Corn ZmmtRF2a gene and application of cloning primer in regulating kernel development

Abstract

The present application relates to corn ZmmtRF2a Gene and its cloning primer in the application of regulating grain development. By using the natural grain mutant found in the process of corn field breeding selection line crk5 , through phenotype analysis, linkage analysis and map-based cloning technology, the target gene causing grain mutation crk5 ZmmtRF2a The gene encodes mitochondrial-located peptide chain release factor 2a, which affects mitochondrial function, regulates corn embryo and endosperm development and grain storage material accumulation. crk5 In the application, ZmmtRF2a The 5th-7th exon is deleted, and the encoded truncated protein lacks GGQ and SPF motifs, which are essential for the release activity of RF, mitochondrial binding and stop codon recognition. The present application provides technical support for the utilization of the gene and opens up a new way.​

Description

[0001] This invention is patent application number 202311351629.6, filed on October 18, 2023, entitled "Genes Regulating Maize Kernel Development". ZmmtRF2a The divisional application is for “the protein it encodes, its functional markers and their applications”. Technical Field

[0002] This invention relates to the field of assisted molecular breeding technology, specifically to maize. ZmmtRF2a Application of genes and their cloning primers in regulating grain development. Background Technology

[0003] corn( Zea mays L. As my country's highest-yielding crop, maize has enormous production potential and is an important food, feed, chemical, and energy crop, playing an irreplaceable role in industrial and agricultural production and ensuring national food security. Maize kernel size is a crucial factor in maize yield, and the molecular mechanisms of its formation and development have attracted widespread attention. Although numerous genes regulating maize kernel development and filling have been cloned, the precise regulation of kernel filling by key genes and molecular mechanisms remains to be further elucidated due to the extremely complex biological process of maize embryo and endosperm development.

[0004] Mitochondria are the power source of eukaryotic cells, and their dysfunction often leads to insufficient energy supply, resulting in growth defects in human cells and plants. Most mitochondrial genes related to maize kernel development function at the transcriptional level, while genes affecting kernel development at the translational level are rarely reported. (Kernel mutants) DEK66 It encodes a mitochondrial ribosome assembly factor, has GTPase activity, and may affect mitochondrial protein translation; DEK44 Encoding the mitochondrial large subunit protein L9 (RPL), it is directly involved in mitochondrial translation. RPS35' maturation and translation initiation of mRNA. Mitochondrial protein synthesis mechanisms work synergistically with cytoplasmic protein synthesis mechanisms, which are crucial for cellular homeostasis. Plant mitochondrial translation involves numerous plant-specific PPR proteins and other cofactors. Mitochondrial releasing factors (RFs) and translation termination factors recognize the stop codon at the A site of the small ribosomal subunit, promoting the hydrolysis of the ester bond between the P site tRNA and the nascent polypeptide chain at the active site of the large ribosomal subunit, thereby terminating protein synthesis. In bacteria, there are two types of RFs: 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, promoting 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, such as maize and Arabidopsis thaliana, mtRF exists as mtRF1, mtRF2a, mtRF2b, and mtRF2c. Among them, only mtRF2a contains the GGQ motif, which is essential for ribosome binding and RF release activity, as well as the SPF motif, which is essential for recognizing the UAA / UGA stop codon. However, its specific functions and mechanisms of action are still unclear.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The inventors utilized a natural grain mutant discovered during maize field breeding and selection. crk5 Cloning key genes affecting mitochondrial translation ZmmtRF2a This gene encodes peptide chain release factor 2a, which is involved in mitochondrial ribosome translation. ZmmtRF2a Mutations lead to defects in embryo and endosperm development, causing seeds to lose their ability to germinate.

[0007] The first aspect of this disclosure is that, through phenotypic and cytological analysis, it was discovered... crk5 The mutant kernels had a 56% lower 100-kernel weight compared to the wild type, abnormal endosperm development, embryonic malformation, inability to germinate normally, and reduced intracellular proliferation in the endosperm basal transfer layer cells. Genetic analysis and map-based cloning methods were used to identify factors affecting maize kernel development. ZmmtRF2a The gene, whose cDNA sequence is shown in SEQ ID NO.1.

