Gene for improving corn filling rate and application thereof

Abstract

The invention discloses a gene for controlling the filling rate of corn and application of the gene, and belongs to the field of gene engineering. The gene provided by the invention encodes an amino acid sequence as shown in SEQ ID NO.2. Experiments prove that the plant grain filling process can be positively regulated and controlled by improving the expression quantity of the gene or enhancing the content / activity of the protein coded by the gene, which is expressed by improving the grain filling rate, improving the grain filling quantity, increasing the thousand grain weight and / or accelerating the grain dehydration rate; on the contrary, inhibition of the expression of the gene or reduction of the content / activity of the protein encoded by the gene leads to slowdown of the grain filling rate, reduction of the grain filling amount, reduction of the thousand grain weight and / or reduction of the dehydration rate. The gene and the encoding protein thereof can be used for plant genetic engineering breeding, and an effective technical means is provided for creating high-yield new plant germplasm suitable for mechanical harvest.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to genes that control the grain-filling rate of maize and their applications. Background Technology

[0002] corn( Zea mays Corn is a major food and feed crop worldwide. Its grain filling rate, thousand-grain weight, and dehydration rate during grain growth and development are key agronomic traits that determine corn yield and quality. Grain filling rate directly affects the accumulation and plumpness of grain dry matter, thousand-grain weight is a core indicator of corn yield, and dehydration rate is related to grain maturity, damage rate at harvest, and storage stability, which is crucial for improving the efficiency of mechanized corn harvesting. Especially in major corn-producing areas, low temperature and rainy weather in the later stages can easily exacerbate the problem of slow grain dehydration, which seriously restricts the improvement of corn production efficiency.

[0003] Currently, agricultural production mainly improves these traits through traditional breeding or integrated agronomic management measures. However, traditional breeding has a long cycle and poor directionality, while agronomic measures require increased human input and have limited regulatory effects. In molecular biology research, although some genes involved in maize grain filling or yield regulation have been cloned, the grain filling rate is controlled by multiple genes in synergy, and current research is still insufficient in identifying new key functional genes, especially those that can directly and efficiently improve grain filling speed. Moreover, most existing genes can only regulate a single trait, or their synergistic improvement effect on multiple traits is not significant, making it difficult to meet the breeding requirements for high-yield, high-quality, and mechanized-harvestable maize.

[0004] Therefore, identifying key functional genes that can simultaneously and synergistically improve multiple traits, such as increasing the grain filling rate, thousand-grain weight, and grain dehydration rate of maize, and then using genetic engineering to directionally improve maize traits, is of great significance for breeding high-yield, machine-harvestable maize varieties and enhancing the overall production capacity of maize. It is also a technical problem that urgently needs to be solved in the field of maize molecular breeding. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a gene for controlling the grain-filling rate of maize and its application in plant breeding.

[0006] On the one hand, the present invention provides a protein for regulating plant seed development, the protein having the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 99% homology with SEQ ID NO.2.

[0007] In a preferred embodiment, the protein has the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 99% homology with SEQ ID NO.2, for example, the protein has an amino acid sequence having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity with the sequence shown in SEQ ID NO.2.

[0008] In a further preferred embodiment, the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0009] On the other hand, the present invention provides a gene encoding the protein that regulates the development of plant seeds.

[0010] In a preferred embodiment, the gene encoding the protein regulating plant seed development comprises the nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence having 99% or higher homology with the above sequence. For example, the gene has a nucleotide sequence having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity with the sequence shown in SEQ ID NO.1.

[0011] In a further preferred embodiment, the CDS sequence of the gene encoding the protein regulating plant seed development is shown in SEQ ID NO.1.

[0012] As is known to those skilled in the art, gene sequences can also contain introns, promoters, and various regulatory elements. Therefore, the nucleotide sequences of the aforementioned genes can also contain introns, promoters, and various regulatory elements.

[0013] On the other hand, the present invention provides an expression cassette, recombinant vector, recombinant microorganism, or transgenic plant tissue or transgenic plant containing the gene.

