Method for cultivating dwarf lodging-resistant corn variety based on d9 gene knockout

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a method for cultivating a dwarf lodging-resistant corn variety based on d9 gene knockout, which comprises the following steps: S1, determining a target gene: finding a homologous gene d9 of wheat green revolution semi-dwarf genes Rht-B1 and Rht-D1 in corn by utilizing a bioinformatics analysis method; s2, construction of a gene editing vector: constructing the gene editing vector for knocking out the d9 gene, wherein the vector comprises a targeted editing element aiming at the d9 gene; s3, transforming corn cells: introducing the constructed gene editing vector into the corn cells; s4, obtaining a mutant plant; and S5, screening and culturing. The method is reasonable in design, a knockout mutant of the d9 gene is created by utilizing a gene editing technology, and a genetic material capable of remarkably reducing the corn plant height is obtained; through knockout of the d9 gene, the corn plant height can be effectively reduced, the lodging resistance of corn is improved, a new candidate gene and an effective method are provided for corn breeding, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and in particular to a method for breeding a short-stalk lodging-resistant maize variety based on d9 gene knockout. BACKGROUND

[0002] Maize is an important food crop, feed crop and industrial raw material in China, and plays a vital role in agricultural production. With the growth of population and economic development, the demand for maize is increasing. However, lodging is a serious problem that affects yield and quality during maize production. Lodging not only causes maize plants to break and grains to fall off, but also increases the difficulty and cost of harvesting, causing great losses to agricultural production.

[0003] Breeding short-stalk lodging-resistant maize varieties is one of the effective ways to solve the problem of maize lodging. Traditional breeding methods mainly select varieties with short-stalk lodging-resistant characteristics through hybridization breeding, but this method has the disadvantages of long breeding cycle and poor directionality. With the development of genetic engineering technology, the use of gene editing technology for directional modification of maize genes provides a new idea and method for breeding short-stalk lodging-resistant maize varieties.

[0004] The discovery and use of the semi-dwarf green revolution genes Rht-B1 and Rht-D1 in wheat greatly improved the lodging resistance and yield of wheat, so it is necessary to find similar genes in maize and edit them. The d9 gene is an important gene in maize, and research shows that it is related to the regulation of maize plant height. By knocking out the d9 gene in maize using gene editing technology, a short-stalk lodging-resistant maize variety can be obtained, providing a new technical means for maize lodging resistance breeding. Therefore, we propose a method for breeding a short-stalk lodging-resistant maize variety based on d9 gene knockout. SUMMARY

[0005] The purpose of the present application is to solve the problems existing in the prior art, and to provide a method for breeding a short-stalk lodging-resistant maize variety based on d9 gene knockout.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A method for breeding a short-stalk lodging-resistant maize variety based on d9 gene knockout, comprising the following steps:

[0008] S1: Determine the target gene: use bioinformatics analysis method to find the homologous gene d9 of the semi-dwarf green revolution genes Rht-B1 and Rht-D1 in wheat in maize;

[0009] S2: Gene editing vector construction: construct a gene editing vector for knocking out the d9 gene, which contains a targeted editing element for the d9 gene;

[0010] S3: Transforming corn cells: introducing the constructed gene editing vector into corn cells;

[0011] S4: Obtaining mutant plants: culturing and screening the transformed corn cells to obtain corn mutant plants with d9 gene knockout;

[0012] S5: Screening and breeding: measuring the plant height and evaluating the resistance to lodging of the obtained corn mutant plants, screening out plants with significantly reduced plant height and strong resistance to lodging, and then breeding into stable genetic dwarf corn varieties with resistance to lodging.

[0013] Preferably, using the bioinformatics tool BLAST, the amino acid sequences of wheat Rht-B1 and Rht-D1 genes are homologously aligned with the sequences in the corn genome database, and candidate genes d9 with homology ≥70% are screened out.

[0014] The sequence structure of d9 gene is analyzed to determine its coding region (CDS), promoter region and key functional domain, and the targeted region for gene editing is determined.

