Application of ZmGLU13 gene and related biological materials thereof in regulation and control of corn kernel traits

Abstract

The invention belongs to the technical field of corn molecular breeding, and particularly relates to application of a ZmGLU13 gene and a related biological material thereof in regulation of corn kernel traits. The nucleotide sequence of the ZmGLU13 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein coded by the gene is as shown in SEQ ID NO.3; experimental results show that after the ZmGLU13 gene is deleted in the corn gene, the grain weight, the starch content and the protein of the corn can be reduced, and after the ZmGLU13 gene is over-expressed in the corn gene, the grain weight, the starch content and the protein content of the corn can be remarkably improved; the ZmGLU13 gene or a related biological material containing the ZmGLU13 gene can be used for carrying out genetic improvement on the corn yield and the grain quality.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of corn molecular breeding, and particularly relates to ZmGLU13 Application of a gene and related biological materials in regulating corn kernel traits. BACKGROUND

[0002] As one of the three major crops in the world, corn plays a vital role in the global agricultural, economic and food fields. It not only supplies important food needs for humans and animals, but also plays a key role in biofuels and industrial raw materials. Starch in corn kernels accounts for about 70% of the dry weight, so the yield and quality of starch directly affect the total yield and application value of corn. Understanding and revealing the genetic regulation mechanism of corn starch synthesis is crucial for improving the yield and starch quality of corn.

[0003] Under the current situation of continuous reduction of arable land, increasing yield level has become the fundamental way to solve the contradiction between supply and demand of corn. Kernel is the main harvest target in the production process of corn, and increasing kernel yield is the ultimate goal of corn high-yield breeding. Therefore, mining key genes that regulate corn kernel traits can provide more genetic resources for corn breeding, which is of great significance to promote the breeding process of new corn varieties and ensure high and stable yield of corn. SUMMARY

[0004] To solve the above problems, the application provides ZmGLU13 Application of a gene and related biological materials in regulating corn kernel traits, after overexpression of the ZmGLU13 gene in the corn gene, the grain weight, starch content and protein content of the corn can be improved, and the ZmGLU13 gene can be used for genetic improvement of corn kernel quality.

[0005] To achieve the above purpose, the specific technical solutions of the application are as follows: The first aspect of the application provides a kind of ZmGLU13 Application of a gene and related biological materials in regulating corn kernel traits, the nucleotide sequence of the ZmGLU13 gene is as shown in SEQ ID NO. 1;The trait is agronomic trait and / or quality.

[0006] Further, the expression amount of the ZmGLU13 gene in corn is improved by transgenic technology or gene editing technology to improve the agronomic traits and / or quality of corn kernels.

[0007] Further, the agronomic trait is any one or several of kernel length, kernel width and hundred kernel weight;The quality is starch content and / or protein content.

[0008] Further, the starch content is any one or several of amylose content, amylopectin content and total starch content.

[0009] Further, the biological material comprises any one of the following: a. the ZmGLU13 gene encodes a protein, and the amino acid sequence of the protein is shown as SEQ ID NO. 3; b. a recombinant expression vector comprising the ZmGLU13 gene; c. a recombinant microbial strain comprising the ZmGLU13 gene.

[0010] The second aspect of the present application provides a method for cultivating a high-yield transgenic maize, by increasing the expression amount of the ZmGLU13 gene in the maize of interest, to obtain a high-yield transgenic maize; or increasing the expression amount of the protein encoded by the ZmGLU13 gene in the maize of interest, to obtain a high-yield transgenic maize.

[0011] Further, the yield of the transgenic maize is greater than that of the maize of interest.

[0012] The third aspect of the present application provides a method for cultivating a transgenic maize with high starch content and / or high protein content, comprising the following steps: constructing a recombinant expression vector comprising the ZmGLU13 gene of claim 1, introducing the recombinant expression vector into Agrobacterium, to obtain a recombinant Agrobacterium comprising the ZmGLU13 gene; introducing the ZmGLU13 gene into the genome of the maize of interest by Agrobacterium infection, to make the ZmGLU13 gene overexpressed, to obtain a transgenic maize.

[0013] Further, the starch content and / or protein content of the transgenic maize is greater than that of the maize of interest.

[0014] Further, the Agrobacterium is Agrobacterium EHA105.

