Corn kernel development regulation gene ZmRH51, encoding protein thereof, functional marker and application of corn kernel development regulation gene ZmRH51

Abstract

The invention discloses a corn kernel development regulation gene ZmRH51 as well as an encoding protein, a functional marker and an application of the corn kernel development regulation gene ZmRH51. According to the application, a corn kernel mutant m223 is taken as a basic material, heterozygote of the corn kernel mutant m223 is hybridized with Zheng 58 (Z58) to construct an F2 segregation population, then phenotype identification and cytological observation are performed on the segregation population of the offspring, the gene ZmRH51 for controlling the mutation phenotype of the m223 is obtained through genetic analysis, BSR-seq analysis and map-based cloning, the gene is positioned in a nucleolus region, and the mutation phenotype of the m223 is controlled. The ZmRH51 is used for coding RNA helicase of a DEAD-Box gene family, and research shows that ZmRH51 participates in ribosome assembly and can influence processing of pre-rRNA in corn kernels; the excellent allelic variation genotype of the excavated ZmRH51 gene in the aspect of grain thickness is obtained through further excavation, the grain thickness and hundred-grain weight of the ZmRH51 gene can be increased through the utilization of the gene, and then the yield is increased. In addition, distribution and utilization potentials of different haplotypes in a selfing line are researched, and technical support and excellent germplasm resources are provided for utilization of the 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 ZnRH51 , its encoded protein, functional markers and its applications. Background Art

[0002] Corn (Zea mays L.) belongs to the genus Zea of ​​the Poaceae family. It is native to the Americas. Since it was introduced to my country, it has played an important role in increasing my country's grain production. With the development of my country's economy and the continuous growth of its population, the contradiction between a large population and limited land has become more intense. It is very necessary to increase grain production per unit area. Relevant studies have shown that the number of ears per unit area, the number of grains per ear, and the weight of 100 grains are the factors that directly affect corn yield. The grain weight of corn is the most direct factor affecting corn yield. Corn kernels are also storage organs that provide nutrition for seed germination. Therefore, discovering genes related to corn kernel development can not only analyze the biological basis of kernel development, but also provide a theoretical basis and excellent alleles for high-yield breeding of corn.

[0003] The cloned grain mutant genes are mainly involved in the following biological processes: (1) The biological processes involved in PPR proteins affect the regulation of grain development. PPR proteins are divided into P class consisting of only 35 amino acid structural units (e.g. dek2, dek35, dek37, emp4, emp10 PPR proteins encoded by genes such as ) and other PLSs composed of longer (L) or shorter (S) structural units (such as dek39, emp7, emp17, emp18 (2) Genes that affect the metabolism and transport of sugar, starch, protein and other substances are involved in the regulation of corn grain development, such as Wx, Shrunken2, ZmMdh4, O1, O2, O5 and other genes; (3) genes related to the cell cycle are involved in the regulation of maize grain development, such as RBR family, Dek15、ZmRIBA1 In addition, there are genes related to ribosome biogenesis ZmUrb2 , ZmShrek1 , auxin-related genes dek18 , ZlUT Co-regulate grain development.

[0004] In plants, abnormal ribosome synthesis can lead to abnormal plant development. Normal ribosome synthesis is also important for the normal development of corn kernels. ZmUrb2The mutation of the gene leads to the obstruction of pre-rRNA processing in corn kernels, resulting in a significant lag in the development and filling of the mutant kernels, a smaller embryo, a reduced number of endosperm cells, and a significantly reduced plant height. The ZmShrek1 gene encodes a yeast PWP1 homologous protein WD40, which is mainly involved in the splicing of the pre-rRNA A3 site. The deletion of this site leads to a significant accumulation of 27SA2 pre-rRNA and a significant reduction in the synthesis of 5.8S and 25SrRNA, ultimately leading to phenotypes such as delayed embryo and endosperm development and a significant decrease in 100-grain weight in the mutant kernels.

[0005] 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

[0006] The inventors of this application used corn kernel mutants m223 The heterozygotes were crossed with Zheng 58 (Z58) to construct F2 segregation populations. The offspring segregation populations were phenotypically identified and cytologically observed. The control was obtained through genetic analysis, BSR-seq analysis combined with positional cloning. m223 Genes with mutant phenotypes ZnRH51 , which is located in the nucleolus region and encodes an RNA helicase of the DEAD-Box gene family. Studies have shown that ZnRH51 It is involved in the assembly of ribosomes and can affect the processing of pre-rRNA in corn kernels. 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.

