Maize kernel development regulatory gene ZmRRP45B, its encoded protein, SNP markers, functional markers and their applications

Abstract

The present invention discloses a corn kernel development regulating gene ZmRRP45B , its encoded protein, SNP marker, molecular marker and its application. This application uses the natural mutant of corn kernels found in the process of field breeding and selection. l168 Cloning of key genes affecting RNA exosome function ZmRRP45B This gene encodes the RNA exosome core subunit RRP45, regulates the development of corn embryo and endosperm, affects the accumulation of grain storage materials, and has important utilization value for improving corn yield and quality; ZmRRP45B The identification of excellent haplotypes and analysis of their utilization pathways provide technical support and excellent germplasm resources for the utilization of the gene. The use of the genes and molecular markers disclosed in the present invention application can facilitate the creation of high-yield or high-quality breeding materials for corn, shorten the breeding cycle of new crop varieties, reduce breeding costs, and improve breeding efficiency.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering technology, and specifically to maize kernel development regulating genes ZmRRP45B , its encoded protein, SNP markers, functional markers and their applications. Background Art

[0002] corn (Zea mays L. ) is an important food and feed crop, playing an irreplaceable role in ensuring national food security and national economic development. Grain is the main organ of corn yield, and the molecular mechanism of its formation and development has received extensive attention. Although many genes that regulate corn grain development and filling have been cloned, since corn embryo and endosperm development is an extremely complex biological process, the node genes and molecular mechanisms that accurately regulate grain development and filling still need to be further analyzed.

[0003] RNA decay plays an important role in maintaining the dynamic balance of life activities of organisms and controlling the quality of gene expression. The biological evolution process has formed a precise RNA regulatory mechanism, which eliminates abnormal transcripts through degradation and maintains the normal development of biological organisms. The RNA degradation mechanism can also participate in the degradation of normal mRNA, regulate the abundance of gene expression, and maintain the dynamic balance of transcripts. RNA degradation requires the action of 5'→3' or 3'→5' nuclease, among which 3'→5' RNA degradation is mainly completed by exosomes (EXOSOME), which plays an important role in RNA maturation and quality control. Studies have shown that defects in the exosome-mediated RNA degradation process lead to a variety of physiological and reproductive defects in animals and plants. However, the mechanism of action of plant RNA exosomes in regulating phenotypes still needs further study.

[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 present invention applies to utilize the natural mutants of kernels found in the process of corn field breeding and selection l168 , cloned the key gene affecting RNA degradation ZmRRP45B , which encodes a core subunit of the RNA exosome complex. Further studies have shown that ZmRRP45B Genes regulate the development of corn embryo and endosperm, affect the accumulation of grain storage materials, and can provide new genetic resources for breeding high-yield and high-quality corn varieties.

[0006] In the first aspect disclosed in the present application, phenotypic and cytological analysis was performed to find l168The 100-grain weight of the mutant was 75.75% lower than that of the wild type, the embryo and endosperm were abnormally developed, and the proliferation of cells in the basal transfer layer of the endosperm was reduced. The control gene of the mutant was obtained through genetic analysis and positional cloning. ZmRRP45B A gene having a CDS sequence as shown in SEQ ID NO.1.

[0007] The second aspect disclosed in the present application relates to a grain development regulating gene ZmRRP45B 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 ZmRRP45B The primer pair for the gene has sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0009] The fourth aspect disclosed in the present application relates to a corn kernel phenotypic mutation SNP marker, based on the fourth version of the corn B73 reference genome, and there is a G deletion at the chr5.s_175415208 position.

[0010] The fifth aspect disclosed in the present application relates to a dCAPS molecular marker for identifying phenotypic mutants, including a primer pair as shown in SEQ ID NO.5 and SEQ ID NO.6; and / or a primer pair as shown in SEQ ID NO.7 and SEQ ID NO.8.

[0011] The sixth aspect disclosed in the present application relates to an identification ZmRRP45B The overexpression material molecular marker includes the primer pair shown as SEQ ID NO.9 and SEQ ID NO.10.

[0012] In another aspect disclosed in the present application, the corn kernel development regulating gene ZmRRP45B , the amplification primers, the corn kernel phenotypic mutation SNP markers or the dCAPS molecular markers are used in the regulation of kernel size and / or corn embryo and endosperm developmental traits.

[0013] Another aspect disclosed in the present application relates to a SNP marker that is significantly associated with corn kernel type and storage material content. Based on the fourth version of the corn B73 reference genome, A / T, A / C, and G / A mutations exist at positions chr5.s_175413975, chr5.s_175413829, and chr5.s_175418484, respectively.