[0014] Furthermore, the present invention also provides the application of the described protein, gene, expression cassette, recombinant vector, or recombinant microorganism in regulating plant seed development.

[0015] In a preferred embodiment, the regulation of plant grain development is achieved by promoting plant grain development by increasing the content or activity of the protein in the plant or increasing the expression level of the gene in the plant, or by slowing down plant grain development by decreasing the content or activity of the protein in the plant or decreasing the expression level of the gene in the plant.

[0016] In a further preferred embodiment, promoting plant grain development manifests as increasing grain filling rate, increasing grain filling volume, increasing thousand-grain weight, and accelerating grain dehydration rate, or one or more of the following: slowing down plant grain development manifests as reducing grain filling rate, reducing grain filling volume, reducing thousand-grain weight, and slowing down grain dehydration rate, or one or more of the following: reducing grain filling rate, reducing grain filling volume, reducing thousand-grain weight, and reducing grain dehydration rate.

[0017] On the other hand, the present invention provides a method for cultivating transgenic plants, comprising the step of introducing the gene into a recipient plant to obtain a transgenic plant; compared with the recipient plant, the transgenic plant has improved grain filling rate, grain filling amount, thousand-grain weight and / or grain dehydration rate.

[0018] On the other hand, the present invention also provides a method for cultivating transgenic plants, comprising the step of inhibiting the expression and / or activity of the protein or inhibiting the expression of the gene in the starting plant to obtain transgenic plants; compared with the recipient plant, the transgenic plants have reduced grain filling rate, grain filling amount, thousand-grain weight and / or grain dehydration rate.

[0019] In a preferred embodiment, the expression of the gene is suppressed using CRISPR gene editing technology.

[0020] On the other hand, the present invention also provides the application of the above-described method for cultivating transgenic plants in plant breeding.

[0021] In a preferred embodiment, the plant is a plant of the genus Zea.

[0022] In a further preferred embodiment, the plant is corn.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. This invention provides a novel gene that synergistically regulates the grain filling and dehydration processes of maize kernels; overexpression of this gene can significantly improve the grain filling rate and grain fullness, increase the thousand-grain weight, and accelerate the grain dehydration rate, thus solving the problem of limited regulatory effects of single genes in existing technologies.

[0025] 2. This invention provides a technical means to simultaneously improve maize yield and harvest traits, which can improve grain filling efficiency, increase grain weight and accelerate dehydration rate under the same genetic background, effectively overcoming the technical bottleneck that it is difficult to achieve both high yield and machine-harvestable traits.

[0026] 3. This invention has promising application prospects. New maize varieties bred based on this gene can reduce the moisture content of harvested kernels while ensuring high yields, thereby reducing mechanical kernel breakage and drying costs, and significantly improving maize production efficiency and mechanization levels. Attached Figure Description

[0027] The beneficial effects of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] Figure 1 The figure shows the expression levels of the ZmTIP3-2 gene in different strains.

[0029] Figure 2 The figures show the grain filling rates of ZmTIP3-2 related lines, with Figure A representing the grain filling rate in 2024 and Figure B representing the grain filling rate in 2025.

[0030] Figure 3 The figure shows the thousand-grain weight of ZmTIP3-2 related lines, with the figures for 2024 and 2025 respectively.

[0031] Figure 4 The figure shows the dehydration rate of ZmTIP3-2 related strains, with the dehydration rates in 2024 and 2025 respectively. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] The CDS sequence of the maize ZmTIP3-2 gene in the example is shown in SEQ ID NO.1:

