises four core members (MEA, FIS2, FIE, and MSI1). Mutations in any of these components cause the central nucleus of the maternal sporophyte to divide and produce autonomous seeds without double fertilization. Furthermore, studies have found that rice osfie2 single mutants and osfie1 osfie2 double mutants both produce autonomous embryos and endosperms at high frequencies. These autonomous seeds can be used for apomixis or to maintain heterosis, offering valuable breeding applications. PRC2 also plays an important role in maize kernel development. Autonomous embryos and endosperms also occur in maize, and this autonomous phenotype has been widely exploited in haploid induction breeding techniques. The first key haploid induction gene in maize, ZmPLA1 / MTL / NLD, has an induction rate of only 2%, while another key gene, ZmDMP, increases the induction rate to 10%. Therefore, how to further improve the haploid induction rate and achieve rapid maize breeding is a key technical issue that needs to be addressed urgently. Technical issues

[0004] The purpose of this application is to provide a corn grain development regulatory gene ZmFIE2, its encoding protein, Indel molecular marker and its application, so as to solve the current technical problems of low corn haploid induction rate, long breeding cycle and low corn yield. Technical Solutions

[0005] In order to solve the above technical problems, this application intends to adopt the following technical solutions:

[0006] The first aspect of the present application is to provide an Indel molecular marker for detecting zmfie2 mutants, comprising the following Indel primers:

[0007] Outer primer pairs:

[0008] OF: 5'-CGCGACACTAGTAACGGTCTACA-3';

[0009] OR: 5'-CATGAAGTTGAACCCGATAGCAT-3';

[0010] Inner primer pair:

[0011] IF: 5'-TGTGACGCCTATTTACCTCAGA-3';

[0012] IR: 5'-GGTGAGCCATCAACATGGTC-3'.

[0013] In a second aspect of the present application, it is proposed to apply the above-mentioned maize grain development regulatory gene ZmFIE2, the encoded protein or the InDel molecular marker to the regulation of maize grain size and / or maize embryo and endosperm development.

[0014] In another aspect of the present application, the above-mentioned maize grain development regulatory gene ZmFIE2, the encoded protein or Indel molecular marker is applied to the regulation of maize H3K27me3 modification level and / or endosperm cell division.

[0015] In another aspect of the present application, the above-mentioned maize grain development regulatory gene ZmFIE2, the encoded protein or Indel molecular marker is applied to the induction of unfertilized spontaneous haploid embryos and / or diploid endosperm of maize.

[0016] In another aspect of the present application, the above-mentioned maize grain development regulatory gene ZmFIE2, the encoded protein or Indel molecular marker is applied to the breeding of maize grain size varieties / lines and maize haploid and / or diploid varieties / lines. Beneficial effects

[0017] 1. We found that the epigenetic regulation of maize ZmFIE2 by cell cycle progression affects maize embryo and endosperm development, thereby affecting the accumulation of grain storage substances, which is of great value for improving maize yield.

[0018] 2. We verified that inactivation of ZmFIE2 function can produce an autonomous apomixis phenotype, which can lay the foundation for breeding applications of high-frequency haploid induction and fixed hybrid vigor.

[0019] 3. Using multiple zmfie2 allelic mutants created, we found that the functional loss of ZmFIE2 can directly determine endosperm cell division and apomixis, which will provide new genetic resources and new approaches for achieving high-frequency haploid induction and hybrid vigor fixation in maize. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a phenotypic analysis of zmfie2-1 mutant grains in an embodiment of the present application; in the figure, A is the phenotype of the F2 separated ear of the allelic mutant zmfie2-1, wherein the red arrow indicates the mutant zmfie2-1 grain; B is a comparison of the embryonic surface and dorsal embryonic surface of the wild-type grain (WT) and the mutant zmfie2-1 grain at maturity, as well as the longitudinal section of the grain, wherein the red dotted line indicates the embryo of the grain; bar = 1 cm; C is a paraffin section of the mutant and wild-type grains on the same ear 10 days, 12 days and 15 days after pollination, Bar = 0.2 cm; DE is the embryo of the mutant grain 12 days after pollination (D) and the embryo of the mutant grain 15 days after pollination (E), Bar = 500 μm; FG is the endosperm base transfer layer of the fie2-1 mutant grain (F) and the wild-type grain (G) 12 days after pollination, Bar = 100 μm; HI is the comparison of endosperm filling material between fie2-1 mutant kernels (H) and wild-type kernels (I) 15 days after pollination, Bar = 500 μm.

