Application of maize MYBR47 gene in regulating grain traits and variety selection and transgenic plant breeding method

Abstract

The application relates to the technical field of genetic engineering, in particular to application of a maize MYBR47 gene in regulation of grain traits and variety selection and a transgenic plant cultivation method, wherein the sequence of the MYBR47 gene is shown as SEQ ID NO:1. A nitrogen response gene MYBR47 is found in the maize, the MYBR47 mutation can affect the grain size, starch and protein content; the yeast single hybridization result shows that the gene regulates the development of the maize grain by affecting cell division / expansion. The gene MYBR47 can be used to cultivate new varieties with different grain sizes and improved nitrogen utilization efficiency, and lays a foundation for the application practice of maize breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the maize MYBR47 gene in regulating grain traits and variety selection, and methods for cultivating transgenic plants. Background Technology

[0002] Nitrogen is an essential nutrient element for crop growth and development, playing a crucial role in its yield and quality. However, excessive application of nitrogen fertilizer not only causes environmental pollution and resource waste but also negatively impacts crop quality.

[0003] Maize is my country's largest grain crop, with broad application value and development prospects. Starch and protein are important components of maize kernel endosperm and the main storage forms of carbon and nitrogen, determining kernel yield and quality. Studying the development process of maize kernels and the regulatory mechanisms of carbon and nitrogen accumulation is one of the important ways to achieve synergistic improvement of maize yield and quality. Nitrogen fertilizer application plays a significant role in the accumulation of starch and protein in maize kernels, affecting maize yield and quality. Therefore, studying the regulatory mechanisms of maize kernel development and carbon-nitrogen accumulation balance under nitrogen reduction conditions will help create new maize germplasm. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the maize MYBR47 gene in regulating grain traits and variety selection, as well as a method for cultivating transgenic plants.

[0005] The research team discovered the nitrogen-responsive gene MYBR47 in maize. Homologous genes of this gene showed significantly increased expression levels under low-nitrogen conditions in rice, sorghum, barley, and wheat. Further subcellular localization revealed that exogenous nitrate inhibits MYBR47 entry into the cell nucleus. MYBR47 mutations affect kernel size, starch, and protein content. Yeast one-hybrid results showed that this gene regulates maize kernel development by influencing cell division / expansion. Simultaneously, superior haplotypes of MYBR47 were screened from 95 natural maize populations, contributing to improved maize yield. MYBR47 can be used to breed new maize varieties with different kernel sizes and improved nitrogen use efficiency.

[0006] Specifically, the present invention provides the following technical solution:

[0007] The application of the maize MYBR47 gene in regulating grain traits and variety breeding, wherein the sequence of the MYBR47 gene is shown in SEQ ID NO: 1.

[0008] The maize MYBR47 gene can also be used to regulate kernel size traits and to breed varieties or lines with kernel size.

[0009] The MYBR47 gene can also be used to regulate the total starch and / or protein content of grains, and for breeding varieties or lines with different total starch and / or protein contents. Specifically, overexpression and / or knockout of the MYBR47 gene can be used to cultivate varieties with different total starch and / or protein contents.

[0010] A method for cultivating MYBR47 transgenic plants includes the following steps:

[0011] (1) Construction of overexpression vector: Using genomic DNA from B73 plant leaves as a template, PCR amplification was performed using primer pair F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-GGTCCAGAATCCATGTGTCT-3', as shown in SEQ ID NO: 6-7, to obtain the corresponding PCR amplification products; the PCR amplification products were ligated into the overexpression vector driven by the ubi promoter and Sanger sequencing was performed; the sequencing results were compared with the MYBR47 gene sequence; the constructed overexpression vector was handed over to a gene company for transformation;

[0012] (2) Obtaining and identifying homozygous MYBR47 mutants

[0013] Using genomic DNA from T1 generation transgenic maize leaves as templates, PCR amplification was performed using primer pairs consisting of primer F: 5'-GTCCCTTGGCTCTGAAACCA-3' and primer R: 5'-TCGAGGCCTGCTTCCTGTTA-3', as shown in SEQ ID NO: 8-9. The reaction procedure during amplification is shown in the table below, and the corresponding PCR amplification products were obtained. The PCR amplification products were then subjected to Sanger sequencing. The sequencing results were compared with the target site sequence of the MYBR47 gene edited by the CAS9 protein to select plants with heterozygous or homozygous frameshift mutations in the target site region.

