Application of maize Zm00001d016228 gene or its encoded protein in regulating grain traits

Abstract

The present invention discloses the use of the maize Zm00001d016228 gene or its encoded protein in regulating kernel traits. Maize 100-kernel weight is a key factor affecting maize yield and seed yield. As maize 100-kernel weight increases, seed yield increases, and yield increases. Experimental verification demonstrates that overexpressing the Zm00001d016228 gene in maize significantly increases 100-kernel weight. This provides a sound theoretical basis and direction for genetic improvement of 100-kernel weight and cultivation of high seed yield traits and high yields. Its application in the biobreeding process for improving maize kernel traits has promising prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of maize molecular breeding, and in particular to the application of the maize Zm00001d016228 gene or its encoded protein in regulating grain traits. Background Art

[0002] Maize is an important food, feed, and energy crop. Achieving large-scale yield increases is crucial for ensuring national food security. Kernel traits, including kernel length, kernel width, and 100-kernel weight, are key yield components and play a crucial role in yield improvement. Maize kernel traits are typically quantitative traits and are susceptible to environmental conditions. Traditional breeding methods for germplasm selection suffer from long cycles and low efficiency. Therefore, discovering new genes controlling maize kernel development, gaining a deeper understanding of the molecular mechanisms underlying kernel development, and genetically analyzing the regulatory mechanisms of kernel development can provide more optimized breeding strategies, shorten the breeding cycle, and improve breeding efficiency. Therefore, identifying key genes controlling maize kernel development is not only of theoretical significance but also holds broad application prospects. The maize Zm00001d016228 gene, encoding a protein containing an NHL domain, has been identified through QTL mapping. Currently, the role of Zm00001d016228 in regulating 100-kernel weight has not been reported. Summary of the Invention

[0003] Therefore, based on the above background, the present invention provides the application of the maize Zm00001d016228 gene or its encoded protein in regulating grain traits. This gene has important theoretical significance and practical application value in improving maize grains and cultivating new high-yield maize varieties, and provides a new direction for the subsequent cultivation of new high-yield maize lines and the acquisition of germplasm resources.

[0004] The first technical solution of the present invention provides: Zm00001d016228 gene related to corn grain traits, the nucleotide sequence of the coding region of the Zm00001d016228 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Zm00001d016228 gene is shown in SEQ ID NO.2.

[0005] Furthermore, the corn kernel traits include corn 100-kernel weight.

[0006] The second technical solution of the present invention provides the use of the Zm00001d016228 gene related to corn kernel traits, or a product overexpressing the Zm00001d016228 gene in regulating corn kernel traits.

[0007] Furthermore, the regulation refers to overexpressing the Zm00001d016228 gene in corn materials to improve corn grain traits.

[0008] Furthermore, the product overexpressing the Zm00001d016228 gene is one of the following:

[0009] ① an expression cassette containing the Zm00001d016228 gene;

[0010] ② A recombinant expression vector containing the Zm00001d016228 gene;

[0011] ③ A genetically engineered bacterium containing the Zm00001d016228 gene.

[0012] The third technical solution of the present invention provides the application of the Zm00001d016228 gene and the protein encoded therein, or the product overexpressing the Zm00001d016228 gene, wherein the application includes at least one of the following:

[0013] 1) Application in improving corn kernel traits;

[0014] 2) Application in corn breeding or assisted breeding.

[0015] The fourth technical solution of the present invention provides the use of the Zm00001d016228 gene or its encoded protein in identifying, breeding, or assisting in the breeding of corn plants, lines, or varieties with excellent grain traits.

[0016] The fifth technical solution of the present invention provides a method for cultivating high 100-grain weight corn, which comprises transferring the corn gene Zm00001d016228 into corn embryos, and then culturing the embryos to obtain a transgenic corn line overexpressing the Zm00001d016228 gene.

[0017] Furthermore, the obtained transgenic lines overexpressing the maize gene Zm00001d016228 are subjected to continuous self-pollination or hybridization to obtain homozygous offspring stably overexpressing the Zm00001d016228 gene.

[0018] The sixth technical solution of the present invention provides a breeding method for high 100-kernel weight corn, in which hybrid breeding is performed by overexpressing the Zm00001d016228 gene in at least one parent.