[0007] In the first aspect disclosed in the present application, phenotypic and cytological analysis was performed to find m223 The thickness of the mutant kernels decreased by 28%, and the 100-kernel weight decreased by 44%. The embryonic development of the mutant kernels was abnormal, and the stem apical meristem and root meristem could not be differentiated. The endoproliferation of the endosperm basal transfer layer was reduced. Genetic analysis and positional cloning methods were used to obtain the genes that affect the development of corn kernels. ZnRH51 The gene, whose DNA sequence is shown in SEQ ID NO.1, and whose cDNA sequence is shown in SEQ ID NO.2.

[0008] The second aspect disclosed in the present application relates to a grain development regulating gene ZnRH51 The encoded protein has an amino acid sequence as shown in SEQ ID NO.4.

[0009] In a third aspect disclosed in the present application, the corn kernel development regulating gene ZnRH51 Or the protein encoded by the same is used in the preparation of reagents for regulating the kernel thickness trait and / or the 100-grain weight trait of corn kernels.

[0010] In a fourth aspect disclosed in the present application, the corn kernel development regulating gene ZnRH51 Or the protein encoded by the same is used in preparing reagents for regulating corn ribosome synthesis and / or cell cycle.

[0011] based on ZnRH51 The three SNPs in the gene segment that are significantly associated with grain thickness, chr5.s_3927416, chr5.s_3930501, and chr5.s_3930830, can be used to design corresponding molecular markers to identify genotypes and select favorable haplotypes with excellent performance in 100-grain weight and grain thickness.

[0012] 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: (1) Detection and determination of corn strain materials ZnRH51 Gene B73V4: genotypes of chr5.s_3927416, chr5.s_3930501, chr5.s_3930830; (2) Select corn varieties with G, T, and T genotypes at the chr5.s_3927416, chr5.s_3930501, and chr5.s_3930830 loci, respectively, for B73V4, and self-pollinate or hybridize to select favorable (high 100-grain weight, thick grain) haploid varieties.

[0013] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: Positional cloning ZnRH51 The gene is located in the nucleolus region and encodes an RNA helicase of the DEAD-Box gene family, which is involved in the processing of pre-rRNA in plants; interaction protein analysis shows that ZmRH51 interacts with three ribosome assembly factors ZmNop15, ZmRlp7p-1, and ZmRlp7p-2 in the nucleolus, indicating that ZmRH51 is involved in the assembly of ribosomes; studies have shown that ZnRH51 Gene mutations affect the transcription of genes related to ribosome generation and cell cycle, and further exploration revealed ZnRH51 The excellent allele variant genotype of the gene in terms of grain thickness can be used for corn variety breeding, which is helpful to improve its grain thickness and 100-grain weight, thereby increasing yield. The analysis of the function of this gene provides a theoretical basis for studying the regulation of grain development by RNA helicase in corn through participating in ribosome biosynthesis, which is of great significance for increasing corn yield and ensuring food production safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is one of the comparisons between wild-type grains and mutant grains in one embodiment of the present application; A: m223 The original material separated ears, the red arrows indicate the mutants (the same below), the scale bar is 1 cm; B: Z58× m223 F3 separated ear, scale bar is 1 cm; C: Comparison of grain length, width and thickness between wild-type and mutant grains, scale bar is 1 cm; D: Comparison of measurement data of grain length, width and thickness between wild-type and mutant grains; E: Comparison of 100-grain weight between wild-type and mutant grains; Note: All graphs with error bars are mean values ​​plus / minus standard errors, ** indicates that wild-type and mutant m223 The significance between them was P < 0.01, *** indicates wild type and mutant m223 The significance between the two groups was P < 0.001 (Student's t test).

[0015] Figure 2 This is the second comparison between the wild-type grain and the mutant grain in an embodiment of the present application; in the figure, A: the wild-type and mutant grains on the light box m223 Comparison; B: Wild-type grains and mutant grains m223 Longitudinal section, En is endosperm, Em is embryo, scale bar is 2 mm; C: wild-type grains and mutant grains m223 Comparison of germination ability after 4 days of dark culture; scale bar is 1 cm; D: Dynamic development of grains on F4 ears. Scale bar is 2 cm; The red arrow points to the mutant.

[0016] Figure 3 The wild type and mutant at different days after pollination in an embodiment of this application m223 Paraffin section observation; the first row shows complete kernels at different days after pollination, with a scale of 1000m; the second row shows the development of embryos at different days after pollination, with a scale of 200m; the third row shows the development of the base endosperm transfer layer, with a scale of 100m.

