Application of corn MYBR47 gene in regulation and control of grain traits and variety breeding and transgenic plant cultivation method

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a corn MYBR47 gene in regulation and control of grain traits and variety breeding and a transgenic plant cultivation method, and the sequence of the MYBR47 gene is shown as SEQ ID NO: 1. According to the application, a nitrogen response gene MYBR47 is found in corn, and MYBR47 mutation can influence the grain size, starch content and protein content; yeast one-hybridization results show that the gene regulates and controls development of corn kernels by influencing cell division / expansion. By utilizing the gene MYBR47, new varieties with different grain sizes and improved nitrogen utilization efficiency can be cultivated, and a foundation is laid for application practice of corn breeding.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and specifically to the application of corn MYBR47 gene in regulating grain traits and variety breeding, and a transgenic plant breeding method. Background Art

[0002] Nitrogen is an important nutrient element required for crop growth and development, and plays an important role in the formation of its yield and quality. However, excessive application of nitrogen fertilizer not only causes environmental pollution and waste of resources, but also affects the quality of crops.

[0003] Corn is the largest grain crop in my country, with broad application value and development prospects. Starch and protein are important components of the endosperm of corn kernels, and are also the main storage forms of carbon and nitrogen, which determine the yield and quality of kernels. Studying the regulatory mechanism of corn kernel development and carbon and nitrogen accumulation is one of the important ways to achieve coordinated improvement of corn yield and quality. The application of nitrogen fertilizer plays an important role in the accumulation of starch and protein in corn kernels, affecting the yield and quality of corn. Therefore, studying the regulatory mechanism of corn kernel development and carbon and nitrogen accumulation balance under nitrogen reduction conditions will help create new corn germplasm. Summary of the invention

[0004] The purpose of the present invention is to provide an application of corn MYBR47 gene in regulating grain traits and variety breeding and a transgenic plant breeding method.

[0005] The research team of the present invention discovered the nitrogen-responsive gene MYBR47 in corn. The homologous genes of this gene in rice, sorghum, barley and wheat all showed significantly increased expression under low nitrogen conditions. Further subcellular localization revealed that exogenous nitrate inhibited MYBR47 from entering the cell nucleus. MYBR47 mutations affect grain size, starch and protein content. Yeast single hybridization results showed that this gene regulates the development of corn grains by affecting cell division / expansion. At the same time, 95 corn natural population materials were used to screen out the excellent haplotypes of MYBR47, which provided help in improving corn yield. MYBR47 can be used to cultivate new corn varieties with different grain sizes and improved nitrogen utilization efficiency.

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

[0007] Application of corn 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 grain size traits and select grain size varieties or lines.

[0009] The rice MYBR47 gene can also be used to regulate the total starch and / or protein content traits of grains, and to breed varieties or lines with different total starch and / or protein contents of grains. Varieties with different total starch and / or protein contents of grains can be cultivated by overexpressing and / or knocking out the MYBR47 gene.

[0010] A method for cultivating a MYBR47 transgenic plant comprises the following steps:

[0011] (1) Construction of overexpression vector: Using genomic DNA from leaves of B73 plants as a template, a primer pair consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-GGTCCAGAATCCATGTGTCT-3', as shown in SEQ ID NO: 6-7, was used for PCR amplification to obtain the corresponding PCR amplification product; the PCR amplification product was connected to 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 delivered to a gene company for transformation;

[0012] (2) Acquisition and identification of MYBR47 homozygous mutants

[0013] The genomic DNA of T1 transgenic corn leaves was used as a template, and a primer pair consisting of primer F: 5'-GTCCCTTGGCTCTGAAACCA-3' and primer R: 5'-TCGAGGCCTGCTTCCTGTTA-3', as shown in SEQID NO: 8-9, was used for PCR amplification. The reaction procedure during the amplification process is shown in the following table, and the corresponding PCR amplification products were obtained; the PCR amplification products were subjected to Sanger sequencing. The sequencing results were compared with the target site sequence edited by the CAS9 protein on the MYBR47 gene, and plants with heterozygous or homozygous frameshift mutations in the target site region were selected;

