A method of reducing the moisture content of corn kernels

Abstract

The application discloses a method for reducing the water content of corn kernels, and relates to the precise mutation of a ZmRPG protein in a corn inbred line Y97 by using a CRISPR / Cas12Y7 gene editing system, so as to obtain a new ZmRPG mutant capable of significantly reducing the water content of corn kernels, and provide a new technical means and excellent germplasm resource for corn mechanized kernel harvesting breeding.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding technology, and more specifically, relates to a method for reducing the moisture content of corn kernels. Background Technology

[0002] Corn (Zea mays L.) has firmly established itself as my country's largest crop, serving as a staple food, feed, and industrial raw material. my country's demand for corn is increasing year by year, and there are also higher requirements for the diversity of corn varieties. As a crucial food crop, mechanized corn production is a key link in my country's agricultural development. Mechanized corn kernel harvesting is paramount for improving production efficiency, saving costs, and freeing up rural labor in the corn production process. As a raw material for feed production, the quality of corn kernels is crucial and directly affects my country's feed industry chain. Achieving mechanized kernel harvesting and improving corn kernel quality both meet the significant needs of my country's agricultural production.

[0003] Basic research on maize kernel moisture content, a limiting factor for mechanized harvesting and improving maize kernel quality, has lagged behind. Furthermore, due to a long breeding process, breeders have not paid sufficient attention to the related traits of maize kernel dehydration, resulting in some maize varieties dehydrating slowly in the later stages of physiological maturity, leading to high kernel moisture content. This is detrimental to mechanized harvesting and also affects maize quality during transportation and storage, even posing risks to human and animal safety. Cloning genes related to maize kernel dehydration and understanding their functional mechanisms is of great significance for both maize production and basic research.

[0004] Utilizing gene editing technology to specifically induce targeted mutations in genes related to maize kernel dehydration, and creating rapidly dehydrated maize materials, has significant application value. However, currently there are relatively few genes involved in maize kernel water content, making it more difficult to study the biological mechanisms of maize kernels. ZmRPG is a newly discovered small peptide that affects the dehydration rate of maize kernels. The extent to which different amino acid sites of this peptide affect the dehydration rate is still largely unknown, and research on site-directed mutagenesis at different sites of this peptide is very limited (only one patent, CN118684748A). A Cell paper (Yu et al., Cell, 2024, DOI:10.1016 / j.cell.2024.10.030) from the same team (Yan Jianbing's research group at Huazhong Agricultural University) and the same gene (ZmRPG / microRPG1) records that frameshift mutations of this gene reduce kernel moisture content by 1.8%-17.0% (average 7.1%) at harvest. This patent aims to obtain mutant materials with better effects on reducing maize kernel moisture content by precisely deleting different amino acid sites of this small peptide in different maize varieties. Summary of the Invention

[0005] To address at least one deficiency or improvement need in the existing technology, this invention provides a method for reducing the moisture content of corn kernels. The method involves using genetic engineering techniques to edit specific key sites of the target gene ZmRPG in corn. The edited protein undergoes deletion of specific amino acid sites, and the corn is further cultivated and screened to obtain a rapid dehydration material.

[0006] To achieve the above objective, according to one aspect of the present invention, a method for reducing the moisture content of corn kernels is provided, characterized in that the 7th amino acid of the protein in the sequence shown in SEQ ID NO. 1 of corn is deleted.

[0007] Furthermore, the deletion of the 7th amino acid was achieved through gene editing techniques targeting the sequence shown in SEQ ID NO.5 or its reverse complementary sequence.

[0008] Furthermore, the method specifically includes: 1) Design target sites for the gene encoded by the protein sequence shown in SEQ ID NO. 1 in maize, and design gRNA sequences based on the target sites, and link them to a gene editing backbone vector carrying the Cas12Y7 protein-encoding gene to obtain a gene editing vector; the gRNA sequence is shown in SEQ ID NO. 5.

[0009] 2) The gene editing vector described in step 1) was introduced into maize plants using Agrobacterium-mediated genetic transformation to achieve a deletion of the 7th amino acid in the protein of the sequence shown in SEQ ID NO. 1 in maize; 3) Perform phenotypic identification of grain moisture content on the corn plants obtained in step 2) and screen corn plants with significantly reduced grain moisture content.