[0021] Figure 2 is a grain phenotype analysis of zmfie2-2 and zmfie2-3 mutants in an embodiment of the present application; in the figure, A is the grain phenotype of the zmfie2-2 mutant on the mature separated ears after self-pollination; B is a schematic diagram of the editing method of the zmfie2-2 mutant, and the red line indicates the missing amino acid; C is the PCR product after Indel primer amplification, and the electrophoresis band in 12% nucleic acid PAGE gel; D is the grain phenotype of the zmfie2-3 mutant on the mature separated ears after self-pollination; EF is the reciprocal cross phenotype of the zmfie2-2 and zmfie2-3 mutants 12 days after pollination; Bars = 1 cm.

[0022] Figure 3 is a cytological analysis of wild-type and mutant kernels in an example of the present application; in the figure, A is a comparative observation of the whole kernel and embryo of zmfie2-2 and WT paraffin sections on detached ears 10 days, 12 days and 15 days after pollination; BD are paraffin sections of the basal transfer layer (BETL) of zmfie2-2 and WT endosperm 12 days after pollination; Bars = 100 μm.

[0023] Figure 4 is a transmission electron microscopy observation of the endosperm of wild-type and mutant grains in one embodiment of the present application; in the figure, AB is a transmission electron microscopy observation of the whole endosperm cells of zmfie2-2 and WT 15 days after pollination; CD is a transmission electron microscopy observation of the endoplasmic reticulum and protein bodies of zmfie2-2 and WT 15 days after pollination.

[0024] Figure 5 shows the detection of zmfie2-2 and WT protein levels and H3K27me3 modification levels in an embodiment of the present application; in the figure, A is immunoblotting detection using a ZmFIE2-specific antibody in WT and zmfie2-2, with tubulin as an internal reference; B is immunoblotting detection using an H3K27me3-specific antibody in WT and zmfie2-2, with H3 as an internal reference; C is immunofluorescence detection using an H3K27me3-specific antibody in corn kernels 12 days after pollination in WT and zmfie2-2.

[0025] Figure 6 is an analysis of the biological processes that ZmFIE2 may be involved in in one embodiment of the present application; in the figure, A is a volcano plot of differentially expressed genes in the mutant compared with the wild type; the blue dots are genes with downregulated expression, and the red dots are genes with upregulated expression; B is an entry in the GO annotation related to the cell cycle and cell division; C is a heat map of the differential expression of fie2 relative to WT, RBR1 target genes and other cell cycle-related genes in the transcriptome data 12 days after pollination, and the color bar represents the difference fold value.

[0026] FIG7 is an analysis of the ploidy of endosperm cell cycle regulated by ZmFIE2 in one embodiment of the present application; FIG

[0027] AD are flow cytometric analyses of endosperm cells at 7 (A), 8 (B), 10 (C), and 12 (D) days after pollination (AP), where 3C represents G1, 6C represents G2 / M, and the interval between 3C and 6C represents S phase; three independent biological replicates were performed for each phase; *, p < 0.05; **, p < 0.01; ***, p < 0.001; E is the expression level analysis of cell cycle-related genes in WT and fie2 endosperm at 7, 8, and 10 days after pollination; the internal reference gene is UBQ; F is immunoblot analysis of CDKA and RBR1 protein enrichment in WT and fie2 endosperm at 8, 10, 12, and 15 days after pollination; the internal reference gene is TUB.