[0008] A second aspect of this disclosure relates to a seed development regulatory gene. ZmmtRF2a The encoded protein has the amino acid sequence shown in SEQ ID NO.2.

[0009] A third aspect of this disclosure relates to an amplification method. ZmmtRF2a The cDNA primer pairs are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0010] Another aspect of this disclosure is the regulation of maize kernel development genes. ZmmtRF2a It is applied to the genetic engineering regulation of the 100-kernel weight trait in maize.

[0011] Another aspect of this disclosure provides a method for breeding superior haplotypes with a 100-kernel weight in maize, comprising the following steps: (1) Testing to determine maize strains ZmmtRF2a Genotypes at the following loci: chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, and chr5.s_218528368 in gene B73V4. (2) Select maize line materials with genotypes GG, CC, GG, and CC respectively at the loci B73V4: chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, and chr5.s_218528368, and self-pollinate or crossbreed them to breed advantageous haplotype lines.

[0012] One or more technical solutions provided in the embodiments of this disclosure have at least one of the following technical effects or advantages: Targeted cloning ZmmtRF2a Genes influence mitochondrial function and regulate the development of maize embryo and endosperm, as well as the accumulation of storage substances in the kernels. Attached Figure Description

[0013] Figure 1 As one embodiment of this disclosure crk5 Seed phenotypic analysis of mutants; 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 the grain. En: endosperm; Em: embryo; (F)WT and crk5 The weight of 100 grains (n ​​= 3) is the average value ± SD; (G) WT and crk5 Bar chart of endosperm starch content (n = 3), values ​​are mean ± SD; (H)WT and crk5 number of starch granules / 10 3 Pixels (n = 5). Values ​​are average ± SD; (IJ)WT (I) and crk5(J) Scanning electron microscopy (SEM) image of the central region of mature endosperm, scale bar, 50 µm; (K) WT and crk5 Seed germination, scale bar, 1cm; *** P <0.001, * P <0.05, Student's t test.

[0014] Figure 2 This is a cytological analysis of wild-type and mutant seeds in one embodiment of this disclosure; in the figure, (AI) 10 days post-pollination (A) and 15 days post-pollination (BI) WT and crk5 Paraffin sections of grains, scale bars: 1 mm (AB), 200 µm (C), 100 µm (DE), 50 µm (FG), 200 µm (HI); En: endosperm; Em: embryo; (JM) 15 days post-pollination WT (J and L) and crk5 Transmission electron microscopy (TEM) observation of starch endosperm (K and M), scale bar, 10 µm (JK), 2 µm (LM); SG: starch granules; PB: protein bodies.

[0015] Figure 3 As one embodiment of this disclosure ZmmtRF2a Map-based cloning of genes, schematic diagrams of gene structure and protein structure; in the diagram, A: ZmmtRF2a Map-based cloning of genes; B: ZmmtRF2a Schematic diagram of gene structure; C: Schematic diagram of ZmmtRF2a protein structure.

[0016] 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 one embodiment of this disclosure; in the diagram, Indel primer pairs are used. crk5 and WT crk5 The site was detected by RT-PCR.

[0017] Figure 5 Phylogenetic and subcellular localization analysis of ZmmtRF2a in one embodiment of this disclosure; In the figure, A: amino acid sequence alignment of ZmmtRF2a in maize, Arabidopsis thaliana, humans and Saccharomyces cerevisiae; where 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 Implementation

[0018] Unless otherwise specified, all instruments and equipment used in the following embodiments are conventional instruments and equipment; all reagents and materials used are commercially available conventional products; and all experimental and detection methods used (such as genomic DNA extraction, RNA extraction and reverse transcription, RT-PCR, corn kernel paraffin sections, etc.) are conventional methods unless otherwise specified.