[0019] The beneficial effects achieved by the present invention are as follows:

[0020] Corn 100-kernel weight is a key factor influencing corn yield and seed yield. As corn 100-kernel weight increases, seed yield increases, leading to higher yields. The present invention has experimentally demonstrated that overexpressing the Zm00001d016228 gene in corn significantly increases 100-kernel weight. This provides a sound theoretical foundation and direction for genetically improving 100-kernel weight and cultivating high seed yield traits and high yields. Its application in biobreeding for improving corn kernel traits holds promise. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attachment Figure 1 This is the positive identification of gene overexpression plants in the examples of the present invention.

[0022] Attachment Figure 2 This is a grain comparison diagram of the overexpression mutant of the present invention and the B73 inbred line in the examples of the present invention.

[0023] Attachment Figure 3 Statistical diagram of grain phenotypes of overexpression mutant lines. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0026] Example 1: Construction and identification of Zm00001d016228 gene overexpression strain

[0027] 1. Construction of Zm00001d016228 gene overexpression vector

[0028] The CDS sequence of the Zm00001d016228 gene (SEQ ID No. 1, 1548 bp) was cloned from B73 leaves, and it was found that the gene encodes a protein consisting of 516 amino acids (SEQ ID No. 2).

[0029] The CDS sequence of the gene was transferred into the plant overexpression vector 35s-pMDC83-GFP-Gate using the LR reaction of the Gateway system. The recombinant vector pMDC83-Zm00001d016228 containing the strong 13S promoter was transformed into Agrobacterium AGL1. In collaboration with Weimi Company, B73 transgenic materials were created, and T0 generation overexpression transgenic seedlings were obtained. The DNA of the T0 generation transformed plants was extracted and the genotype was identified.

[0030] The CDS sequence of cloned Zm00001d016228 (SEQ ID No. 1)

[0031]

[0032] Amino acid sequence of the protein encoded by Zm00001d016228 (SEQ ID No. 2)

[0033] MGLRSPLVLLLLLLLFLAATFRPSAAALAPPTGSIVKQLSSVVRWPRAAPSTHGPKQPGHPQY

[0034] ADGHVGVALQFESGYFVETLVEGDKLGVTPHTVRVSPVEGGELLAVDSAHSNIVRITPPLSE

[0035] YSRGRLVAGSFQGHSGHIDGKPSDARFKRPTGVAVDDMGNVYVADTANLAIRKIGESGVTTI

[0036] AGGKSNIPGYRDGPSEDAKFSTDFDVVYVKKMCSLLVIDRGNAALRKISLPQEDCTYQDSA

[0037] LLSSVPYFIINFVLFIYIDLILVIGAVVAGYIFSGFQHGFGFSGSEKVEAPENEQHESSTIGKPPL

[0038] VVESLKEEPGAGWPSLGTLIADLLKLAIEGVGKLLLSVVPQRMQHGKRKTDLTPLRDRLVM

[0039] PEDREETAAAAQKLSSTPMRPETAHAPNPVTETAAPKAPKSTKPSKLRDSSTLSSKHRSSKR

[0040] QEYADFYGTSEPAPVGAAAKVPKDRLRHRHHHREKSGEVAYGAAHHDLKPAEAKPADYSDPSYDPYVRSKYAAESGYRY.

[0041] 2. Identification of overexpression clones of Zm00001d016228 gene

[0042] DNA from Zm00001d016228 transgenic maize plants was extracted using the CTAB method. The specific steps are as follows:

[0043] (1) Take about 200 mg of fresh leaves and add them to a 2 ml centrifuge tube. Grind them into powder using liquid nitrogen cryo-grinding or a grinder.

[0044] (2) Add 600 μl of 2× CTAB preheated at 65°C, mix well, and keep in a 65°C water bath for 20 min, shaking up and down 2-3 times during this period to ensure complete cell lysis.

[0045] (3) Add 600 μl of chloroform / isoamyl alcohol (24:1), mix thoroughly by inverting for 1 min, and then let it stand for 3-5 min to allow the layers to separate.

[0046] (4) Centrifuge at 10,000 rpm for 10 minutes and carefully transfer the supernatant to a new 1.5 ml centrifuge tube.

[0047] (5) Add an equal volume of -20°C pre-cooled isopropanol, mix thoroughly by inverting, and place at -20°C for 30 minutes to precipitate the DNA.

