Maize kernel development regulatory gene zmRH51, encoded protein, functional marker and application thereof

Abstract

This invention application discloses a gene regulating maize kernel development. ZmRH51 This application focuses on the encoded proteins, functional markers, and applications of maize kernel mutants. m223 Using this as the basic material, its heterozygotes were crossed with Zheng 58 (Z58) to construct the F2 segregating population. Phenotypic identification and cytological observation were performed on the progeny segregating population. Controlling populations were obtained through genetic analysis, BSR-seq analysis, and map-based cloning. m223 Genes with mutant phenotypes ZmRH51 This gene is located in the nucleolar region and encodes an RNA helicase belonging to the DEAD-Box gene family. Studies have shown that... ZmRH51 It participates in ribosome assembly and can influence the processing of pre-rRNA in maize kernels; and further mining yields... ZmRH51 The superior allelic variant genotype of this gene in terms of grain thickness can be utilized to improve grain thickness and 100-grain weight, thereby increasing yield. Furthermore, 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.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological genetic engineering, and particularly relates to a maize kernel development regulation gene ZmRH51 , an encoded protein thereof, a functional marker and application thereof. BACKGROUND

[0002] Maize (Zea mays L.) belongs to the plant of Poaceae and Zea, and is originally from America. Since being introduced into China, maize has played an important role in improving the grain yield of China. With the development of economy and the continuous growth of population in China, the contradiction between more population and less land is more intense, and it is necessary to improve the grain yield per unit area. Related researches show that the number of ears per unit area, the number of grains per ear and the weight of 100 grains are direct factors affecting the yield of maize, and the weight of maize grain is the most direct factor affecting the yield of maize, and the maize grain is also a storage organ to provide nutrition for seed germination. Therefore, exploring the maize kernel development related genes can not only analyze the biological basis of kernel development, but also provide a theoretical basis and excellent alleles for high-yield breeding of maize.

[0003] At present, the cloned kernel mutant genes mainly involve the following biological processes: (1) the biological process involving PPR protein affects the regulation of kernel development, the PPR protein is divided into P class (such as PPR protein encoded by dek2, dek35, dek37, emp4, emp10 gene) composed of only 35 amino acid structural units and PLS class (such as dek39, emp7, emp17, emp18 gene) containing longer (L) or shorter (S) structural units; (2) the genes affecting the metabolism and transportation of substances such as sugar, starch and protein participate in the regulation of maize kernel development, such as Wx, Shrunken2, ZmMdh4, 01, 02, 05 gene; (3) the genes related to cell cycle participate in the regulation of maize kernel development, such as RBR family, Dek15, ZmRIBA1 gene. In addition, ribosome biosynthesis related genes ZmUrb2 , ZmShrek1 auxin related genes dek18 , ZmEHD1 also jointly regulate the kernel development.

[0004] In plants, abnormal ribosome synthesis will cause the plant body to be unable to develop normally. Normal synthesis of ribosomes is also important for the normal development of maize kernels, ZmUrb2The mutation of the gene causes the pre-rRNA processing in the corn kernel to be blocked, so that the mutant kernel development and plumping are obviously delayed, the embryo is small, the endosperm cell number is reduced, and the plant height is obviously reduced; the ZmShrek1 gene encodes a yeast PWP1 homologous protein WD40, the gene is mainly involved in the cutting of the pre-rRNA A3 site, the deletion of the site causes the 27SA2 pre-rRNA to be obviously accumulated, the synthesis of 5.8S and 25S rRNA is obviously reduced, and finally causes the mutant kernel to have the phenotypes of delayed embryo and endosperm development, and significantly reduced hundred-grain weight.

[0005] The information disclosed in this section is only for the purpose of enhancing the understanding of the background of the present disclosure and should not be considered as admitting or implying in any form that this information constitutes the prior art known to those skilled in the art. SUMMARY

[0006] The present application is based on a corn kernel mutant m223 , and a hybrid of the heterozygote and Zheng 58 (Z58) is used to construct an F2 separation population, then the offspring separation population is subjected to phenotype identification and cytological observation, and a gene controlling the mutant phenotype is obtained through genetic analysis, BSR-seq analysis and map-based cloning. m223 ZmRH51 The gene is located in the nucleolus region, and it encodes an RNA helicase of the DEAD-Box gene family, researches show that ZmRH51 the gene is involved in ribosome assembly and can affect the pre-rRNA processing in the corn kernel; in addition, the distribution and utilization potential of different haplotypes in inbred lines are studied, which provides technical support and excellent germplasm resources for the utilization of the gene.