[0015] Preferably, the construction of gene editing vector includes sgRNA design: according to the sequence of the targeted region of d9 gene, 2-3 specific sgRNAs are designed using CRISPR design tool to ensure no off-target risk in corn genome and off-target probability ≤0.01%; sgRNA sequence needs to contain 20bp core region complementary to target sequence and PAM sequence (NGG).

[0016] Preferably, the construction of gene editing vector also includes vector assembly: using pUbi as the basic vector, the following elements are inserted in the direction from RB to LB:

[0017] Promoter: corn ubiquitin promoter (pUbi) driving Cas9 protein expression;

[0018] Nuclease gene: Cas9 coding gene (carrying nuclear localization signal NLS);

[0019] sgRNA expression cassette: containing U6 promoter, designed sgRNA sequence and terminator;

[0020] Screening marker gene: hygromycin resistance gene (Hpt) driven by 35S promoter (CaMV35S);

[0021] Border sequence: T-DNA left and right border (RB, LB) to ensure integration of vector fragment into corn genome.

[0022] Vector verification: enzyme digestion identification and sequencing verification to ensure correct vector construction.

[0023] Preferably, the gene editing vector is introduced into the corn recipient cell by using the Agrobacterium-mediated method in S3, and the Agrobacterium strain is EHA105.

[0024] Preferably, the co-culture is as follows: the disinfected young embryo is mixed with the Agrobacterium bacterial solution, and co-cultured at 28 DEG C in the dark for 2-3 days, and the pH of the culture medium is maintained at 5.2 during the period.

[0025] The screening culture is as follows: after the co-culture, the young embryo is transferred to the MS screening culture medium containing 50 mg / L hygromycin and 250 mg / L cephalosporin, and cultured at 25 DEG C in the dark, and the culture medium is replaced every 2 weeks, and the resistant callus is obtained by continuous screening for 3-4 generations.

[0026] Preferably, obtaining the mutant plant comprises differentiation culture, rooting culture and molecular identification.

[0027] Preferably, the differentiation culture is as follows: the resistant callus is transferred to the differentiation culture medium, and cultured at 25 DEG C under the condition of 16 h light / 8 h dark, so as to induce differentiation of the bud.

[0028] The rooting culture is as follows: when the bud is grown to 3-5 cm, the bud is cut and transferred to the rooting culture medium, and the culture is continued until the root is formed to form a complete plant.

[0029] The molecular identification is as follows: the genomic DNA of the regenerated plant is extracted, the target editing region of the d9 gene is amplified by PCR, and the amplification product is sequenced and analyzed to determine whether base insertion, deletion or replacement occurs, and the d9 gene knockout homozygous mutant is screened out.

[0030] Preferably, the plant height is measured as follows: the positive mutant is planted in the field together with the wild type (WT), and the plant height is measured at the mature stage, and the average value is calculated by repeating 3 times, and the mutant with a plant height reduction of more than 30% compared with the wild type is screened out.

[0031] The lodging resistance is evaluated as follows: at the corn filling stage, the wind speed is 10-12 m / s, the rainfall is 20 mm / h, and the duration is 30 min, the lodging rate is calculated, the number of lodging plants / the total number of plants*100%, and the mutant with a lodging rate of less than or equal to 10% is selected.

[0032] Preferably, the genetic stability is verified as follows: the selected excellent mutant is selfed and propagated, and the plant height and lodging resistance are observed to determine whether the genetic inheritance is stable, and finally the short-stalk lodging-resistant corn variety is cultivated.

[0033] The beneficial effects of the present application are as follows:

[0034] 1. The d9 gene knockout mutant is created by using the gene editing technology, and the corn genetic material with a significantly reduced plant height is successfully obtained, thereby providing a new candidate gene resource for corn short-stalk breeding.