[0010] Furthermore, in step 1), the gene editing backbone vector is Wimi-Cas12Y7.

[0011] Furthermore, in step 2), the maize plant is the maize inbred line Y97.

[0012] Furthermore, the present invention also provides a maize ZmRPG mutant, which is obtained by deleting the 7th amino acid of the maize protein sequence shown in SEQ ID NO. 1, and the amino acid sequence of the mutant is shown in SEQ ID NO. 13.

[0013] Furthermore, the present invention also provides a method for creating maize plant cells, or plant seeds, or plant tissues, or plant parts with reduced water content, the method comprising the step of introducing the ZmRPG mutant into maize plant cells, plant seeds, plant tissues, plant parts or plants.

[0014] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: This invention marks the first time that the ZmRPG gene has been directionally edited in the maize inbred line Y97 using the CRISPR / Cas12Y7 gene editing system, resulting in a novel ZmRPG mutant with a site-directed amino acid deletion mutation (a deletion mutation was performed at the seventh amino acid position of the ZmRPG protein), and the edited type RPG97-KO3. RPG97-KO3 maize plants exhibited a stable rapid dehydration phenotype across multiple locations and generations, with kernel moisture content reduced by 17.19–24.20% compared to the wild-type control, averaging a reduction of 20.7%. Compared to the rapid dehydration effect of ZmRPG mutants described in existing technologies (average 7.1%), this application achieved unexpected technical results, demonstrating a significantly superior effect in reducing maize kernel moisture content, and possessing significant breeding application value. This invention provides a method for reducing maize kernel moisture content, offering a new technical means and excellent germplasm resources for mechanized maize grain harvesting breeding. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the transcription and translation of the target gene ZmRPG micropeptide provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the gene editing vector provided in an embodiment of the present invention; Figure 3 This is a comparison diagram of the protein domains of RPG97-KO3 before and after editing obtained in the embodiments of the present invention; the left is the ZmRPG protein domain before mutation, and the right is the ZmRPG protein domain after mutation.

[0016] Figure 4 This is a comparison of the kernels of RPG97-KO3 corn and control Y97 corn after harvest, according to an embodiment of the present invention; the left image is of corn kernels from control Y97, and the right image is of corn kernels from RPG97-KO3. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0018] Example 1. Construction to obtain a target ZmRPG Gene knockout vector (1) sgRNA targeting sequence design This invention uses precise gene editing technology to design mutations and deletions at multiple amino acid sites in the maize endogenous ZmRPG protein (SEQ ID NO.1), as shown in Table 1. ZmRPG In-frame deletion mutations at three different amino acid sites on the amino acid sequence ， The nucleotide sequence of the ZmRPG protein is shown in SEQ ID NO.2.

[0019] To accurately perform in-frame deletion mutations at the ZmRPG amino acid sites in Table 1, the gene editing tool used in this embodiment is the CRISPR / Cas12 Y7 system. ZmRPG Editing target points were designed on the reading frame, selecting targets with high target scores, low target miss rates, and suitable locations. The DNA sequences of the deleted amino acid sites and corresponding target regions designed in this invention are shown in Table 1. Table 1 ZmRPG Deletion of amino acid sites and gRNA

[0020] The gRNA molecule sequence in this invention was obtained through artificial synthesis.

[0021] (2) Construction of gene editing vector Wimi-Cas12Y7-ZmRPG The gRNA was ligated into the gene-editing backbone vector Wimi-Cas12Y7 via homologous recombination cloning, resulting in the gene-editing vector Wimi-Cas12Y7-ZmRPG. This vector contains Cas12Y7, gRNA, and bar genes. The Cas12Y7 gene is driven by the maize UBI promoter, the gRNA by the ZmU6 promoter, and the bar gene by the CaMV 35S promoter. This vector is derived from *E. coli*, and the backbone vector is a universal vector that has been used internationally for many years; it is non-pathogenic and will not evolve into a pathogenic one. Its diagram is shown below. Figure 2 As shown.