[0014] Another aspect disclosed in the present application relates to a method for identifying a gene ZmRRP45BThe dCAPS molecular markers of excellent haplotypes include primer pairs as shown in SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, and SEQ ID NO.17 and SEQ ID NO.18.

[0015] In another aspect disclosed in the present application, the corn kernel development regulating gene ZmRRP45B , the SNP marker or the dCAPS molecular marker is used in the breeding of inbred lines for corn grain yield and quality traits.

[0016] In another aspect disclosed in the present application, a method for identifying a gene is provided. ZmRRP45B The method for obtaining excellent haplotypes comprises the following steps:

[0017] The extracted DNA of the material to be identified is used as a template, and the PCR products containing the chr5.s_175413829 and chr5.s_175418484 SNP sites are amplified by PCR using the outer primer pair. Then, the second round of amplification is performed using the inner primer pair and the diluted PCR product as a template. The PCR products obtained in the second round are respectively treated with restriction endonucleases Sac I and Spe I digestion, and gel electrophoresis detection, AAA and TAA haplotypes are two higher bands; ACG and TCG haplotypes are two lower bands; ACA and TCA haplotypes are Sac I digested with enzyme to form a lower band. Spe After digestion with I, the AAG and TAG haplotypes are higher. Sac I was a higher band after digestion. Spe I was used as a lower band after enzyme digestion; AAA and TAA haplotypes were selected as breeding materials for improving starch content.

[0018] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages:

[0019] 1. Positional Cloning ZmRRP45B Genes regulate the development of corn embryo and endosperm, affecting the accumulation of grain storage materials and having important application value in improving corn grain yield and quality.

[0020] 2. ZmRRP45B The identification of excellent haplotypes and analysis of their utilization pathways provide technical support and excellent germplasm resources for the utilization of this gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 In one embodiment of this applicationl168 Analysis of mutant grain phenotypes; in the figure, A: Grain phenotypes on mature detached ears; B: Comparison of wild-type and mutant grains on the same ear 7 days after pollination; C: Comparison of wild-type and mutant grains on the same ear 15 days after pollination; D: Comparison of wild-type and mutant grains 20 days after pollination; E: Comparison of longitudinal sections of wild-type and mutant grains; F: Comparison of grain length, grain width and grain thickness phenotypes between wild-type and mutant grains; G: Difference in 100-grain weight between wild-type and mutant grains; H: Statistical difference in grain length, grain width and grain thickness between wild-type and mutant grains; I: Difference in grain germination between wild-type and mutant; J: Representative plants of wild-type and mutant in the adult stage: K: Difference in germination rate between wild-type and mutant grains; L: Difference in starch content between wild-type and mutant grains.

[0022] Figure 2 This is a cytological analysis of wild-type and mutant grains in one embodiment of the present application; in the figure, the first row is a comparison of paraffin sections of mutant and wild-type grains 8 to 15 days after pollination; the second row is a comparison of embryos of mutant and wild-type grains; and the third row is a comparison of endosperm basal transfer layers of mutant and wild-type.

[0023] Figure 3 In one embodiment of this application ZmRRP45B Map-based cloning, gene structure and function verification of genes; in the figure, A: ZmRRP45B Map-based cloning of genes; B: ZmRRP45B Schematic diagram of the gene structure; C: ZmRRP45B Schematic diagram of protein structure; D: ZmRRP45B Function backfill verification.

[0024] Figure 4 The molecular marker map of dCAPS1-F1 / R1 and dCAPS1-F2 / R2 and their enzyme digestion and the natural population verification map of the mutation site in an embodiment of the present application, A: dCAPS1-F2 / R2 PCR product in the restriction endonuclease N i Electrophoresis bands in 10% nucleic acid PAGE gel after enzyme digestion, B: 178 natural populations in ZmRRP45B ( rrp45b ) Verification of the chr5.s_175415208 site on the gene.

[0025] Figure 5 is the result of the grain trait association analysis in one embodiment of the present application; in the figure, the upper ZmRRP45B Manhattan plot of the association analysis between SNPs and grain traits; in the middle is ZmRRP45BSchematic diagram of the gene structure; below is the LD block diagram of the SNP; 100KW represents the 100-grain weight trait, KW represents the grain width trait, KNPE represents the number of grains per ear trait, Oil represents the oil content trait of the grain, PC represents the protein content trait of the grain, and SC represents the starch content trait of the grain.

