Maize ZmmtRF2a gene mutation marker, identification primer and application of corn ZmmtRF2a gene mutation marker in regulation and control of grain development

Abstract

The invention relates to a corn ZmmtRF2a gene mutation marker, an identification primer and application of the corn ZmmtRF2a gene mutation marker in regulation and control of grain development. A natural grain mutant crk5 found in a corn field breeding line selection process is utilized, a target gene ZmmtRF2a causing crk5 grain mutation is obtained through phenotypic analysis, linkage analysis and map-based cloning technologies, the gene encodes a peptide chain release factor 2a for mitochondrial localization, the peptide chain release factor 2a affects the mitochondrial function, and the gene can be used for inducing the mutation of the crk5 grain. The development of corn embryos and endosperm and the accumulation of grain storage substances are regulated and controlled. In crk5, ZmmtRF2a lacks 5th to 7th exons, the encoded truncated protein does not have GGQ and SPF motifs, the motifs are crucial to release activity of RF, mitochondrial binding and termination codon recognition, technical support is provided for utilization of the gene, and a new way is opened up.

Description

[0001] This invention is a divisional application of the patent application with the application number 202311351629.6, the application date of October 18, 2023, and the invention title of "Maize Kernel Development Regulation Gene ZmmtRF2a , Its Encoded Protein, Functional Marker and Application". Technical Field

[0002] This invention relates to the field of assisted molecular breeding technology, and specifically relates to maize ZmmtRF2a gene mutation markers, identification primers and their applications in regulating kernel development. Background Art

[0003] Maize ([[]] Zea mays L. ) is currently the crop with the highest yield in China, with great production potential. It is an important food, feed, chemical and energy crop, and plays an irreplaceable role in industrial and agricultural production and ensuring national food security. Maize kernel size is an important component factor of maize yield, and the molecular mechanism of its formation and development has received extensive attention. Although many genes regulating maize kernel development and filling have been cloned, due to the extremely complex biological process of maize embryo and endosperm development, the key genes and molecular mechanisms for precisely regulating kernel filling still need to be further analyzed.

[0004] Mitochondria are the power source of eukaryotic cells, and their dysfunction often leads to insufficient effective energy supply, resulting in growth defects in human cells and plants. Mitochondrial genes related to maize kernel development mostly play a role at the transcriptional regulation level, and there are few reports on genes affecting kernel development at the translational level. The kernel mutant DEK66 encodes a mitochondrial ribosome assembly factor and has GTPase activity, which may affect mitochondrial protein translation; DEK44 encodes the mitochondrial large subunit protein L9 (RPL), which is directly involved in the rps3 ​5'-Maturation and Translation Initiation of mRNA. The mitochondrial protein synthesis mechanism works in concert with the cytoplasmic protein synthesis mechanism and is crucial for cellular homeostasis. Plant mitochondrial translation involves many plant-specific PPR proteins and other cofactors. Mitochondrial release factor (RF) cooperates with translation termination factors to recognize the stop codon at the A-site of the small ribosomal subunit, promoting the hydrolysis of the ester bond between the tRNA at the P-site and the nascent polypeptide chain at the active site of the large ribosomal subunit, thereby terminating 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-canonical 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 necessary for ribosome binding and RF release activity, as well as the SPF motif necessary for recognizing UAA / UGA stop codons, but its specific functions and mechanisms of action are still unclear.

[0005] The information disclosed in this background section is only for enhancing the understanding of the background of the present disclosure and should not be regarded as an admission or an implication in any form that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] The inventors of the present invention utilized a natural mutant of grains discovered during the process of maize field breeding and line selection crk5 to clone the key gene affecting mitochondrial translation ZmmtRF2a , which encodes peptide chain release factor 2a during mitochondrial ribosome translation. ZmmtRF2a The mutation results in defects in embryo and endosperm development, and the seeds lose their germination ability.

