Molecular marker related to corn kernel starch content and application thereof

Abstract

The invention discloses a molecular marker related to the starch content of corn kernels and application of the molecular marker, and relates to the technical field of biology. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO.3. According to the invention, a key gene ZmEnd1 for regulating and controlling the corn starch content is successfully excavated for the first time, and two key SNP (Single Nucleotide Polymorphism) sites, namely SNP-4-245033385 and SNP-4-2450333215, located in a ZmEnd1 gene promoter region are found, and the two sites are obviously associated with the corn grain starch content. Based on the research results, the invention further develops a molecular marker closely related to the corn kernel starch content. By utilizing the molecular marker, researchers can quickly and accurately detect the genetic characteristics of the starch content in the corn kernels, so that powerful technical support is provided for breeding of corn varieties with high starch content.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a molecular marker related to the starch content of corn kernels and an application thereof. Background Art

[0002] Maize (Zea mays L.) is one of the most important food crops and an indispensable source of feed and industrial raw material. The kernel, the most important harvested organ of the maize plant, usually contains more than 70% of its dry weight, which is the most important form of energy storage in the kernel. In cereals such as maize and rice, starch content not only determines the weight and volume of the kernel, but also affects processing performance and the quality of the final product. Therefore, exploring the genes that regulate kernel starch content and deeply analyzing the molecular mechanisms of starch synthesis and accumulation are of great theoretical and practical significance for increasing maize yield and improving kernel quality. However, traditional breeding methods rely on phenotypic selection, which is limited by the time-consuming and high cost of kernel starch content detection and the interference of environmental factors, making it difficult to achieve efficient and accurate genetic improvement.

[0003] With the rapid development of molecular biology technology, the use of molecular marker technology can significantly improve breeding efficiency. Molecular marker technology is a tool that reveals the genetic differences of biological individuals or populations at the molecular level. It is based on the sequence differences at specific DNA sites between different individuals. It is closely linked to the target traits and can quickly, efficiently and accurately select the target traits, shortening the breeding cycle. Among them, genome-wide association analysis (GWAS) has been widely used in the study of the genetic basis of complex quantitative traits in plants. Therefore, identifying genes related to the regulation of corn kernel starch content and developing their molecular markers are of great significance to corn genetic improvement. Summary of the Invention

[0004] The present invention aims to provide a molecular marker related to corn kernel starch content and its application to address the aforementioned problems of the prior art. Using this molecular marker, researchers can quickly and accurately detect the genetic characteristics of corn kernel starch content, thereby providing strong technical support for the breeding of high-starch corn varieties.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a molecular marker related to the starch content of corn kernels, whose nucleotide sequence is shown in SEQ ID NO.3. The molecular marker includes two SNP sites, SNP-4-245033215 and SNP-4-245033385; the SNP-4-245033215 is located at the 1751st base of the nucleotide sequence; and the SNP-4-245033385 is located at the 1921st base of the nucleotide sequence.

[0007] The present invention also provides a dCAPS primer set, comprising an upstream primer dCAPS-F having a nucleotide sequence as shown in SEQ ID NO.26, an upstream primer dCAPS-F-mutation having a nucleotide sequence as shown in SEQ ID NO.27, and a downstream primer dCAPS-R having a nucleotide sequence as shown in SEQ ID NO.28.

[0008] The present invention also provides the use of the dCAPS primer set in preparing a detection product for identifying the starch content trait of corn kernels.

[0009] Furthermore, the detection product is a kit.

[0010] The present invention also provides a detection product for identifying the starch content trait of corn kernels, comprising the above-mentioned dCAPS primer set.

[0011] Furthermore, the detection product is a kit.

[0012] The present invention also provides the application of the above molecular marker in identifying the starch content trait of corn kernels.

[0013] The present invention also provides the use of the dCAPS primer set in identifying the starch content trait of corn kernels.

[0014] The present invention also provides the use of the above-mentioned detection product in identifying the starch content trait of corn kernels.

[0015] The present invention also provides a method for identifying the starch content of corn kernels, comprising the following steps:

[0016] Extracting genomic DNA of the sample to be tested;

[0017] The genomic DNA was used as a template and PCR amplification was performed using the dCAPS primer set to obtain the haplotype of the sample to be tested, and the starch content trait of the corn kernels was determined: the starch content of the corn kernels of the AA haplotype was higher than that of the GG haplotype.

[0018] The present invention discloses the following technical effects:

[0019] This study successfully identified ZmEnd1, a key gene that regulates corn starch content, for the first time. This discovery provides important clues for further research into the mechanism of starch metabolism in corn kernels. The study also identified two key SNPs in the promoter region of the ZmEnd1 gene: SNP-4-245033385 and SNP-4-245033215. These two SNPs are significantly associated with corn kernel starch content.