[0014] temperature time 95℃ 3min 95℃ 30s 60℃ 30s 72℃ 1min Go to step2 34cycles 72℃ 5min 16℃ ∞

[0015] (3) Identification of MYBR47 overexpressing plants

[0016] The genomic DNA of T1 transgenic corn leaves was used as a template. Since the overexpression vector was fused with a flag tag, the identification primers were designed to combine the primers on the gene with the primers on the flag, specifically a primer pair consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-CTTGTCATCGTCATCCTTG-3', as shown in SEQ ID NO: 10-11, and PCR amplification was performed. The reaction procedure during the amplification process was shown in the table above, and the corresponding PCR amplification product was obtained; according to the amplification results, the plants with the correct bands were selected. At the same time, quantitative PCR primers were designed, specifically a primer pair consisting of primer F: 5'-CATCAAGTCTGCTGAGGAGG-3' and primer R: 5'-GTACGGCACGTGGCCTGATTC-3', as shown in SEQ ID NO: 12-13, and QPCR detection was performed. Plants with significantly increased MYBR47 expression levels were MYBR47 transgenic plants.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention cloned the gene MYBR47 that regulates corn kernel size. Overexpression and knockout of the gene MYBR47 will affect the protein content of corn kernels. New varieties with different kernel protein contents can be cultivated using it, laying a foundation for the application practice of corn breeding.

[0019] (2) The use of the MYBR47 gene can facilitate the creation of high-yield corn breeding materials, shorten the breeding cycle of new crop varieties, reduce breeding costs, and improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results showed that the MYBR47 gene was induced to be highly expressed in corn, rice, sorghum, barley and wheat under low nitrogen conditions, and that nitrate affects the subcellular localization of MYBR47.

[0021] Figure 2 Effects of MYBR47 on grain size, 100-grain weight and protein content. A: Comparison of grain width between wild type and mutant, scale bar is 1 cm; B: Comparison of 100-grain weight between wild type and mutant; C: Comparison of grain protein content between wild type and mutant; D: Comparison of grain width between wild type and overexpressed, scale bar is 1 cm; E: Comparison of 100-grain weight between wild type and overexpressed; F: Comparison of grain protein content between wild type and overexpressed.

[0022] Figure 3 Interaction of MYBR47 with promoters of cell division / size-related genes.

[0023] Figure 4MYBR47 haplotype analysis. A: MYBR47 variable site and haplotype analysis; B: Comparison of 100-grain weight between two haplotypes of MYBR47. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 unless otherwise specified; the tests or detection methods involved are all conventional methods unless otherwise specified.

[0026] Example 1 Nitrate affects the expression and nuclear localization of MYBR47

[0027] The research team of the present invention found that the expression level of MYBR47 was significantly increased under low nitrogen treatment conditions when the maize inbred line B73 was treated with high nitrogen (10mM KNO3) and low nitrogen (0.4mM KNO3). Further research found that the expression level of MYBR47 was highest in the grain, 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 homologous genes in rice, sorghum, wheat and barley crops were also related to nitrogen levels ( Figure 1 B). The 35S::MYBR47-GFP vector was constructed and transient expression experiments were performed on corn leaf protoplasts. Figure 1 As shown in C, when no nitrate treatment was added, MYBR47 was distributed in the nucleus and cytoplasm; when nitrate treatment was added, MYBR47 was mainly distributed in the cytoplasm. This indicates that nitrate changes the nuclear localization of MYBR47.

[0028] Example 2 MYBR47 affects grain size and carbon-nitrogen balance

[0029] In the DNA of corn B73, the nucleotide sequence of the MYBR47 gene is shown as SEQ ID NO: 1, and the cDNA sequence is shown as 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] In order to study the function of MYBR47, MYBR47 was knocked out and overexpressed. First, a 23bp target sequence (shown in SEQ ID NO: 4) was designed in the CDS region near the translation start codon of the MYBR47 gene, specifically: 5'-CCTCACGCAGCTCCTCTCCCAAC-3'. In order to improve the editing efficiency, a 23bp target sequence (shown in SEQ ID NO: 5) was also designed at other positions in the CDS region of the MYBR47 gene, specifically: 5'-GAAGAGGCACTCGCATACTACGG-3'. The Crispr / Cas9 vector construction and gene transformation process were completed by Weimi Company.

