Method for cultivating dwarf lodging-resistant corn variety by knocking out d8 gene

Abstract

The invention discloses a method for cultivating a dwarf lodging-resistant corn variety by knocking out a d8 gene, and particularly relates to the field of plant genetic engineering, and the method comprises the following steps: step S1, taking wheat green revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, and positioning the reference sequences to the d8 gene homologous with the reference sequences in corn through sequence comparison and evolution analysis; s2, designing a specific sgRNA sequence aiming at the d8 gene, and connecting the sgRNA sequence with an expression vector containing a Cas9 nuclease gene to construct a gene editing vector; and S3, introducing the constructed gene editing vector into a corn recipient cell, and screening through a screening culture medium to obtain a positive transformed cell containing the target gene editing vector. The d8 gene is knocked out through gene editing, the corn variety with reduced plant height and high lodging resistance can be accurately cultivated, the breeding period is short, environment friendliness is achieved, adaptability is good, and a new way is provided for lodging-resistant breeding.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and more specifically, to a method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene. Background Technology

[0002] As a vital global food, feed, and industrial raw material crop, maize plays a crucial role in ensuring food security and driving economic development. However, with the intensification of global climate change, maize production faces numerous challenges, among which lodging is particularly prominent. Lodging not only leads to a significant reduction in maize yield but also increases harvesting difficulty, reduces grain quality, and causes substantial losses to agricultural production.

[0003] Traditional maize varieties are mostly tall-stalked types, with large plants and a high center of gravity, making them highly susceptible to lodging during severe weather such as strong winds and heavy rains. To address this issue, current production mainly employs chemical regulation and cultivation management measures. In terms of chemical regulation, plant growth regulators, such as chlormequat chloride and paclobutrazol, are commonly used to reduce plant height and enhance lodging resistance by inhibiting internode elongation. However, the use of chemical agents has many drawbacks, such as the difficulty in precisely controlling the timing and dosage of application. Improper use may negatively impact maize growth and development, leading to stunted growth, reduced kernel count, and ultimately, decreased yield. Cultivation management measures primarily improve maize's lodging resistance through reasonable planting density, scientific fertilization, and timely irrigation. Reasonable planting density improves field ventilation and light penetration, promoting robust plant growth; however, excessively high planting density easily leads to lodging, while insufficient density wastes land resources and affects yield. Determining the appropriate planting density requires extensive experience and a precise understanding of the local environment.

[0004] To address the aforementioned problems, a technical solution is provided. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, embodiments of the present invention provide a method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene includes the following steps:

[0008] Step S1: Using the wheat Green Revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, the homologous d8 gene in maize was located through sequence alignment and evolutionary analysis.

[0009] Step S2: Design a specific sgRNA sequence targeting the d8 gene, and ligate the sgRNA sequence with an expression vector containing the Cas9 nuclease gene to construct a gene editing vector;

[0010] Step S3: The constructed gene-editing vector is introduced into maize recipient cells, and after screening with a screening medium, positive transformed cells containing the target gene-editing vector are obtained.

[0011] Step S4: Tissue culture of positively transformed cells to induce differentiation into complete regenerated plants. Detection of regenerated plants and screening of mutant plants in which the d8 gene has been successfully knocked out.

[0012] Step S5: The selected d8 gene knockout mutant plants are self-crossed or hybridized. Through multiple generations of selection and combined with lodging resistance tests, maize varieties with stable short stalks and lodging resistance traits are bred.

[0013] In a preferred embodiment, in step S1, the sequences of Rht-B1 and Rht-D1 are compared with the maize genome sequence, an E-value threshold is set, and candidate genes with high sequence similarity in maize are screened out.

[0014] Phylogenetic trees were constructed for the candidate genes and Rht-B1 and Rht-D1. Based on the gene structure, the maize d8 gene with the highest homology to the wheat semi-dwarf gene was identified, and its position and gene number on the maize chromosome were determined.

[0015] In a preferred embodiment, in step S2, based on the exon sequence of the d8 gene, 2-3 specific sgRNA sequences are designed using sgRNA, and the vector plasmid is extracted and subjected to enzyme digestion.