[0019] Example 1: crk5 Phenotypic analysis of mutants crk5 This is a grain mutant discovered by the inventors during field breeding and selection (collected by Tang Jihua in Yuanyang, Henan Province in August 2017). Observation of wild-type (WT) and mutant grains on the same mature ear revealed... crk5 The grains are smaller than those of the wild type, and the embryo and endosperm development is delayed. crk5 The mature kernels are significantly smaller than WT in length, width and thickness. Figure 1 AE), resulting in a 56% reduction in 100-seed weight compared to the wild type. Figure 1 F). Longitudinal section of the kernels shows that, compared to WT, crk5 The embryo becomes smaller and the endosperm decreases. Figure 1 E), starch decreased by 9% ( Figure 1 G). Furthermore, compared to 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 embryos and endosperm prevent their seeds from germinating. Figure 1 K). Observe for 10-15 days after pollination. crk5 Paraffin sections of WT seeds were found crk5 The embryo showed no obvious differentiation, and the proliferation of cells in the basal transfer layer of the endosperm was reduced (see...). Figure 2 ).

[0020] Example 2: ZmmtRF2a Map cloning by crk5 Using the F2 segregating population derived from the inbred line Zheng 58 as material, the target gene was located within a physical region of approximately 112 kb on chromosome 5 of maize through genetic analysis and map-based cloning (see [link to original text]). Figure 3 A). The linkage markers and their sequences used in map-based cloning are shown in Table 1. Gene annotation and sequencing analysis within candidate regions indicate that... Zm00001d018330 The gene (cDNA sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) is the target gene, containing 7 exons. crk5 middle Zm00001d018330 The deletion of exons 5 through 7 results in the loss of the C-terminal amino acid sequence in the protein it encodes (see...). Figure 3 B).

[0021] Zm00001d018330 The methods for cloning genes are as follows: 1) Using a plant polysaccharide and polyphenol RNA extraction kit, RNA was extracted from the endosperm of mutants and wild-types 13 days after pollination, respectively, according to the kit instructions. The total RNA was fully dissolved in RNase-free double-distilled water, and any possible DNA residues were removed with DNase I.

[0022] 2) RNA quality was initially assessed using agarose gel electrophoresis. RNA concentration and absorbance at 260 nm and 280 nm were measured using a NanoDrop instrument. Based on OD... 260 / 280 Whether the value is between 1.8 and 2.2 (28S : 18S > 1.0) determines the integrity and purity of the RNA.

[0023] 3) The obtained RNA was used as a template for reverse transcription, and the resulting cDNA was aliquoted and stored at -20°C for later use.

[0024] 4) Based on the maize B73 reference genome Zm00001d018330 Transcript sequence, design amplification primers: F: 5'-AGGACTGAGGAGCTATCCCG-3' (SEQ ID No. 3); R: 5'-TTCCTGGGTGCAACACTGTC-3' (SEQ ID No. 4).

[0025] Table 1 ZmmtRF2a Primers and sequences used for cloning .

[0026] Example 3: ZmmtRF2a Development of molecular markers for mutation sites against ZmmtRF2a For exon deletions, easily detectable PCR molecular markers CRK5-5U-F / E4-R and CRK5-5U-F / E5-R were developed, as detailed below: ZmmtRF2a The mutation site is located before chr5:218,526,636 in the fourth version of the maize B73 reference genome. The following indel primers were designed: F: 5'-AGGACTGAGGAGCTATCCCG-3' (SEQ ID No. 3); R: 5'-TTCATGACCATTGCAGCCCA-3' (SEQ ID No. 5); R: 5'-CCATCAACTTTGATAGTGGCCCT-3' (SEQ ID No. 6).

[0027] Among them, CRK5-5U-F (SEQ ID No. 3) is the right-side primer, which can specifically amplify the left-side primers CRK5-E4-R (SEQ ID No. 5) and CRK5-E5-R (SEQ ID No. 6), respectively. ZmmtRF2a The cDNA sequence between the 5' UTR and exon 4 / exon 5 (see [link]). Figure 4 ).

[0028] against crk5 Molecular marker detection of mutants includes the following steps: (1) The reaction system is as follows: 1 μL cDNA template (30-50 ng / μL); 0.4 μL Left primer (10 μM); 0.4 μL Right primer (10 μM); 3.2 μL ddH2O; 5 μL 2×PCR Mix.