[0020] Based on these research results, the present invention further developed a molecular marker closely related to the starch content of corn kernels. By utilizing this molecular marker, researchers can quickly and accurately detect the genetic characteristics of starch content in corn kernels, thus providing strong technical support for the breeding of high-starch corn varieties. This technological breakthrough not only improves the efficiency of corn genetic improvement but also lays a solid foundation for the targeted improvement of corn quality, possessing significant scientific value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Figure 2 is the result of detecting significant SNPs controlling grain starch content in four environments and BLUP; (a) is the Manhattan plot of SNPs significantly associated with grain starch content, and the red boxes are the sites co-located in the four environments and BLUP; (b) is the QQ plot of SNPs significantly associated with grain starch content; 2020XSBN, 2021CZ, 2021YA, and 2021XSBN represent Xishuangbanna Dai Autonomous Prefecture, Yunnan Province in 2020, Chongzhou City, Sichuan Province in 2021, Ya'an City, Sichuan Province in 2021, and Xishuangbanna Dai Autonomous Prefecture, Yunnan Province in 2021, respectively; (c) is the expression pattern of candidate genes in different tissues;

[0023] Figure 2 The figures are the detection results of the differences in transcriptional activity caused by the variation of the ZmEnd1 promoter; among them, (a) is the association analysis result of the ZmEnd1 candidate gene; (b)-(d) are the phenotypic values ​​of grain starch content of different haplotypes in Chongzhou City (CZ), Sichuan Province in 2021, Ya'an City (YA), Sichuan Province in 2021 and Best Linear Unbiased Prediction (BLUP), respectively; *** represents significant differences at the P<0.001 level; ** represents significant differences at the P<0.01 level; (e) is the fluorescence imaging result of ZmEnd1 promoter activity; (f) is the quantitative comparison result of Hap1 and Hap2 promoter activity; *** represents significant differences at the P<0.001 level.

[0024] Figure 3Figure 2 is a diagram of dCAPS marker development; (a) is a gel electrophoresis diagram of dCAPS identification of different haplotypes; (b) is the location of the enzyme cleavage site introduction, the red base is the location of the marker, the black sequence is the sequence of the promoter marker, the blue sequence is the F primer with a mutation site, and the bold base is the mutated base; (c) is the NcoⅠ enzyme recognition site.

[0025] Figure 4 Figure 2 is the result of ZmEnd1 expression pattern analysis; (a) is the result of ZmEnd1 expression pattern analysis in embryo, endosperm and grain of B73 inbred line at different stages; (b) is the result of in situ hybridization of ZmEnd1 in grains of B73 inbred line 15 DAP after pollination; (c) is the expression of ZmEnd1 in different parts and at different developmental stages of grains; different letters represent significant differences; (d) is the statistical graph of ZmEnd1 expression at different developmental stages of grains of different haplotypes; ***, ** and * represent significant differences at P < 0.001, P < 0.01 and P < 0.05, respectively;

[0026] Figure 5 Results of detecting the subcellular localization of the ZmEnd1 gene in tobacco leaves; (a) is a schematic diagram of the pCAMBIA1305-35S-eGFP and pCAMBIA1305-35S-eGFP-ZmEnd1 vectors; (b) is the subcellular localization of the ZmEnd1 gene in tobacco leaves; the scale bar is 30 μm; (c) is the fluorescence detection result of ZmEnd1 in the chloroplast channel; the scale bar is 30 μm;

[0027] Figure 6 Results of maize yield trait testing between the ZmEnd1 mutant and the B73 inbred line; (a) is a phenotypic trait comparison diagram; (b)-(g) are statistical diagrams of ear length, 100-grain weight, grain length, grain width, ear diameter, and grain thickness, respectively; ***, **, and * represent significant differences at the P < 0.001, P < 0.01, and P < 0.05 levels, respectively; ns indicates no significant difference.

[0028] Figure 7 Statistical graphs of the contents of corn kernels; (a)-(d) are statistical graphs of the contents of UDP-glucose, starch, glucose-1-P, and trehalose-6-phosphate, respectively;

[0029] Figure 8 The following are the results of scanning electron microscopy observations of corn kernels of the ZmEnd1 mutant and the B73 inbred line; (a) is a scanning electron micrograph of corn kernels at different stages; (b)-(d) are statistical graphs of starch granule diameters at 15, 20, and 25 days post-application phases, respectively;

[0030] Figure 9Figures are the results of phenotypic identification of heterologous expression of the ZmEnd1 gene in rice; (a) is a comparison of yield traits of heterologous expression of ZmEnd1 in rice; Nip, wild-type rice Nipponbare; (b) is the semi-quantitative analysis result of ZmEnd1 in transgenic grains; (c) is the detection result of 1000-grain weight of rice overexpressing ZmEnd1 and wild-type Nipponbare; (d) is the detection result of starch content in grains of rice overexpressing ZmEnd1 and wild-type Nipponbare; (e) is the detection result of glucose-1-phosphate content in grains of rice overexpressing ZmEnd1 and wild-type Nipponbare; ***, ** and * represent significant differences at P<0.001, P<0.01 and P<0.05, respectively. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The primer information involved in the present invention is shown in Table 1.