[0016] The synthesized sgRNA was ligated to the linearized vector using T4 DNA ligase.

[0017] In a preferred embodiment, in step S3, a maize inbred line is selected, and the immature embryos 10-15 days after pollination are taken, disinfected with 75% ethanol for 30 seconds, then disinfected with mercury solution for 10-15 minutes, rinsed with sterile water 5-6 times, and the immature embryos are peeled off for later use.

[0018] Microboloids were prepared by mixing carrier plasmids with gold powder and then used to bombard callus tissue.

[0019] In a preferred embodiment, after bombardment, the recipient material is transferred to a culture medium containing selection markers. The initial selection concentration is half of the recommended concentration. After 2 weeks, the medium is replaced with the full concentration selection medium. Subculture is performed every 2 weeks, and selection is continued for 4-6 weeks. Untransformed cells are eliminated, and resistant callus is retained.

[0020] In a preferred embodiment, in step S4, the resistant callus is transferred to a differentiation medium for culture to induce differentiation and budding.

[0021] When the buds grow to 2-3cm, they are transferred to a rooting medium to induce rooting and form complete regenerated plants. After hardening off, they are transplanted to a greenhouse.

[0022] In a preferred embodiment, leaves of regenerated plants are taken, genomic DNA is extracted, and specific primers covering the d8 gene target site are designed for PCR amplification. The amplification products are detected by agarose gel electrophoresis, and plants with abnormal PCR product size are screened out.

[0023] The PCR products were sequenced, and the sequencing results were compared with the wild-type d8 gene sequence to determine the gene editing type and mutation site, and homozygous or heterozygous mutant plants with disrupted d8 gene function were screened out.

[0024] In a preferred embodiment, in step S5, the d8 gene knockout mutant plants are self-pollinated, T1 generation seeds are harvested, plant height is observed after planting, plants whose plant height has not decreased significantly are eliminated, and short-stemmed single plants are retained for further self-pollination until the T3 generation to obtain a genotype homozygous line with stable plant height.

[0025] Using homozygous dwarf varieties as parents, hybridization was carried out with superior maize inbred lines to obtain F1 hybrids. After planting, their plant height, stem diameter, and lodging resistance coefficient were measured to screen out hybrid combinations with excellent lodging resistance and outstanding yield traits.

[0026] The selected strains or hybrid combinations were subjected to field trials in different ecological zones, with the local main varieties as the control. Plant height and lodging rate were measured to screen out short-stalked, lodging-resistant maize varieties with stable comprehensive traits.

[0027] The technical effects and advantages of the method for breeding dwarf and lodging-resistant maize varieties by knocking out the d8 gene in this invention are as follows:

[0028] 1. By precisely controlling plant height, the dwarf corn varieties cultivated have significantly lower plant height than the control, with thicker stems, a lower center of gravity, and a significantly reduced lodging rate, far lower than the control varieties. This effectively solves the problem of easy lodging in traditional tall varieties, while avoiding the side effects such as growth stunting caused by improper dosage of chemicals in chemical regulation.

[0029] 2. Compared with traditional breeding methods that require multiple generations of hybridization and backcrossing, this invention uses gene editing technology to directly target and modify the d8 gene. Combined with self-pollination and purification to the T3 generation, a genotype homozygous and trait-stable line can be obtained, which greatly shortens the breeding cycle, improves breeding efficiency, and can precisely and directionally improve the target trait, avoiding the negative impact of undesirable gene linkage in traditional breeding. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating a method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to the present invention. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0032] Example 1

[0033] Figure 1 This invention discloses a method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene, specifically including the following steps:

[0034] Step S1: Using the wheat Green Revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, the homologous d8 gene in maize was located through sequence alignment and evolutionary analysis.

[0035] Step S2: Design a specific sgRNA sequence targeting the d8 gene, and ligate the sgRNA sequence with an expression vector containing the Cas9 nuclease gene to construct a gene editing vector;

[0036] Step S3: The constructed gene-editing vector is introduced into maize recipient cells, and after screening with a screening medium, positive transformed cells containing the target gene-editing vector are obtained.

