SNP molecular marker related to corn leaf width, negative regulatory factor and application thereof

Abstract

The application belongs to the field of crop genetic breeding, and relates to a corn leaf width regulating factor, in particular to a SNP molecular marker related to corn leaf width regulation, a negative regulating factor and application thereof. Zm OXS3) is a protein composed of 171 amino acids, encoding oxidative stress 3 related protein, and its molecular biological function has not been reported in corn. The application studies the molecular biological function of the gene in regulating corn leaf width, and provides a potential new gene resource for breeding new excellent high-yield corn varieties with suitable leaf width.

Description

Technical Field

[0001] This application belongs to the field of crop genetics and breeding, and relates to the regulatory factors of maize leaf width, specifically a SNP molecular marker and negative regulatory factor related to the regulation of maize leaf width and their applications. Background Technology

[0002] Corn, as one of my country's three major staple foods, plays a crucial role in the country's food security. Corn planting density is a key factor influencing yield per unit area. Reasonable planting density primarily increases yield by improving the effective utilization of light energy within the plant population, optimizing leaf area index and plant growth relationships, and leveraging the population's yield advantages. To achieve reasonable planting density, selecting a suitable plant type becomes paramount, leading to the concept of an ideal plant type. Through years of practice and exploration, people have continuously summarized and generalized the ideal plant type, ultimately reaching a consensus: leaves above the ear point upwards with a smaller and narrower leaf angle, which is beneficial for improving light distribution within the plant population; leaves below the ear have a larger and more expansive leaf angle, effectively intercepting light energy. Generally speaking, breeders typically select breeds based on traits such as leaf type, root type, stem type, and ear type, as these traits are relatively easy to observe directly with the naked eye, improving breeding efficiency. In corn, leaf size and shape influence the canopy structure of the corn population, thereby affecting organic matter synthesis; therefore, they are important components in regulating plant type. In cornfields, if the spacing between corn plants is too small, their leaves will crowd and overlap, resulting in the lower leaves receiving almost no sunlight, and even the upper leaves not being able to completely block sunlight. This easily induces shade-avoidance syndrome (SAS), which can lead to significant yield reduction in severe cases. Variations in leaf width affect leaf shape, and mitigating the shading effect between individual plants can be addressed by selecting plant types with appropriate leaf widths.

[0003] Leaf width is a complex quantitative trait controlled by multiple genes. To date, numerous quantitative trait loci (QTLs) for leaf width have been identified using different genetic populations. Simultaneously, several genes and mutants associated with maize leaf width have been identified. For example, studies have found that NS1 and NS2 expression in SAM affects leaf polarity formation, with the mutants ns1 and ns2 exhibiting narrower mature leaves and leaf sheaths. RGD2 encodes an AGO7-type protein essential for the synthesis of ta-siARF, which regulates ARF transcription through ta-siARF, affecting auxin distribution and leading to narrower, curled leaves in its mutants. ZmNL4 encodes a kelch-repeat superfamily protein; knocking out ZmNL4 through CRISPR / Cas9 editing significantly reduces maize leaf width. Therefore, by identifying genes regulating maize leaf width and developing suitable germplasm for breeding new maize varieties with ideal plant architectures, reliable and superior germplasm can be provided for selecting maize varieties with ideal plant architectures. Summary of the Invention

[0004] To further explore key genes related to leaf width, this invention proposes a SNP molecular marker, a negative regulator, and their applications related to maize leaf width.

[0005] The technical solution of this invention is implemented as follows: A negative regulator of maize leaf width, the negative regulator encoding an Oxidativestress 3-related protein.

[0006] Furthermore, the negative regulatory factor is a nucleotide sequence that is more than 90% similar to the coding region of SEQ ID No. 1 from 1 to 516 bp.

[0007] Furthermore, the coding region sequence of the negative regulatory factor is shown as 1-516bp in SEQ ID No. 1, and is named... ZmOXS3 Gene.