[0017] Figure 4 This is the BSR-Seq result analysis in one embodiment of the present application.

[0018] Figure 5 In one embodiment of this application m223 Positional cloning diagram.

[0019] Figure 6 In one embodiment of this application m223 Mutants Mu Insertion site.

[0020] Figure 7 In one embodiment of this application ZnRH51 Gene expression analysis; A: Kernels and different tissues at different developmental stages of maize ZnRH51qRT-PCR analysis of gene expression; B: 10 DAP, 13 DAP, ZnRH51 Gene expression qRT-PCR analysis; ZmActin was used as an internal reference.

[0021] Figure 8 In one embodiment of this application ZnRH51 Subcellular localization of genes, scale bar is 20 μm.

[0022] Fig. 9 Analysis and verification of ZmRH51 interacting proteins in one embodiment of the present application; A: Yeast two-hybrid experiment shows that ZmRH51 interacts with ZmNop15, ZmRlp7p-1, and ZmRlp7p-2; B: BLuc shows that ZmRH51 interacts with ZmNop15, ZmRlp7p-1, and ZmRlp7p-2 (the fluorescence signal intensity represents the interaction affinity between them); C: BiFC shows that ZmRH51 interacts with ZmNop15, ZmRlp7p-1, and ZmRlp7p-2.

[0023] Fig.10 In one embodiment of this application, RT-qPCR analysis Zmrh51 Relative rRNA level detection of mutants and WT; A: 45S rDNA structure diagram (Liu et al; 2022); B: WT and Zmrh51 The ratio is 1, which is indicated by a dotted line. Total RNA was extracted from 13 DAP kernels. ZmActin2 was used as an internal reference; Values ​​are mean ± SD (n = 3 biologically independent samples); **** p < 0.0001; *** p 0.001; ** p< 0.01; Ns, no significant difference; t test.

[0024] Fig.11 This is one of the GO enrichment analysis maps in one embodiment of the present application; A: scatter plot of biological process items; B: scatter plot of cellular component items; C: scatter plot of molecular function items.

[0025] Fig.12 This is the second GO enrichment analysis map in the first embodiment of this application; D: RT-qPCR analysis to verify RNA-seq data; WT and m223 Total RNA was extracted from 10DAP grains of Rhizopus edulis; ZmActin2 was used as an internal reference; Values ​​are mean ± SD (n = 3 biologically independent samples); *** p < 0.001; ** p < 0.01; * p < 0.05.

[0026] Fig.13 This is a KEGG pathway analysis in one embodiment of the present application.

[0027] Fig.14 In one embodiment of this application ZnRH51 Haplotype analysis of genes; A: ZnRH51 Five haplotypes composed of SNPs with significant differences in grain thickness; B: Statistical analysis of grain thickness of five haplotypes; C: Statistical analysis of 100-grain weight of five haplotypes. The same lowercase letters on the bar graph indicate no significant difference at the p<0.05 level, and different lowercase letters indicate significant difference at the p<0.05 level. DETAILED DESCRIPTION

[0028] The main sources of experimental materials involved in the following examples are: m223 The mutants were derived from the UniformMu mutant library of maize. The F2 segregating population was a hybrid of the high-quality inbred line Z58 maintained in the laboratory and m223 The F1 population was formed in the South Breeding Base of Henan Agricultural University in Sanya, Hainan in the winter of 2020. m223 The mutant cannot grow normally. Therefore, when assembling the F1 population, the normal grains in the original material separated ears are self-pollinated and hybridized with Z58. The hybrid plant corresponding to the heterozygous genotype is the F1 ear. The F2 segregating population is formed by self-pollination of the F1 ears at the Yuanyang Base of Henan Agricultural University in Yuanyang, Henan in the spring of 2021. The specific assembly route is as follows: .

[0029] The maize genome was extracted using the SLS method; TransZol Plant polysaccharide and polyphenol RNA extraction kit (Beijing Quanshijin Company) was used to extract corn kernel RNA; the RNA reverse transcription kit was the All-in-One First-Strand Synthesis MasterMix (with dsDNase) kit produced by Kemix; the construction of yeast two-hybrid, subcellular localization, BLUC, BiFc and other experimental vectors was constructed by homologous recombination method using Fusion plus rapid homologous recombination premix produced by Kemix; other reagents and materials involved were conventional products on the market unless otherwise specified; the experiments and detection methods involved (such as RT-PCR, corn kernel paraffin sections, etc.) were conventional methods unless otherwise specified; the instruments and equipment involved were conventional instruments and equipment unless otherwise specified.