[0032] Acquisition of MYBR47 overexpression materials:

[0033] (1) Construction of overexpression vector: Using the genomic DNA of the leaves of B73 plants as a template, a primer pair consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-GGTCCAGAATCCATGTGTCT-3' (shown in SEQ ID NO: 6-7) was used for PCR amplification to obtain the corresponding PCR amplification product. The PCR amplification product was connected to the ubi promoter-driven overexpression vector (fused with a flag tag) 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 delivered to Weimi Company for transformation.

[0034] (2) Acquisition and identification of MYBR47 homozygous mutants

[0035] The genomic DNA of T1 transgenic corn leaves was used as a template, and a primer pair consisting of primer F: 5'-GTCCCTTGGCTCTGAAACCA-3' and primer R: 5'-TCGAGGCCTGCTTCCTGTTA-3' (shown in SEQ ID NO: 8-9) was used for PCR amplification. The reaction program during the amplification process is shown in Table 1, and the corresponding PCR amplification products were obtained. The PCR amplification products were subjected to Sanger sequencing. The sequencing results were compared with the target site sequence edited by CAS9 protein on the MYBR47 gene (shown in SEQ ID NO: 1), and plants with heterozygous or homozygous frameshift mutations in the target site region were selected.

[0036] Table 1 PCR reaction procedure

[0037] temperature time 95℃ 3min 95℃ 30s 60℃ 30s 72℃ 1min Go to step2 34cycles 72℃ 5min 16℃ ∞

[0038] Among them, the results of homozygous mutation types are as follows Figure 2A obtained two types of mybr47 homozygous mutations, named -76bp and +77bp (WT is maize inbred line B73). The MYBR47 genes of the two homologous chromosomes of -76bp had the same mutation, and there was a 76-base deletion in the MYBR47 genes on both homologous chromosomes, which caused frameshift and premature termination of protein translation, resulting in the loss of MYBR47 protein function. The MYBR47 genes of the two homologous chromosomes of +77bp had the same mutation, and there was a 77-base insertion in the MYBR47 genes on both homologous chromosomes, which caused frameshift and premature termination of protein translation, resulting in the loss of MYBR47 protein function.

[0039] (3) Identification of MYBR47 overexpressing plants

[0040] The genomic DNA of T1 transgenic corn leaves was used as a template. Since the overexpression vector was fused with a flag tag, the identification primers were designed to combine the primers on the gene with the primers on the flag. Specifically, the primer pair consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-CTTGTCATCGTCATCCTTG-3' (shown in SEQ ID NO: 10-11) was used for PCR amplification. The reaction procedure during the amplification process is shown in Table 1, and the corresponding PCR amplification products were obtained. According to the amplification results, the plants with the correct bands were selected. At the same time, quantitative PCR primers were designed, specifically the primer pair consisting of primer F: 5'-CATCAAGTCTGCTGAGGAGG-3' and primer R: 5'-GTACGGCACGTGGCCTGATTC-3' (shown in SEQ ID NO: 12-13) was used for QPCR detection, and the plants with significantly increased MYBR47 expression were listed as overexpression materials and named OE1 and OE2 respectively.

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

[0042] The planting materials were B73, -76bp, +77bp, OE1 and OE2, and they were self-pollinated. The grains in the middle of the mature ear were taken to observe the grain size. Specifically, 10 grains were randomly selected and placed side by side, and photos were taken to observe the changes in width and length. At the same time, the 100-grain weight of the grains was measured. The results are as follows: Figure 2 B and Figure 2 As shown in E, the 100-grain weight of the mutant grains was significantly increased, while that of the overexpressed grains was significantly decreased.

[0043] The protein content of the grains was determined by near infrared spectroscopy. Figure 2 C and Figure 2 As shown in F, the protein content of the mutant grains was significantly increased, while the protein content of the overexpression grains was significantly decreased.

