Method for creating maize dwarfing materials by using gene editing technology

Abstract

The present invention provides a method for creating maize dwarfing materials by using gene editing technology, which includes designing CRISPR / Cas9-based sgRNAs targeting the target genes ZmGA20ox3 / ZmGA20ox5 in maize, ligating the DNA fragments encoding the sgRNAs into a vector carrying Cas, transforming maize with the constructed vector to achieve site-directed mutagenesis of the genes ZmGA20ox3 / ZmGA20ox5, and further obtaining transgenic maize plants with loss-of-function of the corresponding genes. The present invention for the first time reveals the biological functions of the maize ZmGA20ox3 / ZmGA20ox5 genes, performs gene editing on the maize ZmGA20ox3 / ZmGA20ox5 genes by CRISPR / Cas9 technology, and further screens to obtain mutant materials without transgenic insertion fragments. These maize dwarfing materials have important breeding value.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and genetic breeding, and in particular to a method for creating corn dwarfing materials using gene editing technology. Background Art

[0002] The increase in corn yield in the 20th century mainly relied on increasing the planting density of corn per unit area (Duvick et al., 2010), but too high a density is accompanied by the risk of lodging, which may reduce yield. Therefore, reducing corn plant height to a certain extent can help increase corn yield. In the late 1960s, the world-famous "Green Revolution" set off a craze that semi-dwarf plants can increase yields. During the "Green Revolution", semi-dwarf rice and wheat high-yield varieties were successfully created. In the past few decades, many corn dwarf mutants have been identified, but they have not been fully applied to corn breeding.

[0003] Gibberellic acid (GAs) is a natural tetracyclic diterpene carboxylic acid that plays an important role in the growth and development of plants, such as seed germination, stem elongation, and flower formation. More than 130 types of gibberellins have been discovered, of which four are active, namely GA1, GA3, GA4, and GA7 (Hedden et al., 2000). There are two forms of GA in plants, one in the free form, which participates in the plant growth metabolic pathway, and the other in the bound form, which is inactive. The normal content of GA in plants is maintained by the dynamic balance between bound GA and free GA (Kawaide., 2006). The biosynthesis of GA includes the following steps: 1) geranylgeranyl diphosphate is used to synthesize ent-kaurene in protoplasms; 2) ent-kaurene is oxidized to generate GA12 on the endoplasmic reticulum, and GA12 is further converted to GA53; 3) GA12 and GA53 enter the cytoplasm and generate active GA molecules under the action of a series of gibberellin oxidases (Teng et al., 2012).

[0004] CRISPR / Cas9 technology can accurately edit specific parts of the genome, which can achieve gene knockout or insert specific fragments. It has been successfully applied to genome editing of different plants, accelerating gene function research and crop molecular genetic improvement. The corn dwarf mutant material obtained through CRISPR / Cas9 technology is expected to be applied to corn breeding. Summary of the invention

[0005] The purpose of the present invention is to provide a method for creating corn dwarfing materials using gene editing technology.

[0006] In order to achieve the object of the present invention, in a first aspect, the present invention provides a gene for controlling plant height, including maize ZmGA20ox3 and / or ZmGA20ox5 genes, wherein the maize ZmGA20ox3 gene is a gene encoding the following protein (a) or (b):

[0007] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0008] (b) A protein derived from (a) in which one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 1 and which has the same function.

[0009] The maize ZmGA20ox5 gene is a gene encoding the following protein (a') or (b'):

[0010] (a′) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0011] (b′) A protein derived from (a′) having the same function as that of SEQ ID NO: 2, wherein one or more amino acids are substituted, deleted or added.

[0012] In a second aspect, the present invention provides an application of corn ZmGA20ox3 and / or ZmGA20ox5 genes in the breeding of corn dwarfing materials, which utilizes genetic engineering methods to perform site-directed mutations on corn ZmGA20ox3 and / or ZmGA20ox5 genes, thereby rendering the corn ZmGA20ox3 and / or ZmGA20ox5 genes functionally deficient, thereby achieving genetic improvement of corn plant height (reducing plant height).