[0015] Compared with the prior art, the present application has the following beneficial effects: The present application discloses ZmGLU13 application of a gene and related biological material in regulating maize kernel traits, ZmGLU13 the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and after increasing the expression amount of the ZmGLU13 gene in maize, the kernel weight (the kernel width, kernel length and hundred kernel weight of maize are all significantly higher than those of wild type maize), starch content and protein content of maize can be significantly increased. Therefore,ZmGLU13 Genes can effectively improve the yield and quality of corn kernels. Figure 1 Genes can be used to genetically modify the kernel traits of corn. Attached Figure Description

[0016] 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.

[0017] Figure 1 A schematic diagram illustrating the identification of CRISPR / Cas9 transgenic plants KO-1 and KO-2. ZmGLU13 A in the text is Figure 1 A schematic diagram of the knockout sites. Figure 1 In the diagram, B represents the sequencing peak of the PCR product of the transgenic plant KO-1, WT represents the wild-type plant, and the red box shows that KO-1 has a 10bp sequence missing compared to the wild-type plant. Figure 2 In the diagram, C represents the sequencing peak of the PCR product of the transgenic plant KO-2, WT represents the wild-type plant, and the red box shows that KO-1 has a 16bp sequence missing compared to the wild-type plant.

[0018] Figure 2 The variation of starch granules in the natural cross-section of mature kernel endosperm of wild-type (WT), KO-1, and KO-2. Figure 2 In the diagram, A represents an electron microscope. The observation areas of the images in rows a, b, and c are different. The scale bar of the images in row a is 30 μm, the scale bar of the images in row b is 100 μm, and the scale bar of the images in row c is 30 μm. Figure 3 In the graph, B represents a statistical representation of the starch grain size of each endosperm cell from the fourth layer of the aleurone layer. * indicates... P <0.05, ** indicates P <0.01.

[0019] ZmGLU13 for Figure 3 Results of the effects of knockout on kernel length, kernel width and 100-kernel weight of corn. Figure 3 In the graph, A represents the statistical chart of corn kernel length. Figure 3 B in the chart represents the statistical graph of corn kernel width. Figure 4 C in the chart represents the statistical chart of 100 kernels weight of corn. * indicates... P <0.05, ** indicates P <0.01.

[0020] ZmGLU13 forFigure 4 The effects of overexpression on kernel length, kernel width, and 100-kernel weight in maize. Figure 4 A in the figure represents a statistical chart of corn kernel length. Figure 4 B in the figure represents the statistical chart of corn kernel width. Figure 5 C in the chart represents the statistical chart of 100 kernels weight of corn. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0021] ZmGLU13 for Figure 5 Results of the effect of knockout on maize quality traits. Figure 5 In this context, A represents the crude protein content in corn endosperm. Figure 5 In this context, B represents the amylopectin content in the corn endosperm. Figure 5 C in the figure represents the amylose content in corn endosperm. Figure 6 In this context, D represents the total starch content in the corn endosperm; ** indicates... P <0.01.

[0022] ZmGLU13 for Figure 6 Results of the effect of overexpression on maize quality traits. Figure 6 In this context, A represents the crude protein content in corn endosperm. Figure 6 In this context, B represents the amylopectin content in the corn endosperm. Figure 6 C in the figure represents the amylose content in the corn endosperm. Figure 7 In this context, D represents the total starch content in the corn endosperm. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0023] ZmGLU13 for Figure 7 The effect of knockout on the content of reducing sugars and soluble sugars in the grains. Figure 7 In this figure, A represents the result of the soluble sugar content determination. Figure 8 In the figure, B represents the result of the reducing sugar content determination. ** indicates... P <0.01.

[0024] ZmGLU13 for Figure 9 A statistical chart showing gene expression during maize kernel development.

[0025] ZmGLU13 The protein localization results for ZmGLU13 are shown below. 35S::GFP represents the control group plants, illustrating the distribution of green fluorescent protein (GFP) in cells; 35S::ZmGLU13::GFP represents... ZmGLU13Overexpressing plants, showing the localization of ZmGLU13 protein after fusion with GFP. In the figure, DAPI staining (blue) is used to label the cell nucleus, GFP staining (green) is used to show the protein expression location, Merged (merged image) shows the co-localization of protein with cell nucleus, and Bright (bright field image) provides cellular morphological information. Detailed Implementation

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0027] The KN5585 inbred maize line in this invention is from Weimi Biotechnology Co., Ltd.