[0030] Embodiment 1: m223 Phenotypic analysis of mutants m223The mutant originated from the UniformMu mutant pool of maize (W22 genetic background), and the ears of the heterozygous plants showed grain size segregation after self-pollination ( Figure 1 A), after being matched with Z58 background, the self-pollinated ears of heterozygous individual plants also showed stable grain size segregation, and all of them were in line with the theoretical ratio of 3:1 (Table 1). Figure 1 B, C, F3 separated ears were taken for phenotypic statistics and found that compared with the wild type, m223 The mutant kernels showed obvious top wrinkling and thinning phenotypes on the ears. After comparing the kernel length, kernel width and kernel thickness, it was found that there was no significant difference in kernel length and kernel width, while the kernel thickness decreased significantly. The kernel length, kernel width and kernel thickness of the wild type and mutant were measured respectively. Figure 1 D, found in m223 The mutant had a 28% decrease in grain thickness. The 100-grain weight of the wild-type and mutant grains was randomly selected and measured, and it was found that the 100-grain weight of the mutant grains decreased by 44% ( Figure 1 E).

[0031] The seeds were placed on a light box for seed transmittance analysis and it was found that m223 The transmittance of the mutant was slightly reduced ( Figure 2 A); Further observation revealed that compared with the wild type, the longitudinal section area of ​​the hard endosperm of the mutant grains was significantly reduced, the proportion of powdery endosperm was significantly increased, and most of the grains had embryonic abortion and developmental abnormalities ( Figure 2 B); The mutant seeds with embryos were subjected to germination tests and found that their embryos could not develop normally and could not grow roots and buds ( Figure 2 C). To explore m223 The appearance time of mutant phenotype was determined by selecting ears of F4 at different stages after pollination for observation, such as Figure 2 D. At 10 days after pollination (10 DAP), the mutant grain phenotype began to appear, showing slightly smaller grains; at 13 DAP, the phenotypic difference between the wild type and the mutant became significantly larger. At this time, the wild type grains had turned significantly yellow and the grains were plump, while the mutant grains were sluggish and still whitish, and the tops of some grains began to collapse; in the subsequent 15 DAP, 18 DAP, and 20 DAP periods, it was found that the mutant grains were significantly smaller than the wild type, and the grain color was still mainly white and transparent.

[0032] To further explore the cytological differences between wild-type and mutant grains, paraffin sections of grains 10 and 15 days after pollination were observed. Figure 3As shown in the figure: At 10 DAP, the endosperm of wild-type grains was full and the embryo had visible scutellum and leaf primordium; while the top of the mutant grains was obviously collapsed, there was a certain gap between the endosperm and the seed coat, and the embryo was malformed and no differentiation was observed. Compared with the wild-type grains, the intracellular proliferation of the basal transfer layer of the endosperm of the mutant grains was not obvious. At 15 DAP, the scutellum, leaf primordium, stem apical meristem, root apical meristem and hypocotyl structure of the wild-type grain embryo were clearly visible, the top cells of the wild-type grains were denser, and the starch granules were more obvious; while the embryo of the mutant grains was similar to that at 10 DAP, that is, the embryo development stopped.

[0033] Embodiment 2: ZnRH51 Gene cloning 1. m223 Genetic analysis and BSR-seq analysis Randomly select 3 ears with good pollination m223 The number of wild-type and mutant grains was counted in the F4 segregation population constructed with Z58. The results are shown in Table 1. It was found that the ratio of the number of wild-type grains to the number of mutant grains conformed to the Mendelian segregation ratio of 3:1. The results showed that the control m223 The gene for grain traits is a single recessive nuclear gene.

[0034] Table 1 Chi-square test of wild-type and mutant kernels on F4 segregating ears .

[0035] For initial positioning control m223 The location of the mutant kernel gene on the chromosome was analyzed by BSR-Seq. Wild-type kernels at 15 DAP and m223 Each mutant had 36 seeds. TransZol Plant polysaccharide and polyphenol extraction kit was used to extract total RNA from seeds. The RNA of wild-type seeds and m223 Mutant grain RNA was mixed into WT RNA pool in equal amounts. m223 The RNA pool was sent to Beijing Berry Genomics Co., Ltd. for sequencing. Then, the BSR-Seq analysis method developed by Zou et al. was used to detect 48,715 SNP sites. After filtering and screening, 1,702 linked SNP sites were obtained. These SNP sites were mainly enriched between 0.8Mb and 8.0Mb on chromosome 5 ( Figure 4 ).