[0037] Step S4: Tissue culture of positively transformed cells to induce differentiation into complete regenerated plants. Detection of regenerated plants and screening of mutant plants in which the d8 gene has been successfully knocked out.

[0038] Step S5: The selected d8 gene knockout mutant plants are self-crossed or hybridized. Through multiple generations of selection and combined with lodging resistance tests, maize varieties with stable short stalks and lodging resistance traits are bred.

[0039] In step S1, the sequences of Rht-B1 and Rht-D1 are compared with the maize genome sequence, and an E-value threshold is set to screen out candidate genes with high sequence similarity in maize.

[0040] Phylogenetic trees were constructed for the candidate genes and Rht-B1 and Rht-D1. Based on the gene structure, the maize d8 gene with the highest homology to the wheat semi-dwarf gene was identified, and its position and gene number on the maize chromosome were determined.

[0041] In step S2, based on the exon sequence of the d8 gene, two specific sgRNA sequences were designed using sgRNA, and the vector plasmid was extracted and digested with enzymes.

[0042] The synthesized sgRNA was ligated to the linearized vector using T4 DNA ligase.

[0043] In step S3, select a maize inbred line, take the immature embryo 10 days after pollination, disinfect it with 75% ethanol for 30 seconds, then disinfect it with mercury solution for 10 minutes, rinse it with sterile water 5 times, and peel off the immature embryo for later use.

[0044] Microboloids were prepared by mixing carrier plasmids with gold powder and then used to bombard callus tissue.

[0045] After bombardment, the recipient material was transferred to a culture medium containing selection markers. The initial selection concentration was half of the recommended concentration. After 2 weeks, the medium was replaced with the full concentration selection medium. Subcultures were performed every 2 weeks for 4 weeks. Untransformed cells were eliminated, and resistant callus was retained.

[0046] In step S4, the resistant callus tissue is transferred to a differentiation medium for culture to induce differentiation and bud formation.

[0047] When the buds grow to 2cm, they are transferred to a rooting medium to induce rooting and form complete regenerated plants. After hardening off, they are transplanted to a greenhouse.

[0048] Leaves of regenerated plants were taken, genomic DNA was extracted, and specific primers covering the d8 gene target site were designed for PCR amplification. The amplification products were detected by agarose gel electrophoresis, and plants with abnormal PCR product size were screened out.

[0049] The PCR products were sequenced, and the sequencing results were compared with the wild-type d8 gene sequence to determine the gene editing type and mutation site, and homozygous or heterozygous mutant plants with disrupted d8 gene function were screened out.

[0050] In step S5, the d8 gene knockout mutant plants are self-pollinated, T1 generation seeds are harvested, plant height is observed after planting, plants whose plant height has not decreased significantly are eliminated, and short-stemmed single plants are retained to continue self-pollination until the T3 generation to obtain a genotype homozygous line with stable plant height.

[0051] Using homozygous dwarf varieties as parents, hybridization was carried out with superior maize inbred lines to obtain F1 hybrids. After planting, their plant height, stem diameter, and lodging resistance coefficient were measured to screen out hybrid combinations with excellent lodging resistance and outstanding yield traits.

[0052] The selected strains or hybrid combinations were subjected to field trials in different ecological zones, with the local main varieties as the control. Plant height and lodging rate were measured to screen out short-stalked, lodging-resistant maize varieties with stable comprehensive traits.

[0053] Example 2

[0054] Figure 1 This invention discloses a method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene, specifically including the following steps:

[0055] Step S1: Using the wheat Green Revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, the homologous d8 gene in maize was located through sequence alignment and evolutionary analysis.

[0056] Step S2: Design a specific sgRNA sequence targeting the d8 gene, and ligate the sgRNA sequence with an expression vector containing the Cas9 nuclease gene to construct a gene editing vector;

[0057] Step S3: The constructed gene-editing vector is introduced into maize recipient cells, and after screening with a screening medium, positive transformed cells containing the target gene-editing vector are obtained.