[0013] In a third aspect, the present invention provides a method for creating corn dwarfing materials using gene editing technology, designing a CRISPR / Cas9-based sgRNA sequence for the target gene ZmGA20ox3 and / or ZmGA20ox5 in corn, connecting a DNA fragment encoding the sgRNA sequence to a vector carrying Cas, and transforming corn with the constructed vector (such as Agrobacterium-mediated method) to achieve site-directed mutagenesis of the gene ZmGA20ox3 and / or ZmGA20ox5, thereby obtaining transgenic corn plants with ZmGA20ox3 and / or ZmGA20ox5 gene function deficiency.

[0014] Preferably, for the gene ZmGA20ox3, the nucleotide sequences of the sgRNA action site are 5'-GGAGCCATTCCTGTGGCCGC-3' and 5'-CTGTCCTTCGGCTTCCACGA-3'. (SEQ ID NO: 3-4)

[0015] Preferably, for the gene ZmGA20ox5, the nucleotide sequences of the sgRNA action sites are 5’-AGATCCCCGCGCCATTCCTG-3’ and 5’-CTGTCGTTCGGCTACCACGA-3’. (SEQ ID NO:5-6)

[0016] More preferably, the two sgRNA action sites are concatenated onto the same gene editing vector through different expression cassettes.

[0017] In the present invention, the vector carrying Cas9 is pBUE411. The constructed maize transformation vectors are pBUE411-2gR-GA20ox3 and pBUE411-2gR-GA20ox5.

[0018] Optionally, the maize is the inbred line Zong 31.

[0019] In a fourth aspect, the present invention provides a method for creating maize dwarfing materials. Transgenic maize plants are prepared according to the above method, and then the transgenic maize plants are hybridized, backcrossed, self-crossed or asexually propagated, thereby creating maize dwarfing materials.

[0020] Preferably, the created maize dwarfing materials do not contain exogenous inserted fragments.

[0021] The protein encoded by the NOD gene in maize has the function of regulating cell number. The nod mutant has defects in growth and development and cell differentiation, showing narrow leaves and extremely dwarf plant height (Rosa et al., 2017). The maize blh12 / blh14 double mutant shows a dwarf phenotype due to severely shortened internode length, and the mutant tassel develops abnormally resulting in complete sterility (Tsuda et al., 2017). The maize gif1 mutant has narrow leaves, irregularly curved internodes and shorter plants, with increased long branches in the female inflorescence and reduced branches in the male inflorescence (Zhang et al., 2018). The maize dwarfing materials created by the present invention belong to semi-dwarfing materials, with normal internode development and normal seed setting. Compared with maize materials with severe dwarfing, sterility after dwarfing and irregular internode development after dwarfing, they are more suitable for application in hybrid maize production.

[0022] By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0023] The present invention for the first time reveals the biological functions of the maize ZmGA20ox3 and / or ZmGA20ox5 genes, gene edits the maize ZmGA20ox3 and / or ZmGA20ox5 genes through the CRISPR / Cas9 technology, and further screens to obtain mutant materials without transgenic inserted fragments. These maize dwarfing materials have important breeding value. Brief Description of the Drawings

[0024] Figure 1 This is the analysis result of the mutant sequence of the target site of the homozygous mutant in Example 5 of the present invention.

[0025] Figure 2 This is the comparison result of the plant heights of the ZmGA20ox3 gene-edited plants in Example 5 of the present invention. Among them, A: Comparison of the heights of the heterozygous mutants and homozygous mutants with edited sites and wild-type maize plants; B: Column chart of plant heights.