[0022] The specific construction process is as follows: ① The ccdB sequence in the backbone vector Wimi-Cas12Y7 was removed by BsaI digestion. The backbone vector Wimi-Cas12Y7 was digested with BsaI at 37℃ for about 1 hour. A 50 μl digestion system was prepared, containing 1 μg (10 μl) of Wimi-Cas12Y7, 1 μl of BsaI enzyme, 5 μl of BsaI enzyme buffer, and 34 μl of H2O. The digested vector product was recovered from the gel using a DC301 kit (Novozymes PCR gel recovery kit). The intermediate template was amplified using dual-target primers, and then the gel was recovered.

[0023] ② Ligate the enzyme digestion vector product and the PCR product. Ligation system (mixed on ice): 2 μl of the enzyme digestion vector product obtained in step ①, 5 μl of the PCR product obtained in step ①, 1 μl of homologous recombinase, 1 μl of recombinase buffer, and 1 μl of H2O. Transform E. coli by running the Ligase program on the PCR instrument at 50℃ for 20 min.

[0024] ③ The ligation product was transformed into *E. coli* to obtain the gene editing vector Wimi-Cas12Y7-ZmRPG. After removing the competent *E. coli* DH5α cells from the refrigerator, they were quickly placed on ice. After 5 minutes, once the bacterial block had dissolved, the ligation product obtained in step ② was added: 10 μl of ligation product + 100 μl of competent *E. coli* DH5α cells. The mixture was incubated on ice for 25 min, then heat-shocked at 42℃ for 45 s, and placed on ice for 2 min. 100 μl of antibiotic-free LB was added, and the mixture was shaken at 37℃ and 200 rpm for 1 h. The mixture was then plated and incubated with LB + Kana at 37℃ for one day.

[0025] The above-described vector construction process is only one embodiment provided by the present invention. Other conventional gene editing vector construction methods can be used for construction, and no specific or unique limitation is imposed.

[0026] Example 2: Transforming the knockout vector into maize Y97 This invention employs Agrobacterium-mediated genetic transformation to transform the gene editing vector Wimi-Cas12Y7-ZmRPG into the maize inbred line Y97. The specific method and procedure are as follows: 2.1 Callus preparation Embryogenic callus tissues with rapid growth, soft texture, loose and brittle structure, and bright color were selected. These callus tissues can be subcultured for a long time and can maintain their embryogenetic capacity for a considerable period of time, making them ideal recipient materials for Agrobacterium-mediated genetic transformation.

[0027] 2.2 Preparation of Agrobacterium Two days before infection, streak Agrobacterium on LB medium and incubate in the dark at 28°C, or pick single clones and shake them in liquid LB medium. Collect the bacterial cells and transfer them to 50 mL centrifuge tubes. Resuspend the cells in infection medium to achieve an OD concentration of [value missing]. 660 =Between 0.8 and 1.5; The prepared infection solution was activated by shaking the bacteria at 200 rpm for 2 hours at 28°C on a shaker for 2 hours, and then used for infection.

[0028] 2.3 Infection Pretreated callus tissue was selected and transferred to an infection culture medium. Soak the callus tissue in the invasion staining solution for 10-30 minutes, break up the callus tissue clumps, and shake well to ensure that the callus tissue is fully in contact with Agrobacterium tumefaciens. Remove the remaining infection solution and transfer the infected callus tissue into a co-culture medium. Incubate in the dark at 19°C for 3 days.

[0029] 2.4 Resumption of Culture Rinse the callus surface 3-5 times with sterile water containing antibiotics. Once the water is no longer cloudy, discard the liquid and transfer the callus to a Petri dish lined with filter paper. Dry the callus surface in a laminar flow hood. Transfer to recovery medium and incubate in the dark at 28°C for 7-10 days. Then transfer to selection medium.

[0030] 2.5 Screening After recovery culture, the transformed callus tissue was transferred into a selection medium supplemented with antibiotics and cultured in the dark at 28°C for 20-30 days. Transfer the callus tissue into a sterile petri dish, break it up, and then press it thin. On the UV operating table, pick out the brighter callus particles, 3-5 particles are piled together and transferred to a new selection medium; Incubate in the dark at 28°C for 20 days, then transfer to a new screening medium for subculture and propagation. After two cycles (40 days), the callus tissue that emits fluorescence is picked again (without breaking up the callus tissue).