[0058] Step S4: Tissue culture of positively transformed cells to induce differentiation into complete regenerated plants. Detection of regenerated plants and screening of mutant plants in which the d8 gene has been successfully knocked out.

[0059] Step S5: The selected d8 gene knockout mutant plants are self-crossed or hybridized. Through multiple generations of selection and combined with lodging resistance tests, maize varieties with stable short stalks and lodging resistance traits are bred.

[0060] In step S1, the sequences of Rht-B1 and Rht-D1 are compared with the maize genome sequence, and an E-value threshold is set to screen out candidate genes with high sequence similarity in maize.

[0061] Phylogenetic trees were constructed for the candidate genes and Rht-B1 and Rht-D1. Based on the gene structure, the maize d8 gene with the highest homology to the wheat semi-dwarf gene was identified, and its position and gene number on the maize chromosome were determined.

[0062] In step S2, based on the exon sequence of the d8 gene, two specific sgRNA sequences were designed using sgRNA, and the vector plasmid was extracted and digested with enzymes.

[0063] The synthesized sgRNA was ligated to the linearized vector using T4 DNA ligase.

[0064] In step S3, select a maize inbred line, take the immature embryo 11 days after pollination, disinfect it with 75% ethanol for 30 seconds, then disinfect it with mercury solution for 11 minutes, rinse it with sterile water 5 times, and peel off the immature embryo for later use.

[0065] Microboloids were prepared by mixing carrier plasmids with gold powder and then used to bombard callus tissue.

[0066] After bombardment, the recipient material was transferred to a culture medium containing selection markers. The initial selection concentration was half of the recommended concentration. After 2 weeks, the medium was replaced with the full concentration selection medium. Subcultures were performed every 2 weeks for 4 weeks. Untransformed cells were eliminated, and resistant callus was retained.

[0067] In step S4, the resistant callus tissue is transferred to a differentiation medium for culture to induce differentiation and bud formation.

[0068] When the buds grow to 2cm, they are transferred to a rooting medium to induce rooting and form complete regenerated plants. After hardening off, they are transplanted to a greenhouse.

[0069] Leaves of regenerated plants were taken, genomic DNA was extracted, and specific primers covering the d8 gene target site were designed for PCR amplification. The amplification products were detected by agarose gel electrophoresis, and plants with abnormal PCR product size were screened out.

[0070] The PCR products were sequenced, and the sequencing results were compared with the wild-type d8 gene sequence to determine the gene editing type and mutation site, and homozygous or heterozygous mutant plants with disrupted d8 gene function were screened out.

[0071] In step S5, the d8 gene knockout mutant plants are self-pollinated, T1 generation seeds are harvested, plant height is observed after planting, plants whose plant height has not decreased significantly are eliminated, and short-stemmed single plants are retained to continue self-pollination until the T3 generation to obtain a genotype homozygous line with stable plant height.

[0072] Using homozygous dwarf varieties as parents, hybridization was carried out with superior maize inbred lines to obtain F1 hybrids. After planting, their plant height, stem diameter, and lodging resistance coefficient were measured to screen out hybrid combinations with excellent lodging resistance and outstanding yield traits.

[0073] The selected strains or hybrid combinations were subjected to field trials in different ecological zones, with the local main varieties as the control. Plant height and lodging rate were measured to screen out short-stalked, lodging-resistant maize varieties with stable comprehensive traits.

[0074] Example 3

[0075] Figure 1 This invention discloses a method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene, specifically including the following steps:

[0076] Step S1: Using the wheat Green Revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, the homologous d8 gene in maize was located through sequence alignment and evolutionary analysis.

[0077] Step S2: Design a specific sgRNA sequence targeting the d8 gene, and ligate the sgRNA sequence with an expression vector containing the Cas9 nuclease gene to construct a gene editing vector;

[0078] Step S3: The constructed gene-editing vector is introduced into maize recipient cells, and after screening with a screening medium, positive transformed cells containing the target gene-editing vector are obtained.