[0037] Table 1 Primer information

[0038]

[0039]

[0040] Example 1

[0041] 1. Experimental Methods

[0042] 1. Using GWAS to identify genetic loci that regulate starch content in maize grains

[0043] Linked populations were planted in Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (22.02°N, 100.80°E) (2020XSBN), Chongzhou City, Sichuan Province (30.32°N, 103.38°E) (2021CZ), Ya'an City, Sichuan Province (9.59°N, 102.57°E) (2021YA), and Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (22.02°N, 100.80°E) (2021XSBN). A completely randomized block design was used, with 3-m row length and 0.7-m row spacing, and 12 plants per row. Five clusters of uniform growth were collected from each population. Kernels from the middle of the clusters were dried at 60°C for 2 days. Starch content in the kernels was collected using a Botong DA7250 near-infrared spectrometer with three technical replicates. Combined with the high-density genotypes of 969,439 SNP sites already available in the inventor's research group, GWAS analysis was performed using the FarmCPU model in the R package rMVP in R studio. The significant threshold for GWAS was set at P = 0.05 / number of effective markers, i.e., P = 1E -06 .

[0044] 2. Identification of key genes and significant variant sites for grain starch content using candidate gene association analysis

[0045] By comparing a stable SNP (SNP-4-245041154) detected across four GWAS environments and BLUP, eight candidate genes were identified within the LD (=50 Kb) region of this SNP. Analysis of expression patterns in different tissues showed that Zm00001d054004 (named ZmEnd1) was significantly overexpressed in the endosperm.

[0046] The nucleotide sequence of the ZmEnd1 gene is shown in SEQ ID NO.1.

[0047] Candidate gene association analysis was further used to verify the regulatory effect of this gene on the starch content of corn grains. The present invention used the DNA of 285 corn inbred lines of the associated population as a template, and amplified the upstream 2000bp and gene body of the ZmEnd1 gene from the genome by PCR and sequenced. The sequencing results were assembled and aligned using the DNAMAN program (V version 5.2.2, LynnonBio-soft, Canada). DNASP v.5.0 was used to detect nucleotide polymorphisms, including SNPs and InDels. Combining the grain starch content phenotypic data of 285 inbred lines under four environments and BLUP, SNPs / InDels with a minimum allele frequency (MAF) ≥ 0.05 and a heterozygosity rate (HR) < 20% were set, and association analysis was performed based on the FarmCPU model in TASSEL5.0 (the significant marker threshold was set to: P = 0.05 / number of effective markers). In the present invention, the variant sites (P < 0.001) were considered to be significantly correlated with grain starch content. Haplotypes were divided based on the significantly correlated SNPs and InDels. Linkage disequilibrium (LD) decay between adjacent markers was calculated using Haploview software. The significance of grain starch content between different haplotypes was analyzed using a t-test. Haplotypes with higher starch content were defined as superior haplotypes.

[0048] 3. Detecting promoter haplotype activity using dual luciferase assay

[0049] To detect the promoter activity of the two haplotypes, the present invention amplified and sequenced the 2000bp sequence upstream of the transcription start site in the material where the haplotype was located. Using Hind III (AAGCTT) and BamHI (GGATCC) as restriction sites, the two haplotypes of ZmEnd1 were constructed into the pGreenII-0800-LUC vector (P800) by homologous recombination and named P800-Hap1 (AA) and P800-Hap2 (GG). The vector construction primers are detailed in Table 1, and the vector map is detailed in Table 1. Figure 1 The constructed vectors P800-Hap1(AA) and P800-Hap2(GG) were then transformed into Agrobacterium GV3101-p19, respectively. The promoter activity represented by the two haplotypes was verified using a tobacco transient expression system. The specific steps are as follows:

[0050] (1) 30 μL of competent cells transformed with P800-Hap1(AA) and P800-Hap2(GG) vectors were spread on YEP medium containing resistance and cultured in the dark at 28°C for two days.

[0051] (2) Single clones were picked and placed in 1 mL of YEP liquid medium supplemented with the corresponding resistance (Kan and Rif added at a ratio of 1:1000) and cultured at 28°C with a shaker at 200 rpm for 12 h.

[0052] (3) Use PCR technology to perform positive identification on the bacterial solution, and determine whether the inserted band size is correct by electrophoresis, that is, whether the vector is successfully transformed into Agrobacterium competent cells.

[0053] (4) Pipette 100 μL of the positive bacterial suspension into 50 mL of YEP medium containing the resistance agents (Kan and Rif at a ratio of 1:1000) and culture for 18 h. When the OD600 reaches 1.0, centrifuge at 4000 rpm for 10 min to collect the cells. Resuspend the cells in 10 mM MgCl2 (with AS and MES added at a ratio of 1:1000) to an OD600 of 0.8.

[0054] (5) Tobacco seeds were sown in a culture box and cultured for approximately 40 days under conditions of 12 h light / 10 h dark, 25°C, and 60%-70% humidity. Tobacco plants with good growth and smooth leaves were selected and the suspension was injected from the back using a 1 mL syringe. To ensure the accuracy of the experimental results, the suspensions of the two haplotypes were injected into symmetrical locations on the same leaf, with the injection positions alternated for six biological replicates, and the injection areas were marked.

[0055] (6) After the injection, the tobacco leaves were cultured at room temperature for 48 h. The injected leaves were then immersed in 150 μg / mL luciferase substrate solution (D-Luciferin, Potassium Salt) for 15 min. LUC fluorescence was observed using a multifunctional imaging system.

