CN114181966B - Method for creating corn dwarf material based on Zm00001d008708 gene - Google Patents

Abstract

The invention discloses a method for creating a corn dwarf material based on the Zm00001d008708 gene, which belongs to the technical field of genetic engineering. The present invention discloses a method for creating dwarfing maize materials based on the Zm00001d008708 gene. The Zm00001d008708 gene of maize is edited by CRISPR/Cas9 technology, and further screened to obtain mutant materials containing target gene deletion fragments. These maize dwarfing materials have important breeding value.

Description

A method of creating maize dwarf material based on Zm00001d008708 gene

technical field

The invention relates to the technical field of genetic engineering, and more specifically relates to a method for creating dwarfing maize materials based on the Zm00001d008708 gene by using gene editing technology.

Background technique

The improvement of corn production in the 20th century mainly relied on increasing the planting density of corn per unit area. However, if the density is too high, there will be the risk of lodging, which may reduce the yield. Therefore, reducing the plant height of corn to a certain extent will help improve corn yield. At the end of the 1960s, the world-famous "Green Revolution" set off a frenzy that semi-dwarf plants could increase yields. High-yield varieties of semi-dwarf rice and wheat were successfully created during the "Green Revolution". Many maize dwarf mutants have been identified over the past few decades but have not been fully utilized in maize breeding.

The creation of traditional dwarf corn is mainly through the method of backcrossing, which often requires 6 backcrossing generations, which is not only time-consuming, but also often accompanied by gene redundancy, which leads to the introduction of some non-target traits and limits the application of improved materials Effect. Zm00001d008708 is a kind of DUF1421 structural family protein, whose function is unknown at present.

Therefore, it is an urgent problem for those skilled in the art to provide a method for creating a dwarf maize material based on the Zm00001d008708 gene.

Contents of the invention

In view of this, the present invention provides a method for creating dwarf maize materials based on the Zm00001d008708 gene.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for creating dwarf corn material based on the Zm00001d008708 gene, the specific steps are as follows:

(1) Design the sgRNA action site based on CRISPR/Cas9 for the gene Zm00001d008708;

The nucleotide sequence of the sgRNA action site is:

5'-TCCAGCACCAGCGGTTCTTCTGG-3'; SEQ ID NO. 3; and

5'-CCGTGCGGAGGCTGACTGAAAAC-3'; SEQ ID NO.4;

(2) Using pCBC-MT1T2 plasmid as template, MT1T2-F, MT1T2-F0, MT2T2-R0, MT2T2-R as primers, PCR amplifies the target fragment;

The primer sequences are as follows:

MT1T2-F: 5'-AATAATGGTCTCAGGCGCCAGCACCAGCGGTTCTTC-3'; SEQ ID NO.5;

MT1T2-F0: 5'-GCCAGCACCAGCGGTTCTTCGTTTTAGGCTAGAAATAGC-3'; SEQ ID NO.6;

MT1T2-R0: 5'-TGCGGAGGCTGACTGAAAACGCTTCTTGGTGCC-3'; SEQ ID NO.7;

MT1T2-R: 5'-ATTATTGGTCTCTAAACTGCGGAGGCTGACTGAAAA-3'; SEQ ID NO.8;

The PCR reaction system is: pCBC-MT1T2 plasmid 1 μl, MT1T2-F 0.5 μl, MT1T2-F0 0.5 μl, MT1T2-R0 0.5 μl, MT1T2-R 0.5 μl, 2×Mix 10 μl, ddH ₂ O 7 μl; PCR amplification The reaction program was: 98°C for 3 min; 98°C for 30 s, 57°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min, 4°C∞.

(3) Ligate the target fragment obtained in step (2) with the vector pBUE411 to construct the Zm00001d008708 gene editing vector pBUE411-2gR-Zm00001d008708;

The enzyme digestion ligation reaction system is as follows: target fragment 2 μl, pBUE411 2 μl, 10xNEB T4 Buffer 1.5 μl, 10xBSA 1.5 μl, BsaI 1 μl, T4 Ligase 1 μl, ddH2O 6 μl, Total 15 μl;

(4) The gene editing vector pBUE411-2gR-Zm00001d008708 obtained in step (3) was transformed into Agrobacterium LBA4404 for genetic transformation of maize, and the dwarf maize material was obtained through screening and identification.