[0014] temperature time 95℃ 3min 95℃ 30s 60℃ 30s 72℃ 1min Go to step 2 34 cycles 72℃ 5min 16℃ ∞

[0015] (3) Identification of MYBR47 overexpressing plants

[0016] Using genomic DNA from T1 generation transgenic maize leaves as templates, and considering the flag tag fused to the overexpression vector, the identification primers were designed to combine primers from the gene with primers from the flag. Specifically, primer pair F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-CTTGTCATCGTCATCCTTG-3' were used, as shown in SEQ ID NO: 10-11. PCR amplification was performed, and the reaction procedure was shown in the table above, yielding the corresponding PCR amplification products. Based on the amplification results, plants with the correct bands were selected. Simultaneously, quantitative PCR primers were designed, specifically primer pair F: 5'-CATCAAGTCTGCTGAGGAGG-3' and primer R: 5'-GTACGGCACGTGGCCTGATTC-3', as shown in SEQ ID NO: 12-13. qPCR detection was performed, and plants with significantly increased MYBR47 expression were identified as MYBR47 transgenic plants.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] (1) This invention clones the gene MYBR47 that regulates the size of maize kernels. Overexpression and knockout of the gene MYBR47 will affect the protein content of maize kernels. It can be used to breed new varieties with different kernel protein content, laying the foundation for the application practice of maize breeding.

[0019] (2) The use of the MYBR47 gene can facilitate the creation of high-yield maize breeding materials, shorten the breeding cycle of new crop varieties, reduce breeding costs, and improve breeding efficiency. Attached Figure Description

[0020] Figure 1 To investigate the high expression of the MYBR47 gene induced by low nitrogen conditions in maize, rice, sorghum, barley, and wheat, and the effect of nitrate on the subcellular localization of MYBR47.

[0021] Figure 2 The effects of MYBR47 on seed size, 100-seed weight, and protein content are shown in the following figures: A: Comparison of seed width between wild-type and mutant (scale bar: 1 cm); B: Comparison of 100-seed weight between wild-type and mutant; C: Comparison of seed protein content between wild-type and mutant; D: Comparison of seed width between wild-type and overexpressed MYBR47 (scale bar: 1 cm); E: Comparison of 100-seed weight between wild-type and overexpressed MYBR47; F: Comparison of seed protein content between wild-type and overexpressed MYBR47.

[0022] Figure 3 This study investigates the interaction between MYBR47 and promoters of cell division / size-related genes.

[0023] Figure 4For MYBR47 haplotype analysis. A: MYBR47 variant sites and haplotype analysis; B: Comparison of 100-grain weight between the two MYBR47 haplotypes. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test or detection methods involved are all conventional methods unless otherwise specified.

[0026] Example 1: The effect of nitrate on the expression and nuclear localization of MYBR47

[0027] The research team of this invention found that when treating the maize inbred line B73 with high nitrogen (10 mM KNO3) and low nitrogen (0.4 mM KNO3), the expression level of MYBR47 was significantly increased under low nitrogen treatment. Further research revealed that MYBR47 expression was highest in the kernels, and the expression level under low nitrogen conditions was significantly higher than that under high nitrogen conditions. Figure 1 A); it was also found that the expression levels of MYBR47 homologs in rice, sorghum, wheat, and barley were also related to nitrogen levels. Figure 1 B). The 35S::MYBR47-GFP vector was constructed, and transient expression experiments were performed on maize leaf protoplasts. The results are as follows: Figure 1 As shown in Figure C, without nitrate treatment, MYBR47 is distributed in both the nucleus and cytoplasm; with nitrate treatment, MYBR47 is mainly distributed in the cytoplasm. This indicates that nitrate alters the nuclear localization of MYBR47.

[0028] Example 2: The effect of MYBR47 on grain size and carbon-nitrogen balance

[0029] The nucleotide sequence of the MYBR47 gene in the DNA of maize B73 is shown in SEQ ID NO: 1, and the cDNA sequence is shown in SEQ ID NO: 2.

[0030] The MYBR47 gene encodes the protein MYBR47. The amino acid sequence of the protein MYBR47 is shown in SEQ ID NO: 3.