[0008] The aforementioned SNP molecular marker is located at the 3'-UTR of the ZmOXS3 gene, at base C 243 downstream of the stop codon; the nucleotide sequence of the ZmOXS3 gene is the first 516 bp of SEQ ID No. 1; the downstream region of the stop codon is the last 517-1018 bp of SEQ ID No. 1.

[0009] Furthermore, a base mutation (SNP) exists downstream of the stop codon in the ZmOXS3 gene, such as... Figure 1 As shown.

[0010] The SNP molecular marker of the aforementioned negative regulatory factor is located downstream of the stop codon of ZmOXS3. One SNP (C / G) exists in the narrow-leaved inbred line Yu1122 and the broad-leaved inbred line 953. The SNP molecular marker is located at the 243rd base C downstream of the stop codon of the gene ZmOXS3.

[0011] The primer pair used to identify the above-mentioned SNP molecular markers is OXS3-F1 and OXS3-R1, wherein the OXS3-F1 sequence is shown in SEQ ID No. 2 and the OXS3-R1 sequence is shown in SEQ ID No. 3.

[0012] The application of the aforementioned negative regulatory factors in regulating ear height in maize involves the following steps: (1) Construct an overexpression vector for the gene ZmOXS3; (2) The overexpression vector from step (1) was transformed into Agrobacterium competent cells and used to transform maize inbred line B104 to obtain transgenic positive strains with narrower leaves than wild-type B104.

[0013] Preferably, in step (1), the overexpression vector is obtained by using the cDNA of the narrow-leaved inbred line Yu1122 as a template, and using primer pairs designed with the cDNA sequence of OXS3 of maize inbred line B73 as primers to amplify the cDNA of Yu1122 to obtain the cDNA of OXS3, and then constructing the overexpression vector of OXS3.

[0014] Preferably, the primer pair is OXS3-F2 and OXS3-R2, wherein the OXS3-F2 sequence is shown in SEQ ID No. 4 and the OXS3-R2 sequence is shown in SEQ ID No. 5.

[0015] The present invention has the following beneficial effects: 1. The regulatory factor involved in this application is gene Zm00001d038780 ( Zm OXS3 is a 171-amino acid protein encoding an oxidative stress 3-related protein, the amino acid sequence of which is shown in SEQ ID No. 6. Its molecular biological function has not yet been reported in maize. This invention investigated the molecular biological function of this gene in regulating maize leaf width and provides a potential new gene resource for breeding superior, high-yielding maize varieties with suitable leaf widths.

[0016] 2. The purpose of this invention is to provide a gene for controlling the width of maize leaves. ZmOXS3The candidate gene was accurately isolated using genome-wide association analysis. This gene possesses the DNA fragment shown in SEQ ID No. 1. Increased mRNA accumulation of this gene leads to narrower maize leaves; therefore, cloning this gene will contribute to understanding the molecular mechanisms underlying maize leaf width.

[0017] 3. This invention utilizes RNA overexpression technology to promote endogenous RNA expression in maize. ZmOXS3 Gene expression. Specifically, it refers to... ZmOXS3 Genes are fused with other regulatory elements, such as constitutive promoters (e.g., CaMV35S promoter) or organ-specific promoters, to construct gene expression vectors. Through transgenic technology, maize inbred lines with narrower leaves are created for breeding new narrow-leaved maize varieties.

[0018] 4. This invention will be based on Figure 1 Primers designed based on the SNP sequences shown, such as SEQ ID No. 2 and SEQ ID No. 3, can be applied to the identification of maize leaf width. This can predict the leaf width of different germplasm materials and has important practical application significance for maize breeding. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 for ZmOXS3 Evidence of regulation of maize leaf width: (A) Population structure analysis of an associated population of 375 inbred lines; (B) Genome-wide association analysis demonstrating a key region on chromosome 6 associated with leaf width; (C) Association analysis of candidate genes demonstrating a highly significant SNP associated with leaf width; (D) ZmOXS3 The haplotype analysis, the chart at the top shows ZmOXS3 A significantly associated SNP was found downstream of the stop codon. The bottom left plot compares leaf widths of different haplotype maize inbred lines. The bottom right plot compares the inbred lines of haplotype 1 and haplotype 2. ZmOXS3 Expression level. (E) Sequence analysis of the representative inbred line Yu1122 with narrow leaves and the representative inbred line 953 with broad leaves.