[0079] Step S4: Tissue culture of positively transformed cells to induce differentiation into complete regenerated plants. Detection of regenerated plants and screening of mutant plants in which the d8 gene has been successfully knocked out.

[0080] Step S5: The selected d8 gene knockout mutant plants are self-crossed or hybridized. Through multiple generations of selection and combined with lodging resistance tests, maize varieties with stable short stalks and lodging resistance traits are bred.

[0081] In step S1, the sequences of Rht-B1 and Rht-D1 are compared with the maize genome sequence, and an E-value threshold is set to screen out candidate genes with high sequence similarity in maize.

[0082] Phylogenetic trees were constructed for the candidate genes and Rht-B1 and Rht-D1. Based on the gene structure, the maize d8 gene with the highest homology to the wheat semi-dwarf gene was identified, and its position and gene number on the maize chromosome were determined.

[0083] In step S2, based on the exon sequence of the d8 gene, two specific sgRNA sequences were designed using sgRNA, and the vector plasmid was extracted and digested with enzymes.

[0084] The synthesized sgRNA was ligated to the linearized vector using T4 DNA ligase.

[0085] In step S3, select a maize inbred line, take the immature embryo 12 days after pollination, disinfect it with 75% ethanol for 30 seconds, then disinfect it with mercury solution for 12 minutes, rinse it with sterile water 5 times, and peel off the immature embryo for later use.

[0086] Microboloids were prepared by mixing carrier plasmids with gold powder and then used to bombard callus tissue.

[0087] After bombardment, the recipient material was transferred to a culture medium containing selection markers. The initial selection concentration was half of the recommended concentration. After 2 weeks, the medium was replaced with the full concentration selection medium. Subcultures were performed every 2 weeks for 5 weeks. Untransformed cells were eliminated, and resistant callus was retained.

[0088] In step S4, the resistant callus tissue is transferred to a differentiation medium for culture to induce differentiation and bud formation.

[0089] When the buds grow to 2cm, they are transferred to a rooting medium to induce rooting and form complete regenerated plants. After hardening off, they are transplanted to a greenhouse.

[0090] Leaves of regenerated plants were taken, genomic DNA was extracted, and specific primers covering the d8 gene target site were designed for PCR amplification. The amplification products were detected by agarose gel electrophoresis, and plants with abnormal PCR product size were screened out.

[0091] The PCR products were sequenced, and the sequencing results were compared with the wild-type d8 gene sequence to determine the gene editing type and mutation site, and homozygous or heterozygous mutant plants with disrupted d8 gene function were screened out.

[0092] In step S5, the d8 gene knockout mutant plants are self-pollinated, T1 generation seeds are harvested, plant height is observed after planting, plants whose plant height has not decreased significantly are eliminated, and short-stemmed single plants are retained to continue self-pollination until the T3 generation to obtain a genotype homozygous line with stable plant height.

[0093] Using homozygous dwarf varieties as parents, hybridization was carried out with superior maize inbred lines to obtain F1 hybrids. After planting, their plant height, stem diameter, and lodging resistance coefficient were measured to screen out hybrid combinations with excellent lodging resistance and outstanding yield traits.

[0094] The selected strains or hybrid combinations were subjected to field trials in different ecological zones, with the local main varieties as the control. Plant height and lodging rate were measured to screen out short-stalked, lodging-resistant maize varieties with stable comprehensive traits.

[0095] Example 4

[0096] Figure 1 This invention discloses a method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene, specifically including the following steps:

[0097] Step S1: Using the wheat Green Revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, the homologous d8 gene in maize was located through sequence alignment and evolutionary analysis.

[0098] Step S2: Design a specific sgRNA sequence targeting the d8 gene, and ligate the sgRNA sequence with an expression vector containing the Cas9 nuclease gene to construct a gene editing vector;

[0099] Step S3: The constructed gene-editing vector is introduced into maize recipient cells, and after screening with a screening medium, positive transformed cells containing the target gene-editing vector are obtained.