[0026] Figure 3 This is the screening result of the mutant plants without transgenic fragments in Example 6 of the present invention. Detailed Embodiments

[0027] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as those described in the molecular cloning experimental manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0028] Example 1 Design of Editing Sites Based on CRISPR / Cas9

[0029] Target sites were designed through the CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The maize target genes ZmGA20ox3, ZmGA20ox5 and related information were downloaded from the database MaizeGDB (https: / / www.maizegdb.org / ). The exon sequences of the target genes were uploaded to the CRISPR-P website, and the reference genome was selected as the maize genome to obtain potential editing sites adjacent to 5'-NGG. Two target sites were designed for each gene, and they were located on the first exon of the gene. The two target sites (located in different expression cassettes) were concatenated onto the same gene editing vector. The target site sequences of ZmGA20ox3 were 5'-GGAGCCATTCCTGTGGCCGC-3' and 5'-CTGTCCTTCGGCTTCCACGA-3', and the target site sequences of ZmGA20ox5 were 5'-AGATCCCCGCGCCATTCCTG-3' and 5'-CTGTCGTTCGGCTACCACGA-3'.

[0030] Example 2 Construction of Gene Editing Vectors

[0031] Design primers according to the selected editing site and the restriction enzyme sites at the multiple cloning sites of vectors pCBC-MT1T2 and pBUE411. The primer information is shown in Table 1:

[0032] Table 1 Amplification primers

[0033]

[0034]

[0035] Amplify the target fragment by a two-round PCR method. In the first-round PCR reaction, use the first pair of primers MT1-F0 / MT2-R0 to amplify with the pCBC-MT1T2 plasmid as the template. In the second-round PCR reaction, use the second pair of primers MT1-BsF / MT2-BsR to amplify with the first-round amplification product as the template, and concatenate the two target sites to obtain the target fragment.

[0036] The reaction system for the first round is as follows:

[0037]

[0038] The reaction system for the second round is as follows:

[0039]

[0040] The two-round PCR amplification reaction program is as follows: 94°C for 2 min; 98°C for 10 s, 58°C for 30 s, 68°C for 1 min, 35 cycles; 68°C for 7 min, 25°C ∞.

[0041] Then, digest the purified target fragment and the pBUE411 vector with the restriction enzyme BsaI-HF alone. The digested linear vector is treated with 0.2 μL of dephosphorylase CPI at 37°C for 30 min to prevent self-ligation of the vector after digestion.

[0042] The single-enzyme digestion system:

[0043]

[0044]

[0045] The enzyme digestion condition: water bath at 37°C for 2 h.

[0046] The target fragment is ligated to the expression vector with T4 ligase:

[0047] The ligation system:

[0048]

[0049] Ligate at room temperature for 1 h or overnight at 16°C. Respectively construct the gene ZmGA20ox3 and ZmGA20ox5 editing vectors.

[0050] Example 3 Transfer of Gene Editing Vector into Agrobacterium tumefaciens LBA4404

[0051] The gene ZmGA20ox3 and ZmGA20ox5 editing vectors constructed in Example 2 were respectively transferred into Agrobacterium tumefaciens LBA4404. The specific steps are as follows:

[0052] (1) Take 5 μL of plasmid and add it to 200 μL of Agrobacterium competent cells;

[0053] (2) Incubate on ice for 30 min;

[0054] (3) Take out from ice and quickly freeze in liquid nitrogen for 5 min;

[0055] (4) Take out from liquid nitrogen and incubate in a 37 °C water bath for 5 min;

[0056] (5) Place on ice for another 5 min after taking out;

[0057] (6) Add 800 μL of blank YEB medium and recover at 28 °C on a shaker at 200 rpm for 4 - 5 h;

[0058] (7) Centrifuge at 4000 rpm at room temperature for 5 min;

[0059] (8) Discard part of the supernatant, and suspend the precipitate with the remaining about 200 μL of supernatant. Spread it on a YEB solid culture plate containing the corresponding resistance, and incubate it upside down in the dark at 28 °C for 36 h;

[0060] (9) Pick the single colonies grown on the plate and inoculate them into a YEB liquid culture medium containing the corresponding resistance. Incubate them overnight at 28 °C with shaking at 200 rpm; Use the bacterial solution as a template for PCR amplification and identification.