[0056] At the same time, the dual luciferase reporter gene detection kit (Yisheng Biotechnology Co., Ltd., Shanghai) was used for detection. The specific steps are as follows:

[0057] (1) Place the leaf from the injection area in a 2 mL EP tube, add two small steel balls, and quickly freeze in liquid nitrogen. Use a tissue crusher to grind the sample into powder (30 Hz, 60 s).

[0058] (2) After the leaflets are broken, add 300 μL of lysis buffer to the EP tube, shake and mix thoroughly, and place on ice for 5 min to allow the leaves to fully break apart.

[0059] (3) Centrifuge at 12000 rpm for 3 min and transfer the supernatant to a new EP tube for later use.

[0060] (4) Add 50 μL of supernatant to the culture plate, and set up 3 technical replicates for each sample.

[0061] (5) Prepare firefly luciferase (LUC) reaction solution and Renilla luciferase (REN) reaction solution separately. Dilute firefly luciferase substrate (50×) and Renilla luciferase substrate (50×) to 1× working solution with the corresponding buffer. Incubate at room temperature.

[0062] (6) Add 100 μL of firefly luciferase reaction solution to the sample, shake and mix, and use a microplate reader (Varioskan LUX) to detect the activity of firefly luciferase (electrochemiluminescence, 1000 ms).

[0063] (7) Add 100 μL of Renilla luciferase reaction solution, mix gently, and test the activity of Renilla luciferase. The enzyme activity test should be completed within 30 minutes.

[0064] (8) Analyze data.

[0065] 4. dCAPS molecular marker development

[0066] Sequence analysis using dCAPSFinder2.0 (http: / / helix.wustl.edu / dcaps / dcaps.htmL) was performed to select appropriate restriction endonucleases for the two SNPs located in the promoter region of the ZmEnd1 gene that are used to delineate haplotypes. Specific primers were designed based on the selected endonuclease sequences. During primer design, mutations were introduced at appropriate positions to allow the amplified product to be cleaved by the specific restriction endonuclease but not by the other haplotype. PCR reactions were performed using DNA from inbred lines of different haplotypes as templates using Phanta Max Super-Fidelity DNA Polymerase and detected by 1% agarose gel electrophoresis. The specific primers designed in this invention, dCAPS-F / R, are detailed in Table 1. The PCR products were digested with the selected restriction endonucleases (digestion system: 1 μL 10× QuickCut Buffer, 1 μL DNA, 1 μL QuickCut enzyme, 7 μL sterile water) at 37°C for 15 min. Agarose gel electrophoresis is used to detect and observe band patterns. Different haplotypes produce different band combinations due to differences in their SNPs, thus enabling genotyping. The validity and accuracy of dCAPS molecular markers are confirmed by performing fragment amplification and enzyme digestion on multiple inbred lines with different haplotypes and comparing the results of dCAPS molecular markers with known genotype data (such as Sanger sequencing results).

[0067] dCAPS molecular marker (SEQ ID NO.2, negative sense strand):

[0068] (The single underline indicates the position of SNP-4-245033215 (negative strand), the wavy line indicates the primer design site, and the lowercase letters cagtgg indicate the enzyme cleavage site. If the enzyme cleaves two bands, it is haplotype Hap1; if it cannot be cleaved and only has one band, it is haplotype Hap2.)

[0069] Nucleotide sequence of ZmEnd1 gene promoter (SEQ ID NO.3):

[0070] R represents A or G; a single underline corresponds to SNP-4-245033215, and a double underline corresponds to SNP-4-245033385.

[0071] 5. Analysis of the spatiotemporal expression pattern and subcellular localization of ZmEnd1

[0072] Analysis of expression patterns in different tissues: To further explore the expression of ZmEnd1 in various tissues of maize, we collected three biological replicates of each of the root, stem, leaf, ear, tassel, and grain samples from maize inbred line B73 during the filling period and from 8 to 21 DAP, extracted RNA, and reverse transcribed it for qRT-PCR analysis. The primers are listed in Table 1. ZmActin1 gene was used as an internal reference, and 2 -ΔΔCT Methods The relative expression levels of target genes were calculated.

[0073] Subcellular localization: To verify the subcellular localization of the ZmEnd1 gene, the present invention cloned the ZmEnd1 gene into the pCAMBIA2300-eGFP vector to generate the p35S:ZmEnd1-eGFP fusion expression vector. The primers are shown in Table 1. The fusion expression vector was then transformed into the Agrobacterium strain GV3101, and the bacterial solution was cultured to OD 600 =0.8, an Agrobacterium suspension was prepared and injected into tobacco leaves for transient expression. The p35S:eGFP vector was used as a negative control. After 48 h of culture, the eGFP fluorescence signal in the tobacco leaves was detected using a confocal fluorescence microscope (Zeiss LSM 800, Baden-Württemberg, Germany).

[0074] 6. Creation of genetic material for ZmEnd1

[0075] Two maize ZmEnd1 gene EMS premature termination mutants were obtained from the maize EMS mutant website (http: / / www.elabcaas.cnmemd / public / index.html). The B73 inbred line was used as the wild type. DNA from the mutant strains was extracted using a modified SLS method. Specific detection primers were designed based on the location of the mutation site. Using the mutant DNA as a template, a fragment containing the mutation site was amplified. Sequencing was performed to determine homozygous expression. Detection primers are detailed in Table 1.