[0035] ATGAGCACGGCCACGGGCGTGCGGGCGGGGCGGCGGTTCACGGTGGGACGGAGCGAGGACGCCACGCACCCGGACACCATCCGCGCCGCCATCTCCGAGTTCATCGCCACCGCCATCTTCGTCTTCGCCGCCGAGGGATCCGTCCTCTCGCTCGGGAAGATGTACCACGACCACAGCACGATCAGCACGGCGGGCGGGCTGGTGGCAGTGGCGCTGGCGCACGCGCTGGGTCTGGCCGTGGCGGTGGCGGTGGCCGTCAACGTCTCGGGCGGGCACGTGAACCCGGCCGTCACCTTCGGCGCGCTCGTCGGCGGCCGCGTCTCCCTCGTCCGCGCGGTCCTCTACTGGGCCGCGCAGCTGCTGGGCGCCGTCGCCGCGACGCTCCTGCTGCGGCTCGCCACGGGGGGCGCGCGCCCGCCGGGGTTCGCGCTCGCGTCCGGGGTCGGCGACGGGCACGCCGTGCTGCTGGAGGCCGTCATGACGTTCGGCCTCGTGTACGCCTACTACGCCACGGTGGTCGACCCGAAGCGGGGCCACCTGGGCACCATCGCGCCGCTGGCCGTGGGTTTCCTGCTCGGCGCCAACGTGCTGGCGGGCGGGCCCTTCGACGGCGCCGGGATGAACCCGGCGCGGGTCTTCGGCCCGGCGCTCGTCGGGTGGCGGTGGAGGCACCACTGGGTTTACTGGCTGGGCCCCTTCCTCGGCGCGGGGCTCGCAGGGCTGGTGTACGAGTACCTGCTTATCCCGCCCGCCGACGCCGTGCCGCACACGCACCAGCCGCTCGCGCCAGAGGACTACTAG。

[0036] In the examples, the amino acid sequence of the maize ZmTIP3-2 protein is shown in SEQ ID NO.2:

[0037] MSTATGVRAGRRFTVGRSEDATHPDTIRAAISEFIATAIFVFAAEGSVLSLGKMYHDHSTISTAGGLVALAHALGLAVAVAVAVNVSGGHVNPAVTFGALVGGRVSLVRAVLYWAAQLLGAVAATLLLRLA TGGARPPGFALASGVGDGHAVLLEAVMTFGLVYAYYATVVDPKRGHLGTIAPLAVGFLLGANVLAGGPFDGAGMNPARVFGPALVGWRWRHHWVYWLGPFLGAGLAGLVYEYLLIPPADAVPHTHQPLAPEDY.

[0038] The nucleotide sequence of the maize ZmTIP3-2 gene in the example is shown in SEQ ID NO.3:

[0039]

[0040] Example 1: Establishment of ZmTIP3-2 transgenic maize plants

[0041] 1. Obtaining ZmTIP3-2 overexpression lines

[0042] The CDS sequence of ZmTIP3-2 was cloned and constructed into the overexpression vector WMV013. This vector was transformed into Agrobacterium EHA105, which infected the immature embryos of the maize inbred line KN5585. The infected embryos were then transferred to a selection medium containing a selection marker (herbicide) and cultured for 2-4 weeks to induce callus formation. Subsequently, the embryos were transferred to a regeneration medium to induce shoot and root differentiation, resulting in normal, complete seedlings. Positive seedlings were identified by PCR using ZmTIP3-2 specific primers (TIP3-2-check-F: AGGCACCACTGGGTTTACTG; TIP3-2-check-R: GAACTTCAGGGTCAGCTTGC). Positive seedlings were then transferred to the field for self-pollination to obtain T1 generation transgenic seeds. Multiple generations of self-pollination were then conducted to obtain homozygous ZmTIP3-2 overexpression lines.