[0036] 2. m223 Fine positioning To further narrow the candidate interval, 50 pairs of Indel markers were developed in this interval, and the BSA pooling method was used to screen the linkage markers. Five pairs of linkage markers, 5-S08658, 5-S08666, 5-S08668, 5-S08692, and 5-S08693, were screened. The candidate interval was determined to be between 3.58Mb and 4.43Mb using 100 F4 mutant seeds. Subsequently, three more pairs of linkage markers were screened, and a total of 865 plants were used to finally determine the candidate interval to be between 3.9Mb and 4.2Mb, with a physical distance of approximately 352kb.

[0037] 3. Analysis and verification of candidate genes After querying the Gramene website (https: / / ensembl.gramene.org / Zea_mays / ), it is known that there are 11 protein-coding genes in the B73 V4 reference genome within the 3.9Mb-4.2Mb physical interval ( Figure 5 ). Based on the expression changes of these 11 genes, 9 significantly differentially expressed genes were screened, and the insertion of Mutator transposon in each gene was analyzed using the primer combination of each candidate gene and Mu8. Finally, primers 1F and 1R, 1R and Tir67 were used to detect Zm00001d013056 There is a Mutator transposon inserted 485 bp downstream of the gene start codon (B73 V4 Chr5:3927312) Figure 6 B) and is linked to the mutant kernel phenotype.

[0038] To further determine m223 The phenotype of the mutant kernels is indeed caused by Zm00001d013056 EMS mutants caused by transposon insertion of genes were purchased from the MEMD mutant library: EMS4-121b11 , the mutant Zm00001d013056 There is a mutation from C to T at 1397 bp downstream of the gene start codon, the codon changes from CGA to UGA, and the encoded amino acid changes from Arg to the stop codon, causing the gene to terminate prematurely. EMS seeds were amplified using 2F and 2R, and the amplified products were sequenced. Heterozygous seeds were selected for planting, self-pollination and cross-pollination were performed simultaneously. m223 The hybrid seeds of the materials were hybridized. The results showed that EMS4-121b11 After self-pollination of heterozygotes, the phenotype of grain size segregation appeared on the ears, and the segregation ratio was consistent with that of wild type: mutant = 3:1; m223 The hybrid ears of heterozygotes also showed a wild-type: mutant = 3:1 grain size phenotype. Three mutant grains were selected from each of the three hybrid separated ears. Further testing of the mutants in the hybrid separated ears revealed that Zm00001d013056There is a heterozygous Mutator transposon insertion 485 bp downstream of the gene start codon, and a heterozygous mutation from C to T 1397 bp downstream of the start codon. m223 The gene responsible for the mutant kernel phenotype is Zm00001d013056 .

[0039] Embodiment three: ZnRH51 Gene-encoded protein sequence analysis and evolutionary analysis 1. ZnRH51 The gene encodes a protein containing a DEAD-Box domain Zm00001d013056 Gene B73 reference genome version 4 has 5 transcripts. According to the results of BSR-Seq and cDNA amplification, m223 Only the T002 transcript was detected in the material. Zm00001d013056 The CDS sequence of the gene T002 transcript is 1950 bp long (as shown in SEQ ID NO.3), encoding a protein composed of 649 amino acids (as shown in SEQ ID NO.4). The protein domain was predicted using the InterPro website (https: / / www.ebi.ac.uk / interpro / ), and the results showed that the protein contained a DEAD / DEAH box helicase domain, that is, the gene encodes an RNA helicase of the DEAD-Box family. Zm00001d013056 Gene domains and gene annotations will Zm00001d013056 Gene named ZnRH51 .

[0040] 2. ZnRH51 Evolutionary analysis of genes Utilize the OrthoDB website (https: / / www.orthodb.org / ) ZnRH51 The amino acid sequence of the gene was used to predict homologous proteins in Arabidopsis, yeast, and rice, and it was found that there were two highly homologous genes in Arabidopsis. AtRH51 and AtRH27 , the homologous gene in rice is OsRH27 , the homologous gene in yeast is HAS1 . We further used MEGA11 software and GeneDoc software to analyze ZnRH51Comparison of the protein sequences encoded by the three homologous genes revealed that in the two domains unique to DEAD-Box RNA helicase, the four proteins showed high consistency in nine very conserved motifs, indicating that the four proteins may have similar functions. However, compared with Arabidopsis and rice, yeast HAS1 and ZmRH51 differed greatly, and rice OsRH27 had the highest homology with ZmRH51.