[0035] 2. Reducing the plant height of corn by knocking out the d9 gene can effectively improve the lodging resistance of corn and reduce yield loss caused by lodging, which is of great significance to ensure the stability of corn yield;

[0036] 3. The target gene is determined based on bioinformatics analysis, and precise operation is combined with gene editing technology, which provides a direct and effective technical approach for using gene editing technology to breed short-stalk lodging-resistant corn varieties and accelerates the breeding process of corn lodging resistance;

[0037] 4. The d9 gene knockout mutant genetic material obtained has stable short-stalk characteristics and can be used as an excellent parent for subsequent corn variety improvement, providing valuable basic materials for corn breeding work. DETAILED DESCRIPTION

[0038] The embodiment of the present application discloses a method for breeding short-stalk lodging-resistant corn varieties based on d9 gene knockout. A method for breeding short-stalk lodging-resistant corn varieties based on d9 gene knockout comprises the following steps:

[0039] S1: Determine the target gene: use bioinformatics analysis method to find the homologous gene d9 of wheat green revolution semi-dwarf gene Rht-B1 and Rht-D1 in corn;

[0040] S2: Gene editing vector construction: construct a gene editing vector for knocking out the d9 gene, which comprises a targeted editing element for the d9 gene;

[0041] S3: Transforming corn cells: introducing the constructed gene editing vector into corn cells;

[0042] S4: Obtain mutant plants: culture and screen the transformed corn cells to obtain corn mutant plants with the d9 gene knocked out;

[0043] S5: Screening and breeding: measuring the plant height of the obtained corn mutant plants and evaluating the lodging resistance, screening out plants with significantly reduced plant height and strong lodging resistance, and then breeding into stable genetic short-stalk lodging-resistant corn varieties.

[0044] In this embodiment, with the help of bioinformatics tool BLAST, the amino acid sequences of wheat Rht-B1 and Rht-D1 genes are subjected to homology comparison with the sequences in the corn genome database, and the candidate gene d9 with homology ≥70% is screened out. The sequence structure of the d9 gene is analyzed to determine its coding region (CDS), promoter region and key functional domain, and the target region of gene editing is determined. Through precise homology comparison and sequence analysis, the reliability of the target gene d9 is ensured, laying a foundation for the efficiency and specificity of subsequent gene editing.

[0045] In this embodiment, the gene editing vector construction includes sgRNA design: according to the sequence of the target region of d9 gene, 2-3 specific sgRNAs are designed by using CRISPR design tool, ensuring that there is no off-target risk in the corn genome, and the off-target probability is ≤0.01%; the sgRNA sequence needs to contain a 20bp core region complementary to the target sequence and a PAM sequence (NGG). The sgRNA design with low off-target risk can reduce the non-specific interference of gene editing on the corn genome, and ensure the genetic stability and safety of the mutant.

[0046] In this embodiment, the gene editing vector construction also includes vector assembly: the following elements are inserted in the RB to LB direction based on the pUbi as the basic vector:

[0047] Promoter: corn ubiquitin promoter (pUbi), driving Cas9 protein expression;

[0048] Nuclease gene: Cas9 coding gene (carrying nuclear localization signal NLS);

[0049] sgRNA expression cassette: containing U6 promoter, designed sgRNA sequence and terminator;

[0050] Selection marker gene: hygromycin resistance gene (Hpt), driven by 35S promoter (CaMV35S);

[0051] Border sequence: T-DNA left and right border (RB, LB), ensuring the integration of the vector fragment into the corn genome.

[0052] Vector verification: enzyme digestion identification and sequencing verification to ensure correct vector construction; strict vector verification steps can avoid experimental failure caused by incorrect vector construction and improve the success rate of subsequent genetic transformation.

[0053] In this embodiment, the Agrobacterium-mediated method is used to introduce the gene editing vector into the corn recipient cells in S3, and the Agrobacterium strain is EHA105. Co-culture: mix the disinfected young embryo with the Agrobacterium bacterial solution, and co-culture at 28°C in the dark for 2-3 days, maintaining the pH of the culture medium at 5.2 during the period; screening culture: after co-culture, transfer the young embryo to MS screening medium containing 50 mg / L hygromycin and 250 mg / L cefotaxime, and culture at 25°C in the dark, replace the culture medium every 2 weeks, and continuously screen for 3-4 generations to obtain resistant callus. The optimized co-culture conditions improve the interaction efficiency of Agrobacterium and corn young embryo, creating a suitable environment for successful transfer of vector fragments.