[0028] Starch accounts for approximately 70% of the dry weight of maize kernels, and its yield and quality significantly impact its total yield and application value. Revealing the genetic regulatory mechanisms of maize starch synthesis is crucial for improving yield and starch quality. Given the decreasing arable land, increasing yield per unit area is key to resolving the supply-demand imbalance. Grain is the primary harvest target for maize, and increasing grain yield is the ultimate goal of high-yield breeding. Identifying key genes regulating grain traits can provide genetic resources for breeding, advance the selection of new varieties, and ensure high and stable yields.

[0029] This invention provides ZmGLU13 The application of genes and related biological materials in regulating maize kernel traits revealed deletions in maize genes. ZmGLU13 Gene overexpression can reduce kernel weight, starch content, and protein in maize, while overexpression of the maize gene... ZmGLU13 After gene expression, the kernel weight, starch content, and protein content of maize can be significantly increased. Specifically, the overexpressed lines have larger kernel width and length, as well as a higher 100-kernel weight, and the starch and protein content in the kernels is also higher. ZmGLU13 Genes can effectively improve the yield and quality of corn kernels, utilizing... ZmGLU13 Genes can be used to genetically modify the kernel traits of corn.

[0030] Example 1: ZmGLU13 The discovery of its encoded protein The ZmGLU gene family of corn is found in the Arabidopsis and rice databases in the early stage of the application, and a candidate gene for regulating the quality and phenotype of corn is found in KN5585 inbred line (corn inbred line selected and obtained by Shengmibio Biotechnology Co., Ltd.), namely ZmGLU13 gene.

[0031] The nucleotide sequence of the gene in the genomic DNA of KN5585 inbred line is shown as SEQ ID NO. 1; the nucleotide sequence of the gene in the cDNA of KN5585 inbred line is shown as SEQ ID NO. 2; ZmGLU13 The nucleotide sequence of the gene in the genomic DNA of KN5585 inbred line is shown as SEQ ID NO. 1; the nucleotide sequence of the gene in the cDNA of KN5585 inbred line is shown as SEQ ID NO. 2; ZmGLU13 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 3. ZmGLU13

[0032] SEQ ID NO. 1: ​

[0033] SEQ ID NO. 2:

[0034] SEQ ID NO.3: .

[0035] Example 2: ZmGLU13 Application in regulating maize kernel traits 1. ZmGLU13 Obtaining gene knockout maize plants To further verify ZmGLU13 This invention utilizes CRISPR / Cas9 gene editing technology to [address gene function]. ZmGLU13 Gene knockout to obtain ZmGLU13 Gene knockout corn plants.

[0036] Gene editing was performed using standard procedures common in the field, with the maize recipient being the KN5585 inbred line. The obtained gene-edited materials KO-1 and KO-2 were investigated for grain traits and starch content.

[0037] like Figure 1 As shown in A, the two different edited lines have deletions of 10 and 16 bases, respectively, between 0bp and 500bp (compared to the wild type of the KN5585 inbred line).

[0038] KO-1 has the following frameshift mutation compared with the genomic DNA of corn inbred line KN5585: the bases from 187bp to 202bp in the sequence shown in SEQ ID NO. 1 are missing, thereby causing frameshift, resulting in the loss of function of ZmGLU13, and the sequencing results are shown in B in Figure 1

[0039] KO-2 has the following frameshift mutation compared with the genomic DNA of corn inbred line KN5585: the bases from 193bp to 203bp in the sequence shown in SEQ ID NO. 1 are missing, thereby causing frameshift, resulting in the loss of function of ZmGLU13, and the sequencing results are shown in C in Figure 1

