Application of corn korn9 protein or its coding gene in regulating corn ear row number and molecular marker related to corn ear row number

Abstract

The present application relates to the field of biotechnology, and discloses application of corn KRN9 protein or its coding gene in regulating corn ear row number and a molecular marker related to corn ear row number. KRN9 The present application finds that overexpression of the corn KRN9 protein or its coding gene can increase the ear row number of corn, and further provides application of the corn KRN9 protein or its coding gene or biological material containing the coding gene in regulating the ear row number of corn. In addition, the present application also finds a molecular marker related to the ear row number of corn. The present application provides a new method for regulating the ear row number of corn, and is helpful for promoting the development of molecular breeding technology of corn yield.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to application of corn KRN9 protein or its encoding gene in regulating corn ear row number and molecular markers related to corn ear row number. Background Art

[0002] Maize (Zea mays L.) is a major crop that combines food, feed, energy, and industrial raw materials. With the ever-increasing demand for food and energy, further increasing maize yield is a major challenge. Ear row number is a key factor in determining maize yield and is closely related to yield per plant. It is also an important quantitative trait with high heritability, making it a valuable tool for breeding high- and stable-yielding maize varieties. Therefore, identifying new genes associated with ear row number in maize has important theoretical and practical value for molecular design and breeding of maize. Currently, a number of genes associated with ear row number have been discovered in maize through a combination of mutant studies and QTL mapping, and a small number have been confirmed to be applicable to genetic improvement in maize breeding.

[0003] Previous studies have cloned several genes that influence ear row number and elucidated the associated regulatory pathways, but research on epigenetic modifications affecting ear row number is limited. Over 80% of proteins in eukaryotes undergo N-terminal acetylation, yet limited research exists on the role of this modification in regulating crop agronomic traits, with no reports on the role of related genes in ear row number. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a new method for regulating the number of rows of corn ears.

[0005] The present invention provides the use of corn KRN9 protein or its encoding gene, or biological materials containing the encoding gene, in regulating the number of corn ear rows, in breeding corn with a large number of ear rows, and / or in improving high-yield corn germplasm resources.

[0006] The present invention screened a new gene that regulates the number of rows of ears KRN9 , which encodes an N-terminal acetyltransferase that positively regulates the number of corn ear rows.

[0007] By overexpression in maize KRN9 Gene that increases the number of rows of ears in corn.

[0008] In the application of the present invention, the amino acid sequence of the maize KRN9 protein is shown in SEQ ID NO: 3;

[0009] and / or, the DNA sequence encoding the maize KRN9 protein is shown in SEQ ID NO: 1;

[0010] And / or, the biological material is an expression cassette, a vector or a host cell.

[0011] The present invention also provides a method for constructing corn with a large number of ear rows, wherein the corn is expressed or overexpressed by genetic modification, hybridization, backcrossing, selfing or asexual reproduction. KRN9 Gene; the corn KRN9 The CDS sequence of the gene is shown in SEQ ID NO: 2.

[0012] The present invention also provides a molecular marker associated with the number of corn ear rows, which contains a nucleotide sequence in which the polymorphism at position 1963 of the sequence shown in SEQ ID NO: 1 is G / C; when the polymorphic site of the molecular marker is G, it corresponds to a large number of corn ear rows; when the polymorphic site of the molecular marker is C, it corresponds to a small number of corn ear rows.

[0013] The SNP (single nucleotide polymorphism) molecular marker site associated with the number of corn ear rows provided by the present invention is derived from the corn gene KRN9 (Zm00001d044446) is located at the 1963rd nucleotide from the 5' start end of SEQ ID NO. 1 (including its flanking sequences) listed in the present invention, and the nucleotide at this position is G or C. This SNP molecular marker is significantly associated with the number of corn ear rows. Corn germplasm resources with the GG genotype at the SNP site have more ear rows than corn germplasm resources with the CC genotype at the site.

[0014] The present invention also provides a specific primer set for detecting the above molecular markers, which are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0015] The present invention also provides a reagent or a kit containing the above-mentioned specific primer set.