[0054] In this embodiment, obtaining mutant plants includes differentiation culture, rooting culture and molecular identification, differentiation culture: the resistant callus is transferred to differentiation culture medium (MS+2.0 mg / L 6-BA+0.5 mg / L NAA), and is cultured under the condition of 25 DEG C, 16 h illumination / 8 h darkness, so as to induce differentiation of buds; rooting culture: when the bud is 3-5 cm long, the bud is cut off and transferred to rooting culture medium (1 / 2MS+0.5 mg / L IBA), and specific hormone ratio and illumination condition promote bud differentiation of the callus and accelerate the formation process of regenerated plants;

[0055] Molecular identification: genomic DNA of the regenerated plant is extracted, a target editing region of the d9 gene is amplified by PCR, and the amplification product is sequenced and analyzed to determine whether base insertion, deletion or replacement occurs, and a d9 gene knockout homozygous mutant is screened out, the molecular level identification can accurately confirm the knockout effect of the d9 gene, and the genotype of the obtained mutant is accurate, which provides reliable materials for subsequent phenotype analysis.

[0056] In this embodiment, plant height determination: the positive mutant and wild type (WT) are planted in the field, and the plant height is measured at the mature stage, repeated 3 times, and the average value is calculated, and the mutant with plant height reduced by more than 30% compared with the wild type is screened out; lodging resistance evaluation: during the corn filling period, artificial simulation of wind and rain is carried out, the wind speed is 10-12 m / s, the rainfall is 20 mm / h, and the duration is 30 min, the lodging rate is calculated, the number of lodging plants / the total number of plants*100%, and the mutant with a lodging rate of less than or equal to 10% is selected; genetic stability verification: the selected excellent mutant is self-crossed and propagated, and is continuously planted for 3 generations, and whether the plant height and lodging resistance are stably inherited is observed, and finally a dwarf and lodging-resistant corn variety is bred, and strict phenotype screening and genetic stability verification ensure that the finally bred corn variety has stable dwarf characteristics and strong lodging resistance, and meets the needs of agricultural production.

[0057] In the present application, a d9 gene knockout mutant is created by using gene editing technology, and a corn genetic material with significantly reduced plant height is successfully obtained, which provides a new candidate gene resource for corn dwarf breeding, and the d9 gene knockout can effectively improve the lodging resistance of corn and reduce the yield loss caused by lodging, which has important significance for ensuring the stability of corn yield, based on bioinformatics analysis to determine the target gene, combined with gene editing technology for precise operation, which provides a direct and effective technical approach for breeding a dwarf and lodging-resistant corn variety by using gene editing technology, and accelerates the breeding process of corn lodging resistance, and the obtained d9 gene knockout mutant genetic material has stable dwarf characteristics, which can be used as an excellent parent for subsequent corn variety improvement, and provides valuable basic materials for corn breeding.

[0058] The above are all preferred embodiments of the present application, and do not limit the protection scope of the present application, so that: all equivalent changes made according to the structure, shape, principle of the present application should be covered in the protection scope of the present application.

Claims

1. A method for breeding a short-stalked, lodging-resistant maize variety based on d9 gene knockout, characterized in that, The method comprises the following steps: S1: determining a target gene: using bioinformatics analysis method, finding the homologous gene d9 of wheat green revolution semi-dwarf gene Rht-B1 and Rht-D1 in corn; S2: gene editing vector construction: constructing a gene editing vector for knocking out the d9 gene, the vector comprising a targeted editing element for the d9 gene; S3: transforming corn cells: introducing the constructed gene editing vector into corn cells; S4: obtaining mutant plants: culturing and screening the transformed corn cells to obtain corn mutant plants with the d9 gene knocked out; S5: screening and cultivation: determining the plant height of the obtained corn mutant plants and evaluating the lodging resistance, screening plants with significantly reduced plant height and strong lodging resistance, and then cultivating into stable genetically dwarf and lodging-resistant corn varieties.

2. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 1, characterized in that, With the help of bioinformatics tool BLAST, the amino acid sequences of wheat Rht-B1 and Rht-D1 genes are subjected to homology comparison with the sequences in the corn genome database, and candidate genes d9 with homology ≥ 70% are screened out; The sequence structure of the d9 gene is analyzed to determine its coding region (CDS), promoter region and key functional domain, and the targeted region for gene editing is determined.

3. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 1, characterized in that, The gene editing vector construction includes sgRNA design: according to the sequence of the targeted region of the d9 gene, 2-3 specific sgRNAs are designed using the CRISPR design tool to ensure that there is no off-target risk in the corn genome, and the off-target probability is ≤ 0.01%; the sgRNA sequence needs to contain a 20bp core region complementary to the target sequence and a PAM sequence (NGG).

4. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 1, characterized in that, The gene editing vector construction also includes vector assembly: using pUbi as the basic vector, the following elements are inserted in the direction from RB to LB: Promoter: corn ubiquitin promoter (pUbi) driving Cas9 protein expression; Nuclease gene: Cas9 coding gene (carrying nuclear localization signal NLS); sgRNA expression cassette: containing U6 promoter, designed sgRNA sequence and terminator; Selection marker gene: hygromycin resistance gene (Hpt) driven by 35S promoter (CaMV35S); Border sequence: T-DNA left and right border (RB, LB) to ensure integration of the vector fragment into the corn genome. Vector verification: enzyme digestion identification and sequencing verification are performed to ensure correct vector construction.

5. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 1, characterized in that, In S3, the gene editing vector is introduced into corn recipient cells by Agrobacterium-mediated method, and the Agrobacterium strain is EHA105.

6. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 5, characterized in that, Co-culture: mix the disinfected immature embryos with the Agrobacterium bacterial solution, and co-culture at 28°C in the dark for 2-3 days, maintaining the culture medium pH at 5.2 during the period; Screening culture: after co-culture, transfer the immature embryos to MS screening medium containing 50 mg / L hygromycin and 250 mg / L cefotaxime, and culture at 25°C in the dark, replace the culture medium every 2 weeks, and continuously screen for 3-4 generations to obtain resistant calli.

7. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 1, characterized in that, Obtaining mutant plants includes differentiation culture, rooting culture and molecular identification.

8. The method for breeding a short-stalked, lodging-resistant maize variety based on d9 gene knockout according to claim 7, characterized in that, The differentiation culture: the resistant callus was transferred to differentiation medium (MS+2.0 mg / L 6-BA+0.5 mg / L NAA), and cultured at 25℃ with 16h light / 8h dark, to induce differentiation of buds; Rooting culture: when the buds grew to 3-5 cm, the buds were cut and transferred to rooting medium (1 / 2MS+0.5 mg / L IBA), and cultured until roots formed into complete plants; Molecular identification: genomic DNA of the regenerated plants was extracted, the targeted editing region of d9 gene was amplified by PCR, and the amplification product was sequenced and analyzed to determine whether base insertion, deletion or substitution occurred, and d9 gene knockout homozygous mutants were screened.

9. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 1, characterized in that, Plant height determination: the identified mutants and wild type (WT) were planted in the field, and plant height was measured at the mature stage, repeated 3 times, and the average value was calculated to screen mutants with plant height reduced by ≥30% compared with the wild type; Lodging resistance evaluation: at the corn filling stage, artificial simulation of wind and rain was carried out, wind speed was 10-12 m / s, rainfall was 20 mm / h, and the duration was 30 min, the lodging rate was calculated, and mutants with lodging rate ≤10% were selected.

10. The method of breeding a short-stature, lodging-resistant maize variety based on d9 gene knockout according to claim 1, characterized in that, Genetic stability verification: the selected excellent mutants were selfed and propagated, and were continuously planted for 3 generations, and whether the plant height and lodging resistance were stably inherited was observed, and finally a dwarf lodging-resistant corn variety was bred.