[0021] Figure 2Phenotypic identification of the Ds mutant line and the wild-type K17 showed that the leaves of the loss-of-function mutant were wider than those of the wild type. (A) Ds was inserted 317 bp upstream of the ZmOXS3 transcription start site; (B) The leaves of the Ds-inserted mutant were wider than those of the wild-type K17 (upper three leaves of the spike); (C) The expression level of ZmOXS3 in the mutant was significantly lower than that in the wild type.

[0022] Figure 3 The leaves of the ZmOXS3 overexpressing transgenic lines were narrower than those of the wild-type B104. (A) qRT-PCR analysis of ZmOXS3 showed that the expression level of the overexpressing transgenic lines was significantly higher than that of the wild-type B104; (B) The leaves of the ZmOXS3 overexpressing transgenic lines were narrower than those of the wild-type B104. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0025] A negative regulator of maize leaf width, wherein the negative regulator is a protein encoding Oxidativestress 3.

[0026] Furthermore, the negative regulatory factor is a nucleotide sequence that is more than 90% similar to the coding region of SEQ ID No. 1 from 1 to 516 bp.

[0027] Furthermore, the nucleotide sequence of the negative regulatory factor is shown as 1-516 bp in SEQ ID No. 1, and is named... ZmOXS3 Gene.

[0028] Furthermore, a base mutation (SNP) exists in the downstream region of the ZmOXS3 gene, such as... Figure 1 As shown.

[0029] The SNP molecular markers of the aforementioned negative regulatory factors, located downstream of ZmOXS3, show one SNP (C / G) in the narrow-leaved inbred line Yu 1122 and the wide-leaved inbred line 953. The SNP molecular markers are located in... ZmOXS3 The 243rd base C downstream of the stop codon of the gene; where ZmOXS3 The nucleotide sequence of the gene is the first 516 bp of SEQ ID No. 1; the downstream region is the last 517-1018 bp of SEQ ID No. 1.

[0030] Primer pairs used to identify the aforementioned SNP molecular markers, wherein the primer pairs are OXS3 -F1 and OXS3 -R1, where OXS3 The -F1 sequence is shown in SEQ ID No. 2. OXS3 The -R1 sequence is shown in SEQ ID No. 3.

[0031] OXS3 -F1:'5-TACCAGTACCACCAGTAGGCA-3'; OXS3 -R1: '5-ATGTTTTTCTGCCTGGACTGC-3'.

[0032] Example 1 The negative regulator of maize leaf width, ZmOXS3, exhibits polymorphism between the narrow-leaved maize inbred line Yu1122 and the wide-leaved inbred line 953, reflecting variations in leaf width. To further verify this, the following experiments were conducted: An associated population was constructed using 375 maize inbred lines with significant differences in leaf width. These 375 inbred lines were then sequenced using Illumina high-throughput resequencing, achieving an average genome coverage of 7.9-fold. By comparing the sequencing data with the B73 reference genome (V4), a total of 12,987,653 SNPs were identified. Based on these SNP data, genetic structure analysis was performed on the associated population. The 375 inbred lines were divided into four groups: the Lérid group, the Lancaster group, the Tangsipingtou group, and a germplasm group derived from American hybrids. Figure 1 A).

