Corn ZmMYBR37 gene and application thereof in regulation and control of corn kernel size

Abstract

The invention discloses a corn ZmMYBR37 gene and an application of the corn ZmMYBR37 gene in regulating and controlling the size of corn kernels. The nucleotide sequence of the ZmMYBR37 gene is as shown in SEQ ID No: 1 in a sequence table. A mutant material with small grains and reduced hundred-grain weight is separated from a corn EMS mutant library, and a mutation site occurs on a function unknown gene ZmMYBR37, so that protein translation is terminated in advance. The information analysis finds that the gene coding protein has a conservative MYB structural domain, and the gene coding protein is speculated to belong to an MYB transcription factor family. Gene expression analysis finds that the gene is high in constitutive expression in corn and high in expression level in leaves and embryos. The corn material overexpressed with the ZmMYBR37 has the advantages that the grain size is increased, and the hundred-grain weight is obviously increased, so that the ZmMYBR37 has the potential of increasing the corn yield.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological genes, and particularly relates to a corn ZmMYBR37 gene and application thereof in regulating corn kernel size. BACKGROUND

[0002] Corn is the largest crop in planting area in China, and is an important food, feed and industrial raw material, which plays an important role in guaranteeing global food security, economic development and energy utilization. With the increase of global population and rapid economic development, the demand for corn is increasing, and the increase of abiotic stress caused by global climate change brings severe challenges to corn production. Improving corn yield is an important strategy to guarantee food and energy supply.

[0003] Kernel size is an important factor affecting corn yield, and excavating key genes for regulating corn kernel size lays a foundation for cultivating high-yield corn new varieties. Transcription factors affect the growth and development process of plants by regulating the transcription level of downstream target genes. MYB transcription factors are a class of transcription factors containing MYB domains, which widely exist in animals and plants, and are reported to be involved in biological processes such as cell differentiation, secondary metabolism and stress response. However, there is no report on MYB transcription factors regulating kernel size in corn. SUMMARY

[0004] The purpose of the present application is to provide a corn ZmMYBR37 gene and application thereof in regulating corn kernel size.

[0005] The corn ZmMYBR37 gene, the polynucleotide of the ZmMYBR37 gene is as shown in (a), (b), (c) or (d): (a) the polynucleotide as shown in SEQ ID No: 1; or (b) a polynucleotide capable of hybridizing to the complement of SEQ ID No: 1 under stringent hybridization conditions, the protein encoded by the polynucleotide still has the function of regulating corn kernel size; (c) a polynucleotide having at least 90% or more homology with the polynucleotide as shown in SEQ ID No: 1; or (d) a polynucleotide mutant obtained by deleting, substituting or inserting one or more bases on the basis of the polynucleotide as shown in SEQ ID No: 1, and the protein encoded by the polynucleotide mutant still has the function of regulating corn kernel size.

[0006] The ZmMYBR37 protein, the amino acid sequence of the ZmMYBR37 protein is as shown in (a), (b) or (c): (a) the amino acid sequence shown in SEQ ID No: 2 in the sequence listing; or (b) an amino acid having at least 90% or more identity with the amino acid represented by SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids in the protein shown in SEQ ID No: 2, and the protein still has the function of regulating corn kernel size.

[0007] Amplify the ZmMYBR37 Primers for any fragment of the gene.

[0008] Containing the ZmMYBR37 Recombinant gene expression vector.

[0009] described ZmMYBR37 Application of the gene or the ZmMYBR37 protein in regulating corn kernel size.

[0010] A method for enlarging corn kernels, ZmMYBR37 The gene is overexpressed in maize plants.

[0011] Beneficial effects of the present invention: The present invention isolates mutant materials with smaller grains and reduced 100-grain weight from the corn EMS mutant library, and the mutation site occurs in a gene with unknown function. ZmMYBR37 The protein encoding this gene has a conserved MYB domain, which indicates that it belongs to the MYB transcription factor family. Gene expression analysis found that this gene is constitutively expressed in maize, with high expression levels in leaves and embryos. ZmMYBR37 The kernel size and 100-kernel weight of corn materials increased significantly, indicating that ZmMYBR37 Has the potential to increase corn yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 For corn ZmMYBR37 Sequence and tissue expression characteristics; in the figure, A-maize ZmMYBR37 Genome sequence information and ZmMYBR37 Mutation site; B- ZmMYBR37 Sequencing analysis of genomic sequences at the mutation site; C- Analysis of the conserved domain of ZmMYBR37 protein; D- ZmMYBR37 Gene expression levels in different tissues.