[0041] In order to analyze the relationship between ZmRH51 and homologous proteins in other species, we first used BLASP in NCBI to search for homologous sequences of ZmRH51 in 20 species such as sorghum, wheat, rye, and Arabidopsis, and then used the neighbor-joining method (NJ: neighbor-joining method) in MEGA11 software to perform phylogenetic tree analysis on homologous proteins in each species. The results showed that among common species, ZmRH51 in maize was more closely related to homologous proteins in sorghum, followed by homologous proteins in rice, and had greater genetic differences with homologous proteins in Arabidopsis, and the relationship was also more distant.

[0042] Embodiment 4: ZnRH51 Spatiotemporal gene expression analysis To explore ZnRH51 The expression of genes in various tissues of maize was studied by selecting different tissues such as roots, stems, leaves, tassels, and ears of inbred line W22 as well as endosperms at different stages after pollination. ZmRH51-qRT-F / R was used as amplification primer, ZmActin2 was used as internal reference, and 2 -ΔΔCt To calculate the relative expression level for analysis ZnRH51 The expression of genes in various tissues, the results showed ZnRH51 The gene is constitutively expressed. In comparison, the expression level in the grain is lower, and with the increase of pollination days, ZnRH51 The expression level of the gene first decreased and then increased, but the expression level was higher in the filaments, roots, and tassels, indicating that the gene plays an important role in the entire development of the corn plant.

[0043] In order to investigate the ZnRH51 The transcription and expression of genes were analyzed by RT-qPCR after extracting RNA from wild-type and mutant grains at 10 DAP and 13 DAP and reverse transcribed into cDNA. ZnRH51 The expression of the gene was found to be higher in the wild type than in the mutant at 10 DAP and 13 DAP. ZnRH51 The gene expression level gradually decreased, but the difference between the mutant and the wild type gradually increased ( Figure 7 B).

[0044] Example 5: Subcellular localization of ZmRH51 The expression site of ZmRH51 in cells was predicted by Gramene (http: / / ensembl.gramene.org / Zea_mays), and the results showed that ZmRH51 is a protein localized in the cell nucleus. ZnRH51 The gene was constructed into a PRTL vector containing the reporter gene eGFP sequence to express the fusion protein ZmRH51:GFP. Under the drive of the 35S promoter, the OsGHD7:RFP fusion protein was used as a nuclear marker and co-transfected into maize protoplasts for subcellular localization analysis. The results showed that the red fluorescence signal of OsGHD7:RFP covered the fluorescence signal of ZmRH51:GFP, indicating that ZmRH51 was expressed in the nucleus, and the expression site was speculated to be the nucleolus. In order to verify this speculation, the AtHDT1:RFP fusion protein was used as a nuclear nucleolus marker and co-expressed with ZmRH51:eGFP in maize protoplasts; the results showed (see Figure 8 ) The green fluorescence signal of ZmRH51:eGFP fusion protein can overlap with the red fluorescence signal of AtHDT1:RFP fusion protein, indicating that ZmRH51 is a protein localized in the nucleolus.

[0045] Embodiment six: ZnRH51 Analysis and verification of interacting proteins Nop15 and Rlp7p are two assembly factors of the 60S large subunit of the ribosome. HAS1 (homologous protein of ZmRH51) in yeast interacts with Nop15 and Rlp7p and participates in the assembly of the 60S large subunit of the ribosome. In order to verify whether ZmRH51 is involved in the biosynthesis of ribosomes in maize, the corresponding homologous genes of Nop15 and Rlp7 in maize (ZmNop15: Zm00001d050300, ZmRlp7p-1: Zm00001d026254 and ZmRlp7p-2: Zm00001d031703) were cloned, the corresponding protein interaction vectors were constructed, and the interaction was verified by yeast two-hybrid, Bluc, and BiFc experiments (see Fig. 9 ).

[0046] First, ZnRH51The CDS sequence of the gene was connected to the pGBKT7 (BD) vector, and the CDS sequences of the three candidate interacting genes were connected to the pGADT7 (AD) vector. Finally, BD-ZmRH51 and the three genes connected to the AD vector were co-transformed into Y2H yeast competent cells for point-to-point verification. The results showed that the three combinations of AD-ZmRlp7-1+BD-ZmRH51, AD-ZmRlp7p-2+BD-ZmRH51, and AD-ZmNop15+BD-ZmRH51 could all grow in SD-Trp / -Leu / -His medium, indicating that the three proteins interacted with ZmRH51, but it was found that the interaction between ZmNop15 and ZmRH51 was weak.