[0026] Figure 6 The haplotype molecular markers used in one embodiment of the present application; in the figure, A: dCAPS2-F1R1 (chr5.s_175413829) PCR product agarose gel electrophoresis band; B: dCAPS2-F2R2 PCR product agarose gel electrophoresis band; C: dCAPS2-F2R2 PCR product Sac I digestion, electrophoresis bands in 10% nucleic acid PAGE gel; D: dCAPS3-F1R1 (chr5.s_175418484) PCR product agarose gel electrophoresis band; E: dCAPS3-F2R2 PCR product agarose gel electrophoresis band; F: dCAPS3-F2R2 PCR product Spe Electrophoresis bands in 10% nucleic acid PAGE gel after enzyme digestion with I; Note: Lanes 1 (CF3), 2 (B110), and 3 (CIMBL50) are Hap1; Lanes 4 (GEMS50), 5 (CHUAN48-2), and 6 (BY809) are Hap7; Lanes 7 (CIMBL39), 8 (SY1077), and 9 (CIMBL11) are Hap5 / 6; Lanes 10 (CIMBL70), 11 (CIMBL99), and 12 (CML50) are Hap3 / 4. DETAILED DESCRIPTION

[0027] 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 commercially available conventional products unless otherwise specified; the tests and detection methods involved (such as genomic DNA extraction, RNA extraction and reverse transcription, RT-PCR, real-time fluorescence quantitative PCR, BN-PAGE gel electrophoresis, Western blot, corn kernel paraffin sections, etc.) are all conventional methods unless otherwise specified.

[0028] Embodiment 1: l168 Phenotypic analysis of mutants

[0029] l168 The mutant (Tang Jihua, 2020, collected in Yuanyang Science and Education Park of Henan Agricultural University) is a grain mutant discovered during field breeding selection. Observation of mature wild-type and mutant grains on the same ear revealed that, compared with the wild-type, l168The mutant grains became smaller, the endosperm was not fully filled, and the top was slightly collapsed, resulting in a significant decrease in the 100-grain weight of the mutant (75.75% decrease relative to the wild type) (see Figure 1 AH); Most mutant seeds cannot germinate normally, and the mutants grow weakly after germination (see Figure 1 IJ), whose bud rate (24.8%) was lower than that of the wild type (91.7%) (see Figure 1 K), the starch content of the mutant was reduced by 8.98% at the same dry weight (see Figure 1 L).

[0030] 8 to 15 days after pollination (8 to 15 DAP) l168 Paraffin sections of mutant and wild-type kernels were observed and it was found that embryonic development of mutants was delayed and the proliferation of cells in the basal transfer layer of endosperm was reduced (see Figure 2 ).

[0031] Embodiment 2: ZmRRP45B Map-based cloning and functional verification of genes

[0032] use l168 The F2 segregating populations of the maize gene were used as materials. Through genetic analysis and positional cloning, the target gene was gradually located in a physical interval of about 500 kb on the fifth chromosome of maize (see Figure 3 A).

[0033] 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 Zm00001d016770 The gene (CDS sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) is a potential candidate gene, which contains 9 exons. l168 middle Zm00001d016770 The gene has a G deletion at +85bp in exon 7, which causes the 302nd amino acid encoded by it to mutate from valine to a stop codon (see Figure 3 BC).

[0034] Zm00001d016770 The gene cloning method is as follows:

[0035] ①Use the Plant Polysaccharide and Polyphenol RNA Extraction Kit to extract RNA from mutant and wild-type endosperm 10 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.

[0036] ② The 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.

[0037] ③ Use the obtained RNA as a template for reverse transcription, and the obtained cDNA is packaged and stored in a -20℃ refrigerator for later use.

[0038] ④ According to the maize B73 reference genome Zm00001d016770 Transcript sequence, design amplification primers:

[0039] F: 5'-ATGGAGCACCTCCGGTG-3' (SEQ ID No. 3);

[0040] R: 5'-CTAACTCCTGTCACTTTTCTCTT-3' (SEQ ID No. 4).

[0041] To verify this candidate gene and further clarify its biological function, a Zm00001d016770 The overexpression vector was used to transform maize embryos and obtain transgenic seeds. rrp45b The hybridization was performed and the genotype of the F1 ears obtained by the hybridization was identified. rrp45b The kernels were used for self-pollination to obtain F2, and the phenotype and genotype of F2 ears were observed and identified. l168 The mutation site is rrp45b / rrp45b When the transgenic positive plants showed normal grain phenotype, while the transgenic negative plants showed mutant grain phenotype. Zm00001d016770 Can be covered l168 Grain mutant phenotype is produced l168 The functional gene of the kernel mutant phenotype is named ZmRRP45B (See Figure 3 D).