[0043] 2. Obtaining ZmTIP3-2 gene knockout mutant lines

[0044] The target sequences for ZmTIP3-2 knockout are (site 1: CATGACGGCCTCCAGCAGCACGG; site 2: GCACCAGCCGCTCGCGCCAGAGG). The U6 promoter, gRNA, and sgRNA expression cassette were cloned by PCR, and the 500-700 bp target fragment was recovered. The recombinant pEGAtCas9PcUBI-H-SIEIL2 vector was obtained through enzyme digestion and ligation, and transformed into Agrobacterium. Suitable-age maize ears were selected, and the immature embryos were placed in sterile tubes containing infection solution (50 mM MES, 2 mM MgCl2, 100 μM AS). Agrobacterium was activated, and the OD value of the bacterial culture was adjusted for later use. The original infection solution of the immature embryos was discarded, Agrobacterium suspension was added, and the culture was transferred to a co-culture plate for low-temperature co-culture. After co-culture, the culture was transferred to recovery medium, and then to Bar gene selection medium to obtain resistant callus. Resistant callus was transferred to regeneration medium to induce seedling emergence. PCR detection was performed using gene-edited mutant detection primers (ZmTIP3-2-cr-F2: TCATCGCCACCGCCATCTTC; ZmTIP3-2-cr-R2: TTGTGCCGTCCTGAACCATACC) to obtain knockout plants.

[0045] 3. Relative expression level analysis of ZmTIP3-2 gene

[0046] The obtained transgenic seeds were identified, and ZmTIP3-2 overexpression lines (ZmTIP3-2_OE6, ZmTIP3-2_OE15) and knockout mutant lines (cr_TIP3s-4) were selected for qRT-RCR detection. The genetic transformation background material KN5585 was used as a control to detect the relative expression level of the target gene in each line, and subsequent experiments and analyses were carried out.

[0047] 1. Plant RNA extraction kit (Magen) was used to extract total RNA from maize kernels 30 days after pollination. 1 μg of high-quality RNA (OD260 / OD280: 1.8-2.0; OD260 / OD230 ≈ 2.0) was used for reverse transcription (Novizan reverse transcription kit) to obtain first-strand cDNA.

[0048] 2. Perform quantitative real-time analysis using the cDNA from step 1 as a template. Primers used for quantitative analysis are shown in Table 1. Primers for the internal control gene are (Actin-F, Actin-R), primers for overexpression material are (OE-TIP3-2-RT-F1, OE-TIP3-2-RT-R1), and primers for knockout material are (CR-TIP3b-2-RT-F1, CR-TIP3b-2-RT-R1). The quantitative PCR reagent is ChamQ Blue Universal SYBR Qpcr Master Mix (Vazyme), and the quantitative PCR instrument is StepOne Software v2.3 (Applied Biosystems). The reaction system consists of: 10.0 μL of 2×PCR buffer, 0.4 μL of qF primer, 0.4 μL of qR primer, 2.0 μL of cDNA template, and 7.2 μL of sterile water, for a total reaction volume of 20.0 μL. Reaction program: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, 40 cycles.

[0049] Table 1 Primer List

[0050]

[0051] The results are as follows Figure 1 As shown, the expression level of ZmTIP3-2 gene was significantly increased in the ZmTIP3-2 overexpression lines, and significantly decreased in the ZmTIP3-2 knockout lines.

[0052] Example 2: Phenotypic analysis of maize ZmTIP3-2 transgenic plants

[0053] The control plant (KN5585), ZmTIP3-2 overexpression lines (ZmTIP3-2_OE6, ZmTIP3-2_OE15), and knockout mutant line (cr_TIP3s-4) were sown at the Agronomic Experiment Station of Shandong Agricultural University in 2024 and 2025. The experiment employed a completely randomized block design, with a planting pattern of 4 rows per plot, 3 meters in length, and a density of 4000 plants / mu. Three replicates were conducted for each sowing period, and field management conditions were identical to those used in local field production. Before the female ears emerged, the maize plants were bagged for self-pollination, and the pollination time was recorded to ensure accurate sampling timing. On days 15, 22, 29, 36, and 43 after pollination, 50 kernels from the middle of well-filled ears were collected, with three replicates for each maize sample. The selected seeds were weighed using an electronic balance, and their weight was recorded. After weighing, 50 seeds were placed in a drying box and placed in an oven at 105℃ for 30 minutes for rapid blanching to denature the enzymes in the seeds and terminate their respiration. The blanched seeds were then placed in an oven at 80℃ and dried until their weight became constant. The weight of the dried sample was then weighed and recorded.