[0033] Leaf width was determined using 375 inbred lines in Zhengzhou over two seasons. Genome-wide association analysis (rMVP) was performed using a mixed linear model, and candidate regions on chromosome 6 with strong associations were identified. Figure 1 B). Within this region, a SNP significantly associated with leaf width was identified in a gene region containing one gene. Further association analysis of candidate genes showed that the strongest SNP significantly associated with leaf width was identified in the gene region of Zm00001d038780. Figure 1C). The candidate gene within this interval was initially identified as Zm00001d038780. The full-length gene can be searched on MaizeGDB using the gene ID Zm00001d038780. This gene encodes an Oxidative stress 3-related protein located on chromosome 6 and is named ZmOXS3. This gene consists of two exons and one intron. One SNP in the 3'-UTR region significantly affects maize leaf width. Using this single SNP, 375 inbred lines were divided into two haplotypes, and the difference in leaf width between the two haplotypes reached a significant level (…). Figure 1 D); Seven inbred lines were selected from the two haplotypes respectively to analyze the expression level of ZmOXS3, showing that the expression level of haplotype I was significantly lower than that of haplotype II ( Figure 1 D). Using the narrow-leaved inbred line Yu 1122 (haplotype II) and the wide-leaved inbred line 953 (haplotype I) as materials, the gene was cloned using cDNA reverse transcription of mRNA from Yu 1122 and 953 as templates. Simultaneously, using the DNA from these two materials as templates, the downstream SNPs of this gene were isolated using OXS3-F1 and OXS3-R1 assays. Sequence difference analysis showed that there were significantly different SNPs in the downstream sequence of this gene between Yu 1122 and 953. Figure 1 E).

[0034] Example 2 ZmOXS3 Verification of gene function of Ds mutant We found a mutant of ZmOXS3 (Mutan) in our maize Ds mutant library, with Ds insertion in... ZmOXS3 317bp upstream of the gene ( Figure 2 A). To clarify whether the ZmOXS3 mutant affects leaf width, leaf width was assessed in ZmOXS3 mutant and wild-type (WT) lines in Zhengzhou over two seasons. The results showed that the upper leaves of the ZmOXS3 mutant were significantly wider than those of the wild type. Figure 2 B), and the expression level of ZmOXS3 in the mutant was significantly lower than that in the wild type ( Figure 2 C) indicates that this gene negatively regulates the width of maize leaves.

[0035] Example 3 ZmOXS3 The overexpression of transgenic genes was used to verify their gene function. 1. Construction of overexpression vectors according to ZmOXS A pair of primers, OXS3-F2 and OXS3-R2, were designed based on the coding region sequence of the gene, with NcoI and SpeI restriction sites added to the 5' ends of the primers, respectively. These primers were used to amplify the gene from the Yu1122 material. ZmOXS3The cDNA sequence was obtained, and the PCR product was detected by agarose gel electrophoresis. The recovered target PCR product was ligated into the pMD19-T vector, transformed, and single clones were selected. PCR detection and sequencing were performed, and plasmids were extracted from the correctly sequenced bacterial cultures. The plasmid and the pCAMBIA1304 vector plasmid were double-digested with NcoI and SpeI restriction enzymes, respectively. The digested target fragments were recovered separately, and the vector fragment and the target fragment were ligated using T4 ligase. The ligation product was transformed into E. coli, positive clones were identified, and the recombinant plasmid was extracted and named pCAMBIA1304- ZmOXS3 The recombinant plasmid was transformed into Agrobacterium competent cells and used to transform the maize inbred line B104.

[0036] Primer OXS3-F2 (SEQ ID No. 4): '5-ACGGGGGACTCTTGA CCATGG TAATGCTTGGGGACCGCGAG-3', The horizontal line represents the NcoI restriction enzyme site.

[0037] OXS3-R2 (SEQ ID No. 5): 5'- AAGTTCTTCTCCTTTA CTAGTC TACTGGTGGTACTGGTA-3', the underlined part is the SpeI restriction site.