[0013] Figure 2 For wild type and ZmMYBR37 Development of mutant seedlings, plants, and grains; Figure A: Wild type (WT) and mutant ( ZmMYBR37 ) seedlings; Bar = 2.5 cm; B-WT andZmMYBR37 Plant (Bar = 50 cm) and leaf development and mature stage WT and ZmMYBR37 Ear (Bar = 3 cm) and kernel (Bar = 1.5 cm).

[0014] Figure 3 To ZmMYBR37 Overexpression of maize seedling stage ZmMYBR37 Expression level analysis (A) and mature stage kernel phenotype and kernel length, kernel width and 100-kernel weight analysis, Bar = 2 cm (B); **, p<0.05. DETAILED DESCRIPTION

[0015] For the purpose of promoting an understanding of the principles of the application, reference will now be made to the embodiments illustrated in the drawings. It is expressly understood that the drawings are for illustration only and that the application can be embodied in many different forms. In the drawings:

[0016] Example 1 Maize ZmMYBR37 Mutant analysis Maize wild type and ZmMYBR37 Mutant material planting: maize inbred line B73 was used as wild type control material, maize ZmMYBR37 Mutant (EMS4107afd) was ordered from maize EMSDB mutant library, wild type and ZmMYBR37 Mutant seeds were sown in planting pots respectively, and the wild type and mutant seedlings were cultured in a greenhouse with temperature of 25℃ and light cycle of 12h light / 12h dark. When the seedlings grew for 14 days, part of the aboveground stem and leaf tissue and underground root tissue of the wild type and mutant seedlings were taken respectively for weighing analysis of the biomass of the seedlings. The size of the mature kernels and the 100-kernel weight were observed after the maize ear matured. The phenotype observation was set with at least 3 biological replicates.

[0017] Maize ZmMYBR37 Mutant genotype identification: based on the genomic sequence of Zm00001d042756, primers zmmybr37F1: 5'-AGCCGCCGCGGACTTGGTTG-3' (SEQ ID NO: 3) and zmmybr37R1: 5'-GCGAGGCATGGACTTCCAC-3' (SEQ ID NO: 4) were designed on the upstream and downstream of the mutation site, and the DNA of the stem and leaf tissue of the wild type and ZmMYBR37 Mutant, PCR amplification and sequencing were performed, and the sequence differences of the amplification products of the wild type and mutant were compared.

[0018] Maize ZmMYBR37 Mutant mutation site verification and ZmMYBR37 molecular characteristic analysis: the mutant library information showed ZmMYBR37The mutation site is located in the gene Zm00001d042756 on Chr3, and the G at position 137 in the coding region is mutated to A, resulting in the mutation of tryptophan at position 46 in the encoded protein to a stop codon, and the premature termination of protein translation Figure 1 A). The DNA sequence of the mutant seedlings was extracted for genotype identification, and the sequencing results showed ZmMYBR37 The mutation site is consistent with the prediction information of the mutant library Figure 1 B). The encoded protein of Zm00001d042756 has a conserved MYB domain and belongs to MYB-related transcription factor 37 (MYBR37) Figure 1 C), and the qTeller database shows that the gene is constitutively expressed in maize, with high expression levels in leaves and embryos Figure 1 D), but the biological function of maize MYBR37 has not been reported.

[0019] Compared with the wild type, the aboveground and underground biomass of the maize ZmMYBR37 mutant seedlings was significantly reduced Figure 2 A). At the tasseling stage, the average plant height of the wild type maize was 238±5 cm, while the plant height of the maize ZmMYBR37 mutant was significantly reduced, with an average of 196±6 cm; and the leaf of the maize ZmMYBR37 mutant was significantly smaller than that of the wild type, with the width of the wild type ear leaf being 9±0.5 cm, while the width of the ear leaf of the maize ZmMYBR37 mutant was 6.4±0.7 cm; the mature ear and kernel of the maize ZmMYBR37 mutant were significantly smaller than those of the wild type, with the kernel length and width of the wild type being 1.1±0.2 cm and 0.71±0.1 cm, respectively, while the kernel length and width of the maize Figure 2 mutant were 0.7±0.1 cm and 0.71±0.1 cm, respectively ZmMYBR37 B). The 100-kernel weight of the wild type maize was 18.5±0.5 g, while the 100-kernel weight of the maize Figure 2 mutant was significantly reduced, with an average of 13±1.1 g B).