[0007] The first aspect of the present application is that, through phenotype and cytological analysis, it is found that m223 the kernel thickness of the mutant kernel is reduced by 28%, the hundred-grain weight is reduced by 44%, the embryo development of the mutant kernel is abnormal, the stem tip meristem and root meristem cannot be differentiated, and the endosperm basal transfer layer has reduced endosmosis; a gene affecting the corn kernel development is obtained through genetic analysis and map-based cloning, the DNA sequence of the gene is shown as SEQ ID NO. 1, and the cDNA sequence of the gene is shown as SEQ ID NO. 2. ZmRH51

[0008] The second aspect of the present application relates to a protein encoded by a kernel development regulation gene ZmRH51 , and the amino acid sequence of the protein is shown as SEQ ID NO. 4.

[0009] The third aspect of the present application is that the corn kernel development regulation gene ZmRH51 or the protein encoded by the gene is applied in the preparation of a reagent for regulating the kernel thickness trait or / and the hundred-grain weight trait of the corn kernel.​​

[0010] In a fourth aspect of the present disclosure, the corn kernel development regulatory gene ZmRH51 or its encoded protein is used in the preparation of a reagent for regulating corn ribosome synthesis or / and cell cycle.

[0011] Based on ZmRH51 Based on the three SNPs in the gene segment, chr5.s_3927416, chr5.s_3930501, and chr5.s_3930830, which are significantly associated with kernel thickness, corresponding molecular markers can be designed to identify genotypes for selecting advantageous haplotypes with excellent performance in hundred kernel weight and kernel thickness.

[0012] In another aspect of the present disclosure, a method for selecting a corn kernel hundred kernel weight excellent haplotype is provided, comprising the following steps:

[0013] (1) detecting and determining the ZmRH51 genotype of the gene B73V4: chr5.s_3927416, chr5.s_3930501, and chr5.s_3930830 of the corn line material;

[0014] (2) selecting corn line materials with genotypes of G, T, and T at the B73V4: chr5.s_3927416, chr5.s_3930501, and chr5.s_3930830 sites, respectively, and selfing or crossing to select advantageous (high hundred kernel weight and thick kernel) haplotype lines.

[0015] The one or more technical solutions provided in the embodiments of the present disclosure have at least any of the following technical effects or advantages:

[0016] The ZmRH51 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 ribosome assembly. ZmRH51 The mutation of the gene affects the transcription of related genes such as ribosome generation and cell cycle, and further mining ZmRH51 excellent allelic genotypes of the gene in kernel thickness, which are used for corn variety breeding, help to improve kernel thickness and hundred kernel weight, and further improve yield. Analyzing the function of the gene provides a theoretical basis for studying the regulation of kernel development by RNA helicases through ribosome biosynthesis in corn, which has important significance for improving corn yield and ensuring food production safety. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 This is one example of a comparison between wild-type seeds and mutant seeds in an embodiment of this application; A: m223 The original material was separated into ear clusters; the red arrows indicate mutants (the same applies below). The scale bar is 1 cm. B: Z58× m223 F3: Separated ear of grain, scale bar 1cm; C: Comparison of kernel length, width, and thickness between wild-type and mutant kernels, scale bar 1cm; D: Comparison of kernel length, width, and thickness measurements between wild-type and mutant kernels; E: Comparison of 100-kernel weight between wild-type and mutant kernels; Note: All graphs with error bars are average values ​​plus / minus standard error. ** indicates wild-type and mutant. m223 The significance between them was P < 0.01, and *** indicates wild type and mutant. m223 The significance between them was P < 0.001 (Student t-test).

[0018] Figure 2 This is the second comparison between wild-type and mutant seeds in one embodiment of this application; in the figure, A: wild-type and mutant seeds on the lightbox. m223 Comparison; B: Wild-type grains vs. mutant grains m223 Longitudinal section, En represents endosperm, Em represents embryo, scale bar is 2mm; C: wild-type grains and mutant grains m223 Comparison of germination ability after 4 days of dark culture; scale bar is 1cm; dynamic development of grains on D:F4 ears. Scale bar is 2cm; the red arrow indicates the mutant.