[0061] Example 4 Transformation of Maize with Agrobacterium

[0062] Pick single colonies and inoculate them into 5 - 10 mL of YEB liquid medium containing the corresponding antibiotics. Incubate them with shaking at 200 rpm and 28 °C for 8 h; Then inoculate the shaken bacterial solution into fresh YEB liquid medium containing the corresponding antibiotics at a ratio of 1:100, and incubate it overnight with shaking at 200 rpm and 28 °C. The next day, aliquot the shaken bacterial solution overnight into 2 mL centrifuge tubes, and centrifuge at 5000 rpm for 5 min; Resuspend with an infection medium containing 200 μM acetosyringone, and adjust OD 600 = 0.3 for standby.

[0063] Put the young embryos of the receptor material maize inbred line Zong 31 into a 1.5 mL centrifuge tube containing 1 mL of infection medium solution. After 1 h, suck out the infection medium with a pipette and add 1 mL of fresh infection medium. Invert and mix well for 1 min to thoroughly wash the endosperm on the surface of the maize young embryos. Then, suck out the infection medium solution with a pipette and add 0.2 mL of fresh infection medium solution. Place the centrifuge tube containing the maize young embryos on a heater (Hangzhou Bioer Technology Co., Ltd., model CHB-100) and heat shock at 45 °C for 5 min; suck out the infection medium solution with a pipette, add 1 mL of Agrobacterium liquid containing 40 mg / L of acetosyringone, and place for 5 min; take out and blot dry with a sterilized filter paper, and place it on a co-culture medium supplemented with 300 mg / L of cysteine, and co-culture for 3 days under dark conditions at 23 °C. After co-culture, transfer the young embryos to a recovery medium and culture for 7 days under dark conditions.

[0064] After two rounds of screening, resistant calli were obtained. Transfer these calli to a regeneration medium and regenerate plants under strong light. The culture conditions were 28 °C and 16 h of light, and regenerated seedlings would appear soon. When the regenerated seedlings grew to 3 leaves, the seedlings could be transplanted into a rooting medium and cultured indoors. After the seedlings grew new leaves and roots, take out the seedlings from the canning bottle, rinse the medium with tap water, and transplant them into small flower pots mixed with nutrient soil and vermiculite (1:3, volume ratio). When the seedlings grew 2-3 new leaves again, they could be transplanted into a field or a large flower pot.

[0065] For the infection medium, co-culture medium, regeneration medium, and rooting medium used in this example, please refer to CN201710090814.2 for details.

[0066] Identification of the genetically modified maize plants with editing in Example 5

[0067] Extract DNA from the maize leaves that survived after glufosinate screening by the CTAB method, and perform PCR identification with gene-specific primers of ZmGA20ox3 and ZmGA20ox5. The amplified products were detected by 1% agarose gel electrophoresis. The primer sequences used are shown in Table 2:

[0068] Table 2 Amplification primers

[0069]

[0070] The reaction system is as follows:

[0071]

[0072] The reaction procedure was as follows: 94°C for 2 min; 98°C for 10 s, 60°C for 30 s, 68°C for 1 min, for 35 cycles; extension at 68°C for 7 min, 25°C indefinitely. The product size was approximately 1.1 Kb, and the PCR products with the correct amplified product band size were sent for sequencing. Two mutant lines with ZmGA20ox3 gene editing ( Figure 1 , A) and four mutant lines with ZmGA20ox5 gene editing ( Figure 1 , B) were screened. The heterozygous and homozygous mutant materials with editing were significantly shorter than the wild-type maize plants ( Figure 2 , A and B).