[0020] Example 2 Overexpression of Maize ZmMYBR37 Plant Analysis RNA was extracted from leaves of wild-type maize materials, and cDNA was obtained by reverse transcription as a template. Based on the coding region sequence of the Zm00001d042756 gene, primers ZmMYBR37F1: 5'-ATGGACTTCCACCACGGCTG-3' (SEQ ID NO: 5) and ZmMYBR37R1: 5'-TCAAGGGGTGGTGATGTCGTG-3' (SEQ ID NO: 6) were designed, and the coding region of ZmMYBR37 was obtained by PCR amplification. ZmMYBR37 The CDS sequence was constructed into the pCAMBIA3301 vector and driven by the maize Ubi promoter. ZmMYBR37 Ubi-ZmMYB37-pCAMBIA3301 was expressed and introduced into maize B73 inbred line using Agrobacterium transformation. Thirteen positive homozygous transformation events were obtained through three consecutive generations of identification and planting. Transgenic plants were grown in the greenhouse, and leaf materials were extracted and identified using primers ZmMYBR37SF1: 5'-TCAAGGGCTGGAGAAGTACG-3' (SEQ ID NO: 7) and ZmMYBR37SR1: 5'-TGAGCTGCCGGTTGAAGTA-3' (SEQ ID NO: 8). ZmMYBR37 Transcriptional levels of maize ZmMYBR37 Grains harvested after ripening of the overexpressing plants were subjected to grain phenotype and 100-grain weight analysis.

[0021] Detection of ZmMYBR37 overexpression lines in maize using qPCR ZmMYBR37 The transcriptional levels of MYBR37-OE1, MYBR37-OE3 and MYBR37-OE13 plants were found to be Figure 3 The gene transcription level is high ( Figure 3 Compared with the wild type, the mature grains of MYBR37-OE1, MYBR37-OE3, and MYBR37-OE13 were significantly larger (p<0.05). The length and width of the wild type grains were 0.8±0.2 cm and 0.71±0.05 cm, respectively. The length and width of the grains in the overexpression lines were 1.0±0.2 cm and 0.76±0.03 cm, respectively. Compared with the wild type, the 100-grain weight of the grains in the ZmMYBR37 overexpression lines was significantly increased (p<0.05). The 100-grain weight of the wild type grains was 18.7±1.2 g, while the 100-grain weight of the grains in MYBR37-OE1, MYBR37-OE3, and MYBR37-OE13 grains were 22.3±0.8 g, 23.2±0.5 g, and 23.6±0.3 g, respectively. ZmMYBR37 Middle B), indicating overexpression ​ Has the potential to increase corn yields.

[0022] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Corn ZmMYBR37 A gene characterized by described ZmMYBR37 The polynucleotide of the gene is shown as (a), (b), (c) or (d): (a) the polynucleotide shown in SEQ ID No: 1 in the sequence listing; or (b) a polynucleotide that can hybridize with the complementary sequence of SEQ ID No: 1 under stringent hybridization conditions, wherein the protein encoded by the polynucleotide still has the function of regulating corn kernel size; (c) a polynucleotide having at least 90% homology with the polynucleotide represented by SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases in the polynucleotide represented by SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating corn kernel size.

2. ZmMYBR37 protein, characterized in that The amino acid sequence of the ZmMYBR37 protein is shown in (a), (b) or (c): (a) the amino acid sequence shown in SEQ ID No: 2 in the Sequence Listing; or (b) an amino acid having at least 90% or more identity with the amino acid represented by SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids in the protein shown in SEQ ID No: 2, and the protein still has the function of regulating corn kernel size.

3. Amplify the method according to claim 1 ZmMYBR37 Primers for any fragment of the gene.

4. Containing the composition according to claim 1 ZmMYBR37 Recombinant gene expression vector.

5. The method according to claim 1 ZmMYBR37 Use of the gene or the ZmMYBR37 protein according to claim 2 in regulating corn kernel size.

6. A method for enlarging corn kernels, characterized in that: Will ZmMYBR37 The gene is overexpressed in maize plants.

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