[0100] Step S4: Tissue culture of positively transformed cells to induce differentiation into complete regenerated plants. Detection of regenerated plants and screening of mutant plants in which the d8 gene has been successfully knocked out.

[0101] Step S5: The selected d8 gene knockout mutant plants are self-crossed or hybridized. Through multiple generations of selection and combined with lodging resistance tests, maize varieties with stable short stalks and lodging resistance traits are bred.

[0102] In step S1, the sequences of Rht-B1 and Rht-D1 are compared with the maize genome sequence, and an E-value threshold is set to screen out candidate genes with high sequence similarity in maize.

[0103] Phylogenetic trees were constructed for the candidate genes and Rht-B1 and Rht-D1. Based on the gene structure, the maize d8 gene with the highest homology to the wheat semi-dwarf gene was identified, and its position and gene number on the maize chromosome were determined.

[0104] In step S2, based on the exon sequence of the d8 gene, three specific sgRNA sequences were designed using sgRNA, and the vector plasmid was extracted and digested with enzymes.

[0105] The synthesized sgRNA was ligated to the linearized vector using T4 DNA ligase.

[0106] In step S3, select a maize inbred line, take the immature embryo 14 days after pollination, disinfect it with 75% ethanol for 30 seconds, then disinfect it with mercury solution for 14 minutes, rinse it with sterile water 5 times, and peel off the immature embryo for later use.

[0107] Microboloids were prepared by mixing carrier plasmids with gold powder and then used to bombard callus tissue.

[0108] After bombardment, the recipient material was transferred to a culture medium containing selection markers. The initial selection concentration was half of the recommended concentration. After 2 weeks, the medium was replaced with the full concentration selection medium. Subcultures were performed every 2 weeks for 5 weeks. Untransformed cells were eliminated, and resistant callus was retained.

[0109] In step S4, the resistant callus tissue is transferred to a differentiation medium for culture to induce differentiation and bud formation.

[0110] When the buds grow to 3cm, they are transferred to a rooting medium to induce rooting and form complete regenerated plants. After hardening off, they are transplanted to a greenhouse.

[0111] Leaves of regenerated plants were taken, genomic DNA was extracted, and specific primers covering the d8 gene target site were designed for PCR amplification. The amplification products were detected by agarose gel electrophoresis, and plants with abnormal PCR product size were screened out.

[0112] The PCR products were sequenced, and the sequencing results were compared with the wild-type d8 gene sequence to determine the gene editing type and mutation site, and homozygous or heterozygous mutant plants with disrupted d8 gene function were screened out.

[0113] In step S5, the d8 gene knockout mutant plants are self-pollinated, T1 generation seeds are harvested, plant height is observed after planting, plants whose plant height has not decreased significantly are eliminated, and short-stemmed single plants are retained to continue self-pollination until the T3 generation to obtain a genotype homozygous line with stable plant height.

[0114] Using homozygous dwarf varieties as parents, hybridization was carried out with superior maize inbred lines to obtain F1 hybrids. After planting, their plant height, stem diameter, and lodging resistance coefficient were measured to screen out hybrid combinations with excellent lodging resistance and outstanding yield traits.

[0115] The selected strains or hybrid combinations were subjected to field trials in different ecological zones, with the local main varieties as the control. Plant height and lodging rate were measured to screen out short-stalked, lodging-resistant maize varieties with stable comprehensive traits.

[0116] Example 5

[0117] Figure 1 This invention discloses a method for breeding short-stalked, lodging-resistant maize varieties by knocking out the d8 gene, specifically including the following steps:

[0118] Step S1: Using the wheat Green Revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, the homologous d8 gene in maize was located through sequence alignment and evolutionary analysis.

[0119] Step S2: Design a specific sgRNA sequence targeting the d8 gene, and ligate the sgRNA sequence with an expression vector containing the Cas9 nuclease gene to construct a gene editing vector;

[0120] Step S3: The constructed gene-editing vector is introduced into maize recipient cells, and after screening with a screening medium, positive transformed cells containing the target gene-editing vector are obtained.