[0007] In the first aspect of the present disclosure, through phenotypic and cytological analyses, it was found that crk5 the 100-grain weight of the mutant grains was reduced by 56% compared to the wild type, the endosperm development was abnormal, the embryo was deformed and unable to germinate normally, and the proliferation in the cells of the endosperm basal transfer layer decreased; by using genetic analysis and map-based cloning methods, the gene affecting maize grain development was obtained ZmmtRF2a , and its cDNA sequence is shown in SEQ ID NO.1.

[0008] In the second aspect of the present disclosure, it relates to an encoded protein of a gene regulating grain development ZmmtRF2a , and its amino acid sequence is shown in SEQ ID NO.2.

[0009] The third aspect of the present disclosure relates to a pair of cDNA primers for amplifying the ZmmtRF2a , and the sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0010] Another aspect of the present disclosure is to apply the maize grain development regulatory gene ZmmtRF2a in the genetic engineering regulation of the maize grain 100-grain weight trait.

[0011] Another aspect of the present disclosure provides a method for selecting excellent haplotypes of maize grain 100-grain weight, including: (1) Detect and determine the genotypes of the ZmmtRF2a gene B73V4 at the loci of chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, and chr5.s_218528368 in the maize line material; (2) Select the maize line material 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 in sequence, and self-cross or cross to breed a favorable haplotype line.

[0012] One or more technical solutions provided in the embodiments of the present disclosure have at least any one of the following technical effects or advantages: The ZmmtRF2a gene cloned by positional cloning affects mitochondrial function, regulates maize embryo and endosperm development and grain storage substance accumulation. When the ZmmtRF2a gene lacks the 5th to 7th exons, the C-terminal amino acid sequence of the encoded protein will be missing, and then a crk5 mutant is formed. The mature grains of the mutant are significantly smaller than those of the WT in length, width, and thickness, resulting in a decrease in its 100-grain weight compared to the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 For the phenotypic analysis of mutant grains in an embodiment of the present disclosure crk5 ; in the figure, (A-D) are the grain characteristics of the wild type (WT) and crk5 on the same ear, scale bar, 1 cm; (E) are the longitudinal sections of WT and crk5 grains. En: endosperm; Em: embryo; (F) are the 100-grain weights of WT and crk5 (n = 3), the values are mean ± SD; (G) are the bar graphs of the endosperm starch content of WT and crk5 (n = 3), the values are mean ± SD; (H) are WT and crk5Number of starch grains / 10 3 Pixels (n = 5). Values are mean ± SD; (I-J) WT (I) and crk5 (J) Scanning electron microscopy (SEM) image of the central region of the 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 t - test.

[0014] Figure 2 Cytological analysis of wild - type and mutant grains in an embodiment of the present disclosure; in the figure, (A - I) WT and crk5 Paraffin sections of grains, scale bars are 1 mm (A - B), 200 µm (C), 100 µm (D - E), 50 µm (F - G), 200 µm (H - I) respectively; En: endosperm; Em: embryo; (J - M) Transmission electron microscopy (TEM) observation of the starchy endosperm of WT (J and L) and crk5 (K and M) at 15 days after pollination, scale bars are 10 µm (J - K), 2 µm (L - M); SG: starch granule; PB: protein body.

[0015] Figure 3 In an embodiment of the present disclosure ZmmtRF2a Map - based cloning, gene structure and protein structure schematic diagram of the gene; in the figure, A: ZmmtRF2a Map - based cloning of the gene; B: ZmmtRF2a Schematic diagram of the gene structure; C: Schematic diagram of the ZmmtRF2a protein structure.

[0016] Figure 4 CRK5 - 5U - F / 3U - R, CRK5 - 5U - F / E4 - R, CRK5 - 5U - F / E5 - R molecular marker map in an embodiment of the present disclosure; in the figure, using Indel primer pairs crk5 and in WT crk5 sites for RT - PCR detection.

[0017] Figure 5Phylogenetic analysis and subcellular localization analysis of ZmmtRF2a in an embodiment of the present disclosure; in the figure, A: Amino acid sequence alignment of ZmmtRF2a from maize, Arabidopsis thaliana, human, and Saccharomyces cerevisiae; 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 manners

[0018] In the following embodiments, the instrument equipment involved is conventional instrument equipment unless otherwise specified; the reagents and materials involved are commercially available conventional products unless otherwise specified; the test and detection methods involved (such as extraction of genomic DNA, extraction and reverse transcription of RNA, RT-PCR, paraffin section of maize kernels, etc.) are conventional methods unless otherwise specified.