[0076] 7. Determination of Starch Content in ZmEnd1 Mutant Grains

[0077] Starch content in the ZmEnd1 mutant and wild-type grains was determined using the Megazyme K-TSTA total starch measurement kit. Five seeds were randomly selected and weighed. Sample pretreatment: The seeds were ground using a ball mill at 60 Hz for 60 seconds and passed through a 100-mesh sieve. Five 100-mg samples of each sample (accurately weighed) were placed in 2-mL centrifuge tubes. 0.2 mL of 80% (v / v) ethanol in water was added and thoroughly mixed on a vortex mixer to disperse the sample. 2 mL of dimethyl sulfoxide (DMSO) was then immediately added to the centrifuge tubes and mixed again on a vortex mixer. Initial incubation and enzymatic hydrolysis: The centrifuge tubes were placed in a boiling water bath for 5 minutes. Immediately after removal, 3 mL of thermostable α-amylase (30-fold diluted in 50 mM MOPS buffer, pH 7.0, containing 5 mM calcium chloride) was added to the centrifuge tubes and mixed on a vortex mixer for 20 seconds. Return the centrifuge tube to the boiling water bath and incubate for another 6 minutes, vortexing every 2, 4, and 6 minutes to ensure homogenization. Further enzymatic digestion and transfer: Transfer the centrifuge tube to a 50°C water bath, add 4 mL of 200 mM sodium acetate buffer (pH 4.5) containing 5 mM calcium chloride, and then add 0.1 mL of amyloglucosidase (AMG, 3,300 U / mL). Mix the contents of the centrifuge tube with a vortex mixer and incubate in a 50°C water bath for 30 minutes. Centrifugation and absorbance measurement: Transfer the entire reaction solution to a 100 mL volumetric flask (using a funnel as an aid) and rinse any residue from the centrifuge tube. Make up to the mark with 200 mM sodium acetate buffer (pH 4.5) containing 5 mM calcium chloride and shake well. Transfer 2.0 mL of each sample solution to a microcentrifuge tube and centrifuge at 13,000 rpm for 5 minutes. Prepare two aliquots for each sample. Accurately pipette 0.1 mL of supernatant into the bottom of a 16 x 120 mm glass test tube. Add 3.0 mL of GOPOD reagent to each test tube and incubate in a 50°C water bath for 20 min. Finally, measure the absorbance at 510 nm, using a reagent blank as a reference.

[0078] Calculation of starch content:

[0079]

[0080] Where ΔA is the absorbance of the sample solution minus the absorbance of the background blank sample solution; F is the conversion factor for converting absorbance values ​​to μg of glucose (100 μg of glucose divided by the absorbance value measured by 100 μg of glucose); EV is the sample extraction volume of 100 mL; 0.1 represents the sample volume analyzed; D is the sample solution dilution factor; 1 / 1000 represents the conversion from μg to mg; 100 / W represents the conversion to 100 mg of sample, where W is the sample weight (mg); and 162 / 180 is the conversion factor from free glucose to glucose present in starch for the final assay.

[0081] 8. Determination of Glucose-1-Phosphate (G-1-P) Content in the Grains of ZmEnd1 Mutants

[0082] The glucose-1-phosphate content in seeds was determined using a kit from Shanghai Tongwei Biotechnology Co., Ltd. The specific steps were as follows: 5 groups of each sample were weighed, 0.1 g of seed powder per group, and 1 mL of the extract was added to the mixture and homogenized in an ice bath. The mixture was centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was collected and the corresponding reagents (Table 2) were added to a 96-well plate in sequence:

[0083] Table 2 Glucose-1-phosphate determination mixed system

[0084]

[0085] Result calculation: 1PG / G1P content (μg / g) = 836×ΔA÷W×D, where D is the dilution factor, which is 1 when not diluted, and W is the sample mass.

[0086] 9. Determination of uridine diphosphate glucose (UDPG) and trehalose-6-phosphate synthase (T-6-P) content in the seeds of ZmEnd1 mutant

[0087] The uridine diphosphate glucose (UDPG) and trehalose-6-phosphate synthase (T-6-P) content in maize kernels was determined using ELISA kits from Shanghai Tongwei Biotechnology Co., Ltd. The procedure was as follows: 0.1 g of maize kernel powder was accurately weighed and added to 1 mL of pre-chilled extract solution for ice-cold homogenization. The mixture was then centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was collected for later use. The microplate was removed from the aluminum foil pouch after equilibration at room temperature. 50 μL of trehalose-6-phosphate and UDPG standard solutions of varying concentrations were added to the standard wells. 10 μL of the test sample and 40 μL of sample diluent were added to the sample wells. No liquid was added to the blank wells. 100 μL of HRP-conjugated antibodies specific for trehalose-6-phosphate and UDPG were added to all wells except the blank wells. The plate was sealed and incubated at 37°C for 60 minutes. The liquid in the wells was discarded, the wells were patted dry with absorbent paper, and then wash solution was added and allowed to stand for 1 minute. This was repeated five times. Add 50 μL of substrate A and 50 μL of substrate B to each well. Incubate at 37°C in the dark for 15 minutes, then add 50 μL of stop solution. Immediately measure the OD value at 450 nm using a microplate reader. A linear regression curve between the concentration of the standard and the OD value was plotted using Excel. The trehalose-6-phosphate and UDP-glucose content in the sample was calculated using the equation.