[0040] 2、Overexpression ZmGLU13 Obtaining of corn plants Extraction of corn leaf DNA: Take the KN5585 inbred line corn as the sample, put the cut 2cm x 2cm corn leaves into a mortar, add liquid nitrogen and grind, then add 1mL of 65℃ preheated CTAB solution, then transfer to a 2mL centrifuge tube, then put it into a 65℃ water bath for heating for 30min, take out the centrifuge tube and add 1mL of chloroform-isoamyl alcohol mixed solution (the mixed volume ratio of chloroform and isoamyl alcohol is 24:1) in a fume hood, put it into a shaker at 200rpm / min for 15min, mix well, then take it out and put it into a refrigerated centrifuge at 12000rpm / min for 10min; add 500μL of centrifugal supernatant and 500μL of -20℃ pre-cooled isopropanol into a 1.5mL empty centrifuge tube, mix well, then stand at 4℃ for 30min (let the DNA precipitate), put it into a centrifuge at 12000rpm / min for 5min, discard the supernatant; add 500μL of 75v / v% ethanol solution, invert 10 times, put it into a centrifuge at 5000rpm / min for 5min, discard the supernatant, repeat this step 3 times; dry the centrifuge tube naturally, add 50μL of ddH2O to dissolve, and obtain the corn leaf DNA.

[0041] Extraction of total RNA from corn kernels: (1) The enzyme-free gun head, enzyme-free centrifuge tube, mortar and pestle required for RNA extraction need to be sterilized in a high-pressure sterilizer at 121℃ for 30min, and dried in an oven. Wear a mask and ensure that the gloves are clean throughout the RNA extraction process to avoid RNA degradation. ​​(2) Take out the corn kernels stored at -80°C and grind in a pre-cooled mortar (ensure low temperature during grinding, continuously supplement liquid nitrogen). Take 100 mg of plant tissue ground by liquid nitrogen and place it in a pre-cooled centrifuge tube. Immediately add 600 μL Buffer EL, vortex vigorously for 30 s to mix the sample and lysate evenly. Place in a low-temperature refrigerated centrifuge at 12,000 rpm for 5 min. Immediately perform the subsequent operation; (3) Take 500 μL of the supernatant after centrifugation to the adsorption column centrifuge tube, centrifuge at 12,000 rpm for 30 s, and discard the collected filtrate of the adsorption column; (4) Add 250 μL of anhydrous ethanol to the collection tube and mix for 15 s; (5) Transfer the above mixture to a new adsorption column, centrifuge at 12,000 rpm for 30 s, and discard the filtrate; (6) Add 700 μL Buffer RWA to the centrifuge tube containing the adsorption column, centrifuge at 12,000 rpm for 30 s, and discard the filtrate; (7) Add 500 μL Buffer RWB to the centrifuge tube containing the adsorption column, centrifuge at 12,000 rpm for 30 s, and discard the filtrate. Repeat this step once; (8) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min; (9) Transfer the adsorption column to a new RNase-free Collection Tubes 1.5 mL centrifuge tube. Add 50 μL of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12,000 rpm for 1 min. Store the extracted RNA at -80°C.

[0042] Obtain cDNA first strand by RNA reverse transcription: The entire reverse transcription process is performed on ice. First, determine the concentration of the extracted RNA using a micro UV spectrophotometer and dilute it to a concentration of 50 ng / µL. Prepare the following RNA reverse transcription system in an RNase-free centrifuge tube: template RNA 3 µL, 4×gDNA wiper Mix 4 µL, and RNase-free ddH2O to make up to 16 µL. After mixing the RNA reverse transcription system, react at 42°C for 2 min, and immediately cool on ice after the reaction is completed. Add 4 µL of 5×HiScript III qRT Super Mix to the centrifuge tube after the reaction is completed, mix gently, and then perform the reaction in a PCR instrument: 50°C, 15 min; 85°C, 5 s; and immediately cool on ice after the reaction is completed.

[0043] According to the corn ZmGLU13 CDS sequence, design homologous recombination primers GLU13CDS-F and GLU13 CDS-R, primer GLU13 The nucleotide sequence of CDS-F is shown as SEQ ID NO. 4, primer GLU13 The nucleotide sequence of CDS-R is shown as SEQ ID NO. 5, and the cDNA in KN5585 inbred line corn leaf tissue is used as a template to amplify the CDS sequence of the gene by using high-fidelity DNA polymerase.

[0044] SEQ ID NO. 4: 5'-ATGGAGCTGCTGCGACCACGCGG-3'; SEQ ID NO. 5: 5'-GCCGACAGCCTACTACAGAACT-3'.

[0045] PCR reaction system: cDNA template 2 μL, GLU13 CDS-F 1 μL, GLU13 CDS-R 1 μL, 2 × Taq plusMax 5 μL, ddH2O 1 μL.

[0046] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing, 72℃ extension for 1 min, 35 cycles.