[0031] To investigate the function of MYBR47, gene knockout and overexpression of MYBR47 were performed. First, a 23bp target sequence (SEQ ID NO: 4) was designed in the CDS region near the MYBR47 gene translation start codon, specifically: 5'-CCTCACGCAGCTCCTCTCCCAAC-3'. To improve editing efficiency, 23bp target sequences (SEQ ID NO: 5) were also designed at other locations in the MYBR47 gene CDS region, specifically: 5'-GAAGAGGCACTCGCATACTACGG-3'. The construction of the Crispr / Cas9 vector and the gene transformation process were both completed by Weimi Technology Co., Ltd.

[0032] Obtaining MYBR47 overexpression materials:

[0033] (1) Construction of the overexpression vector: Using genomic DNA from B73 plant leaves as a template, PCR amplification was performed using primer pairs consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-GGTCCAGAATCCATGTGTCT-3' (shown in SEQ ID NO: 6-7), yielding the corresponding PCR amplification products. The PCR amplification products were ligated into an overexpression vector (fused with a flag tag) driven by the Ubi promoter and Sanger sequencing was performed. The sequencing results were compared with the sequence of the MYBR47 gene (shown in SEQ ID NO: 1). The constructed overexpression vector was then transformed by Weimi Technology Co., Ltd.

[0034] (2) Obtaining and identifying homozygous MYBR47 mutants

[0035] Using genomic DNA from T1 generation transgenic maize leaves as templates, PCR amplification was performed using primer pairs consisting of primer F: 5'-GTCCCTTGGCTCTGAAACCA-3' and primer R: 5'-TCGAGGCCTGCTTCCTGTTA-3' (shown in SEQ ID NO: 8-9). The reaction procedure during amplification is shown in Table 1, and the corresponding PCR amplification products were obtained. The PCR amplification products were then subjected to Sanger sequencing. The sequencing results were compared with the target site sequence of the MYBR47 gene (shown in SEQ ID NO: 1) edited by the CAS9 protein to select plants with heterozygous or homozygous frameshift mutations in the target site region.

[0036] Table 1. PCR reaction procedure

[0037] temperature time 95℃ 3min 95℃ 30s 60℃ 30s 72℃ 1min Go to step 2 34 cycles 72℃ 5min 16℃ ∞

[0038] Among them, the results of homozygous mutation types are as follows: Figure 2Two homozygous mutations of mybr47 were obtained, named -76bp and +77bp (WT is maize inbred line B73). The -76bp mutation involved the same mutation in the MYBR47 gene on both homologous chromosomes, with a 76-base deletion in the MYBR47 gene on both chromosomes. This caused frameshift and premature termination of protein translation, resulting in the loss of MYBR47 protein function. Similarly, the +77bp mutation involved the same mutation in the MYBR47 gene on both homologous chromosomes, with a 77-base insertion in the MYBR47 gene on both chromosomes. This also caused frameshift and premature termination of protein translation, leading to the loss of MYBR47 protein function.

[0039] (3) Identification of MYBR47 overexpressing plants

[0040] Using genomic DNA from T1 generation transgenic maize leaves as templates, and considering the flag tag fused to the overexpression vector, the identification primers were designed to combine primers from the gene with primers from the flag. Specifically, the primer pair consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-CTTGTCATCGTCATCCTTG-3' (SEQ ID NO: 10-11) was used for PCR amplification. The reaction procedure during amplification is shown in Table 1, and the corresponding PCR amplification products were obtained. Based on the amplification results, plants with the correct bands were selected. Simultaneously, quantitative PCR primers were designed, specifically the primer pair consisting of primer F: 5'-CATCAAGTCTGCTGAGGAGG-3' and primer R: 5'-GTACGGCACGTGGCCTGATTC-3' (SEQ ID NO: 12-13), for qPCR detection. Plants with significantly increased MYBR47 expression were identified as overexpression materials and named OE1 and OE2, respectively.

[0041] (4) Observation of seed size and determination of protein content

[0042] The planting materials were B73, -76bp, +77bp, OE1, and OE2, which were self-pollinated. Kernels from the middle of mature ears were collected for kernel size observation. Specifically, 10 kernels were randomly selected and placed side-by-side, and photographs were taken to observe changes in width and length. The weight of 100 kernels was also measured. The results are as follows: Figure 2 B and Figure 2 As shown in E, the mutant grain weight per 100 grains increased significantly, while the overexpression grain weight per 100 grains decreased significantly.

[0043] The protein content of the grains was determined using near-infrared spectroscopy, and the results are as follows: Figure 2 C and Figure 2 As shown in F, the mutant grain protein content is significantly increased, while the overexpression grain protein content is significantly decreased.

