Maize kernel development regulatory gene ZmmtRF2a, its encoded protein, functional markers and applications

Abstract

The present invention discloses a corn kernel development regulating gene ZmmtRF2a , its encoded protein, functional marker and its application. The present invention applies to utilize the natural mutant of corn kernels found in the process of corn field breeding and selection crk5 Through phenotypic analysis, linkage analysis and positional cloning technology, the crk5 Target gene for kernel mutation ZmmtRF2a This gene encodes the mitochondrial-localized peptide chain release factor 2a, which affects mitochondrial function, regulates the development of corn embryo and endosperm, and the accumulation of grain storage materials. crk5 middle, ZmmtRF2a Exons 5-7 are missing, and the encoded truncated protein lacks GGQ and SPF motifs, which are essential for RF release activity, mitochondrial binding, and stop codon recognition. In addition, the distribution and utilization potential of different haplotypes in inbred lines were studied, providing technical support and excellent germplasm resources for the utilization of this gene.

Description

Technical Field

[0001] The present invention relates to the field of biological genetic engineering technology, and specifically to maize kernel development regulating genes ZmmtRF2a , its encoded protein, functional markers and its applications. Background Art

[0002] corn( Zea mays L. ) As the crop with the highest yield in my country, it has huge production potential and is an important food, feed, chemical and energy crop. It plays an irreplaceable role in industrial and agricultural production and ensuring national food security. Corn kernel size is an important component of corn yield, and the molecular mechanism of its formation and development has received widespread attention. Although many genes that regulate corn kernel development and filling have been cloned, since the development of corn embryo and endosperm is an extremely complex biological process, the node genes and molecular mechanisms for precise regulation of grain filling still need to be further analyzed.

[0003] Mitochondria are the powerhouse of eukaryotic cells, and their dysfunction often leads to insufficient effective energy supply and growth defects in human cells and plants. Mitochondrial genes related to maize kernel development mostly play a role at the transcriptional regulatory level, while there are few reports on genes that affect kernel development at the translational level. DEK66 Encodes a mitochondrial ribosome assembly factor with GTPase activity, which may affect mitochondrial protein translation; DEK44 Encodes the mitochondrial large subunit protein L9 (RPL), which is directly involved in mitochondrial translation rps35' maturation of mRNA and initiation of translation. The mitochondrial protein synthesis machinery works in concert with the cytoplasmic protein synthesis machinery and is essential for cell homeostasis. Plant mitochondrial translation involves many plant-specific PPR proteins and other auxiliary factors. Mitochondrial release factors (RFs) cooperate with translation termination factors to recognize the stop codon at the A site of the small ribosome subunit, promote the hydrolysis of the ester bond between the P site tRNA and the nascent polypeptide chain at the active site of the large ribosome subunit, and terminate protein synthesis. There are two types of RFs in bacteria, RF1 recognizes the stop codons UAG and UAA, and RF2 recognizes the stop codons UGA and UAA. Human mitochondrial RF1a (mtRF1a) or mtRF1L (mtRF1-like) recognizes the stop codons UAG or UAA and promotes ribosome release, while mtRF1 specifically recognizes the non-traditional stop codons AGA and AGG on cytochrome C oxidase subunit 1 (COX1) mRNA. In monocotyledonous and dicotyledonous plants represented by maize and Arabidopsis, mtRFs include mtRF1, mtRF2a, mtRF2b, and mtRF2c. Among them, only mtRF2a contains the GGQ motif required for ribosome binding and RF release activity, and the SPF motif required for recognizing the UAA / UGA stop codon, but its specific function and mechanism of action are still unclear.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] The inventors of this application used natural mutants of corn kernels found during the field breeding process to crk5 Cloning of key genes affecting mitochondrial translation ZmmtRF2a , which encodes peptide chain release factor 2a during mitochondrial ribosome translation. ZmmtRF2a The mutation causes defects in embryo and endosperm development and the seeds lose the ability to germinate.

[0006] In the first aspect disclosed in the present application, phenotypic and cytological analysis was performed to find crk5 The 100-grain weight of the mutant was 56% lower than that of the wild type, the endosperm development was abnormal, the embryo was deformed, and it could not germinate normally. The proliferation of cells in the basal transfer layer of the endosperm was reduced. Genetic analysis and positional cloning methods were used to obtain the genes that affect corn kernel development. ZmmtRF2a The gene, whose cDNA sequence is shown in SEQ ID NO.1.