[0028] Figure 8 is a phenotypic observation of autonomously developed grains on an unfertilized ear in one embodiment of the present application; in the figure, A is a picture of an unfertilized ear 7 days after silking; Bar=1 cm; B is a partially enlarged picture of an unfertilized ear 7 days after silking; CE are enlarged pictures of autonomously developed grains on an unfertilized ear 7 days after silking; Bar=0.5 cm; FG are semi-thin section observation pictures of autonomously developed grains 12 days after silking; Bar=200μm. Best Mode for Carrying Out the Invention

[0029] Type here the best mode description paragraph of the invention. Modes for Carrying Out the Invention

[0030] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0031] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents unless otherwise specified; the test methods involved are all conventional methods unless otherwise specified.

[0032] Example 1: Post-pollination phenotypic analysis of zmfie2 mutants

[0033] Observation of wild-type and mutant kernels from the same mature heterozygous ear (fie2-1, a Uniform-Mu (UFMu-03254) transposon insertion mutant purchased from a US germplasm center; fie2-2, created at Weimi Technology; and fie2-3, derived from an EMS-induced mutant library; all ears collected by Wang Yongyan in Zhengzhou, Henan Province, in July 2023) revealed that wild-type and mutant kernels could be clearly distinguished on separate ears, with approximately 25% exhibiting wrinkled kernels (see Figures 1A and 2A,D). Compared to the wild type, mature ears of zmfie2-2 and zmfie2-3 displayed two mutant types, with zmfie2-m2 exhibiting severe wrinkling (Figures 2A,D). Further reciprocal crosses between zmfie2-2 and zmfie2-3 showed that both mutant kernels were produced in the reciprocal cross ears 12 days after pollination (see Figure 2E, F).

[0034] Paraffin sections of zmfie2 mutant and wild-type kernels were observed 10, 12, and 15 days after pollination. Cytological analysis revealed that the zmfie2 mutant kernels were significantly wrinkled and smaller, with a clear gap between the endosperm and seed coat of the mutant kernels. This feature was more obvious 10 days after pollination. The development of the embryo and endosperm lagged significantly behind that of the wild type, with the embryo development severely impaired, and the scutellum and first leaf primordium unable to differentiate normally (see Figures 1, 2, and 3). There was also reduced proliferation within the cells of the endosperm basal transfer layer, and fewer starch granules and protein bodies were filled in the endosperm compared to the wild type (see Figures 1, 2, and 3).

[0035] Example 2: Design of ZmFIE2 amplification primers

[0036] Based on the genome of the maize kernel development regulatory gene ZmFIE2, a ZmFIE2 amplification primer was designed. The nucleotide sequence of the primer is as follows:

[0037] Upstream primer: 5′-ATGGCGAAGCTGGGCCCG-3′ (SEQ ID No. 3);

[0038] Downstream primer: 5′-TCAGTTTCTGGAGCTCGGAT-3′ (SEQ ID No. 4).

[0039] Example 3: Design and identification of indel marker primers for ZmFIE2 mutation sites

[0040] Develop an easily detectable PCR molecular marker Indel for the base deletion of ZmFIE2 as follows: Design Indel primers based on the ZmFIE2 reference genome in the third version of the maize B73 reference genome:

[0041] OF: 5' - CGCGACACTAGTAACGGTCTACA- 3' (SEQ ID No. 5);

[0042] OR: 5' - CATGAAGTTGAACCCGATAGCAT- 3' (SEQ ID No. 6);

[0043] IF: 5' - TGTGACGCCTATTTACCTCAGA- 3' (SEQ ID No. 7);

[0044] IR: 5' -GGTGAGCCATCAACATGGTC- 3' (SEQ ID No. 8).

[0045] Among them, OF / OR is the outer primer pair, which can specifically amplify the ZmFIE2 gene sequence; IF / IR is the inner primer pair, and the second round of amplification is performed using the PCR product diluted 10 times of OF / OR as the template.