[0048] (6) Centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, add 0.5 ml of 70% ethanol solution, and wash the DNA precipitate by turning it upside down (if the DNA precipitate slides or floats, centrifuge at 12,000 rpm for 5 minutes after washing to allow the DNA to adhere to the bottom of the tube), then discard the ethanol solution (be careful not to discard the white DNA precipitate at the bottom of the centrifuge tube).

[0049] (7) After discarding the ethanol, dry the DNA pellet at room temperature.

[0050] (8) Add 200 μl of pure water to dissolve the DNA and store at 4°C until use.

[0051] The 2×CTAB solution was prepared as follows:

[0052] CTAB powder: 4 g, NaCl: 16.364 g, 1 M Tris-HCl: 20 ml (PH = 8.0), 0.5 M

[0053] EDTA: 8 ml, dissolve in ultrapure water, and then dilute to 200 ml, autoclave and add mercaptoethanol.

[0054] The obtained genomic DNA was amplified by PCR using detection primers. 2 μl of transgenic corn DNA was used as a template and the detection primers were used:

[0055] F: 5-CCATCGTCAACCACTACATCGAGACA-3' (SEQ ID NO: 4)

[0056] R: 5'-CTTCAGCAGGTGGGTGTAGAGCGT-3' (SEQ ID NO: 5)

[0057] PCR amplification was performed under the following conditions: preheating at 95°C for 5 min, 25-30 amplification cycles (denaturation at 98°C for 10 sec, annealing at 65°C for 30 sec, and extension at 72°C for 30 sec / kb), and extension at 72°C for 2 min.

[0058] The amplification system is: 5 μL 5×Q5 Reaction Buffer, 0.5 μL 10 mM dNTPs, 1.25 μL 10 μM primer, 0.25 μL Q5 High-Fidelity DNA Polymerase and template (1 ng to 1 μg), and ddH2O is added to make up to 25 μL.

[0059] The target fragment of 1548 bp was amplified (see Figure 1 ), and sequencing proved that the fragment was the recombinant vector NHL-Zm00001d016228 fragment, proving that the recombinant vector had been transferred into corn. According to the pure line calculation method, if more than 10 individual plants were positive, the line was pure, so they were named NHL-OE1, NHL-OE2, and NHL-OE3.

[0060] Example 2: Verification of the effect of overexpression of Zm00001d016228 gene on regulating 100-kernel weight of maize

[0061] Three strains (OE1, OE2, and OE3) overexpressing the Zm00001d016228 gene, along with a wild-type strain, were planted in the experimental fields of the Guizhou Arid Grain Research Institute. Grains were harvested upon maturity and their phenotypes were analyzed. Statistical data were analyzed using SPSS software using a one-way analysis of variance (ANOVA).

[0062] The results are shown in Table 1.

[0063] Table 1 Statistics of 100-kernel weight of different corn strains

[0064] strain 100-grain weight (g) B73 26.9 OE1 <![CDATA[35.5 * ]]> OE2 <![CDATA[31.5 * ]]> OE3 <![CDATA[29.5 * ]]>

[0065] Notes in the table: * indicates significant difference compared with wild-type B73.

[0066] As shown in Table 1, compared with the wild-type B73, the 100-grain weight of the Zm00001d016228 overexpression lines was significantly increased, ranging from 9.67 to 31.97%, indicating that Zm00001d016228 plays an important role in regulating the 100-grain weight of maize.

[0067] In summary, combined with the positive identification results and field phenotypic identification results, it was shown that Zm00001d016228 could significantly increase the 100-grain weight of corn and could be used for genetic improvement of 100-grain weight of corn and to increase seed yield and yield.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Overexpression Zm00001d016228 The application of the gene product in increasing the 100-kernel weight of corn is characterized in that: described Zm00001d016228 The nucleotide sequence of the coding region of a gene is SEQ ID NO. 1 As shown; Overexpression Zm00001d016228 The product of a gene is one of the following: ① Zm00001d016228 overexpression cassette of the gene; ②Containing the Zm00001d016228 Recombinant expression vector of gene; ③Containing the Zm00001d016228 The genetically engineered bacteria are Agrobacterium.

2. A method for cultivating high 100-kernel weight corn, characterized in that: Maize gene Zm00001d016228 After the gene was transformed into maize embryos, it was overexpressed after culture. Zm00001d016228 Genetic maize transgenic lines, the Zm00001d016228 The nucleotide sequence of the coding region of a gene is SEQ ID NO. 1 shown.

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