[0047] The amplification product is subjected to agarose gel electrophoresis, the target band is recovered and connected with a linearized recombinant vector to obtain a recombinant expression vector WMV012-BAR-ubi+ ZmGLU13 +GFP, and the vector construction method is a conventional vector construction method. The recombinant expression vector constructed is transformed into E. coli DH5α by heat shock method, and after sequencing, a positive clone is obtained, and the recombinant plasmid is extracted by using a plasmid extraction kit. The recombinant plasmid is transformed into Agrobacterium EHA105 by electroporation, and colony PCR verification is performed, and the recombinant Agrobacterium containing ZmGLU13 is obtained.

[0048] Through the method of Agrobacterium infection of immature embryos, the gene ZmGLU13Transformation: Freshly peeled 1mm long immature corn embryos were selected as transformation material. The embryos were placed in a solution containing 1.8 mL of recombinant Agrobacterium-1, and 150 immature embryos were treated within 30 minutes to acclimate them to the culture environment and improve transformation efficiency. The suspension was removed, and the remaining corn embryos were placed in a tube. Another 1.0 mL of recombinant Agrobacterium-1,000 solution was added, and the tube was incubated for 5 minutes. The embryos in the centrifuge tube were then resuspended and transferred to a co-culture medium. Excess Agrobacterium-1,000 solution was removed from the surface using a pipette, and the embryos were co-cultured in the dark at 23°C for 3 days. After co-culture, the embryos were transferred to a selection medium and incubated in the dark at 28°C for 6 days. Finally, they were transferred to a selection medium containing diammonium phosphate and cultured for 14 days. The resistant callus tissue was transferred to differentiation medium and cultured under light for 21 days (25℃, 5000 lux). The differentiated seedlings were transferred to rooting medium and cultured under light until rooting (25℃, 5000 lux) to obtain regenerated seedlings. DNA was extracted from the leaves of the regenerated seedlings for PCR detection. The positive regenerated seedlings were cultured and then transplanted into a greenhouse. The offspring seeds were harvested after 3 to 4 months (maize genetic transformation was completed at Weimi Biotechnology) to obtain OE1 and OE2 overexpressing transgenic maize lines.

[0049] 3. ZmGLU13 Effects on maize agronomic traits (1) Observation of starch granules in corn kernels and starch granules in endosperm by ZmGLU13 The deletion mutant maize lines KO-1 and KO-2 were used as the experimental groups, and the wild-type maize inbred line KN5585 (WT) was used as the control group. The test maize lines KO-1, KO-2, and WT were planted and self-pollinated. When the endosperm matured, the kernel surface was fractured to remove foreign matter, and the samples were then conductively treated: the samples were placed tightly on a conductive carbon film double-sided adhesive and sputtered with gold for 30 seconds on an ion sputtering stage. The morphology of the starch granules was observed under a scanning electron microscope, and the size of the starch granules was recorded.

[0050] The shape of starch granules was observed under a scanning electron microscope. Figure 2 In section A, the statistical results of the starch grain size of each endosperm cell in the fourth layer of the aleurone layer are shown in [the table below]. Figure 2 B in the middle.

[0051] from Figure 2 It can be seen that the starch granules in WT have low plumpness, are loosely and irregularly arranged, uneven in size, and are spherical; the starch granules in KO-1 exhibit a transitional shape from round to polygonal, are tightly and irregularly arranged, and have larger gaps between starch granules in some areas; the starch granules in KO-2 are generally regular polygons with clear edges, uniform size distribution, and are tightly packed. Measurements of starch granule size revealed that the starch granules in WT are significantly larger than those in KO-1, while KO-2 shows a significant difference compared to WT. This indicates...ZmGLU13 The size of the starch granule in the corn endosperm is significantly reduced after gene mutation, and it is presumed that ZmGLU13 The size of the starch granule in the corn endosperm is significantly reduced after gene mutation, and it is presumed that

[0052] (2) Determination of grain length, grain width and 100-grain weight of corn kernels The present application further determines ZmGLU13 The grain length, grain width and 100-grain weight of the kernels of the knockout strain, ZmGLU13 the overexpression strain and the wild-type corn strain, and the results are shown in Figure 3 and Figure 4 .

[0053] As can be seen from Figure 3 , compared with the wild type, the grain length of KO-1 and KO-2 is smaller, the grain width is smaller, and the 100-grain weight is reduced, indicating that ZmGLU13 The grain length, grain width and 100-grain weight of the corn kernels are significantly reduced after the gene deletion mutation.