[0073] Example 6 PCR Detection of T-DNA Fragments in Homozygous Mutants

[0074] Three pairs of specific primers were designed according to the T-DNA sequence inserted into the maize genome, and PCR amplification was performed using the genomic DNA of homozygous mutant leaves as a template. The sequences of the three pairs of primers are shown in Table 3:

[0075] Table 3 Amplification Primers

[0076]

[0077] The reaction system was as follows:

[0078]

[0079] The reaction procedure was as follows: 95°C for 5 min; 95°C for 30 s, 59°C for 30 s, 72°C for 1 min, for 35 cycles; extension at 72°C for 7 min, 25°C indefinitely.

[0080] PCR amplification was performed using the genomic DNA of the leaves of the ga20ox3 homozygous mutant mut1 as a template. The amplified products were detected by 1% agarose gel electrophoresis, and it was found that there was no T-DNA insertion in the genomes of a total of 4 transgenic maize plants, namely No. 2, No. 3, No. 7, and No. 8 ( Figure 3 ). These maize dwarfing materials have important breeding value.

[0081] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

[0082] References

[0083] [1] Duvick D.N., Smith J.S.C., Cooper M., et al. Long-term selection in a commercial hybrid maize breeding program. Plant Breeding, 2010, 24: 109 - 151.

[0084] [2] Hedden P., Phillips A.L.. Gibberellin metabolism: new insights revealed by the genes. Trends in Plant Science, 2000, 5(12): 523 - 530.

[0085] [3] Kawaide H.. Biochemical and molecular analyses of gibberellin biosynthesis in fungi. Journal of the Agricultural Chemical Society of Japan, 2006, 70(3): 583 - 590.

[0086] [4] Teng F., Zhai L., Liu R., et al. ZmGA3ox2, a candidate gene for a major QTL, qPH3.1, for plant height in maize. The Plant Journal, 2012, 73(3): 405 - 416.

[0087] [5] Rosa M., Abraham J., María J., et al. The maize MID - COMPLEMENTING ACTIVITY homolog CELL NUMBER REGULATOR13 / NARROW ODD DWARF coordinates organ growth and tissue patterning. The Plant Cell, 2017, 29(3): 474 - 490.

[0088] [6]Tsuda K.,Abraham-Juarez M.J.,Maeno A.,et al.KNOTTED1 cofactors,BLH12 and BLH14,regulate internode patterning and vein anastomosis in maize.The Plant Cell,2017,29(5):1105-1118.