[0038] 2. Genetic transformation A method for genetic transformation of maize mediated by Agrobacterium was adopted (referencing Wang Ping'an, Creation of Transgenic Maize Materials Resistant to Maize Maize Based on RNA Interference, Master's Thesis, Henan Agricultural University, 2011). Using immature embryos of 1.2-1.8 mm as recipient material, maize immature embryos were infected with Agrobacterium LBA4404 under conditions of OD600 value of 0.5 and infection time of 10 minutes. After co-culture, resting culture, callus induction, low-pressure screening of callus (PPT concentration 3 mg / L), and high-pressure screening (PPT concentration 6 mg / L), regenerated plants were obtained. Phenotypic screening of regenerated plants was performed at a herbicide concentration of 200 mg / L (PPT). Seedlings sensitive to herbicides were removed. Total DNA was extracted from leaves of herbicide-insensitive seedlings for PCR detection of plant marker genes (Bar) and target genes. Positive plants were transplanted to the field and self-pollinated to obtain T1 generation seeds, completing the identification process of the transgenic T1 generation.

[0039] In this embodiment, a total of 6 independent transgenic positive plants were obtained. Genetically stable transgenic positive plants were obtained through multiple generations of continuous self-pollination. Phenotypic identification in the field showed that the leaf width of the positive transgenic lines was significantly narrower than that of the wild type. Figure 3 A).

[0040] ZmOXS3 We examined the expression of the ZmOXS3 gene because it narrowed maize leaves in transgenic lines and widened maize leaves in wild-type B104.

[0041] ZmOXS4 expression was analyzed using qRT-PCR (referencing Zhang Jun, Map-based Cloning and Functional Analysis of ZmCLA4 Gene Related to Maize Leaf Angle, Doctoral Dissertation, Henan Agricultural University, 2014). The results showed that ZmOXS4 expression was present in maize leaves at the 10-leaf stage. ZmOXS3 The expression level in the overexpressing transgenic lines was significantly higher than that in wild-type B104. Figure 3 B), indicating ZmOXS3 High expression levels of this substance lead to narrower maize leaves, thus suggesting... ZmOXS3 It acts as a negative regulator of maize leaf width.

[0042] This demonstrates that gene overexpression technology can stimulate endogenous genes in maize. ZmOXS3 Increased gene expression leads to narrower corn leaves, proving the control of... ZmOXS3 Gene expression can be used to cultivate inbred lines with narrow leaves as basic breeding materials, and to cultivate hybrids with suitable leaf widths for production and application.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Overexpression ZmOXS3 The application of genes in narrowing maize leaves is characterized by: The ZmOXS3 The gene encodes the amino acid sequence shown in SEQ ID No.

6.

2. The overexpression according to claim 1 ZmOXS3 The application of genes in narrowing maize leaves is characterized by: The ZmOXS3 The gene is more than 90% similar to the coding region of SEQ ID No. 1, from 1 to 516 bp.

3. The overexpression according to claim 1 ZmOXS3 The application of genes in narrowing maize leaves is characterized by: The ZmOXS3 The nucleotide sequence of the gene is shown in SEQ ID No. 1, 1-516 bp.

4. The overexpression according to any one of claims 1-3 ZmOXS3 The application of genes in narrowing maize leaves is characterized by, The steps are as follows: (1) Construction ZmOXS3 Gene overexpression vectors; (2) The overexpression vector in step (1) was transformed into Agrobacterium competent cells and used to transform maize inbred line B104 to obtain transgenic positive strains with narrower maize leaves compared with maize inbred line B104.

5. The overexpression according to claim 4 ZmOXS3 The application of genes in narrowing maize leaves is characterized by: The construction step in step (1) is as follows: According to ZmOXS3 Overexpression primer pairs were designed for the coding region of the gene. Using cDNA from the inbred line Yu1122 as a template, PCR amplification was performed. The PCR amplification product was then inserted into the pCAMBIA1304 vector via the intermediate vector pMD18-T to obtain pCAMBIA1304-ZmOXS3, which is the overexpression vector.

6. The overexpression according to claim 5 ZmOXS3 The application of genes in narrowing maize leaves is characterized by: The overexpression primer pair is OXS3-F1 and OXS3-R1, wherein the OXS3-F1 sequence is shown in SEQ ID No. 4 and the OXS3-R1 sequence is shown in SEQ ID No. 5.

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