[0054] As can be seen from Figure 4 , compared with the wild type, ZmGLU13 the grain width of the corn kernels of the overexpression transgenic corn strain is larger, the grain length is larger, and the 100-grain weight is further increased. It indicates that ZmGLU13 The overexpression of the gene can significantly increase the corn kernel weight, increase the grain width, and increase the 100-grain weight, thereby proving that ZmGLU13 The gene has an important biological function in regulating the development of corn kernels.

[0055] 4、 ZmGLU13 Effect on corn quality traits (1) Determination of crude protein content in corn endosperm Crude protein determination: according to the steps of the national standard "GB / T 24318-2009 Determination of total nitrogen content and crude protein content in feed raw materials by Dumas combustion method", the Dumas nitrogen determination instrument (NDA702) produced by VELP Company of Italy is used to determine the crude protein content in the corn endosperm, and the protein coefficient is 6.25.

[0056] Specifically: 0.1g~0.3g of corn kernel powder is weighed and wrapped in a tin foil square sheet (or nitrogen-free paper) specially used for Dumas instrument, and is measured. The instrument is placed in the measured substance under the determination conditions according to the steps of the instrument use instruction book. The measured sample will be quantitatively combusted under the standardization condition of 800℃. The instrument automatically amplifies and converts the detection signal, and transmits the data to the external microprocessor for processing.

[0057] The determination results of the crude protein content in the kernels are shown in Figure 5 A and Figure 6The results in Table 1 show that, compared with WT, the crude protein content in the seeds of KO-1 and KO-2 is reduced, and the difference between KO-2 and WT is extremely significant; on the contrary, the crude protein content in the seeds of OE1 and OE2 is extremely significantly increased compared with WT.

[0058] (2) Determination of starch content in maize endosperm The tested maize (maize inbred line KN5585, KO-1, KO-2, OE1, OE2) was planted, and mature seeds were harvested.

[0059] The amylose content in the endosperm of mature seeds was detected according to the method of GB / T 15683-2008 Rice-Determination of amylose content, and the amylopectin content in the endosperm of mature seeds was detected according to the method of DB 7648-87 Determination of amylose in rice, maize and millet seeds, and the total starch content was calculated. The detection results of starch content in the endosperm are shown in Table 2. Figure 5 The results in Table 2 show that, compared with WT, the amylose content, amylopectin content and total starch content in the endosperm of KO-1 and KO-2 are reduced, and the difference between KO-2 and WT is extremely significant; on the contrary, the amylose content, amylopectin content and total starch content in the endosperm of OE1 and OE2 are extremely significantly increased compared with WT. Figure 6 The results in Table 2 show that, compared with WT, the amylose content, amylopectin content and total starch content in the endosperm of KO-1 and KO-2 are reduced, and the difference between KO-2 and WT is extremely significant; on the contrary, the amylose content, amylopectin content and total starch content in the endosperm of OE1 and OE2 are extremely significantly increased compared with WT.

[0060] The results show that, in terms of starch content, the KO or overexpression of ZmGLU13 The amylose content, amylopectin content and total starch content in the endosperm of KO-1 and KO-2 are reduced compared with WT, and the difference between KO-2 and WT is extremely significant.

[0061] (3) Determination of reducing sugar and soluble sugar content Determination of reducing sugar in seeds: 0.1000 g of maize seed powder was weighed into a 2 mL centrifuge tube, 2 mL of distilled water was added, and then it was extracted in a 50°C water bath for 20 minutes, shaking every 4 minutes, and then vortexed for 10 minutes, followed by centrifugation at 10000 r / min for 10 minutes at room temperature to collect the supernatant. The supernatant was transferred to a 10 mL glass test tube (the operation was repeated twice after adding 2 mL of distilled water), and the supernatant of the two extractions was combined and diluted to 10 mL. 2.50 mL of the diluted solution was taken in a glass test tube, 1.50 mL of DNS reagent was added, shaken well, heated in a boiling water bath for 5 minutes, immediately immersed in cold water to cool to room temperature, then diluted to 10 mL with distilled water, shaken well, and then the absorbance was measured at 540 nm.