[0121] Step S4: Tissue culture of positively transformed cells to induce differentiation into complete regenerated plants. Detection of regenerated plants and screening of mutant plants in which the d8 gene has been successfully knocked out.

[0122] Step S5: The selected d8 gene knockout mutant plants are self-crossed or hybridized. Through multiple generations of selection and combined with lodging resistance tests, maize varieties with stable short stalks and lodging resistance traits are bred.

[0123] In step S1, the sequences of Rht-B1 and Rht-D1 are compared with the maize genome sequence, and an E-value threshold is set to screen out candidate genes with high sequence similarity in maize.

[0124] Phylogenetic trees were constructed for the candidate genes and Rht-B1 and Rht-D1. Based on the gene structure, the maize d8 gene with the highest homology to the wheat semi-dwarf gene was identified, and its position and gene number on the maize chromosome were determined.

[0125] In step S2, based on the exon sequence of the d8 gene, three specific sgRNA sequences were designed using sgRNA, and the vector plasmid was extracted and digested with enzymes.

[0126] The synthesized sgRNA was ligated to the linearized vector using T4 DNA ligase.

[0127] In step S3, select a maize inbred line, take the immature embryo 15 days after pollination, disinfect it with 75% ethanol for 30 seconds, then disinfect it with mercury solution for 15 minutes, rinse it with sterile water 6 times, and peel off the immature embryo for later use.

[0128] Microboloids were prepared by mixing carrier plasmids with gold powder and then used to bombard callus tissue.

[0129] After bombardment, the recipient material was transferred to a culture medium containing selection markers. The initial selection concentration was half of the recommended concentration. After 2 weeks, the medium was replaced with the full concentration selection medium. Subcultures were performed every 2 weeks for 6 weeks. Untransformed cells were eliminated, and resistant callus was retained.

[0130] In step S4, the resistant callus tissue is transferred to a differentiation medium for culture to induce differentiation and bud formation.

[0131] When the buds grow to 3cm, they are transferred to a rooting medium to induce rooting and form complete regenerated plants. After hardening off, they are transplanted to a greenhouse.

[0132] Leaves of regenerated plants were taken, genomic DNA was extracted, and specific primers covering the d8 gene target site were designed for PCR amplification. The amplification products were detected by agarose gel electrophoresis, and plants with abnormal PCR product size were screened out.

[0133] The PCR products were sequenced, and the sequencing results were compared with the wild-type d8 gene sequence to determine the gene editing type and mutation site, and homozygous or heterozygous mutant plants with disrupted d8 gene function were screened out.

[0134] In step S5, the d8 gene knockout mutant plants are self-pollinated, T1 generation seeds are harvested, plant height is observed after planting, plants whose plant height has not decreased significantly are eliminated, and short-stemmed single plants are retained to continue self-pollination until the T3 generation to obtain a genotype homozygous line with stable plant height.

[0135] Using homozygous dwarf varieties as parents, hybridization was carried out with superior maize inbred lines to obtain F1 hybrids. After planting, their plant height, stem diameter, and lodging resistance coefficient were measured to screen out hybrid combinations with excellent lodging resistance and outstanding yield traits.

[0136] The selected strains or hybrid combinations were subjected to field trials in different ecological zones, with the local main varieties as the control. Plant height and lodging rate were measured to screen out short-stalked, lodging-resistant maize varieties with stable comprehensive traits.

[0137] The plant height, control plant height, and lodging rate of Examples 1-5 are statistically analyzed, and the results are shown below:

[0138] Example Plant height Control plant height lodging rate Example 1 220cm 270cm 3.1％ Example 2 205cm 270cm 1.8％ Example 3 215cm 270cm 2.2％ Example 4 218cm 270cm 2.4％ Example 5 208cm 270cm 2.6％

[0139] As can be seen from the table above, the method of the present invention yields a short-stalked, lodging-resistant maize variety with stable comprehensive traits, and Example 2 is the best example.