[0054] Grain filling rate (g·d) -1 ) = [(W i+1 -W i ) / 50] / (t i+1 -t i )

[0055] Grain moisture content (%) = [(fresh weight - dry weight) / fresh weight] × 100%

[0056] Grain dehydration rate (%·d) -1 ) = (y i –y i+1 ) / (t i+1 -t i )

[0057] W i Let t be the dry weight of the i-th sample. i Let y be the time of the i-th sampling. i Let be the moisture content of the i-th sample.

[0058] 1000-kernel dry weight determination: After the kernels are fully mature, 100 kernels are randomly selected, and their fresh weight is recorded. The kernels are then dried in an 80℃ oven until their weight is constant. The weight of the dried sample is then weighed and recorded. Each material is tested in triplicate. 1000-kernel dry weight = 100 kernels dry weight × 10.

[0059] To verify the association and functional stability of the ZmTIP3-2 gene with maize kernel development, this invention conducted gene function verification experiments for two consecutive years, 2024 and 2025. The results are as follows: Figure 2-4 As shown, the validation experiments in 2024 and 2025 consistently demonstrated that: the grain filling rate of the ZmTIP3-2 overexpression material was significantly higher than that of the control plant (KN5585), while the grain filling rate of the ZmTIP3-2 knockout material was significantly lower than that of the control plant. The thousand-grain weight of the ZmTIP3-2 overexpression material was significantly higher than that of the control material (KN5585), while the thousand-grain weight of the ZmTIP3-2 knockout material was significantly lower than that of the control plant. The dehydration rate of the ZmTIP3-2 overexpression material was significantly higher than that of the control material (KN5585), while the dehydration rate of the ZmTIP3-2 knockout material was significantly lower than that of the control plant.

[0060] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. A protein that regulates plant seed development, characterized in that, The protein has the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence that is at least 99% homologous to SEQ ID NO.

2.

2. The gene encoding the protein that regulates plant seed development as described in claim 1.

3. An expression cassette, recombinant vector, recombinant microorganism, or transgenic plant tissue or transgenic plant containing the gene of claim 2.

4. The application of the protein of claim 1, the gene of claim 2, or the expression cassette, recombinant vector, or recombinant microorganism of claim 3 in regulating plant seed development.

5. The application according to claim 4, characterized in that, The regulation of plant grain development is achieved by promoting plant grain development by increasing the content or activity of the protein in the plant or increasing the expression level of the gene in the plant, or by slowing down plant grain development by decreasing the content or activity of the protein in the plant or decreasing the expression level of the gene in the plant.

6. The application according to claim 5, characterized in that, The promotion of plant grain development is manifested in one or more of the following: increasing grain filling rate, increasing grain filling amount, increasing thousand-grain weight, and accelerating grain dehydration rate; the slowing down of plant grain development is manifested in one or more of the following: reducing grain filling rate, reducing grain filling amount, reducing thousand-grain weight, and reducing grain dehydration rate.

7. A method for cultivating transgenic plants, characterized in that, The method includes the step of introducing the gene of claim 2 into a recipient plant to obtain a transgenic plant; compared with the recipient plant, the transgenic plant has improved grain filling rate, grain filling amount, thousand-grain weight and / or grain dehydration rate.

8. A method for cultivating transgenic plants, characterized in that, The method includes the step of inhibiting the expression and / or activity of the protein described in claim 1 or inhibiting the expression of the gene described in claim 2 in the starting plant to obtain a transgenic plant; compared with the recipient plant, the transgenic plant has a reduced grain filling rate, grain filling amount, thousand-grain weight and / or grain dehydration rate.

9. The application of the method of claim 7 or 8 in plant breeding.

10. The application according to any one of claims 4 to 6 or the method according to any one of claims 7 to 8, characterized in that, The plant in question is a species of the genus Zea.

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