[0019] Figure 3 Wild-type and mutant strains at different post-pollination days in one embodiment of this application. m223 Paraffin sections were observed; the first row shows complete grains at different days after pollination, with a scale bar of 1000m; the second row shows embryo development at different days after pollination, with a scale bar of 200m; the third row shows the development of the endosperm transfer layer at the base, with a scale bar of 100m.

[0020] Figure 4 This is an analysis of BSR-Seq results in one embodiment of this application.

[0021] Figure 5 As shown in one embodiment of this application m223 The bit-clone image.

[0022] Figure 6 As shown in one embodiment of this application m223 mutant Mu Insertion site.

[0023] Figure 7 As shown in one embodiment of this application ZmRH51Gene expression analysis; A: Maize kernel at different developmental stages and different tissues ZmRH51 Gene expression qRT-PCR analysis; B: 10 DAP, 13 DAP, ZmRH51 Gene expression qRT-PCR analysis; ZmActin was used as internal control.

[0024] Figure 8 For the analysis and verification of ZmRH51 interacting proteins in an embodiment of the present application ZmRH51 Subcellular localization of genes, scale bar, 20 μm.

[0025] Figure 9 For the analysis and verification of ZmRH51 interacting proteins in an embodiment of the present application; A: Yeast two-hybrid experiment shows that ZmRH51 interacts with ZmNop15, ZmRlp7p-1, ZmRlp7p-2; B: BLuc shows that ZmRH51 interacts with ZmNop15, ZmRlp7p-1, ZmRlp7p-2 (fluorescent signal intensity represents the interaction affinity between them); C: BiFC shows that ZmRH51 interacts with ZmNop15, ZmRlp7p-1, ZmRlp7p-2.

[0026] Figure 10 RT-qPCR analysis in an embodiment of the present application Zmrh51 Detection of relative rRNA levels of mutants and WT; A: 45S rDNA structure diagram (Liu et al; 2022); B: The ratio of 45S rDNA to 18S rDNA in WT and Zmrh51 is 1, indicated by a dotted line. Total RNA was extracted from 13 DAP kernels. ZmActin2 was used as internal control; values are mean ± standard deviation (n = 3 biological independent samples); **** p<0.0001; *** p 0.001; ** p<0.01; Ns, no significant difference; t test.

[0027] Figure 11 One of the GO enrichment analysis maps in an embodiment of the present application; A: Biological process item scatter plot; B: Cell component item scatter plot; C: Molecular function item scatter plot.

[0028] Figure 12 The second GO enrichment analysis map in an embodiment of the present application; D: Verification of RNA-seq data by RT-qPCR analysis; total RNA was extracted from 10 DAP kernels in WT and m223 ; ZmActin2 was used as internal control; values are mean ± standard deviation (n = 3 biological independent samples); *** p<0.001; ** p<0.01; * p<0.05.

[0029] Figure 13 KEGG pathway analysis for an embodiment of the present application.

[0030] Figure 14 KEGG pathway analysis for an embodiment of the present application ZmRH51 Haplotype analysis of genes; A: ZmRH51 Five haplotypes composed of SNPs with significant difference in kernel thickness of genes; B: Statistical analysis of kernel thickness of five haplotypes; C: Statistical analysis of 1000-grain weight of five haplotypes. The same lowercase letters on the column chart 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

[0031] The main test materials involved in the following examples are as follows:

[0032] m223 The mutants are from the maize UniformMu mutant library. The F2 separation population is obtained by selfing the F1 of the cross between the laboratory-preserved elite inbred line Z58 and the mutant. m223 The F1 population is configured in the winter of 2020 in the Nanfeng base of Henan Agricultural University in Sanya, Hainan. Due to the abnormal growth of the mutants, the original material is separated from the normal kernels in the ear and selfed at the same time as the cross with Z58, and the hybrid genotype corresponds to the F1 ear of the hybrid single plant. The F2 separation population is obtained by selfing the F1 ear in the spring of 2021 in the Yuanyang base of Henan Agricultural University in Yuanyang. The specific configuration route is as follows: m223

[0033] .