[0088] 10. Scanning electron microscopy observation of ZmEnd1 mutants

[0089] ZmEnd1 mutant and wild-type kernels were collected at 15, 20, and 25 DAP. Intact kernels were removed from the ears with a razor blade and fixed in electron microscopy fixative for 24 hours. The fixed kernels were then rinsed three times with 0.1 M phosphate buffer (pH 7.0) for 15 minutes each. The fixed kernels were then immersed in a 1% osmium phosphate buffer solution for 2 hours at room temperature and rinsed again with phosphate buffer three times for 15 minutes. The kernels were then dehydrated in a gradient of 30%, 50%, 70%, 80%, and 90% ethanol for 20 minutes each, followed by two dehydration cycles in anhydrous ethanol for 20 minutes. The kernels were then infiltrated in a mixture of ethanol and isoamyl acetate in equal proportions for 30 minutes, followed by treatment with pure isoamyl acetate for 20 minutes. After drying in a critical point dryer, the kernels were mounted on a sample stage with conductive adhesive and gold-sputtered using an ion sputtering system. Finally, starch granule micrographs were observed and photographed under a scanning electron microscope (SEM).

[0090] 11. In situ hybridization observation of ZmEnd1 mutant grains

[0091] Sample fixation and dehydration: ZmEnd1 mutant and wild-type grains 15 DAP after pollination were selected and fixed overnight at 4°C using pre-cooled 70% FAA solution. The samples were washed three times with 70% ethanol for 30 minutes each time, the fixative was removed, and the samples were operated on ice and dehydrated in a gradient manner using 30%, 50% (30 minutes each), 70%, 85%, and 100% ethanol (1 hour each). After dehydration, the samples were stored in anhydrous ethanol at 4°C overnight and then infiltrated with xylene ethanol solutions of different dilutions (25%, 50%, 75%, and 100%), with 50-100 mL of each concentration taken for 60 minutes. Finally, the infiltration was repeated twice in 100% xylene. Paraffin-embedded sections: At 60°C, use different dilutions of paraffin / xylene solution (25%, 50%, 75%, 100%), take 50-100 mL of each concentration, and soak for 2 hours respectively. Incubate the tissue in 100% paraffin at 60°C overnight. To prevent moisture from penetrating, make sure the beaker where the tissue is placed is well sealed. Transfer the tissue to the mold, pour in the melted paraffin, adjust the position of the tissue and add more melted paraffin. After cooling, place the mold on ice for 5-10 seconds to allow the stainless steel embedding box to shrink, then remove the wax block with a spatula and use a microtome to cut sections with a thickness of 8-10 μm.

[0092] RNA hybridization and imaging: Digest sections in a working solution containing 10 μg / mL proteinase K for 10-15 minutes at room temperature. This step helps improve probe penetration. Wash sections twice with PBS for 5 minutes each and then fix with 4% paraformaldehyde for 10 minutes at room temperature to inactivate proteinase K. Cover sections with prehybridization buffer (containing 50% formamide, 10% dextran sulfate, 1× SSC, 0.1% Tween-20, 50 μg / mL heparin, and 50 μg / mL yeast tRNA) and incubate at 42°C for 1 hour to reduce nonspecific binding. Add labeled RNA probe (usually digoxigenin or biotin) to the hybridization buffer to a final concentration of approximately 1 ng / μL. Add the hybridization solution dropwise to the sections, cover with a coverslip, and place in a humidified chamber. Apply the probe to the tissue sections and incubate overnight at 50°C. After hybridization, the sections were washed with 0.2×SSC, treated with RNase, incubated with anti-digoxigenin antibody at room temperature for 2 h, then washed with a buffer containing 1% bovine serum albumin, incubated with freshly dissolved NBT / BCIP solution for 12-15 h, and then imaged and observed under a microscope.

[0093] 12. Heterologous Expression of ZmEnd1 Gene in Rice

[0094] To further investigate the gene function of ZmEnd1, particularly its impact on crop yield components and grain starch synthesis, ZmEnd1 was heterologously expressed in the elite rice inbred line Nipponbare (Nip). Using the pCUB vector as the backbone, Bar as the selection marker, and BamH1 (GGATCC) restriction site, the complete CDS sequence of ZmEnd1 was cloned into the vector via homologous recombination to create the pCUB:ZmEnd1 recombinant plasmid. The recombinant plasmid was transformed into Nip via Agrobacterium-mediated transfection, and ZmEnd1-overexpressing lines were identified by PCR. Transgenic lines were propagated to the T2 generation and tested positive by PCR. Semi-quantitative analysis of ZmEnd1 expression in the grains of each transgenic line was performed, and phenotypes were characterized. Vector construction primers are detailed in Table 1.