[0046] Functional molecular marker detection of the paternally imprinted gene ZmFIE2 includes the following steps:

[0047] (1) Prepare a 10 µL reaction system, including the following components:

[0048] DNA template (50 ng / µL) 1.0 µL

[0049] OF(10µmol / L) 0.5 µL

[0050] OR (10µmol / L) 0.5 µL

[0051] 2×PCR Mix 5μL

[0052] ddH2O 3 µL;

[0053] (2) The PCR amplification procedure is:

[0054]

[0055] (3) Prepare a 10 µL reaction system, including the following components:

[0056] 1 µL of PCR product diluted 10-fold from DNA template (1)

[0057] IF (10µmol / L) 0.5 µL

[0058] IR (10µmol / L) 0.5 µL

[0059] 2×PCR Mix 5μL

[0060] ddH2O 3 µL;

[0061] (4) The PCR amplification procedure is:

[0062]

[0063] Then, 12% nucleic acid PAGE gel was used for detection, and 3 μL of wild-type material and mutant were loaded into each well. The detection bands of the wild-type material and mutant are shown in Figure 2C, and the mutant is a lower band (see Figure 2C).

[0064] Example 4: Detection of protein levels and H3K27me3 modification levels in zmfie2 mutants and WT wild-type plants

[0065] To examine protein levels in zmfie2 mutants and wild-type plants, a ZmFIE2-specific antibody was further developed. Western blotting was used to analyze ZmFIE2 protein expression in the endosperm of maize kernels 12 days after pollination. ZmFIE2 protein expression was undetectable in the mutant (Figure 5A). To verify the effect of ZmFIE2 mutation on H3K27me3 methylation, immunoblotting and immunofluorescence were used to analyze H3K27me3 methylation in the endosperm of wild-type and mutant kernels. The results showed that loss of ZmFIE2 function in the mutant resulted in a dramatic decrease in H3K27me3 methylation in the endosperm cells (Figure 5BC).

[0066] Example 5: ZmFIE2 regulates the cell cycle progression of endosperm cells

[0067] To further explore the gene expression differences between the wild type (WT) and the mutant (fie2) and their possible involvement in biological processes, RNA-seq analysis was performed using endosperm cells at 12 days of age. A total of 2,394 differentially expressed genes were screened. Compared with the WT, 1,323 genes were upregulated and 1,071 genes were downregulated in the mutant fie2 (Figure 6 A). Further GSEA enrichment analysis of these DEGs revealed that these differentially expressed genes were mainly enriched in the cell cycle and cell division items (Figure 6 B). Further analysis of the DEGs in these entries revealed (Figure 6C) that the DNA replication factors MINICHROMOSOME MAINTENANCE (MCM4, MCM5, MCM6, and MCM7) and Cyclin-Dependent Kinases (CDC2, CYCA2;1, CYCA3;1, and CYCD5-3), which are negatively regulated by RBR1, were all upregulated; Origin recognition complex subunit 6 (ORC6), as a key factor in initiating DNA replication, binds to the MCM complex to maintain cell cycle and proliferation, and is upregulated in fie2 mutants; ROA2 and ROA3 are homologous genes of MCM3 and MCM6, respectively, which play a key role in maintaining the cell cycle, and their expression levels are significantly upregulated in fie2 mutants.