[0019] Example 1: crk5 Phenotypic analysis of the mutant crk5 It is a grain mutant discovered by the present inventor during the process of breeding and selecting lines in the field (collected from Yuanyang, Henan by Tang Jihua in August 2017). Observing the wild-type (WT) and mutant grains on the same mature ear, it was found that crk5 the grains of crk5 are smaller than those of the wild-type, and the development of the embryo and endosperm is delayed. Figure 1 The mature grains of Figure 1 are significantly smaller than those of the WT in length, width, and thickness ( crk5 A-E), resulting in a 56% reduction in 100-grain weight compared to the wild-type ( Figure 1 F). Longitudinal sectioning of the grains showed that compared with the WT, Figure 1 the embryo of crk5 is smaller and the endosperm is reduced ( Figure 1 E), and the starch content is reduced by 9% ( Figure 1 G). In addition, compared with the round starch grains and dense matrix in the WT endosperm, crk5 the starch grains of crk5 are irregular in shape and the number is reduced by 22.44% ( Figure 2 H-J). The defective embryo and endosperm cause its seeds to be unable to germinate (

[0020] Example 2: ZmmtRF2a Map-based cloning of Using the F2 segregation population derived from the cross between crk5 and the backbone inbred line Zheng 58 as the material, through genetic analysis and map-based cloning, the target gene was located within a physical interval of approximately 112 kb on chromosome 5 of maize (seeFigure 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 region indicated that Zm00001d018330 the gene (cDNA sequence as shown in SEQ ID NO.1 and amino acid sequence as shown in SEQ ID NO.2) is the target gene, which contains 7 exons and crk5 in Zm00001d018330 exons 5 - 7 are deleted, resulting in the deletion of the C-terminal amino acid sequence of the encoded protein (see Figure 3 B).

[0021] Table 1 ZmmtRF2a Primers and Sequences Used for Cloning

[0022] Zm00001d018330 The cloning method of the gene is as follows: 1) Using a plant polysaccharide and polyphenol RNA extraction kit, extract the RNA of the mutant and wild-type endosperm 13 days after pollination according to the kit instructions. Dissolve the total RNA thoroughly with RNase-free double-distilled water and remove possible DNA residues with DNaseI.

[0023] 2) Initially detect the RNA quality by agarose gel electrophoresis and detect the RNA concentration and the optical absorption values of the RNA at 260 nm and 280 nm using a NanoDrop instrument. Judge the integrity and purity of the RNA according to whether the OD 260 / 280 is between 1.8 and 2.2 (28S:18S > 1.0).

[0024] 3) Use the obtained RNA as a template for reverse transcription, and store the obtained cDNA in aliquots at -20 °C in a refrigerator for later use.

[0025] 4) According to the Zm00001d018330 transcript sequence in the maize B73 reference genome, design amplification primers: F: 5’-AGGACTGAGGAGCTATCCCG- 3’ (SEQ ID No.3); R: 5’-TTCCTGGGTGCAACACTGTC -3’ (SEQ ID No.4).

[0026] Example 3: ZmmtRF2a Development of Molecular Markers for Mutation Sites For ZmmtRF2a exon deletions, develop easily detectable PCR molecular markers CRK5-5U-F / E4-R and CRK5-5U-F / E5-R, specifically as follows: ZmmtRF2aThe 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: 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 primer, which can specifically amplify the cDNA sequence between the 5’UTR and exon 4 / exon 5 with the left primers CRK5-E4-R (SEQ ID No.5) / CRK5-E5-R (SEQ ID No.6) ZmmtRF2a (see Figure 4 ).