[0034] The corn genome is extracted by SLS method; TransZol The corn kernel RNA is extracted by Plant polysaccharide polyphenol RNA extraction kit (Beijing Quanshi Gold Company). The RNA reverse transcription kit is All-in-One First-Strand Synthesis MasterMix (with dsDNase) kit produced by Kemix Company. The construction of experimental vectors such as yeast two-hybrid, subcellular localization, BLUC, and BiFc is carried out by homologous recombination method using Fusion plus rapid homologous recombination premix produced by Kemix Company. Other reagents and materials involved, if not specified, are commercial regular products. The test and detection methods involved, such as RT-PCR and corn kernel paraffin section, if not specified, are regular methods. The instruments and equipment involved, if not specified, are regular instruments and equipment.

[0035] Example 1 m223 ​Phenotypic analysis of mutants

[0036] m223 The mutant originated from the maize UniformMu mutant library (W22 genetic background), and its heterozygous single plants showed kernel size segregation after self-pollination. Figure 1 A) After pairing with the Z58 background, the self-pollinated ears of heterozygous single plants also showed stable grain size segregation, all conforming to the theoretical ratio of 3:1 (Table 1). Figure 1 Phenotypic analysis of F3 segregating ears from B and C revealed that, compared to the wild type, m223 The mutant kernels exhibited obvious phenotypic characteristics such as tip wrinkling and thinning on the ear. A comparison of kernel length, width, and thickness revealed no significant difference in kernel length and width, but a significant decrease in kernel thickness. Kernel length, width, and thickness were measured separately for the wild type and the mutant. Figure 1 D, found in m223 The grain thickness of the mutant decreased by 28%. Randomly selected wild-type and mutant grains were weighed to determine their 100-grain weight, revealing that the mutant grain weight decreased by 44%. Figure 1 E).

[0037] Seeds were placed on a light box for light transmittance analysis, and it was found that... m223 The light transmittance of the mutant is slightly decreased. Figure 2 A); Further observation revealed that, compared to the wild type, the longitudinal section area of ​​the hard endosperm in the mutant grains was significantly reduced, while the proportion of the floury endosperm was significantly increased. Most of the grains exhibited embryonic abortion and developmental abnormalities. Figure 2 B); Germination experiments on mutant seeds with embryos revealed that the embryos could not develop normally and could not grow roots or shoots. Figure 2 C). To investigate m223 The timing of mutant phenotype appearance was determined by observing ears of fruit at various stages after F4 pollination, such as... Figure 2 D. At 10 days post-pollination (10 DAP), the mutant seed phenotype began to appear, showing slightly smaller seeds. At 13 DAP, the phenotypic difference between the wild type and the mutant became significantly larger. At this time, the wild type seeds had turned noticeably yellow and were plump, while the mutant seeds developed slowly, remained whitish, and some seeds began to collapse at the top. At the subsequent 15 DAP, 18 DAP, and 20 DAP periods, it was found that the mutant seeds were significantly smaller than the wild type, and the seed color remained mainly white and transparent.

[0038] To further investigate the cytological differences between wild-type and mutant seeds, paraffin sections of seeds were observed 10 and 15 days after pollination. Figure 3As shown: at 10 DAP, the wild-type grains had a fully filled endosperm, and the embryo showed visible scutellum and leaf primordia; while the mutant grains showed obvious collapse at the top, with a certain gap between the endosperm and seed coat, and the embryo was malformed and showed no differentiation. Compared with wild-type grains, the mutant grains did not show obvious intracellular proliferation in the basal transfer layer cells of the endosperm. At 15 DAP, the scutellum, leaf primordia, shoot apical meristem, root apical meristem, and hypocotyl structures of the wild-type grain embryo were clearly visible, and the apical cells of the wild-type grain were more dense, with more prominent starch granules; while the mutant grain embryo was similar to that at 10 DAP, i.e., embryo development had stopped.

[0039] Example 2: ZmRH51 Cloning of genes

[0040] 1. m223 Genetic analysis and BSR-seq analysis

[0041] Three well-pollinated ears of rice were randomly selected. m223 The F4 segregating population constructed with Z58 was analyzed, and the number of wild-type and mutant kernels was counted. The results are shown in Table 1. The ratio of wild-type kernels to mutant kernels conformed to a Mendelian segregation ratio of 3:1, indicating that controlling... m223 The gene for the grain trait is a single recessive nuclear gene.

[0042] Table 1. Chi-square test of wild-type and mutant kernels on F4 segregating ears.

[0043] .