[0068] To further clarify the regulation of the endosperm cell cycle by ZmFIE2, endosperm cells from wild-type and fie2 mutant kernels were analyzed by flow cytometry (FCM). At 7 days after pollination (DAP), when most endosperm cells are in mitosis, the proportion of endosperm cells in G1 and S phases was significantly reduced in fie2 compared with the WT, while the proportion of cells in G2M phase was increased (Figure 7A). At 8 days after pollination (DAP), endosperm cells complete mitosis and enter the endoreduplication phase, at which point the endosperm cell number is determined. Compared with the WT, the proportion of 3C and 6C endosperm cells in fie2 significantly increased, while the proportion of 12C, 24C, and 48C high-ploidy endosperm cells was significantly decreased (Figure 7B). At 10 days after pollination (DAP), endosperm cells fully entered endoreduplication. Compared with the WT, the proportion of 6C endosperm cells in fie2 decreased, while the proportion of 3C, 12C, 24C, and 48C endosperm cells decreased (Figure 7C). At 12 days after pollination, compared with the WT, the proportion of endosperm cells at 6C in fie2 cells was significantly increased, while the proportions of endosperm cells at 3C, 12C, 24C, 48C, and 96C cells were all decreased (Figure 7D). This result indicates that endosperm cells in fie2 cells accumulate a large amount of G2M during mitosis, delaying entry into endoreduplication and disrupting cell cycle progression. Next, qRT-PCR was used to analyze the expression of cell cycle-specific genes and cyclin-dependent kinase 1 (CDKA;1 replication complex formation) (Figure 7E). Analysis of endosperm cells at 7, 8, and 10 days after pollination revealed that at 7 days after pollination, compared with the WT, the expression of CYCD2;1 (G1→S) and CDKA;1 was lower in fie2, while the expression of CYCA1;2 (S→G2) and CYCA1;1 (G2→M) remained largely unchanged. However, the expression of CYCB1;3 (G2→M) and CYCD5;1 increased. At 8 and 10 days after pollination, compared with the WT, the expression of all specific genes in fie2 decreased significantly, except for CYCB1;3, which increased at 8 days after pollination. This result indicates abnormal G2→M mitosis in fie2 endosperm cells, consistent with the flow cytometry results. Western blotting experiments were further used to analyze the protein levels of CDKA;1 and RBR1 in endosperm cells at 8, 10, 12, and 15 days after pollination. It was found that CDKA;1 and RBR1 were significantly downregulated in endosperm cells at 8, 10, and 12 days after pollination, while there was no significant difference in CDKA;1 in endosperm cells at 15 days after pollination (Figure 7F).Further analysis of the transcriptome data revealed that the DNA replication factors MCM4, MCM5, MCM6, and MCM7, as well as cyclin-dependent kinases, which are negatively regulated by RBR1, were upregulated (Figure 6C). This result was consistent with the immunoblotting results. Therefore, the functional defect of ZmFIE2 caused the disorder of mitosis and endoreduplication in endosperm cells.

[0069] Example 6: Phenotypic analysis of autonomously developed grains of the zmfie2-2 mutant

[0070] Because the fie mutant in Arabidopsis produces autonomously developing grains even without fertilization, female ears identified as harboring the zmfie2-2 mutation were bagged and their silking was inspected daily. Silking was considered the first day when it reached approximately 10 cm. Unpollinated ears were then removed and observed on days 7 (see Figure 8 A-E) and 12 (see Figure 8 F, G). The results demonstrated that fie2-2 does produce autonomously developing endosperm in unpollinated ears, providing new insights into improving haploid induction rates.

Claims

1. A maize kernel development regulatory gene ZmFIE2, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. A protein encoded by the maize kernel development regulatory gene ZmFIE2, whose amino acid sequence is shown in SEQ ID NO.

2.

3. An Indel molecular marker for detecting zmfie2 mutants, characterized in that: Includes the following Indel primers: Outer primer pairs: OF: 5'-CGCGACACTAGTAACGGTCTACA-3'; OR: 5'-CATGAAGTTGAACCCGATAGCAT-3'; Inner primer pair: IF: 5'-TGTGACGCCTATTTACCTCAGA-3'; IR: 5'-GGTGAGCCATCAACATGGTC-3'.

4. Use of the maize kernel development regulating gene ZmFIE2 according to claim 1, the encoded protein according to claim 2, or the Indel molecular marker according to claim 3 in regulating maize kernel size and / or maize embryo and endosperm development.

5. Use of the maize kernel development regulatory gene ZmFIE2 according to claim 1, the encoded protein according to claim 2, or the Indel molecular marker according to claim 3 in regulating maize H3K27me3 modification levels and / or endosperm cell division.

6. Use of the maize kernel development regulatory gene ZmFIE2 according to claim 1, the protein encoded by the maize kernel development regulatory gene ZmFIE2 according to claim 2, or the Indel molecular marker according to claim 3 in inducing unfertilized spontaneous haploid embryos and / or diploid endosperm in maize.

7. Use of the maize kernel development regulatory gene ZmFIE2 according to claim 1, the encoded protein according to claim 2, or the Indel molecular marker according to claim 3 in the breeding of maize varieties / lines with large or small kernel traits.

8. Use of the maize kernel development regulating gene ZmFIE2 according to claim 1, the encoded protein according to claim 2, or the Indel molecular marker according to claim 3 in the breeding of maize haploid and / or diploid varieties / lines.

Images