[0062] Determination of soluble sugars in corn kernels: Weigh 0.1000 g of corn kernel powder into a 2 mL centrifuge tube, add 2 mL of 80 v / v% ethanol solution, and extract in an 80 °C water bath for 20 min, shaking and mixing every 4 minutes. Remove and vortex for 10 min, then centrifuge at 10000 r / min at room temperature for 10 min to collect the supernatant. Transfer the supernatant to a 10 mL glass test tube (repeat this operation twice), combine the extracted supernatants, and make up to 10 mL. Take 2 mL of the above diluted solution into a glass test tube, add 5 mL of anthrone reagent, shake well, heat in a boiling water bath for 10 min, cool to room temperature, and measure the absorbance at a wavelength of 620 nm.

[0063] The results of the determination of soluble sugar content and reducing sugar content in the grains are shown in the figure. Figure 7 Regarding the determination of soluble sugar content in kernels, there was no significant difference between KO-1, KO-2 and WT; regarding the determination of reducing sugar content in kernels, there was no significant difference between KO-1 and WT, while KO-2 showed a highly significant difference compared to WT.

[0064] Example 3: ZmGLU13 Analysis of spatiotemporal gene expression patterns 1. Real-time quantitative detection of corn ZmGLU13 The relative expression level of the gene needs to be determined. Maize is a homozygous vector and the maize inbred line KN5585.

[0065] Total RNA was extracted from corn kernels at different post-pollination stages and reverse transcribed into cDNA to obtain the cDNA of the corn kernels to be tested. Real-time quantitative PCR was used to detect the cDNA in the corn kernels. ZmGLU13 Relative gene expression levels (in terms of) Tublin (Relative processing of all samples was performed using internal reference genes).

[0066] PCR reaction system: 2µL cDNA template, 0.4µL 10µM upstream primer, 0.4µL 10µM downstream primer, 10µL Master Mix, and RNase-free H2O to a final volume of 20µL.

[0067] The primer sequences are as follows: Upstream primer: 5'-AGGTTCTCGATTGCCTGGTC-3', SEQ ID NO.6; Downstream primer: 5'-GTGCGGCAAATGACGACCATA-3', SEQ ID NO.7.

[0068] PCR amplification procedure as follows: 95℃ denaturation 15 min; 95℃ denaturation 10 s, 60℃ annealing 20 s, 72℃ extension 20 s, 40 cycles; 55℃ 30 s, 81 cycles.

[0069] The relative expression of the genes in the tested corns is shown in Table 1. ZmGLU13 The relative expression of the genes in the tested corns is shown in Table 1. Figure 8 , ZmGLU13 The expression of the gene showed a trend of first decreasing and then rising with time, and the expression was the highest at 35 days after pollination and the lowest at 20 days after pollination. This expression pattern implied that the gene might play a more critical role in the later development stage of the grain, especially in the synthesis and accumulation of starch and sugar substances. ZmGLU13 The expression of the gene showed a trend of first decreasing and then rising with time, and the expression was the highest at 35 days after pollination and the lowest at 20 days after pollination. This expression pattern implied that the gene might play a more critical role in the later development stage of the grain, especially in the synthesis and accumulation of starch and sugar substances.

[0070] 2. Subcellular localization prediction of ZmGLU13 The cDNA of the model corn inbred line KN5585 at the three-leaf stage was used as a template, and the PCAMBIA1301-EGFP vector was digested with I restriction endonuclease. The digested PCAMBIA1301-EGFP vector was connected with the above-mentioned cDNA to obtain a recombinant expression vector PCAMBIA1301-EGFP- Nco . ZmGLU13

[0071] The PCAMBIA1301-EGFP- ZmGLU13 was transformed into E. coli competent cells by heat shock freezing, and then transferred to LB liquid medium for 37℃ culture for 1 h, and then transferred to LB solid medium containing 100 mg / L kanamycin for 37℃ overnight culture to screen positive strains; the PCAMBIA1301-EGFP- ZmGLU13 in the positive strains was extracted, and the PCAMBIA1301-EGFP- ZmGLU13 was transformed into Agrobacterium by heat shock, and then cultured in antibiotic-free LB liquid medium at 28℃ for 1 h, and then cultured on LB solid medium containing 100 mg / L kanamycin and 50 mg / L rifampicin at 28℃ for 2 days, and then identified by PCR to obtain recombinant bacteria containing ZmGLU13 .