[0044] For initial positioning control m223 The chromosomal location of the mutant grain gene was analyzed using BSR-Seq. Fifteen DAP wild-type grains and [other genes] were selected from the F4 segregating ears. m223 Each mutant seed contains 36 grains. TransZol Plant polysaccharide and polyphenol extraction kit was used to extract total RNA from seeds. The RNA from each qualified wild-type seed sample was then separated into... m223 Equal amounts of mutant seed RNA were mixed to form a WT RNA pool and m223 The RNA pool was sent to Beijing Berry Genomics Co., Ltd. for sequencing. Subsequently, using the BSR-Seq analysis method developed by Zou et al., 48,715 SNP loci were detected. After filtering and screening, 1,702 linked SNP loci were obtained. These SNP loci were mainly enriched in chromosome 5 between 0.8 Mb and 8.0 Mb. Figure 4 ).

[0045] 2. m223 fine positioning

[0046] To further narrow down the candidate interval, 50 pairs of Indel markers were developed within this interval, and linked markers were screened using the BSA pooling method. Five linked marker pairs, 5-S08658, 5-S08666, 5-S08668, 5-S08692, and 5-S08693, were screened. Using 100 mutant grains from F4, the candidate interval was determined to be between 3.58 Mb and 4.43 Mb. Subsequently, three more linked marker pairs were screened, and a total of 865 individual plants were used to finally determine the candidate interval to be between 3.9 Mb and 4.2 Mb, with a physical distance of approximately 352 kb.

[0047] 3. Analysis and validation of candidate genes

[0048] According to the Gramene website (https: / / ensembl.gramene.org / Zea_mays / ), the B73 V4 reference genome contains 11 protein-coding genes within the 3.9Mb-4.2Mb physical region. Figure 5 Based on the expression changes of these 11 genes, 9 differentially expressed genes were screened. Simultaneously, the insertion of Mutator transposons in each gene was analyzed using specific primers for each candidate gene and primer combinations for Mu8. Finally, primers 1F and 1R, and 1R and Tir67 were used to detect the insertion of Mutator transposons in these genes. Zm00001d013056 There is an insertion of a Mutator transposon 485 bp downstream of the gene start codon (B73 V4 Chr5:3927312). Figure 6 B), and is linked to the mutant grain phenotype.

[0049] To further determine m223 The phenotype of mutant seeds is indeed due to Zm00001d013056 Caused by transposon insertion in the gene; the EMS mutant was purchased from the MEMD mutant library. EMS4-121b11 The mutant is in Zm00001d013056 A mutation from C to T exists 1397 bp downstream of the start codon, changing the codon from CGA to UGA and altering the encoded amino acid from Arg to a stop codon, causing premature gene termination. EMS seeds were amplified using 2F and 2R methods, and the amplification products were sequenced. Heterozygous seeds were selected for planting and self-pollination with... m223 The materials were hybridized with heterozygous seeds. The results showed that... EMS4-121b11 After self-pollination of heterozygotes, a phenotype of grain size segregation appeared on the ears, with a segregation ratio consistent with wild type: mutant = 3:1; m223 The ears of hybrids from heterozygotes also exhibited a wild-type:mutant ratio of 3:1 kernel size phenotype. Three mutant kernels were selected from each of the three segregating hybrid ears, and further analysis of the mutants in the segregating hybrid ears revealed that... Zm00001d013056A heterozygous Mutator transposon insertion was found 485 bp downstream of the start codon, and a heterozygous C-to-T mutation was found 1397 bp downstream of the start codon. These results indicate that the gene controls… m223 The gene responsible for the mutant grain phenotype is Zm00001d013056 .

[0050] Example 3: ZmRH51 Gene-encoded protein sequence analysis and evolutionary analysis

[0051] 1. ZmRH51 The gene encodes a protein containing a DEAD-Box domain.

[0052] Zm00001d013056 The B73 gene, reference genome fourth edition, contains five transcripts. Based on BSR-Seq and cDNA amplification results, in m223 Only the T002 transcript was detected in the material. Zm00001d013056 The full-length CDS sequence of the T002 transcript is 1950 bp (as shown in SEQ ID NO.3), encoding a protein composed of 649 amino acids (as shown in SEQ ID NO.4). Using the InterPro website (https: / / www.ebi.ac.uk / interpro / ), the predicted domains of this protein show that it contains a DEAD / DEAH box helicase domain, meaning the gene encodes an RNA helicase belonging to the DEAD-Box family. Therefore, based on... Zm00001d013056 Gene domains and gene annotation will Zm00001d013056 Gene naming ZmRH51 .