[0095] 2. Experimental Results

[0096] 1. Identification of the key gene ZmEnd1 controlling starch content in maize kernels using GWAS

[0097] Using GWAS, we identified a significant SNP marker (SNP-4-245041154, P = 5.45E) that controls corn starch content in four environments (Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (2020XSBN), Chongzhou City, Sichuan Province (2021CZ), Ya'an City, Sichuan Province (2021YA), and Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (2021XSBN)) under BLUP. -16 )( Figure 1 (a) and (b)). The 50Kb range upstream and downstream was used as the candidate gene screening interval, and a total of 8 candidate genes were identified. The expression pattern analysis of these 8 genes revealed that Zm00001d054004 was highly expressed in the endosperm ( Figure 1 In (c), it was named ZmEnd1 and further verified.

[0098] 2. Identification of genetic variants of ZmEnd1 affecting grain starch content using candidate gene association analysis

[0099] The present invention combined the starch content of grains from four environments and BLUP with the promoter and gene body variation sites of Zm00001d054004 gene to conduct candidate gene association analysis. A total of three significant SNPs ( Figure 2(a)). A SNP (SNP-4-245029222) was identified in the fifth exon, which caused a synonymous mutation (TCG / TCC) of serine (Ser); SNP-4-245033385 and SNP-4-245033215 were both located in the promoter region, of which SNP-4-245033385 was significantly associated with starch content in 2021YA, 2021CZ and BLUP environments, and SNP-4-245033215 was significantly associated with the starch content phenotype of 2021CZ ( Figure 2 (a)). Based on the two SNPs SNP-4-245033385 and SNP-4-245033215, 285 inbred lines were divided into two main haplotypes: Hap 1 (AA) and Hap2 (GG). The starch content of grains of Hap1 in 2021CZ, 2021YA, and BLUP environments was significantly (P<0.001) higher than that of Hap2 ( Figure 2 (b)-(d)), thus Hap1 was identified as the superior haplotype for corn kernel starch content. The LUC / REN value of Hap1 was significantly higher than that of Hap2 by the dual-luciferase reporter gene assay (Dual-LUCiferaseReporter Assay), which further confirmed that the promoter activity of Hap1 was significantly higher than that of Hap2 ( Figure 2 (e) and (f)).

[0100] Specific primers were designed for the SNP-4-245033215 marker, and the restriction site Nco1 was introduced. The genomic DNA of different haplotype materials was used as a template to amplify the fragment (Hap1: B73, SCL16, SCL33, SCL116, SCL138; Hap2: SCL6, SCL90, SCL114, SCL128, SCL133), and the amplified product was digested with the Nco1 restriction endonuclease. Due to the introduction of the restriction site, a recognition site appeared in Hap1 and was digested by the enzyme, while Hap2 was not digested by the enzyme. In the gel electrophoresis results, the digested Hap1 gene fragment appeared as two bands. The results of multiple different haplotype materials proved that different haplotypes can be identified by this dCAPS molecular marker ( Figure 3 ).

[0101] 3. Analysis of ZmEnd1 Expression Pattern

[0102] To investigate the expression changes and differences of ZmEnd1 in different parts of grains and during developmental stages, quantitative analysis was performed. The results showed that among different parts, ZmEnd1 was highly expressed in grains at 10 DAP, 12 DAP, 14 DAP, and 21 DAP after pollination, and was expressed at lower levels in tissues such as roots, stems, and leaves. In grains, expression gradually increased with developmental time, peaked at 12 DAP after pollination, and decreased during the grain filling period until grain maturity ( Figure 4 (c)). It was also found that ZmEnd1 was highly expressed in the endosperm of the grain, with a trend of high expression in the early stage of endosperm development, low expression at 10-20 DAP after pollination, and then high expression again, reaching the highest at 34 DAP. The expression in the embryo was relatively stable ( Figure 4 In (a)). The present invention also found that the expression level of ZmEnd1 of Hap1 was significantly higher than that of Hap2 ( Figure 4 In addition, in situ hybridization results showed that ZmEnd1 was highly expressed mainly in the embryo and endosperm of the grains 15 DAP after pollination, which was consistent with the expression level ( Figure 4 (b)).

[0103] 4. Subcellular Localization Analysis of ZmEnd1-encoded Protein

[0104] like Figure 5 As shown in (a), in order to clarify the expression of ZmEnd1 in subcellular, the present invention constructed a pCAMBIA1305-35S-eGFP-ZmEnd1 fusion expression vector, and used a non-fusion eGFP expression vector as a negative control. Figure 5 As shown in (b), after the tobacco leaves were transformed, the ZmEnd1 protein was observed under a laser confocal microscope and found to be specifically expressed in the cell nucleus and cell membrane. Figure 5 As shown in (c), chloroplast autofluorescence detection revealed that the ZmEnd1 protein was not expressed in chloroplasts. These results indicate that the ZmEnd1 protein is localized in the nucleus and cell membrane.

[0105] 5. Mutation of ZmEnd1 reduces grain starch synthesis

[0106] In order to investigate the effect of maize ZmEnd1 gene loss of function on maize grains, two STOP-GAINED (premature termination) mutants of the ZmEnd1 gene were constructed in the B73 inbred line background: zmend1-1 and zmend1-2. The ear length, ear diameter, 100-grain weight, grain length, and grain width of the two EMS mutant lines zmend1-1 and zmend1-2 and the wild type B73 were measured. The statistical results showed that the 100-grain weight and grain length of the mutants zmend1-1 and zmend1-2 were significantly lower than those of the B73 inbred line, while the ear length and grain thickness were also lower than those of B73; there was no significant difference in grain width and ear diameter between the mutants zmend1-1 and zmend1-2 and the B73 inbred line ( Figure 6 ).