[0047] To verify the authenticity of the interaction between these three genes in yeast, a luciferase complementation experiment was performed. ZnRH51 The CDS sequence of the gene was inserted into the N-Luc vector, and the CDS of the three candidate interacting genes were inserted into the C-Luc vector. Subsequently, the N-Luc vector and C-Luc vector fused with the CDS sequences of different genes were transformed into the Agrobacterium tumefaciens GV3101 strain for culture and the bacterial solution was injected into tobacco for luciferase complementation verification. The results also showed that the three proteins interacted with ZmRH51.

[0048] In addition, ZnRH51 The CDS sequence of the gene was inserted into the PXY104-cYFP vector, and the CDS sequences of the three candidate interacting genes were inserted into the PXY106-nYFP vector for bimolecular fluorescence complementation experiments. The final results were consistent with the results of the yeast two-hybrid experiment and the BLuc experiment. At the same time, in order to explore the location of the interaction between ZmRH51 and the three interacting proteins in the cell, a nucleolus-specific expression gene fused with the RFP reporter gene was used. AtHDT1 The gene was used as a nucleolus marker and was transferred into tobacco cells together with PXY104-cYFP vector and PXY106-nYFP vector with different CDS sequences for expression. The results showed that ZmRH5 interacted with the three proteins in the nucleolus.

[0049] The above three experiments show that ZmNop15, ZmRlp7p-1, and ZmRlp7p-2 all interact with ZmRH51 in the nucleolus, and are very likely to participate in the assembly of ribosomes, just like in yeast.

[0050] Embodiment seven: ZnRH51 Effects of Gene Mutation on Maize Pre-rRNA Ribosome assembly begins with 45S pre-rRNA transcribed by RNA polymerase I in eukaryotes. RT-qPCR was used to detect the accumulation of various intermediates in the pre-rRNA processing in the wild type and mutants to analyze whether ZmRH51 affects the processing of pre-rRNA. The 45S pre-rRNA is transcribed as a large precursor composed of 18S, 5.8S and 25S rRNAs, separated by ITS1 and ITS2, and flanked by 5' ETS and 3' ETS. 45S pre-rRNA, various intermediates and total rRNA were detected by specific primers. The results showed that the signals amplified by the primer combination located in the P-P', ITS1 and ITS2 segments were significantly accumulated in the mutant, and the detection signals in the 18S, 5.8S and 25S segments were significantly reduced ( Fig.10 ), which indicates that the processing of pre-rRNA is affected in the mutant.

[0051] Embodiment eight: m223 Transcriptome analysis of mutants 1. Differentially expressed gene analysis: To study ZnRH51 The biological functions of genes in seed development were compared by RNA sequencing between WT and m223 Transcriptome data (RNA-Seq) of mutant grains. Among the 29,226 genes identified by RNA-seq, q<0.01, fold change>2 were used as screening conditions for significantly differentially expressed genes, and a total of 6,489 significantly differentially expressed genes (DEGs) were detected, of which 3,295 genes were m223 The expression levels of 3194 genes were up-regulated in the mutant grains, while the expression levels of 3194 genes were down-regulated.

[0052] 2. GO enrichment analysis: GO (Gene Ontology) analysis showed that these differentially expressed genes were enriched in 178 items (q<0.01). In biological process, rRNA metabolism (GO:0016072, q=9.4765E-8), rRNA processing (GO:0006364, q=9.4765E-8), ribosome biogenesis (GO:0042254, q=1.0338E-8), cell cycle progression (GO:0022402, q=3.83368E-8) were highly significant; in molecular function, ATPase activity (GO:0015631, q=9.45E-5) and ATP-dependent helicase activity (GO:0008026, q=8.12E-4) were highly significant; in cellular component ... metabolism (GO:0006364, q=9.4765E-8), ribosome biogenesis (GO:0042254, q=1.0338E-8), cell cycle progression (GO:0022402, q= Component), the items without membrane in the cell (GO:0043228, GO:0043232, q=2.67E-10, q=2.67E-10), kinesin complex (GO:0005871, q=1.35E-8), and nucleolus (GO:0005730, q=1.93E-6) were the most significant ( Fig.11 ). To validate the differentially expressed genes detected by RNA-seq, RT-qPCR was performed on 14 genes from ribosome biogenesis entries, and the results were basically consistent with the RNA-seq data ( Fig.12 ).