[0089] [7]Zhang D.,Sun W.,Singh R.,et al.GRF-interacting factor1(gif1)regulates shoot architecture and meristem determinacy in Maize.The Plant Cell,2018 30(2):360-374. Sequence Listing <110> Institute of Crop Science, Chinese Academy of Agricultural Sciences <120> Method for Creating Maize Dwarfing Materials Using Gene Editing Technology <130> KHP191111788.7 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 386 <212> PRT <213> Zea mays <400> 1 Met Asp Ala Ser Pro Thr Pro Pro Leu Pro Leu Arg Ala Pro Thr Pro 1 5 10 15 Ser Ile Asp Leu Pro Ala Gly Lys Asp Arg Ala Asp Ala Ala Ala Asn 20 25 30 Lys Ala Ala Ala Val Phe Asp Leu Arg Arg Glu Pro Lys Ile Pro Glu 35 40 45 Pro Phe Leu Trp Pro His Glu Glu Ala Arg Pro Thr Ser Ala Ala Glu 50 55 60 Leu Glu Val Pro Val Val Asp Val Gly Val Leu Arg Asn Gly Asp Gly 65 70 75 80 Ala Gly Leu Arg Arg Ala Ala Ala Gln Val Ala Ala Ala Cys Ala Thr 85 90 95 His Gly Phe Phe Gln Val Cys Gly His Gly Val Asp Ala Ala Leu Gly 100 105 110 Arg Ala Ala Leu Asp Gly Ala Ser Asp Phe Phe Arg Leu Pro Leu Ala 115 120 125 Glu Lys Gln Arg Ala Arg Arg Val Pro Gly Thr Val Ser Gly Tyr Thr 130 135 140 Ser Ala His Ala Asp Arg Phe Ala Ser Lys Leu Pro Trp Lys Glu Thr 145 150 155 160 Leu Ser Phe Gly Phe His Asp Gly Ala Ala Ala Pro Val Val Val Asp 165 170 175 Tyr Phe Thr Gly Thr Leu Gly Gln Asp Phe Glu Pro Val Gly Arg Val 180 185 190 Tyr Gln Arg Tyr Cys Glu Glu Met Lys Glu Leu Ser Leu Thr Ile Met 195 200 205 Glu Leu Leu Glu Leu Ser Leu Gly Val Glu Arg Gly Tyr Tyr Arg Glu 210 215 220 Phe Phe Glu Asp Ser Arg Ser Ile Met Arg Cys Asn Tyr Tyr Pro Pro 225 230 235 240 Cys Pro Val Pro Glu Arg Thr Leu Gly Thr Gly Pro His Cys Asp Pro 245 250 255 Thr Ala Leu Thr Ile Leu Leu Gln Asp Asp Val Gly Gly Leu Glu Val 260 265 270 Leu Val Asp Gly Glu Trp Arg Pro Val Arg Pro Val Pro Gly Ala Met 275 280 285 Val Ile Asn Ile Gly Asp Thr Phe Met Ala Leu Ser Asn Gly Arg Tyr 290 295 300 Lys Ser Cys Leu His Arg Ala Val Val Asn Arg Arg Gln Glu Arg Gln 305 310 315 320 Ser Leu Ala Phe Phe Leu Cys Pro Arg Glu Asp Arg Val Val Arg Pro 325 330 335 Pro Ala Ser Ala Ala Pro Arg Gln Tyr Pro Asp Phe Thr Trp Ala Asp 340 345 350 Leu Met Arg Phe Thr Gln Arg His Tyr Arg Ala Asp Thr Arg Thr Leu 355 360 365 Asp Ala Phe Thr Arg Trp Leu Ser His Gly Pro Ala Ala Ala Ala Pro 370 375 380 Cys Thr 385 <210> 2 <211> 417 <212> PRT <213> Zea mays <400> 2 Met Val Ser Gln Glu Arg Gln Glu Pro Ala Val Pro Ser Ser Ser Ser 1 5 10 15 Ser Ser Ala Lys Arg Ala Ala Thr Ser Met Asp Ala Ser Pro Ala Pro 20 25 30 Pro Leu Leu Leu Arg Ala Pro Thr Pro Ser Pro Ser Ile Asp Leu Pro 35 40 45 Ala Gly Lys Asp Lys Ala Asp Ala Ala Ala Ser Lys Ala Gly Ala Ala 50 55 60 Val Phe Asp Leu Arg Arg Glu Pro Lys Ile Pro Ala Pro Phe Leu Trp 65 70 75 80 Pro Gln Glu Glu Ala Arg Pro Ser Ser Ala Ala Glu Leu Glu Val Pro 85 90 95 Met Val Asp Val Gly Val Leu Arg Asn Gly Asp Arg Ala Gly Leu Arg 100 105 110 Arg Ala Ala Ala Gln Val Ala Ala Ala Cys Ala Thr His Gly Phe Phe 115 120 125 Gln Val Cys Gly His Gly Val Asp Ala Ala Leu Gly Arg Ala Ala Leu 130 135 140 Asp Gly Ala Ser Asp Phe Phe Arg Leu Pro Leu Ala Glu Lys Gln Arg 145 150 155 160 Ala Arg Arg Val Pro Gly Thr Val Ser Gly Tyr Thr Ser Ala His Ala 165 170 175 Asp Arg Phe Ala Ala Lys Leu Pro Trp Lys Glu Thr Leu Ser Phe Gly 180 185 190 Tyr His Asp Gly Ala Ala Ser Pro Val Val Val Asp Tyr Phe Val Gly 195 200 205 Thr Leu Gly Gln Asp Phe Glu Pro Met Gly Trp Val Tyr Gln Arg Tyr 210 215 220 Cys Glu Glu Met Lys Glu Leu Ser Leu Thr Ile Met Glu Leu Leu Glu 225 230 235 240 Leu Ser Leu Gly Val Glu Leu Arg Gly Tyr Tyr Arg Glu Phe Phe Glu 245 250 255 Asp Ser Arg Ser Ile Met Arg Cys Asn Tyr Tyr Pro Pro Cys Pro Glu 260 265 270 Pro Glu Arg Thr Leu Gly Thr Gly Pro His Cys Asp Pro Thr Ala Leu 275 280 285 Thr Ile Leu Leu Gln Asp Asp Val Gly Gly Leu Glu Val Leu Val Asp 290 295 300 Gly Glu Trp Arg Pro Val Arg Pro Val Pro Gly Ala Met Val Ile Asn 305 310 315 320 Ile Gly Asp Thr Phe Met Ala Leu Ser Asn Gly Arg Tyr Lys Ser Cys 325 330 335 Leu His Arg Ala Val Val Asn Gln Arg Arg Ala Arg Arg Ser Leu Ala 340 345 350 Phe Phe Leu Cys Pro Arg Glu Asp Arg Val Val Arg Pro Pro Ala Ser 355 360 365 Ala Ala Pro Arg Arg Tyr Pro Asp Phe Thr Trp Ala Asp Leu Met Arg 370 375 380 Phe Thr Gln Arg His Tyr Arg Ala Asp Thr Arg Thr Leu Asp Ala Phe 385 390 395 400 Thr Arg Trp Leu Ser His Gly Pro Ala Gln Ala Ala Ala Pro Pro Cys 405 410 415 Thr <210> 3 <211> 20 <212> DNA <213> Zea mays <400> 3 ggagccattc ctgtggccgc 20 <210> 4 <211> 20 <212> DNA <213> Zea mays <400> 4 ctgtccttcg gcttccacga 20 <210> 5 <211> 20 <212> DNA <213> Zea mays <400> 5 agatccccgc gccattcctg 20 <210> 6 <211> 20 <212> DNA <213> Zea mays <400> 6 ctgtcgttcg gctaccacga 20