[0107] In plant sink tissues, UDP-Glc pyrophosphorylase (UGPase) converts UDP-Glc produced by SuSy into glucose-1-P to meet metabolic needs. The contents of uridine diphosphate glucose (UDPG), glucose-1-phosphate (G-1-P), starch, and trehalose-6-phosphate (T-6-P) in seeds of B73 and zmend1-1 and zmend1-2 mutants were measured. The results showed that compared with wild-type B73, the starch content and G-1-P in the mutants were significantly reduced, while UDPG was significantly increased ( Figure 7 (a)-(c)); Trehalose-6-phosphate (T-6-P) showed no significant difference compared with the wild type ( Figure 7 (d)).

[0108] Taken together, these results suggest that the ZmEnd1 gene is involved in regulating the biological pathway of starch synthesis in maize kernels. Loss of this gene function weakens the UDPase-catalyzed conversion of UDPG and pyrophosphate into G-1-P and uridine triphosphate (UTP), hindering the synthesis of the downstream product, G-1-P, and the final product, starch. Simultaneously, the upstream product, UDPG, accumulates due to reduced conversion, resulting in significantly higher UDPG content in the mutant compared to B73. Furthermore, the ZmEnd1 mutation does not affect the accumulation and metabolism of intermediates in other synthesis pathways.

[0109] 6. Scanning electron microscopy observation of ZmEnd1 mutant grains

[0110] Scanning electron microscopy was performed on the grains of mutants zmend1-1, zmend1-2 and wild type B73 at 15, 20 and 25 DAP after pollination. The results showed that as the grain filling progressed, the number and size of starch granules in B73 grains were significantly higher than those in mutants ( Figure 8At 25 days post-production (DAP), B73 exhibited relatively mature, regular polygonal starch granules, fully filled with protective structures formed by alcohol-soluble proteins. In contrast, the mutant starch granules remained in the developmental ellipsoidal or irregular polygonal form, with numerous intracellular voids. This suggests that the ZmEnd1 gene mutation leads to reduced starch granule size and developmental delay in maize kernels.

[0111] 7. Heterologous expression of the ZmEnd1 gene in rice promotes grain starch biosynthesis

[0112] To further explore the gene function of ZmEnd1, especially its effects on crop yield components and grain starch synthesis, ZmEnd1 was heterologously expressed in the elite rice inbred line Nipponbare. The transgenic lines were propagated to the T2 generation and tested positive by PCR. The expression level of ZmEnd1 in the grains of each transgenic line was semi-quantitatively determined, as shown in Figure 2. Figure 9 As shown in (b), the expression level of ZmEnd1 gene in transgenic lines was significantly higher than that in wild-type Nip.

[0113] like Figure 9 As shown in (c), compared with the wild type Nip, the thousand-grain weight of the transgenic line was significantly higher than that of the wild type; Figure 9 As shown in (a), (d) and (e), the starch content and G-1-P content of rice grains were significantly higher than those of wild-type Nip.

[0114] Experimental results showed that heterologous expression of the ZmEnd1 gene in rice can promote the synthesis of G-1-P by increasing the expression of UDP-glucose pyrophosphorylase (UDPase) in the starch synthesis pathway, thereby increasing starch accumulation, which ultimately manifests as an increase in the thousand-grain weight of rice.

[0115] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A molecular marker related to the starch content of corn kernels, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.3, and the molecular marker includes two SNP sites, SNP-4-245033215 and SNP-4-245033385; the SNP-4-245033215 is located at the 1751st base of the nucleotide sequence; and the SNP-4-245033385 is located at the 1921st base of the nucleotide sequence.

2. A dCAPS primer set, characterized in that: The primer comprises an upstream primer dCAPS-F whose nucleotide sequence is shown as SEQ ID NO.26, an upstream primer dCAPS-F-mutation whose nucleotide sequence is shown as SEQ ID NO.27, and a downstream primer dCAPS-R whose nucleotide sequence is shown as SEQ ID NO.

28.

3. Use of the dCAPS primer set according to claim 2 in preparing a detection product for identifying the starch content trait of corn kernels.

4. The use according to claim 3, characterized in that The detection product is a test kit.

5. A detection product for identifying the starch content of corn kernels, characterized in that: Comprising the dCAPS primer set according to claim 2.

6. The detection product according to claim 5, characterized in that: The detection product is a test kit.

7. Use of the molecular marker according to claim 1 in identifying the starch content trait of corn kernels.

8. Use of the dCAPS primer set according to claim 2 in identifying the starch content trait of corn kernels.

9. Use of the detection product according to claim 5 or 6 in identifying the starch content of corn kernels.

10. A method for identifying the starch content of corn kernels, characterized in that: The following steps are involved: Extracting genomic DNA of the sample to be tested; The genomic DNA is used as a template and PCR amplification is performed using the dCAPS primer set of claim 2 to obtain the haplotype of the sample to be tested, and the starch content trait of the corn kernels is determined: the starch content of the corn kernels of the AA haplotype is higher than that of the GG haplotype.

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