[0007] The second aspect disclosed in the present application relates to a grain development regulating gene ZmmtRF2a The encoded protein has an amino acid sequence as shown in SEQ ID NO.2.

[0008] The third aspect disclosed in the present application relates to a method for amplifying the ZmmtRF2a The cDNA primer pairs have sequences as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0009] In another aspect disclosed in the present application, the corn kernel development regulating gene ZmmtRF2a It is used in the genetic engineering regulation of the 100-grain weight trait of corn grains.

[0010] In another aspect disclosed in the present application, a method for breeding an excellent haplotype of 100-grain weight of corn kernels is provided, comprising the following steps:

[0011] (1) Detection and determination of corn strain materials ZmmtRF2a Gene B73V4: genotypes of loci chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, chr5.s_218528368;

[0012] (2) Select corn varieties with genotypes of GG, CC, GG, and CC at loci B73V4: chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, and chr5.s_218528368, respectively, and self-pollinate or hybridize to select favorable haploid varieties.

[0013] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0014] Positional cloning ZmmtRF2a The gene affects mitochondrial function, regulates the development of maize embryo and endosperm, and the accumulation of storage materials in the grain. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 In one embodiment of this application crk5 Analysis of mutant grain phenotypes; in the figure, (AD) wild type (WT) and crk5 Grain characteristics of the same ear, scale bar, 1 cm; (E) WT and crk5 Longitudinal section of kernel. En: endosperm; Em: embryo; (F) WT and crk5 100-grain weight of WT and crk5 Endosperm starch content bar graph (n = 3), values ​​are mean ± SD; (H) WT vs. crk5 Number of starch granules / 10 3pixels (n = 5). Values ​​are mean ± SD; (IJ) WT (I) and crk5 (J) Scanning electron microscopy (SEM) image of the central area of ​​mature endosperm, scale bar, 50 µm; (K) WT and crk5 Seed germination, scale bar, 1 cm; *** P <0.001, * P <0.05, Student's t test.

[0016] Figure 2 Cytological analysis of wild-type and mutant grains in an embodiment of the present application; in the figure, (AI) 10 days after pollination (DAP) (A) and 15 days after pollination (BI) WT and crk5 Paraffin sections of seeds, scale bars are 1 mm (AB), 200 µm (C), 100 µm (DE), 50 µm (FG), and 200 µm (HI); En: endosperm; Em: embryo; (JM) WT (J and L) and crk5 Transmission electron microscopy (TEM) observations of starchy endosperm (K and M); scale bars, 10 µm (JK), 2 µm (LM); SG: starch granules; PB: protein bodies.

[0017] Figure 3 In one embodiment of this application ZmmtRF2a Schematic diagram of gene positional cloning, gene structure and protein structure; in the figure, A: ZmmtRF2a Map-based cloning of genes; B: ZmmtRF2a Schematic diagram of the gene structure; C: Schematic diagram of the ZmmtRF2a protein structure.

[0018] Figure 4 This is a molecular marker diagram of CRK5-5U-F / 3U-R, CRK5-5U-F / E4-R, and CRK5-5U-F / E5-R in an embodiment of the present application; in the figure, Indel primer pairs are used crk5 and WT crk5 The loci were tested by RT-PCR.

[0019] Figure 5 This is the phylogenetic analysis and subcellular localization analysis of ZmmtRF2a in one embodiment of the present application; in the figure, A: amino acid sequence alignment of ZmmtRF2a in maize, Arabidopsis, human and Saccharomyces cerevisiae; wherein the red rectangle represents the SPF / PXT domain, and the blue rectangle represents the GGQ domain; B: phylogenetic tree of ZmmtRF2a; C: subcellular localization of ZmmtRF2a in maize protoplasts; scale bar, 5 µm. DETAILED DESCRIPTION

[0020] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents and materials involved are all conventional commercial products unless otherwise specified; the tests and detection methods involved (such as genomic DNA extraction, RNA extraction and reverse transcription, RT-PCR, corn kernel paraffin sections, etc.) are all conventional methods unless otherwise specified.