Claims

1. Method for creating maize dwarfing materials using gene editing technology, Characterized in that, Design a CRISPR / Cas9-based sgRNA sequence targeting the target gene ZmGA20ox5 in maize, ligate the DNA fragment encoding the sgRNA sequence to a vector carrying Cas, transform maize with the constructed vector to achieve site-directed mutagenesis of the gene ZmGA20ox5, and then obtain transgenic maize plants with loss of function of the ZmGA20ox5 gene; The amino acid sequence of the protein encoded by the gene ZmGA20ox5 is shown in SEQ ID NO: 2; For the gene ZmGA20ox5, the nucleotide sequences of the sgRNA action sites are 5’-AGATCCCCGCGCCATTCCTG-3’ and 5’-CTGTCGTTCGGCTACCACGA-3’; The vector carrying Cas is pBUE411; The transformation is to transform maize by the Agrobacterium-mediated method; The maize is the inbred line Zong 31.

2. A method for creating maize dwarfing materials, Characterized in that, Prepare transgenic maize plants according to the method described in claim 1, and then hybridize, backcross, self-cross or asexually reproduce the transgenic maize plants to create maize dwarfing materials.

3. According to the method described in claim 2, Characterized in that, The created maize dwarfing materials do not contain exogenous insertion fragments.

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