[0016] The present invention also provides any of the following applications of the above-mentioned molecular marker or specific primer set or reagent or kit:

[0017] (1) Application in identifying the number of rows of corn ears;

[0018] (2) Application in molecular marker-assisted breeding of maize with many ears and rows;

[0019] (3) Application in the improvement of germplasm resources for the number of rows of corn ears.

[0020] The present invention also provides a method for identifying the phenotype of the number of ear rows of corn, which uses the above-mentioned specific primer set to perform PCR amplification on the genomic DNA of the corn to be tested, and analyzes the genotype of the above-mentioned molecular marker in the PCR amplification product. If the genotype of the polymorphic site of the molecular marker is GG, the number of ear rows of the corn to be tested is large; if the genotype of the polymorphic site of the molecular marker is CC, the number of ear rows of the corn to be tested is small.

[0021] The beneficial effects of the present invention are at least:

[0022] The present invention provides a new gene for regulating the number of corn ear rows KRN9 , and found a SNP marker related to the number of corn ear rows in this gene, which can provide a new means for high-yield corn breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 图1 Corn in the embodiment of the present invention KRN9 Results of gene function research. In the figure, A is the construction KRN9 Schematic diagram of overexpression vector. B is the overexpression material KRN9 Relative gene expression. C shows representative photos of the number of ear rows in representative overexpression materials OE1 and OE2 and the control material WT. D shows the statistical results of the number of ear rows in the overexpression materials and the control material.

[0024] 图2 Corn in the embodiment of the present invention KRN9 The results of gene expression pattern analysis. KRN9 Tissue expression in B73. B shows the in situ hybridization results in B73 young ears, with a scale bar of 200 μm.

[0025] 图3 Figure 1 shows the results of a protein characterization analysis of maize KRN9, as described in the examples of the present invention. Figure A shows the clustering of KRN9 protein sequences with N-terminal acetyltransferases from other monocots. Figure B shows the KRN9 protein localization results, with the top row showing results from nuclear localization experiments and the bottom row showing results from endoplasmic reticulum localization experiments. Scale bar: 20 μm.

[0026] 图4 Corn in the embodiment of the present invention KRN9 The haplotype analysis results of the gene. In the figure, A is carried in the AM368 population KRN9 The statistical results of the number of ear rows of materials with different haplotypes. KRN9 Relative expression results in young ears carrying different haplotypes.

[0027] In each figure (if any), represent P ≤0.05, represent P ≤0.01, represent P ≤0.001, represent P ≤0.0001. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0030] Example 1 Corn KRN9 Gene function research

[0031] Using maize inbred line B73 cDNA as template, primers SEQ ID NO: 6 and SEQ ID NO: 7 were used to generate the KRN9 The coding region of the gene was amplified and connected to the overexpression vector (CaMV35S promoter, pCambia3301 vector backbone) ( 图1 A in ), get KRN9 Gene overexpression vector.

[0032] described KRN9 The gene is located at the end of chromosome 3, and its DNA sequence is shown in SEQ ID NO: 1; the DNA sequence corresponding to the CDS is shown in SEQ ID NO: 2; and the amino acid sequence is shown in SEQ ID NO: 3.

[0033] The above KRN9 The gene overexpression vector was transformed into Agrobacterium, and the KRN9 The gene overexpression vector was transformed into the maize inbred line B104, and the OE lines were positively detected using primers SEQ ID NO: 8 and SEQ ID NO: 9, obtaining four independent T1 generation positive transgenic maize ears.

[0034] RNA was extracted from the young ears of the T2 generation positive transgenic materials of the above transgenic lines (OE1-OE4) and the recipient material B104, and the primers SEQ ID NO:4 and SEQ ID NO:5 were used to determine the expression of the nucleotide sequence of the transgenic material. KRN9 The results showed that the expression level of each transgenic material KRN9 The expression levels of genes were significantly increased compared with the control B104 (WT) ( 图1 B in ).