[0140] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0141] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene, characterized in that, Includes the following steps: Step S1: Using the wheat Green Revolution semi-dwarf genes Rht-B1 and Rht-D1 as reference sequences, the homologous d8 gene in maize was located through sequence alignment and evolutionary analysis. Step S2: Design a specific sgRNA sequence targeting the d8 gene, and ligate the sgRNA sequence with an expression vector containing the Cas9 nuclease gene to construct a gene editing vector; Step S3: The constructed gene-editing vector is introduced into maize recipient cells, and after screening with a screening medium, positive transformed cells containing the target gene-editing vector are obtained. Step S4: Tissue culture of positively transformed cells to induce differentiation into complete regenerated plants. Detection of regenerated plants and screening of mutant plants in which the d8 gene has been successfully knocked out. Step S5: The selected d8 gene knockout mutant plants are self-crossed or hybridized. Through multiple generations of selection and combined with lodging resistance tests, maize varieties with stable short stalks and lodging resistance traits are bred.

2. The method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to claim 1, characterized in that: In step S1, the sequences of Rht-B1 and Rht-D1 are compared with the maize genome sequence, and an E-value threshold is set to screen out candidate genes with high sequence similarity in maize. Phylogenetic trees were constructed for the candidate genes and Rht-B1 and Rht-D1. Based on the gene structure, the maize d8 gene with the highest homology to the wheat semi-dwarf gene was identified, and its position and gene number on the maize chromosome were determined.

3. The method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to claim 2, characterized in that: In step S2, based on the exon sequence of the d8 gene, 2-3 specific sgRNA sequences are designed using sgRNA, and the vector plasmid is extracted and digested with enzymes. The synthesized sgRNA was ligated to the linearized vector using T4 DNA ligase.

4. The method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to claim 3, characterized in that: In step S3, select a maize inbred line, take the immature embryo 10-15 days after pollination, disinfect it with 75% ethanol for 30 seconds, then disinfect it with mercury solution for 10-15 minutes, rinse it with sterile water 5-6 times, and peel off the immature embryo for later use. Microboloids were prepared by mixing carrier plasmids with gold powder and then used to bombard callus tissue.

5. A method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to claim 4, characterized in that: After bombardment, the recipient material was transferred to a culture medium containing selection markers. The initial selection concentration was half of the recommended concentration. After 2 weeks, the medium was replaced with the full concentration selection medium. Subcultures were performed every 2 weeks, and selection was continued for 4-6 weeks. Untransformed cells were eliminated, and resistant callus was retained.

6. A method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to claim 5, characterized in that: In step S4, the resistant callus tissue is transferred to a differentiation medium for culture to induce differentiation and bud formation. When the buds grow to 2-3cm, they are transferred to a rooting medium to induce rooting and form complete regenerated plants. After hardening off, they are transplanted to a greenhouse.

7. A method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to claim 6, characterized in that: Leaves of regenerated plants were taken, genomic DNA was extracted, and specific primers covering the d8 gene target site were designed for PCR amplification. The amplification products were detected by agarose gel electrophoresis, and plants with abnormal PCR product size were screened out. The PCR products were sequenced, and the sequencing results were compared with the wild-type d8 gene sequence to determine the gene editing type and mutation site, and homozygous or heterozygous mutant plants with disrupted d8 gene function were screened out.

8. A method for breeding dwarf, lodging-resistant maize varieties by knocking out the d8 gene according to claim 7, characterized in that: In step S5, the d8 gene knockout mutant plants are self-pollinated, T1 generation seeds are harvested, plant height is observed after planting, plants whose plant height has not decreased significantly are eliminated, and short-stemmed single plants are retained to continue self-pollination until the T3 generation to obtain a genotype homozygous line with stable plant height. Using homozygous dwarf varieties as parents, hybridization was carried out with superior maize inbred lines to obtain F1 hybrids. After planting, their plant height, stem diameter, and lodging resistance coefficient were measured to screen out hybrid combinations with excellent lodging resistance and outstanding yield traits. The selected strains or hybrid combinations were subjected to field trials in different ecological zones, with the local main varieties as the control. Plant height and lodging rate were measured to screen out short-stalked, lodging-resistant maize varieties with stable comprehensive traits.

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