[0042] ZmRRP45B The transgenic detection primers are as follows:

[0043] F: 5'- GCCCTGCCTTCATACGCTA-3' (SEQ ID No. 9);

[0044] R: 5'-TAGGGCTTTCTCGGTGGTGTAT-3' (SEQ ID No. 10).

[0045] Table 1 ZmRRP45B Primers and sequences used for cloning

[0046] .

[0047] Example 3: Corn ZmRRP45B Development of molecular markers for mutation sites

[0048] against ZmRRP45B The single base mutation of dCAPS1 was developed to be easily detected by PCR molecular markers, as follows:

[0049] l168 The actual position of the mutation site in the fourth version of the maize B73 reference genome is 175,415,208 on chromosome 5. The following dCAPS primers were designed:

[0050] dCAPS1-F1: 5'-AGGAAGCCACTATCACTATGGTTG-3' (SEQ ID No. 5);

[0051] dCAPS1-R1: 5'-AACAGTACCCGGCAAATCAG- 3' (SEQID No. 6);

[0052] dCAPS1-F2: 5'-CCTTTTTATACTTTGCTGGTTCT-3' (SEQID No. 7);

[0053] dCAPS1-R2: 5'-CCTCAGAAAATTAAGTTCACACATAT-3' (SEQ ID No. 8).

[0054] Among them, dCAPS1-F1R1 is the outer primer pair, which can specifically amplify ZmRRP45B Gene sequence; dCAPS1-F2R2 was used as the inner primer pair, and the PCR product of dCAPS1-F1R1 diluted 10 times was used as the template for the second round of amplification.

[0055] against l168 Molecular marker detection of mutation sites includes the following steps:

[0056] (1) The reaction system is:

[0057] DNA template (30-50 ng / μL) 1 μL;

[0058] Left primer (10 μM) 0.5μL;

[0059] Right primer (10 μM) 0.5 μL;

[0060] ddH2O 2 μL;

[0061] 2×PCR Mix 5 μL.

[0062] (2) The PCR amplification procedure for dCAPS1-F1R1 is as follows:

[0063] 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 at 72℃ for 5 min; Soak at 25℃.

[0064] (3) The PCR amplification procedure for dCAPS1-F2R2 is as follows:

[0065] 1 cycle: pre-denaturation at 95℃ for 3 min; 35 cycles: denaturation at 95℃ for 15 s, annealing at 58℃ for 20 s, extension at 72℃ for 15 s; Delay at 72℃ for 5 min; Soak at 25℃.

[0066] The PCR product of dCAPS1-F2R2 was diluted 10 times and cleaved with restriction endonucleases. N I digestion, and then detected by 10% nucleic acid PAGE gel, the wild-type material was digested into smaller bands (see Figure 4 ).

[0067] Embodiment 4: ZmRRP45B Candidate gene association analysis

[0068] To clarify ZmRRP45B The utilization pathway of the gene was to use 129 SNPs (single nucleotide polymorphism sites) (B73V4: Chr5:175412532-175422968) of the gene in 471 inbred lines as genotypes, and candidate gene association analysis was performed based on the grain phenotype, population structure (Q) and kinship (K) of the population. p A total of 45 SNPs were detected with a threshold value of <7.75E-03 (1 / 129) that were significantly associated with grain width, grain thickness, 100-grain weight, number of grains per row, grain starch, grain protein, and grain oil content (see Table 2).

[0069] Table 2 ZmRRP45B Results of candidate gene association analysis

[0070] .

[0071] Embodiment five: ZmRRP45B Excellent haplotype identification

[0072] Among the 45 significantly associated SNPs, ZmRRP45BThe chr5.s_175413975 in the 5'UTR and chr5.s_175413829 and chr5.s_175418484 in the 3'UTR of the gene were the three SNPs most significantly associated with grain traits (p < 4.41E-03, see Figure 5 ). Based on these three SNPs, haplotype analysis was performed. Among the 471 inbred lines, there were 7 combinations (Hap1 AAA, Hap2TAA, Hap3 ACA, Hap4 TCA, Hap5 AAG, Hap6 TAG, Hap7 ACG), and the differences in the associated significant traits all reached extremely significant levels. Taking grain starch content as an example, the mean values ​​of grain starch content of Hap2 and Hap3, and Hap5 and Hap6 were close, and they were calculated according to one type of haplotype respectively ( p = 1.49E-08) (see Table 2): Hap1 (4 copies), Hap2 / 3 (195 copies), Hap4 (2 copies), Hap5 / 6 (69 copies), Hap7 (146 copies). Among them, the average starch content of the haplotype Hap1 with excellent starch content is 66.567%, and its distribution frequency in inbred lines is low (2 / 471), which is a rare haplotype and has potential for use in breeding and improvement; the average starch content of the unfavorable haplotype Hap4 is 62.338%, and its distribution frequency in inbred lines is extremely low (2 / 471), which is a rare haplotype; most inbred lines are selected haplotypes with higher distribution frequencies, and their average starch content is between 65% and 66% (see Table 3).