[0044] Example 3: Interaction between MYBR47 and promoters of cell cycle-related genes

[0045] The promoter sequences of CCS52b, CDC202, CDC45a, CDC45b, CNR1, and CNR5 were constructed into the pHIS2.1 vector, and the CDS sequence of MYBR47 was constructed into the pGADT7 vector. MYBR47, along with CCS52b, CDC202, CDC45a, CDC45b, CNR1, and CNR5, were transformed into the Y187 yeast strain, and yeast one-hybrid assays were performed. Results are as follows: Figure 3 As shown, MYBR47 interacts with cell division-related genes CCS52b, CDC202, CDC45a, CDC45b, CNR1, and CNR5.

[0046] Example 4: Optimal Haplotype Analysis of MYBR47, a Gene Regulating Seed Size

[0047] Using the R-package geneHapR, correlation analysis was performed on the 100-kernel weight of the MYBR47 gene and SNPs in the upstream and downstream 1000bp regions. Results are as follows: Figure 4 As shown in Figure A, at a threshold of P < 0.01, a total of 7 SNPs were significantly associated with 100-grain weight. The first haplotype, represented by RF1708, included 51 samples, while the second haplotype, represented by C103, included 44 samples. Statistical analysis demonstrated a significant difference in 100-grain weight between haplotype 1 and haplotype 2 (P = 0.018). These results prove that the second haplotype of the MYBR47 gene, Hap2, is a superior allele for 100-grain weight.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Corn MYBR47 The application of genes in regulating maize kernel traits and variety breeding is characterized by: The MYBR47 The gene sequence is shown in SEQ ID NO: 1; MYBR47 Genes interact with CCS52b, a gene related to cell division. The application is in regulating the total protein content trait in grains, and in the breeding of maize varieties or lines with different total protein contents in grains.

2. The corn according to claim 1 MYBR47 The application of genes in regulating maize kernel traits and variety breeding is characterized by: By overexpressing and / or knocking out genes MYBR47 In order to cultivate corn varieties with different total protein content in the kernels.

3. A kind MYBR47 The method for cultivating genetically modified corn is characterized by, Includes the following steps: (1) Construction of the overexpression vector: Using genomic DNA from leaves of maize inbred line B73 as a template, PCR amplification was performed using primer pairs consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-GGTCCAGAATCCATGTGTCT-3', as shown in SEQ ID NO: 6-7, to obtain the corresponding PCR amplification products; the PCR amplification products were ligated into the overexpression vector driven by the Ubi promoter and Sanger sequencing was performed; the sequencing results were compared with... MYBR47 Gene sequences were compared; the constructed overexpression vector was transformed; (2) Obtaining and identifying homozygous MYBR47 mutants right MYBR47 Gene knockout was performed; using genomic DNA from T1 generation transgenic maize leaves as templates, PCR amplification was carried out using primer pairs consisting of primer F: 5'-GTCCCTTGGCTCTGAAACCA-3' and primer R: 5'-TCGAGGCCTGCTTCCTGTTA-3', as shown in SEQ ID NO: 8-9. The reaction procedure during amplification is shown in the table below, and the corresponding PCR amplification products were obtained. The PCR amplification products were then subjected to Sanger sequencing; the sequencing results were compared with... MYBR47 By comparing the target site sequences of genes edited by the CAS9 protein, plants with heterozygous or homozygous frameshift mutations in the target site region were selected. The protein content of the seeds of the mutant plants was significantly increased. ； (3) Identification of MYBR47 overexpressing plants Using genomic DNA from T1 generation transgenic maize leaves as templates, and considering the flag tag fused to the overexpression vector, the identification primers were designed to combine primers from the gene with primers from the flag. Specifically, primer pair F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-CTTGTCATCGTCATCCTTG-3' were used, as shown in SEQ ID NO: 10-11. PCR amplification was performed, and the reaction procedure was shown in the table above, yielding the corresponding PCR amplification products. Based on the amplification results, plants with the correct bands were selected. Simultaneously, quantitative PCR primers were designed, specifically primer pair F: 5'-CATCAAGTCTGCTGAGGAGG-3' and primer R: 5'-GTACGGCACGTGGCCTGATTC-3', as shown in SEQ ID NO: 12-13. qPCR detection was performed, and plants with significantly increased MYBR47 expression were identified as... MYBR47 Overexpression in plants resulted in a significant decrease in the protein content of the seeds.

Images