[0053] 3. KEGG enrichment analysis: Among the metabolic pathways enriched by KEGG, eukaryotic ribosome biosynthesis was the most significant (zma03008, p=1.6E-05), and pathways such as phenylpropanoid synthesis, plant hormone signal transduction, and aminoacyl-tRNA biosynthesis were also significantly enriched ( Fig.13 A); At the same time, the metabolic pathway of sugar and starch was also enriched, so the expression of related genes in this pathway was analyzed, and it was found that in this pathway, a total of 61 significantly differentially expressed genes were enriched, of which 23 genes were upregulated and 38 genes were downregulated ( Fig.13 B).

[0054] The above results show that ZnRH51 Mutations in the gene will affect the biosynthesis of ribosomes in plants. As a member of the DEAD-Box RNA helicase family, mutations in the gene encoding ZmRH51 will also affect the expression of related genes encoding ATPase and ATP-dependent helicase. At the same time, defects in this gene will also lead to the normal synthesis of substances such as sugar and starch in the grains.

[0055] Embodiment nine: ZnRH51 Examples of how genes are used in corn kernel development ZnRH51 Gene mutation causes corn kernel thickness to decrease significantly, in order to mine ZnRH51 The excellent allele variation of the gene in kernel thickness was studied by selecting 397 corn natural population materials with broad representation and combining them with ZnRH51 SNP information within the gene segment is used to analyze the grain thickness trait ZnRH51 Candidate gene association analysis. Among the 397 natural maize population materials used in this study, ZnRH51 There are 76 SNPs in the gene segment. With p < 0.01 as the threshold, 3 SNPs were screened out that were significantly associated with grain thickness, chr5.s_3927416 (p = 0.67 × 10 -2 ), chr5.s_3930501(p=0.33×10 -2 ), chr5.s_3930830(p=0.16×10 -2 ). Among them, SNP3927416 is located in the 1st intron, SNP3930501 is located in the 9th intron, and SNP3930830 is located in the 11th intron. Based on these three significant SNPs, 397 natural populations can be divided into 5 haplotypes (Hap) ( Fig.14 A). There is no significant difference in grain thickness between Hap1, Hap2, Hap3, Hap4 and Hap5. There is no significant difference in grain thickness between Hap1 and Hap2, but the grain thickness of both Hap1 and Hap2 is significantly thicker than that of Hap3 and Hap4, which is 3.25% and 6.03% thicker than that of Hap3 and Hap4 ( Fig.14 B).

[0056] In order to analyze whether the change in grain thickness among the five haplotypes would affect the 100-grain weight, statistical analysis of the 100-grain weight of the five haplotype materials was performed. The results showed that Hap2 was a favorable haplotype that also performed well in 100-grain weight. Compared with Hap3 and Hap5, Hap2 increased 100-grain weight by 3.89% and 11.64%, respectively ( Fig.14 C).

[0057] The above results show that ZnRH51 The Hap2 haplotype of the gene is a favorable haplotype that performs well in both kernel thickness and 100-kernel weight. ZnRH51 The excellent haplotype material of the Hap2 gene helps to increase its grain thickness and 100-grain weight, thereby increasing yield.

[0058] 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.

Claims

1. A gene regulating corn kernel development ZnRH51 , whose DNA sequence is shown in SEQ ID NO.

1.

2. The corn kernel development regulating gene according to claim 1 ZnRH51 The encoded protein has an amino acid sequence as shown in SEQ ID NO.

4.

3. The corn kernel development regulating gene according to claim 1 ZnRH51 Or the use of the encoded protein according to claim 2 in the preparation of a reagent for regulating the kernel thickness trait and / or the 100-kernel weight trait of corn seeds.

4. The corn kernel development regulating gene according to claim 1 ZnRH51 Or the encoded protein according to claim 2 is used in the preparation of a reagent for regulating corn ribosome synthesis and / or cell cycle.

5. A SNP molecular marker, based on the fourth version of the corn B73 reference genome, has a G / A variation at the chr5.s_3927416 site, a C / T variation at the chr5.s_3930501 site, and a C / T variation at the chr5.s_3930830 site.

6. The corn kernel development regulating gene according to claim 1 ZnRH51 Or the use of the SNP molecular marker described in claim 5 in breeding varieties / lines related to corn kernel thickness traits and / or 100-grain weight traits.

7. A corn breeding method, characterized in that: The steps include: (1) Detection and determination of corn strain materials ZnRH51 Gene B73V4: genotypes of chr5.s_3927416, chr5.s_3930501, chr5.s_3930830; (2) Select corn varieties with genotypes of G, T, and T at loci B73V4, chr5.s_3927416, chr5.s_3930501, and chr5.s_3930830, respectively, and self-pollinate or hybridize to select favorable haploid varieties.

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