[0028] For the molecular marker detection of crk5 the mutant, the following steps are included: (1) The reaction system is: 1 μL of cDNA template (30 - 50 ng / μL); 0.4 μL of Left primer (10 μM); 0.4 μL of Right primer (10 μM); 3.2 μL of ddH2O; 5 μL of 2×PCR Mix.

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

[0030] Then, it is detected with 1% agarose gel. Bands can be seen in both the wild-type and mutant materials of CRK5-5U-F / CRK5-E4-R; bands can be seen in the wild-type of CRK5-5U-F / CRK5-E5-R, but no band can be seen in the mutant (see Figure 4 ).

[0031] Example 4: ZmmtRF2a Candidate gene association analysis of To analyze and study ZmmtRF2aFor the utilization pathway of the gene, single nucleotide polymorphism sites (SNPs) within the gene and the 3 kb upstream and 2 kb downstream regions were extracted from 440 inbred lines, with a total of 36 SNPs (B73V4: Chr5:218521712 - 218530263). An association analysis was performed with the 100 - kernel weight of the inbred lines, and 4 SNPs (B73V4: chr5.s_218528166, chr5.s_218528168, chr5.s_218528169, chr5.s_218528368) significantly associated with grain length and 100 - kernel weight were detected (see Table 2).

[0032] Table 2 ZmmtRF2a Association analysis of the gene

[0033] Example 5: ZmmtRF2a Identification of excellent haplotypes For the 4 SNPs significantly associated with 100 - kernel weight of grains (see Table 2), there are 2 combinations in 440 inbred lines (see Table 3): Hap1 (6 lines), Hap2 (434 lines). Among them, the average 100 - kernel weight of Hap1 is 18.86 g, and its distribution frequency in inbred lines is extremely low (6 / 440), belonging to a rare haplotype; the haplotype of most inbred lines is Hap2, with a high distribution frequency (434 / 440), and the average 100 - kernel weight is 21.56 g, belonging to a favorable haplotype (see Table 3).

[0034] Table 3 ZmmtRF2a Haplotype analysis of the gene

[0035] Example 6: ZmmtRF2a Phylogenetic tree and subcellular localization Protein sequence analysis showed that ZmmtRF2a is highly homologous to bacterial RF2 (E - value 5e - 117, homology 48.31%, https: / / blast.ncbi.nlm.nih.gov / ), and its homology with human mtRF1a is 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 Figure 5 B). ZmmtRF2a contains the GGQ motif required for ribosome binding and release, as well as the SPF motif for recognizing stop codons (see Figure 5 A). However, in the mutant crk5 ZmmtRF2a lacks these two motifs.

[0036] Bioinformatics analysis by TargetP showed that ZmmtRF2a lacks a mitochondrial targeting signal peptide. Construction35S 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 mitochondrially localized protein encoded by a nuclear gene.

[0037] Although some preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

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

Claims

1. A kind of corn ZmmtRF2a Gene mutation marker, based on Zm00001d018330 The gene lacks the 5th to 7th exons, resulting in the deletion of the C-terminal amino acid sequence of the encoded protein.

2. A kind of corn ZmmtRF2a Gene mutation identification primer, including Indel primers shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID No.

6.

3. The corn according to claim 1 ZmmtRF2a Gene mutation marker or the identification primer according to claim 2 in the corn kernel mutant crk5 Application in at least one of the following (1) to (7): (1) Regulating the corn kernel size trait or preparing a reagent for regulating the corn kernel size trait; (2) Regulating the corn kernel yield trait or preparing a reagent for regulating the kernel yield trait; (3) Regulating the corn starch content trait or preparing a reagent for regulating the corn starch content trait; (4) Breeding varieties / lines with corn kernel size traits or preparing reagents for breeding varieties / lines with corn kernel size traits; (5) Breeding varieties / lines with corn kernel yield traits or preparing reagents for breeding varieties / lines with corn kernel yield traits; (6) Breeding corn starch content traits or preparing reagents for breeding corn starch content traits; (7) Identifying corn kernel mutants crk5 or preparing a reagent for identifying corn kernel mutants crk5 ; The corn kernel yield trait is at least one of grain width, grain thickness, and 100-grain weight.

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