[0031] 2.6 Differentiation and Rooting After heat shock, the callus tissue was transferred to a predifferentiation medium and cultured in the dark at 28°C for 10 days, and then cultured in the light at 28°C for 10 days. Transfer to differentiation medium, and when the regenerated shoots grow to 3-5 cm, transfer to rooting medium; After the seedlings have developed a large number of strong roots, harden them off and transplant them.

[0032] 2.7 Hardening off seedlings and transplanting After rooting, remove the sealing film from the rooting bottle and harden the seedlings in the culture medium for 2-3 days; Wash the roots of the culture medium and transplant them into sterilized nutrient soil. Harden them in the room for 7 days. Transplant in greenhouses or open fields.

[0033] Example 3: Analysis of the editing of mutant target genes in gene-edited maize plants. This invention, through preliminary steps such as gene editing vector construction and target design, and maize transformation, obtained 36 positive T0 generation gene-edited maize lines (RPG-K01 to RPG-K03) in 2024. In 2025, excellent gene-editing Δ... ZmRPG Maize lines. During the intermediate-scale trial conducted in the 2024 maize planting season, T2~T4 generations were planted in the field. ZmRPG DNA was extracted from seedlings of maize plants in the T1 generation to detect gene editing. Primers were designed to amplify the target editing region. The primer sequences are shown in Table 2. The PCR reaction system and reaction procedure are shown in Tables 3 and 4.

[0034] Table 2 Primer list for PCR amplification detection of edit sites

[0035] Table 3 PCR reaction system

[0036] Table 4 PCR reaction procedure

[0037] After recovering the PCR products, sequencing was performed. The sequencing results of the gene-edited plants were compared with the genome of wild-type Y97. Materials with base substitutions, insertions, or deletions were considered positive editing materials; otherwise, they were considered negative materials. The final sequencing results successfully screened the target gene. ZmRPG Materials with precisely deleted mutations, target genes ZmRPG Successfully gene-edited, RPG97-KO1 has a 3 bp (AGG) deletion in its nucleotide sequence compared to the wild type, as shown in SEQ ID NO.8. The mutated RPG97-KO1... ZmRPG The protein has a deletion at amino acid position 30, and its amino acid sequence is shown in SEQ ID NO.9; RPG97-KO2 has a 3 bp (AAC) deletion compared to the wild-type nucleotide sequence, and its nucleotide sequence is shown in SEQ ID NO.10. The mutated protein... ZmRPG The protein has a deletion at amino acid position 13, and its amino acid sequence is shown in SEQ ID NO.11; RPG97-KO3 has a 3bp (CAT) deletion compared to the wild-type nucleotide sequence, and its nucleotide sequence is shown in SEQ ID NO.12. The mutated protein... ZmRPG The 7th amino acid of the protein is deleted; the amino acid sequence is shown in SEQ ID NO.13. Preliminary statistics and comparisons were performed on the moisture content of maize kernels in T1 generation RPG97-KO1~RPG97-KO3 plants. The results are shown in Table 5. The results show that among the three mutants RPG97-KO1~RPG97-KO3, RPG97-KO3 maize material had the lowest kernel moisture content, demonstrating the best effect in reducing maize kernel moisture content compared to the wild-type control. RPG97-KO2 was the second most effective. Preliminary screening revealed RPG97-KO3 as the mutant with the best effect. Target gene. ZmRPG The protein domains also changed accordingly after editing. A comparison of the protein domains before and after editing the RPG97-KO3 target gene is shown in the figure below. Figure 3 As shown.