[0021] Embodiment 1: crk5 Phenotypic analysis of mutants

[0022] crk5 It is a grain mutant discovered by the inventors during the field breeding selection process (collected by Tang Jihua in Yuanyang, Henan in August 2017). By observing the wild type (WT) and mutant grains on the same mature ear, it was found that crk5 The grains are smaller than those of the wild type, and the embryo and endosperm development are delayed. crk5 The length, width and thickness of mature grains of WT were significantly smaller than those of WT ( Figure 1 AE), resulting in a 56% decrease in 100-grain weight compared with the wild type ( Figure 1 F). The longitudinal section of the kernel showed that compared with WT, crk5 The embryo becomes smaller and the endosperm decreases ( Figure 1 E), starch reduced by 9% ( Figure 1 G). In addition, compared with the round starch granules and dense matrix in WT endosperm, crk5 The starch granules were irregular in shape and their number decreased by 22.44% ( Figure 1 HJ). Defective embryo and endosperm result in the inability of the seeds to germinate ( Figure 1 K). Observe 10 to 15 days after pollination crk5 Paraffin sections of WT and WT kernels revealed crk5 The embryos showed no obvious differentiation and the proliferation of cells in the basal transfer layer of the endosperm was reduced (see Figure 2 ).

[0023] Embodiment 2: ZmmtRF2a Positional cloning

[0024] by crk5 The F2 segregating population of the maize gene was used as material, and the target gene was located in the physical interval of about 112 kb on the fifth chromosome of maize by genetic analysis and map-based cloning (see Figure 3 A). The linkage markers and their sequences used in the map-based cloning process are shown in Table 1. Gene annotation and sequencing analysis within the candidate segment showed that Zm00001d018330 The gene (cDNA sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) is the target gene, which contains 7 exons. crk5 middle Zm00001d018330 The deletion of exons 5 to 7 results in the lack of the C-terminal amino acid sequence in the protein it encodes (see Figure 3 B).

[0025] Zm00001d018330 The gene cloning method is as follows:

[0026] 1) Use the Plant Polysaccharide and Polyphenol RNA Extraction Kit to extract RNA from mutant and wild-type endosperm 13 days after pollination according to the kit instructions, fully dissolve the total RNA with RNase-free double distilled water, and use DNaseI to remove possible DNA residues.

[0027] 2) RNA quality was initially tested by agarose gel electrophoresis. The RNA concentration and the absorbance of RNA at 260 nm and 280 nm were tested by NanoDrop instrument. 260 / 280 Whether it is between 1.8 and 2.2 (28S: 18S>1.0) can determine the integrity and purity of the RNA.

[0028] 3) The obtained RNA was used as a template for reverse transcription, and the obtained cDNA was packaged and stored in a -20°C refrigerator for later use.

[0029] 4) Based on the maize B73 reference genome Zm00001d018330 Transcript sequence, design amplification primers:

[0030] F: 5' -AGGACTGAGGAGCTATCCCG- 3' (SEQ ID No. 3);

[0031] R: 5'-TTCCTGGGTGCAACACTGTC-3' (SEQ ID No. 4).

[0032] Table 1 ZmmtRF2a Primers and sequences used for cloning

[0033] .

[0034] Embodiment three: ZmmtRF2a Development of molecular markers for mutation sites

[0035] against ZmmtRF2a The exon deletions were detected by developing PCR molecular markers CRK5-5U-F / E4-R and CRK5-5U-F / E5-R, which are as follows:

[0036] ZmmtRF2a The actual position of the mutation site in the fourth version of the maize B73 reference genome is before chr5:218,526,636. The following Indell primers were designed:

[0037] F: 5'-AGGACTGAGGAGCTATCCCG-3' (SEQ ID No. 3);

[0038] R: 5' -TTCATGACCATTGCAGCCCA- 3' (SEQ ID No. 5);

[0039] R: 5'-CCATCAACTTTGATAGTGGCCCT-3' (SEQ ID No. 6).

[0040] Among them, CRK5-5U-F (SEQ ID No. 3) is the right primer, which can be specifically amplified with the left primers CRK5-E4-R (SEQ ID No. 5) / CRK5-E5-R (SEQ ID No. 6) ZmmtRF2a The cDNA sequence between 5'UTR and exon 4 / exon 5 (see Figure 4 ).