[0035] The ear-row phenotype of negative and positive transgenic materials of T3 generation of OE1-OE4 was identified. The statistical results showed that the overexpression KRN9 The number of ear rows of the genetic material grains was significantly greater than that of the control material (WT) (Table 1 and 图1 C and D in the figure indicate KRN9 The gene has a positive regulatory effect on the number of rows per ear trait.

[0036] Table 1 Phenotypic statistical results of ear row number

[0037]

[0038] Example 2 Corn KRN9 Gene expression pattern analysis

[0039] Using the public data of maize inbred line B73 sequencing (Chen et al., Plant Physiol ., 2014,166:252-264), preliminary analysis KRN9 The tissue expression characteristics of α-amylase were analyzed, and it was found that α-amylase was highly expressed in tissues such as the shoot apex meristem, spikes, grains, and leaves ( 图2 A in ).

[0040] Further, B73 young ears with a length of about 2 mm were taken and designed for KRN9 mRNA probes, using SEQ ID NO: 10 (in situ hybridization sense probe) and SEQ ID NO: 11 (in situ hybridization antisense probe) to conduct in situ hybridization experiments, found KRN9 Expressed in the inflorescence meristem (IM) and spikelet pair meristem (SPM) 图2 B in the figure), which is consistent with its function of regulating the number of ear rows.

[0041] Example 3 Analysis of properties of corn KRN9 protein

[0042] The protein sequence of KRN9 (SEQ ID NO: 3) was compared by BLAST-P and found to be homologous to the N-terminal acetyltransferase reported in Arabidopsis thaliana. Phylogenetic tree analysis showed that it is highly conserved among plants ( 图3 A in ).

[0043] To determine the subcellular localization of KRN9, the CDS fragment of the KRN9 gene was amplified using primers SEQ ID NO:12 and SEQ ID NO:13, and yellow fluorescent protein (YFP, NCBI accession number BBL45167) was fused to the 3′ end of the KRN9 protein. The fusion vector was injected into tobacco leaves via Agrobacterium to transiently express the KRN9 protein, and the fluorescence signal was detected under a confocal laser microscope. The results showed that KRN9 was located in the nucleus, cytoplasm, and endoplasmic reticulum ( 图3 (B) is consistent with the localization results of acetyltransferase in Arabidopsis thaliana.

[0044] Example 4 Corn KRN9 A SNP molecular marker significantly associated with the number of ear rows

[0045] Based on the widely available maize inbred line materials (materials with SNP marker genotypes and ear row number phenotypes in the AM368 population, Li et al., Plant J ., 2022,111:1595-1608) genome-wide SNP markers (Li et al., Plant J ., 2022,111:1595-1608; Fu et al ., Nat Commun ., 2013, 4:2832) as genotype and number of ear rows as phenotype, the general linear model of association analysis software FastQTL was used to identify KRN9 The significant lead SNP in the second intron of the gene is S3_228294434 (located at nucleotide 228294434 on chromosome 3 of the B73 V4 genome, KRN9 1963rd nucleotide of gene (SEQ ID NO: 1)) P This locus has two genotypes, GG and CC. Based on this SNP marker, haplotype analysis of the number of ear rows in the AM368 population was conducted, and it was found that the number of ear rows in materials carrying the GG genotype was significantly more than that in materials carrying the CC genotype ( 图4 GG is considered a superior / enhancing allele.

[0046] Seven inbred lines with extreme ear row numbers (carrying GG and CC alleles, respectively) were selected, and RNA was extracted from young ears of about 2 mm in length. Real-time fluorescence quantitative PCR experiments were performed using primers SEQ ID NO: 4 and SEQ ID NO: 5 to detect KRN9 The expression level of the internal reference gene is ZmActin (Zm00001d010159). The results showed that in the young panicles carrying the GG haplotype, KRN9 The expression level of 图4 B in ).