[0029] (2) The PCR amplification program for CRK5-5U-F / CRK5-E4-R and CRK5-5U-F / CRK5-E5-R is as follows: 1 cycle 95℃ pre-denaturation for 3 min; 35 cycles 95℃ denaturation for 15 s, 58℃ annealing for 20 s, 72℃ extension for 30 s; Delay 72℃ for 5 min; Soak 25℃.

[0030] Then, using 1% agarose gel electrophoresis, bands were observed in both CRK5-5U-F and CRK5-E4-R wild-type and mutant materials; CRK5-5U-F and CRK5-E5-R wild-type materials showed bands, while mutant materials showed no bands (see...). Figure 4 ).

[0031] Example 4: ZmmtRF2a Candidate gene association analysis For analysis and research ZmmtRF2a To investigate the gene utilization pathway, 36 single nucleotide polymorphism (SNPs) were extracted from 440 inbred lines, including the gene and its upstream 3kb and downstream 2kb regions (B73V4: Chr5:218521712-218530263). Association analysis with the 100-grain weight of the inbred lines revealed 4 SNPs (B73V4: chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, chr5.s_218528368) that were significantly associated with grain length and 100-grain weight (see Table 2).

[0032] Table 2 ZmmtRF2a Gene association analysis .

[0033] Example 5: ZmmtRF2a Excellent haplotype identification Four SNPs significantly associated with 100-kernel weight were identified (see Table 2). Two combinations were found among the 440 inbred lines (see Table 3): Hap1 (6 inbred lines) and Hap2 (434 inbred lines). Hap1, with an average 100-kernel weight of 18.86 g, had an extremely low distribution frequency (6 / 440) and was considered a rare haplotype. The majority of inbred lines exhibited the haplotype Hap2, with a high distribution frequency (434 / 440) and an average 100-kernel weight of 21.56 g, making it a favorable haplotype (see Table 3).

[0034] Table 3 ZmmtRF2a Haplotype analysis of genes .

[0035] Example 6: ZmmtRF2a Phylogenetic tree and subcellular localization 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 its homology with human mtRF1a was higher than that with mtRF1 (see...). Figure 5 A). Phylogenetic analysis confirmed that ZmmtFR2a belongs to the plant RF2 branch and is parallel to the human mitochondrial mtRF1a protein (see [link]). Figure 5 B). ZmmtRF2a contains the GGQ motif required for ribosome binding and release, as well as the SPF motif that recognizes the stop codon (see B). Figure 5 A). And in mutants crk5 In ZmmtRF2a, these two motifs are missing.

[0036] TargetP bioinformatics analysis revealed that ZmmtRF2a lacks mitochondrial-targeting signaling peptides. Construction 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 [link to original text]). Figure 5 C). The above results indicate that ZmmtRF2a is a mitochondrial localization protein encoded by a nuclear gene.

[0037] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0038] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of its inventive concept. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. A method for detecting corn ZmmtRF2a The method of gene mutation is characterized by, Includes the following steps: Corn ZmmtRF2a Using the cDNA of the gene as a template, and with CRK5-5U-F (SEQ ID No. 3) as the right primer, amplification was performed using either CRK5-E4-R (SEQ ID No. 5) or CRK5-E5-R (SEQ ID No. 6) as the left primer. ZmmtRF2a The cDNA sequence between the 5' UTR and exon 4 or exon 5; The PCR amplification program was as follows: 1 cycle 95℃ pre-denaturation for 3 min; 35 cycles 95℃ denaturation for 15 s, 58℃ annealing for 20 s, 72℃ extension for 30 s; Delay 72℃ for 5 min; Soak 25℃; Then, using 1% agarose gel electrophoresis, both wild-type and mutant materials amplified by the CRK5-5U-F and CRK5-E4-R primer pairs showed bands; the wild-type amplified by the CRK5-5U-F and CRK5-E5-R primer pairs showed bands, while the mutant showed no bands. corn ZmmtRF2a The cDNA sequence of the gene is shown in SEQ ID NO.1.

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