[0072] The recombinant bacteria were injected into tobacco leaves by injection method, and then cultured in the dark for 1 day, and then placed in normal light for 1 day of continuous culture. The transformed tobacco cells were cultured, and DAPI was injected 1 hour before observation, and finally the transformed tobacco cells were observed using a fluorescence microscope, with tobacco leaf cells transformed with the PCAMBIA1301-EGFP vector as a control.

[0073] The results are shown in Table 2. Figure 9 ​As shown, green fluorescence signal was detected in both nucleus and cell membrane of tobacco leaf cells transformed with PCAMBIA1301-EGFP vector, but only green fluorescence signal was detected on cell membrane of tobacco leaf cells transformed with PCAMBIA1301-EGFP-ZmGLU13, indicating that ZmGLU13 protein was localized to cell membrane. ZmGLU13 As shown, green fluorescence signal was detected in both nucleus and cell membrane of tobacco leaf cells transformed with PCAMBIA1301-EGFP vector, but only green fluorescence signal was detected on cell membrane of tobacco leaf cells transformed with PCAMBIA1301-EGFP-ZmGLU13, indicating that ZmGLU13 protein was localized to cell membrane.

[0074] It should be noted that when numerical ranges are used herein, the numerical range is intended to include each and every number within the range, and any sub-range of numbers within the range. As a result, a numerical range of "1 to 10" is intended to include any number from 1 to 10 (e.g., 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10), as well as sub-ranges such as 2 to 8, 3.5 to 5.5, and 7 to 12, etc. Although the preferred embodiments of the application have been described, those skilled in the art will understand that they can be subjected to many changes and modifications without departing from the spirit and scope of the application. Accordingly, it is intended to include all such changes and modifications insofar as they come within the scope of the claims and their equivalents.

[0075] Obviously, various modifications and changes are possible in the present application without departing from the scope and spirit of the application. Accordingly, it is intended to embrace all such modifications and changes that come within the scope of the claims and their equivalents.

Claims

1. ZmGLU13 The use of genes and related biological materials in regulating maize kernel traits, characterized by, The ZmGLU13 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, Increasing the expression of the genes in corn by transgenic technology or gene editing technology to improve the agronomic traits and / or quality of corn kernels. ZmGLU13 Increasing the expression of the genes in corn by transgenic technology or gene editing technology to improve the agronomic traits and / or quality of corn kernels.

3. Use according to claim 2, characterized in that, The agronomic trait is any one or several of kernel length, kernel width, and hundred kernel weight; the quality is any one or several of starch content and / or protein content.

4. Use according to claim 3, characterized in that, The starch content is any one or several of amylose content, amylopectin content, and total starch content.

5. The use according to claim 1, characterized in that, The biological material includes any one of the following: a. said ZmGLU13 gene encodes a protein; b. comprising said ZmGLU13 recombinant expression vector of the gene; c. a recombinant microbial strain comprising said ZmGLU13 gene.

6. A method of breeding high-yielding transgenic maize plants, characterized in that, increasing the expression of the gene of claim 1 in the corn of interest, resulting in high-yield transgenic corn; or increasing the expression of the protein encoded by the gene of claim 1 in the corn of interest, resulting in high-yield transgenic corn. ZmGLU13 increasing the expression of the gene of claim 1 in the corn of interest, resulting in high-yield transgenic corn; or increasing the expression of the protein encoded by the gene of claim 1 in the corn of interest, resulting in high-yield transgenic corn. ZmGLU13 increasing the expression of the gene of claim 1 in the corn of interest, resulting in high-yield transgenic corn; or increasing the expression of the protein encoded by the 7. A method for breeding a transgenic maize having high starch content and / or high protein content, characterized in that, The method includes the following steps: A recombinant expression vector comprising the gene according to claim 1 is constructed, the recombinant expression vector is introduced into the gene of the corn of interest, the gene is overexpressed, and the transgenic corn is obtained. ZmGLU13 A recombinant expression vector comprising the gene according to claim 1 is constructed, the recombinant expression vector is introduced into the gene of the corn of interest, the gene is overexpressed, and the transgenic corn is obtained. ZmGLU13 A recombinant expression vector comprising the gene according to claim 1 is constructed, the recombin 8. The method of claim 7, wherein, The starch content and / or protein content of the transgenic corn is greater than that of the corn of interest.

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