[0073] Table 3 Haplotype analysis

[0074] .

[0075] Embodiment six: ZmRRP45B Development of molecular markers for superior haplotypes

[0076] Based on the identification of excellent haplotypes for starch content, simple and easy-to-operate PCR markers dCAPS2 and dCAPS3 were developed for the two SNPs chr5.s_175413829 and chr5.s_175418484.

[0077] dCAPS Primers:

[0078] dCAPS2-F1: 5'-CCGTTGTCGTAAATGGGCCTT-3' (SEQ ID No. 11);

[0079] dCAPS2-R1: 5'-TTTGTATGCCCCAGGTGAGT-3' (SEQ ID No. 12);

[0080] dCAPS2-F2: 5'-ATGTATGGCACCCCGGATGAGAGCT-3' (SEQ ID No. 13);

[0081] dCAPS2-R2: 5'-GATTGCGCATGCTACAACTGTATG-3' (SEQ ID No. 14);

[0082] dCAPS3-F1: 5'-AACTCGGTGAATATGGCCAGC-3' (SEQ ID No. 15);

[0083] dCAPS3-R1: 5'-GCCCAAAAGGTAGACGCAAT-3' (SEQ ID No. 16);

[0084] dCAPS3-F2: 5'-TTTCACATCCAAACCGGAAAAACTA-3' (SEQ ID No. 17);

[0085] dCAPS3-R2: 5'-GCTTCACTCTTGCATATTTCGAAC-3' (SEQ ID No. 18).

[0086] Among them, dCAPS2-F1R1 and dCAPS3-F1R1 are outer primer pairs, which can specifically amplify the genomic sequence containing two SNPs (see Figure 6 A and Figure 6 D); dCAPS2-F2R2 and dCAPS3-F2R2 were the inner primer pairs of chr5.s_175413829 and chr5.s_175418484, respectively. The second round of amplification was performed using the PCR products of dCAPS2-F1R1 and dCAPS3-F1R1 diluted 10 times as templates.

[0087] Molecular marker detection for excellent haplotypes includes the following steps:

[0088] (1) The reaction system is:

[0089] DNA template (30-50 ng / μL) 1 μL;

[0090] Left primer (10 μM) 0.5 μL;

[0091] Right primer (10 μM) 0.5 μL;

[0092] ddH2O 2μL;

[0093] 2×PCR Mix 5 μL.

[0094] (2) The PCR amplification procedures for dCAPS2-F1R1 and dCAPS3-F1R1 are as follows:

[0095] 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 at 72℃ for 5 min; Soak at 25℃.

[0096] (3) The PCR amplification procedure for dCAPS2-F2R2 is as follows:

[0097] 1 cycle: pre-denaturation at 95℃ for 3 min; 35 cycles: denaturation at 95℃ for 15 s, annealing at 58℃ for 20 s, extension at 72℃ for 15 s; Delay at 72℃ for 5 min; Soak at 25℃.

[0098] (4) The PCR amplification procedure for dCAPS3-F2R2 is as follows:

[0099] 1 cycle: pre-denaturation at 95℃ for 3 min; 35 cycles: denaturation at 95℃ for 15 s, annealing at 58℃ for 20 s, extension at 72℃ for 15 s; Delay at 72℃ for 5 min; Soak at 25℃.

[0100] The PCR products of dCAPS2-F2R2 and dCAPS2-F3R3 were cleaved with restriction endonucleases Sac I and Spe I was digested and then detected by 10% acrylamide gel electrophoresis. Hap1 and Hap2 were two higher bands; Hap7 had two lower bands; Hap3 and Hap4 were Sac I digested with enzyme to form a lower band. Spe I digestion showed higher bands, Hap5 and Hap6 were Sac I was a higher band after digestion. Spe I digestion results in a lower band (see Figure 6 ).

[0101] 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 application.

[0102] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the invention. Thus, if these modifications and variations to the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. The maize kernel development regulating gene whose CDS sequence is shown in SEQ ID NO.1 ZmRRP45B Application in the regulation of corn kernel size, corn embryo and / or endosperm developmental traits.

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