[0047] This example further selected four corn varieties known in the art: B73 (number of ear rows 16.6), Dan340 (Dan 340, number of ear rows 17.1), 18-599 (number of ear rows 15.0), and Mo17 (number of ear rows 10.4). DNA was extracted from each of them, and PCR amplification was performed with primers SEQ ID NO: 4 and SEQ ID NO: 5. The amplified products were sequenced to obtain the genotypes of the polymorphic sites of the above-mentioned SNP molecular markers of the present invention in each material. The results showed that the genotypes of the corn varieties B73 and Dan340 with many ear rows at the polymorphic sites of the above-mentioned SNP molecular markers were G, while the genotypes of the corn varieties 18-599 and Mo17 with few ear rows at the polymorphic sites of the above-mentioned SNP molecular markers were C. The SNP molecular markers of the present invention can accurately determine varieties with many ear rows.

[0048] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Application of maize KRN9 protein or its encoding gene, or biological materials containing its encoding gene, in regulating the number of maize ear rows; The specific method for regulating the number of corn ear rows is: The expression level of corn KRN9 protein is increased in corn to increase the number of corn ear rows, wherein the amino acid sequence of the corn KRN9 protein is shown in SEQ ID NO: 3; Alternatively, the corn is provided with a nucleotide sequence in which the polymorphism at position 1963 of the sequence shown in SEQ ID NO: 1 is G, so as to increase the number of corn ear rows; Alternatively, the corn may contain a nucleotide sequence in which the polymorphism at position 1963 of the sequence shown in SEQ ID NO: 1 is C to reduce the number of corn ear rows.

2. Application of maize KRN9 protein or its encoding gene, or biological materials containing its encoding gene, in breeding maize with a large number of ear rows; The specific method for breeding corn with many ear rows is: Select corn with high expression of corn KRN9 protein, wherein the amino acid sequence of the corn KRN9 protein is shown in SEQ ID NO: 3; Alternatively, corn containing the nucleotide sequence shown in SEQ ID NO: 1, wherein the polymorphism at position 1963 is G, is selected.

3. Application of maize KRN9 protein or its encoding gene, or biological materials containing its encoding gene, in improving high-yield maize germplasm resources; The specific method for improving the high-yield corn germplasm resources is: The expression level of corn KRN9 protein is increased in corn, wherein the amino acid sequence of the corn KRN9 protein is shown in SEQ ID NO: 3; Alternatively, the corn contains a nucleotide sequence in which the polymorphism at position 1963 is G as shown in SEQ ID NO:

1.

4. The use according to any one of claims 1 to 3, characterized in that The DNA sequence encoding the maize KRN9 protein is shown in SEQ ID NO: 1; And / or, the biological material is an expression cassette, a vector or a host cell.

5. A method for constructing corn with a large number of ear rows, characterized in that: Through transgenic, hybridization, backcrossing, selfing or asexual reproduction, corn expresses or overexpresses corn KRN9 Gene; the corn KRN9 The CDS sequence of the gene is shown in SEQ ID NO:

2.

6. A molecular marker associated with the number of corn ear rows, characterized in that: It contains a nucleotide sequence with a polymorphism of G / C at position 1963 as shown in SEQ ID NO: 1; when the polymorphic site of the molecular marker is G, it corresponds to more rows of corn ears; when the polymorphic site of the molecular marker is C, it corresponds to fewer rows of corn ears.

7. A specific primer set for detecting the molecular marker according to claim 6, characterized in that: As shown in SEQ ID NO:4 and SEQ ID NO:

5.

8. A reagent or kit comprising the specific primer set according to claim 7.

9. Any of the following uses of the molecular marker according to claim 6, the specific primer set according to claim 7, or the reagent or kit according to claim 8: (1) Application in identifying the number of rows of corn ears; (2) Application in molecular marker-assisted breeding of maize with many ears and rows; (3) Application in the improvement of germplasm resources for the number of rows of corn ears.

10. A method for identifying the phenotype of corn ear rows, characterized in that: Using the specific primer set described in claim 7, PCR amplification is performed on the genomic DNA of the corn to be tested, and the genotype of the molecular marker described in claim 6 in the PCR amplification product is analyzed. If the genotype of the polymorphic site of the molecular marker is GG, the number of ear rows of the corn to be tested is large; if the genotype of the polymorphic site of the molecular marker is CC, the number of ear rows of the corn to be tested is small.

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