Application of corn ZmmtRF2a gene and cloning primer thereof in regulation and control of grain development

Abstract

The invention relates to application of a corn ZmmtRF2a gene and a cloning primer thereof in regulation and control of grain development. A natural grain mutant crk5 found in the 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, and the gene encodes a peptide chain release factor 2a positioned by mitochondria, influences the mitochondrial function and regulates and controls development of corn embryos and endosperm and accumulation of grain storage substances. In crk5, ZmmtRF2a lacks the 5th to 7th exons, the encoded truncated protein does not have GGQ and SPF motifs, and the motifs are crucial to release activity of RF, mitochondrial binding and termination codon recognition. The invention provides technical support for the utilization of the gene and opens up a new way.

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 Regulatory 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 the application of maize ZmmtRF2a genes and their cloning primers 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. At present, 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 rps35'-maturation and translation initiation of mRNA. The mitochondrial protein synthesis machinery acts in concert with the cytoplasmic protein synthesis machinery and is essential for cellular homeostasis. Plant mitochondrial translation involves many plant-specific PPR proteins and other cofactors. Mitochondrial release factors (RFs) cooperate with translation termination factors to recognize stop codons at the A-site of the small ribosomal subunit, promoting 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, thus terminating protein synthesis. There are two classes 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 function and mechanism 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 any form of implication that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0006] The inventors of the present disclosure utilized natural mutants of seeds 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 leads to 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-seed weight of mutant seeds was reduced by 56% compared to the wild type, the endosperm development was abnormal, the embryo was malformed and unable to germinate normally, and the proliferation in the cells of the endosperm basal transfer layer was reduced; using genetic analysis and map-based cloning methods, the gene affecting maize seed 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 a protein encoded by a gene regulating seed 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 kernel development regulatory gene ZmmtRF2a in the genetic engineering regulation of the maize kernel 100-kernel weight trait.

[0011] Another aspect of the present disclosure provides a method for selecting excellent haplotypes of maize kernel 100-kernel weight, including the following steps: (1) Detect and determine the ZmmtRF2a genotype of the 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 maize line materials with 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 favorable haplotype lines.

[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 the accumulation of storage substances in kernels. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 For the phenotypic analysis of mutant kernels in an embodiment of the present disclosure crk5 ; in the figure, (A-D) are the kernel 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 kernels. En: endosperm; Em: embryo; (F) are the 100-kernel 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 the number of starch grains / 10 crk5 pixels of WT and 3 (n = 5). The values are mean ± SD; (I-J) are 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 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 of the gene, schematic diagram of the gene structure and protein structure; 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 In an embodiment of the present disclosure, CRK5-5U-F / 3U-R, CRK5-5U-F / E4-R, CRK5-5U-F / E5-R molecular marker map; in the figure, using Indel primer pairs crk5 and in WT crk5 The locus was detected by RT-PCR.

[0017] Figure 5 Phylogenetic 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, 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 mode

[0018] Unless otherwise specified, the instrumentation and equipment involved in the following examples are all conventional instrumentation and equipment; the reagents and materials involved are all commercially available conventional products; the experimental 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 all conventional methods unless otherwise specified.

[0019] Example 1: crk5 Phenotypic analysis of the mutant crk5 The grain mutant was discovered by the inventor during the process of breeding and selecting lines in the field (collected by Tang Jihua in Yuanyang, Henan 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 were smaller than those of the wild type, and the development of the embryo and endosperm was delayed. Figure 1 The mature grains of Figure 1 were 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 the embryo of crk5 was smaller and the endosperm was reduced compared to the WT ( Figure 1 E), and the starch content was reduced by 9% ( Figure 1 G). In addition, compared with the round starch grains and dense matrix in the WT endosperm, the starch grains of crk5 were irregular in shape and the number was reduced by 22.44% ( Figure 1 H-J). The defective embryo and endosperm caused the seeds to be unable to germinate ( Figure 1 K). Observing the paraffin sections of crk5 and WT grains 10-15 days after pollination, it was found that the embryo of crk5 was not significantly differentiated and the intravascular hyperplasia of the endosperm basal transfer layer cells was reduced (see Figure 2 ).

[0020] Example 2: ZmmtRF2a Map-based cloning of Using the F2 segregation population derived from the cross between crk5 and the elite 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 (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 region indicated that the Zm00001d018330 gene (the 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, containing 7 exons, and crk5 in Zm00001d018330 exons 5-7 were deleted, resulting in the deletion of the C-terminal amino acid sequence of the encoded protein (seeFigure 3 B).

[0021] Zm00001d018330 The gene cloning method 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 use DNaseI to remove possible DNA residues.

[0022] 2) Initially detect the RNA quality by agarose gel electrophoresis, and use a NanoDrop instrument to detect the RNA concentration and the light absorption values of the RNA at 260 nm and 280 nm. Determine 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).

[0023] 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.

[0024] 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).

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

[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, as follows: ZmmtRF2a The actual position of the mutation site in the fourth version of the maize B73 reference genome is before chr5:218,526,636. Design the following Indel primers: 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 with the left primers CRK5-E4-R (SEQ ID No.5) / CRK5-E5-R (SEQ ID No.6) ZmmtRF2a the cDNA sequence between the 5’UTR and exon 4 / exon 5 (see Figure 4 ).

[0028] For crk5 the molecular marker detection of mutants, it includes the following steps: (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 of CRK5-5U-F / CRK5-E4-R and CRK5-5U-F / CRK5-E5-R is: 1 cycle of pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s; Delay at 72°C for 5 min; Soak at 25°C.

[0030] Then, it is detected with 1% agarose gel. Both wild-type / mutant materials of CRK5-5U-F / CRK5-E4-R have bands; the wild-type of CRK5-5U-F / CRK5-E5-R has bands, while the mutant has no bands (see Figure 4 ).

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

[0032] Table 2ZmmtRF2a Association analysis of genes

[0033] Example 5: ZmmtRF2a Identification of excellent haplotypes There are 4 SNPs significantly associated with 100 - kernel weight (see Table 2), and 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 genes

[0035] Example 6: ZmmtRF2a Phylogenetic tree and subcellular localization Protein sequence analysis shows 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 confirms 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). In the mutant crk5 , these two motifs of ZmmtRF2a are missing.

[0036] TargetP bioinformatics analysis shows that ZmmtRF2a lacks a mitochondrial targeting signal peptide. Construct 35S Pro ::ZmmtRF2a: eGFP the vector and transiently express it in maize protoplasts. The results show that the eGFP signal overlaps 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, additional changes and modifications can be made to these embodiments by those skilled in the art 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

Maize with a cDNA sequence as shown in SEQ ID NO.1 ZmmtRF2a Use of a gene, a protein encoded by an amino acid sequence as shown in SEQ ID NO.2, or a primer pair with sequences as shown in SEQ ID NO.3 and SEQ ID NO.4 in at least one of the following (1) to (6): (1) Regulating the maize grain size trait or preparing a reagent for regulating the maize grain size trait; (2) Regulating the maize grain yield trait or preparing a reagent for regulating the grain yield trait; (3) Regulating the maize starch content trait or preparing a reagent for regulating the maize starch content trait; (4) Breeding varieties / lines with maize grain size traits or preparing reagents for breeding varieties / lines with maize grain size traits; (5) Breeding varieties / lines with maize grain yield traits or preparing reagents for breeding varieties / lines with maize grain yield traits; (6) Breeding maize starch content traits or preparing reagents for breeding maize starch content traits; The maize grain yield trait is at least one of grain width, grain thickness, and 100-grain weight.

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