[0053] 2. ZmRH51 Evolutionary analysis of genes

[0054] Utilize the OrthoDB website (https: / / www.orthodb.org / ) ZmRH51 Homologous protein prediction analysis of the gene's amino acid sequence in Arabidopsis, yeast, and rice revealed two highly homologous genes in Arabidopsis. AtRH51 and AtRH27 Homologous genes in rice are OsRH27 Homologous genes in yeast are HAS1 Further utilize MEGA11 and GeneDoc software to... ZmRH51Comparison of the protein sequences encoded by these three homologous genes revealed that, within the two domains unique to DEAD-Box RNA helicase, the four proteins exhibited high homology at nine highly conserved motifs, suggesting that the four proteins may have similar functions. However, compared to Arabidopsis and rice, yeast HAS1 and ZmRH51 showed significant differences, while rice OsRH27 showed the highest homology with ZmRH51.

[0055] To analyze the phylogenetic relationships between ZmRH51 and homologous proteins in other species, we first used BLASP in NCBI to identify homologous sequences of ZmRH51 in 20 species, including sorghum, wheat, rye, and Arabidopsis thaliana. Then, we used the neighbor-joining method (NJ) in MEGA11 software to perform phylogenetic tree analysis on the homologous proteins in each species. The results showed that among common species, ZmRH51 in maize was most closely related to its homologous protein in sorghum, followed by its homologous protein in rice, while exhibiting significant genetic differences and a more distant phylogenetic relationship with its homologous protein in Arabidopsis thaliana.

[0056] Example 4: ZmRH51 Spatiotemporal expression analysis of genes

[0057] In order to investigate ZmRH51 To investigate gene expression in various maize tissues, we selected different tissues from the inbred line W22, including roots, stems, leaves, tassels, and ears, as well as the endosperm at different post-pollination stages. ZmRH51-qRT-F / R was used as the amplification primer, and ZmActin2 was used as the internal control. 2... -ΔΔCt To calculate relative expression levels for analysis ZmRH51 The expression of genes in various tissues showed that... ZmRH51 The gene is constitutively expressed, and its expression level is relatively low in the grain, increasing with the number of days since pollination. ZmRH51 The gene expression level first decreased and then increased, but the expression level was higher in parts such as silks, roots, and tassels, indicating that the gene plays an important role in the entire maize plant development process.

[0058] To investigate the differences between wild-type and mutant seeds ZmRH51 To investigate gene transcription and expression, RNA was extracted from 10DAP and 13DAP wild-type and mutant seeds and reverse transcribed into cDNA, which was then analyzed using RT-qPCR. ZmRH51 Gene expression levels were found to be higher in the wild type than in the mutant at 10 DAP and 13 DAP; simultaneously, with increasing post-pollination days, ZmRH51 Gene expression levels gradually decreased, but the difference between mutants and wild-type gradually increased. Figure 7 B).

[0059] Example 5: Subcellular localization of ZmRH51

[0060] The expression location of ZmRH51 in cells was predicted using the Gramene website (http: / / ensembl.gramene.org / Zea_mays), and the results showed that ZmRH51 is a nuclear-localized protein. ZmRH51 The gene was constructed into a PRTL vector containing the reporter gene eGFP sequence to express the fusion protein ZmRH51:GFP. Driven by the 35S promoter, and using the OsGHD7:RFP fusion protein as a nuclear marker, the protein was co-expressed in maize protoplasts for subcellular localization analysis. The results showed that the red fluorescence signal of OsGHD7:RFP overwrote the fluorescence signal of ZmRH51:GFP, indicating that ZmRH51 is expressed in the nucleus, presumably at the nucleolus. To verify this hypothesis, the AtHDT1:RFP fusion protein was used as a nucleolar marker and co-expressed with ZmRH51:eGFP in maize protoplasts; the results showed (see...). Figure 8 The green fluorescence signal of the ZmRH51:eGFP fusion protein can overlap with the red fluorescence signal of the AtHDT1:RFP fusion protein, indicating that ZmRH51 is a nucleolar localized protein.