[0038] Table 5. Moisture content of corn kernels in T1 generation RPG97-KO1~RPG97-KO3 materials

[0039] Example 4 This example provides gene-edited maize △ ZmRPG Application in maize breeding To further investigate the effect of the RPG97-KO3 mutant provided in this invention on the moisture content trait of maize kernels, the inventors planted RPG97-KO3 mutant maize plants and the control Y97 at three experimental sites in Beijing, Gongzhuling (Jilin), and Sanya (Hainan) from May 2025 to October 2024 (three biological replicates were performed for each material at each site). Sampling and moisture content determination methods: When the maize kernels were basically mature, i.e., the milk line disappeared and a black layer appeared at the base of the kernel, samples were taken on the same day to determine the kernel moisture content. The kernel moisture content was determined according to international seed testing regulations and national seed testing procedures. The ears were removed, the husks were peeled off, and the moisture content of the maize ear was randomly measured five times using a PROTIMETER TimberMaster moisture meter. The readings were recorded, and the average value was taken as the moisture content of the maize kernels on that ear. The results are shown in Table 6.

[0040] The results show that, at harvest, the RPG97-KO3 mutant maize plants grown in Beijing, Gongzhuling, and Sanya exhibited significantly lower kernel moisture content in generations T2 through T4 compared to the control Y97. The moisture content of RPG97-KO3 kernels decreased by 17.19% to 24.20%, with an average reduction of 20.7%. This meets the conditions for mechanized harvesting (kernel moisture content ≤25% for the Northeast medium-maturity group and ≤28% for the Huang-Huai-Hai summer-sown group, based on the "National Maize Variety Approval Standard (2021 Revision)"). The target trait is stable and excellent. Furthermore, the target trait remains stable across different generations, indicating that it can be stably inherited.

[0041] Table 6 Comparison of moisture content of RPG97-KO3 T2~T4 generation kernels

[0042] Numerical values ​​are expressed as mean ± standard deviation. Differences in data between different materials were compared using a t-test (α = 0.05). The above results demonstrate that the RPG97-KO3 maize plants obtained by targeted editing of the ZmRPG gene using the CRISPR / Cas12Y7 system exhibited a stable rapid dehydration phenotype across multiple locations and generations, possessing significant breeding application value. The ZmRPG mutant gene, sgRNA, and gene editing method provided by this invention can offer new technical means and superior germplasm resources for mechanized grain harvesting breeding of maize.

[0043] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for reducing the moisture content of corn kernels, characterized in that, In maize inbred line Y97, using the CRISPR / Cas12Y7 gene editing system, the gRNA shown in SEQ ID NO.5 was used as the target sequence to delete the 7th amino acid of the protein in the sequence shown in SEQ ID NO.1 in maize, thereby obtaining the ZmRPG mutant protein with the amino acid sequence shown in SEQ ID NO.

13. The mutant protein leads to a decrease in the water content of maize kernels.

2. The method according to claim 1, characterized in that, The deletion of the 7th amino acid was achieved through gene editing targeting the sequence shown in SEQ ID NO. 5 or its reverse complementary sequence.

3. The method according to claim 1, characterized in that, The method specifically includes: 1) Design target sites for the gene encoding the protein shown in SEQ ID NO. 1 in maize, and design gRNA sequences based on the target sites, and ligate them into a gene editing backbone vector carrying the Cas12Y7 protein encoding gene to obtain a gene editing vector; the gRNA sequence is shown in SEQ ID NO. 5; 2) The gene editing vector described in step 1) was introduced into maize plants using Agrobacterium-mediated genetic transformation to achieve a deletion of the 7th amino acid in the protein of the sequence shown in SEQ ID NO. 1 in maize; 3) Perform phenotypic identification of grain moisture content on the corn plants obtained in step 2) and screen corn plants with significantly reduced grain moisture content.

4. The method according to claim 3, characterized in that, In step 1), the gene editing backbone vector is Wimi-Cas12Y7.

5. A maize ZmRPG mutant, said mutant being prepared by the method of claim 1, the amino acid sequence of said mutant being shown in SEQ ID NO.

13.

6. The method for reducing the moisture content of maize kernels according to any one of claims 1 to 4, or the application of the ZmRPG mutant according to claim 5 in reducing the moisture content of maize kernels.

7. A method for creating corn plant cells, or plant seeds, or plant tissues, or plant parts, or plants with reduced water content, characterized in that, The method includes the step of introducing the ZmRPG mutant of claim 5 into plant cells, plant seeds, plant tissues, plant parts, or a plant, wherein the plant is maize.

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