[0044] Example 3 MYBR47 interacts with 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 and CCS52b, CDC202, CDC45a, CDC45b, CNR1 and CNR5 were transformed into the Y187 yeast strain, respectively, and yeast one-hybrid detection was performed. The results are shown in Figure 3 As shown, it was shown that MYBR47 interacted with the cell division-related genes CCS52b, CDC202, CDC45a, CDC45b, CNR1, and CNR5.

[0046] Example 4 Analysis of the optimal haplotype of the gene MYBR47 regulating grain size

[0047] The R package geneHapR was used to perform association analysis on 100-grain weight using SNPs in the MYBR47 gene and the upstream and downstream 1000 bp segments. Figure 4 As shown in A, under the 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 materials, and the second haplotype represented by C103 included 44 materials. Statistical analysis showed that the 100-grain weight was significantly different between haplotype 1 and haplotype 2 (P = 0.018). These results prove that the second haplotype Hap2 of the MYBR47 gene is an excellent allele for 100-grain weight.

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

Claims

1. Application of corn MYBR47 gene in regulating grain traits and variety breeding, characterized by: The sequence of the MYBR47 gene is shown in SEQ ID NO:

1.

2. The use of the maize MYBR47 gene in regulating grain traits and variety breeding according to claim 1, characterized in that: Including its application in regulating grain size traits, grain size variety or line breeding.

3. The use of the maize MYBR47 gene in regulating grain traits and variety breeding according to claim 1, characterized in that: The invention includes the application in regulating the total starch and / or protein content of grains, and the application in breeding varieties or strains with different total starch and / or different protein content of grains.

4. The use of the maize MYBR47 gene in regulating grain traits and variety breeding according to claim 3, characterized in that: By overexpressing and / or knocking out the MYBR47 gene, varieties with different total starch and / or protein content in the grain can be cultivated.

5. A method for cultivating a MYBR47 transgenic plant, characterized in that: The following steps are involved: (1) Construction of overexpression vector: Using genomic DNA from leaves of B73 plants as a template, a primer pair consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-GGTCCAGAATCCATGTGTCT-3', as shown in SEQ ID NO: 6-7, was used for PCR amplification to obtain the corresponding PCR amplification product; the PCR amplification product was connected to 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 delivered to a gene company for transformation; (2) Acquisition and identification of MYBR47 homozygous mutants The genomic DNA of T1 transgenic corn leaves was used as a template, and a primer pair consisting of primer F: 5'-GTCCCTTGGCTCTGAAACCA-3' and primer R: 5'-TCGAGGCCTGCTTCCTGTTA-3', as shown in SEQID NO: 8-9, was used for PCR amplification. The reaction procedure during the amplification process is shown in the following table, and the corresponding PCR amplification products were obtained; the PCR amplification products were subjected to Sanger sequencing. The sequencing results were compared with the target site sequence edited by the CAS9 protein on the MYBR47 gene, and plants with heterozygous or homozygous frameshift mutations in the target site region were selected; (3) Identification of MYBR47 overexpressing plants The genomic DNA of T1 transgenic corn leaves was used as a template. Since the overexpression vector was fused with a flag tag, the identification primers were designed to combine the primers on the gene with the primers on the flag, specifically a primer pair consisting of primer F: 5'-ATGTCCGCCTCACGCAGCTC-3' and primer R: 5'-CTTGTCATCGTCATCCTTG-3', as shown in SEQ ID NO: 10-11, and PCR amplification was performed. The reaction procedure during the amplification process was shown in the table above, and the corresponding PCR amplification product was obtained; according to the amplification results, the plants with the correct bands were selected. At the same time, quantitative PCR primers were designed, specifically a primer pair consisting of primer F: 5'-CATCAAGTCTGCTGAGGAGG-3' and primer R: 5'-GTACGGCACGTGGCCTGATTC-3', as shown in SEQ ID NO: 12-13, and QPCR detection was performed. Plants with significantly increased MYBR47 expression levels were MYBR47 transgenic plants.

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