[0041] against crk5 The molecular marker detection of mutants includes the following steps:

[0042] (1) The reaction system is:

[0043] cDNA template (30-50 ng / μL) 1 μL

[0044] Left primer (10 μM) 0.4 μL

[0045] Right primer (10 μM) 0.4 μL

[0046] ddH2O 3.2 μL

[0047] 2×PCR Mix 5 μL.

[0048] (2) The PCR amplification procedure for CRK5-5U-F / CRK5-E4-R and CRK5-5U-F / CRK5-E5-R is as follows:

[0049] 1 cycle 95℃ pre-denaturation 3min;

[0050] 35 cycles: 95°C denaturation for 15 s, 58°C annealing for 20 s, 72°C extension for 30 s;

[0051] Delay 72℃ 5min;

[0052] Soak 25℃.

[0053] Then, the 1% agarose gel was used for detection. The CRK5-5U-F / CRK5-E4-R wild type / mutant materials all had bands; the CRK5-5U-F / CRK5-E5-R wild type had a band, but the mutant had no band (see Figure 4 ).

[0054] Embodiment 4: ZmmtRF2a Candidate gene association analysis

[0055] For analysis and research ZmmtRF2a In order to utilize the gene, 36 single nucleotide polymorphism sites (SNPs) were extracted from 440 inbred lines, including this gene and its upstream 3kb and downstream 2kb intervals (B73V4: Chr5:218521712-218530263). Association analysis was performed with the 100-grain weight of the inbred lines, and 4 SNPs (B73V4: chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, chr5.s_218528368) were detected that were significantly associated with grain length and 100-grain weight (see Table 2).

[0056] Table 2 ZmmtRF2a Gene association analysis

[0057] .

[0058] Embodiment five: ZmmtRF2a Excellent haplotype identification

[0059] There were two combinations of the four SNPs significantly associated with 100-grain weight in 440 inbred lines (see Table 2): Hap1 (6) and Hap2 (434). Among them, the average 100-grain weight of Hap1 was 18.86 g, and its distribution frequency in inbred lines was extremely low (6 / 440), which was a rare haplotype; the haplotype of most inbred lines was Hap2, which had a high distribution frequency (434 / 440) and an average 100-grain weight of 21.56 g, which was a favorable haplotype (see Table 3).

[0060] Table 3 ZmmtRF2a Haplotype analysis of genes

[0061] .

[0062] Embodiment six: ZmmtRF2a Phylogenetic tree and subcellular localization of

[0063] Protein sequence analysis showed that ZmmtRF2a was highly homologous to bacterial RF2 (E value 5e-117, homology 48.31%, https: / / blast.ncbi.nlm.nih.gov / ), and had higher homology to human mtRF1a than to mtRF1 (see Figure 5 A). Phylogenetic analysis confirmed that ZmmtFR2a belongs to the plant RF2 branch, parallel to the human mitochondrial mtRF1a protein (see Figure 5 B). ZmmtRF2a contains a GGQ motif required for ribosome binding and release, and an SPF motif for recognition of the stop codon (see Figure 5 A). In the mutant crk5 In ZmmtRF2a, these two motifs are missing.

[0064] TargetP bioinformatics analysis showed that ZmmtRF2a lacked a mitochondrial targeting signal peptide. 35S Pro ::ZmmtRF2a: eGFP The vector was transiently expressed in maize protoplasts and the results showed that the eGFP signal overlapped with the RFP fluorescence of the mitochondrial marker ZmNAD9-RFP (see Figure 5 C). The above results indicate that ZmmtRF2a is a mitochondrial localized protein encoded by a nuclear gene.

[0065] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0066] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations to the present invention fall within the scope of the claims of the present application and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for breeding excellent haplotypes of 100-grain weight of corn, characterized in that: The steps include: (1) Detection and determination of corn strain materials ZmmtRF2a Gene B73V4: genotypes of loci chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, chr5.s_218528368; (2) Select corn line materials with the genotypes of GG, CC, GG, and CC at the loci of B73V4: chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, and chr5.s_218528368, respectively, and self-pollinate or crossbreed to select favorable haploid lines; Said ZmmtRF2a The cDNA sequence of the gene is shown in SEQ ID NO.1.

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