[0061] Example 6: ZmRH51 Analysis and validation of interacting proteins

[0062] Nop15 and Rlp7p are two assembly factors of the 60S large ribosomal subunit. In yeast, HAS1 (a homolog of ZmRH51) interacts with Nop15 and Rlp7p, jointly participating in the assembly of the 60S large ribosomal subunit. To verify whether ZmRH51 is involved in ribosome biosynthesis in maize, the corresponding homologous genes of Nop15 and Rlp7p in maize (ZmNop15: Zm00001d050300, ZmRlp7p-1: Zm00001d026254 and ZmRlp7p-2: Zm00001d031703) were cloned, and corresponding protein interaction vectors were constructed. Interaction was verified using yeast two-hybrid, BLUC, and BiFc experiments (see [link to relevant documentation]). Figure 9 ).

[0063] First, ZmRH51The CDS sequences of the genes were ligated into the pGBKT7 (BD) vector, and the CDS sequences of three candidate interacting genes were ligated into the pGADT7 (AD) vector. Finally, BD-ZmRH51 was co-transformed with the three genes ligated into the AD vector into Y2H yeast competent cells for point-to-point verification. The results showed that the three combinations 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 all three proteins interact with ZmRH51. However, the interaction between ZmNop15 and ZmRH51 was weaker.

[0064] To verify the authenticity of the interaction between these three genes in yeast, a luciferase complementation experiment was conducted. First, the... ZmRH51 The CDS sequences of the genes were inserted into the N-Luc vector, and the CDS sequences of the three candidate interacting genes were inserted into the C-Luc vector. Subsequently, the N-Luc and C-Luc vectors, fused with the different gene CDS sequences, were transformed into Agrobacterium tumefaciens GV3101 strain and cultured. The bacterial culture was then injected into tobacco for luciferase complementation verification. The results also showed that all three proteins interact with ZmRH51.

[0065] In addition, ZmRH51 The CDS sequences of the genes were inserted into the PXY104-cYFP vector, and the CDS sequences of the three candidate interacting genes were inserted into the PXY106-nYFP vector for two-molecule fluorescence complementation experiments. The final results were consistent with those of the yeast two-hybrid experiment and the BLuc experiment. Simultaneously, to investigate the intracellular interaction sites between ZmRH51 and the three interacting proteins, a nucleolar-specific expression gene fused with an RFP reporter gene was used. AtHDT1 The gene, acting as a nucleolar marker, was transferred into tobacco cells along with PXY104-cYFP and PXY106-nYFP vectors containing different CDS sequences for expression. The results showed that ZmRH5 interacts with the three proteins in the nucleolus.

[0066] 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 ribosome assembly, just like yeast.

[0067] Example 7: ZmRH51 The effect of gene mutations on maize pre-rRNA

[0068] Ribosome assembly begins in eukaryotes with the transcription of 45S precursor rRNA by RNA polymerase I. RT-qPCR was used to detect the accumulation of intermediate products during precursor rRNA processing in wild-type and mutant organisms to analyze whether ZmRH51 affects precursor rRNA processing. The 45S precursor rRNA is transcribed into a large precursor composed of 18S, 5.8S, and 25S rRNA, separated by ITS1 and ITS2, flanked by 5' ETS and 3' ETS. Specific primers were used to detect 45S pre-rRNA, various intermediate products, and total rRNA. Results showed that primer combinations amplifying signals located in the P-P', ITS1, and ITS2 regions significantly accumulated in mutants, while detection signals in the 18S, 5.8S, and 25S regions were significantly reduced. Figure 10 This indicates that the processing of precursor rRNA was affected in the mutant.

[0069] Example 8: m223 Transcriptome analysis of mutants

[0070] 1. Differentially expressed gene analysis: In order to study ZmRH51 Biological functions of genes in seed development were compared between WT and WT at 13 DAP using RNA sequencing. m223 Transcriptome data (RNA-Seq) of mutant seeds. Of the 29,226 genes identified by RNA-seq, using q < 0.01 and fold change > 2 as screening criteria for significantly differentially expressed genes, a total of 6,489 significantly differentially expressed genes (DEGs) were detected, of which 3,295 genes were... m223 The expression levels of mutant genes were upregulated in the grains, while the expression levels of 3194 genes were downregulated.

[0071] 2. GO Enrichment Analysis: GO (Gene Ontology) analysis showed that these differentially expressed genes were enriched to 178 items (q<0.01). In biological processes, items related to rRNA metabolism (GO:0016072, q=9.4765E-8), rRNA processing (GO:0006364, q=9.4765E-8), ribosome biosynthesis (GO:0042254, q=1.0338E-8), and cell cycle progression (GO:0022402, q=3.83368E-8) showed high significance. In molecular functions, items related to ATPase activity (GO:0015631, q=9.45E-5) and ATP-dependent helicase activity (GO:0008026, q=8.12E-4) showed high significance. In cellular components… In the component category, the most significant items were non-membrane organelles (GO:0043228, GO:0043232, q=2.67E-10, q=2.67E-10), kinin complex (GO:0005871, q=1.35E-8), and nucleolus (GO:0005730, q=1.93E-6). Figure 11 To validate the differentially expressed genes detected by RNA-seq, RT-qPCR was performed on 14 genes from the ribosome biosynthesis entries, and the results were largely consistent with the RNA-seq data. Figure 12 ).

[0072] 3. KEGG enrichment analysis: Among the metabolic pathways enriched by KEGG, eukaryotic ribosome biosynthesis showed the highest significance (zma03008, p=1.6E-05). Pathways such as phenylpropanol synthesis, plant hormone signal transduction, and aminoacyl-tRNA biosynthesis were also significantly enriched. Figure 13 A); Simultaneously, the metabolic pathways of sugar and starch were also enriched. Analysis of the expression levels of related genes in this pathway revealed a total of 61 significantly differentially expressed genes, of which 23 were upregulated and 38 were downregulated. Figure 13 B).

[0073] The above results indicate that ZmRH51 Mutations in this gene can affect the biosynthesis of ribosomes in plants. As a member of the DEAD-Box RNA helicase family, mutations in the gene encoding ZmRH51 can also affect the expression of genes encoding ATPase and ATP-dependent helicases. Furthermore, defects in this gene can also lead to the normal synthesis of sugars, starches, and other substances in grains.

[0074] Example 9: ZmRH51 Examples of gene application in maize kernel development

[0075] ZmRH51 Gene mutations can lead to a significant reduction in corn kernel thickness, which is necessary for extraction. ZmRH51 Superior allelic variations in kernel thickness were selected from 397 representative natural maize populations, combined with... ZmRH51 SNP information within gene segments is used to determine the grain thickness trait. ZmRH51 Candidate gene association analysis. Of the 397 natural maize populations used in this study, ZmRH51 A total of 76 SNPs were found within the gene segment. Using p < 0.01 as the threshold, three SNPs significantly associated with grain thickness were screened, chr5.s_3927416 (p = 0.67 × 10⁻⁶). -2 ), chr5.s_3930501(p=0.33×10 -2 ), chr5.s_3930830(p=0.16×10 -2 SNP 3927416 is located in intron 1, SNP 3930501 in intron 9, and SNP 3930830 in intron 11. Based on these three significant SNPs, the 397 natural populations can be divided into 5 haplotypes (Hap). Figure 14 A). Compared with Hap5, Hap1, Hap2, Hap3, and Hap4 showed no significant difference in grain thickness. There was no significant difference in grain thickness between Hap1 and Hap2, but both were significantly thicker than Hap3 and Hap4, increasing by 3.25% and 6.03% respectively. Figure 14 B).

[0076] To analyze whether changes in grain thickness among the five haplotypes affected 100-grain weight, statistical analysis of the 100-grain weight was performed on the materials of each of the five haplotypes. The results showed that Hap2 was a favorable haplotype that also exhibited excellent 100-grain weight; compared to Hap3 and Hap5, Hap2 increased the 100-grain weight by 3.89% and 11.64%, respectively. Figure 14 C).

[0077] The above results indicate that ZmRH51 The Hap2 haplotype of the gene is a favorable haplotype that exhibits excellent performance in both kernel thickness and 100-kernel weight. In actual maize breeding, haplotypes containing this gene can be selected in early generations. ZmRH51 The superior haplotype of the Hap2 gene helps to improve grain thickness and 100-grain weight, thereby increasing yield.

[0078] Although some preferred embodiments of this invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

Claims

1. A maize kernel development regulatory gene with a DNA sequence as shown in SEQ ID NO. 1 ZmRH51 Or application of a protein encoded by an amino acid sequence as shown in SEQ ID NO. 4 in preparation of a reagent for regulating maize kernel thickness trait or / and hundred kernel weight trait.

2. A maize kernel development regulatory gene whose DNA sequence is shown as SEQ ID NO. 1 ZmRH51 Use in breeding varieties / lines related to the traits of kernel thickness and / or 100-seed weight of maize.

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