The two sgRNA action sites are connected in series to the same gene editing vector through different expression cassettes.

The vector carrying Cas9 is pBUE411.

The maize is the inbred line Zheng 58.

It can be known from the above-mentioned technical solutions that, compared with the prior art, the present invention discloses a method for creating corn dwarfing materials based on the Zm00001d008708 gene using gene editing technology. The gene editing technology can precisely edit the target gene without external For the introduction of edge genes, the present invention accurately knocks out Zm00001d008708 through gene editing technology, which can accurately reduce the plant height of corn without changing other agronomic traits.

The present invention reveals the biological function of the maize Zm00001d008708 gene for the first time. The maize Zm00001d008708 gene is gene-edited by CRISPR/Cas9 technology, and further screened to obtain mutant materials containing the deletion of the target gene fragment. These maize dwarf materials have important breeding value . The corn dwarfing material created by the invention belongs to the highly dwarfing material, the internodes are shortened but fruity normally, and can be applied to hybrid corn production through reasonable cultivation and management measures.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention, and those skilled in the art can also obtain other drawings according to the provided drawings without creative work.

Fig. 1 accompanying drawing is the sequencing comparison result of the present invention;

Fig. 2 accompanying drawing is the mutant strain of the present invention;

Wherein, A is a mutant maize plant; B is a wild-type maize plant.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The present invention uses gene editing technology to create corn dwarfing materials, designs a CRISPR/Cas9-based sgRNA sequence for the target gene Zm00001d008708 in corn, connects the DNA fragment encoding the sgRNA sequence to a vector carrying Cas9, and uses the constructed vector Maize is transformed (such as Agrobacterium-mediated method) to achieve site-directed mutation of the gene Zm00001d008708, and then obtain maize plants with Zm00001d008708 gene function loss.

The construction of embodiment 1 gene editing vector

(1) The gene controlling the plant height of maize is the Zm00001d008708 gene, and its nucleotide sequence is as follows:

ATGAACGCGTCGCAGTTCATGGACAAGCAGATCCTCGGCCTGGCTGCCTCCGCTTCCCCCTCCGGCGGCGGCGCGGGGGGCGGTGGGGGTGTGGATCTCAGCGATCTGATGATACCGATCCCCCAGGAGGACGCCGAGAACCGCCTCGGTCGCCGGCGTAGCAGCACCCAGCGTCAACGGAACCGCAGACGACATGCTACCCAGTT ATGACTTCCAGCCCATCCGCACTAGTGGCGGCGCCGCGGCCGCCGCCGCGCCTCAGGCCTCGTGGGGGGTCGCTCGACTCCAAGGCACCCTCTGCCTCATACAACCTCCAAGAGTGCTGGTATATTGGAGCCGCATGTGCTGAAGAAAGTTAGTCATGAGGAAGACAAGAGTAACTTTCCTAACTAGTTACTATTGCGGATATTGATCGAACCATGAAGAAG TACTCTGATAACCTTTTGCATGCACTGGAAGGTGTAAGCTCAAGGCTTTCACAGATGGAGGGTAGAACACACCAACTCGAAAACTCTGTTGACGAGTTGAAGTTAACAATCGGTAACTATAATGGTAGCACTGATGGAAAACTGAGGAACCTTGAGAACATGCTCAGGGAGGTCCAAGCAGGTGTGCAGATTTTGCGAGACAAGCAGGAAATTGTCGAGA CACAGCTCCACCTTGCGAAGCTCCAGACAAACAAAACCGATGGCCAATCATCAGAAAATAGTGGGTCTGGACAGGCTGGTTTCAGCAGCAGCCGGTGGTTCCTCCACAAGCAGCCATTCAGCCACAAACAAGTCCTAACCCCTTCGCAACCACCTGCACTTCCTGCCCTTCCTGCTCCAAATGCACCACCTCCACCTCCAACGCTTCAAAACCAATCATCATTACAGTTTCC AAGTCATTTACAACATTCACAGGTACCATCTGTGCCTTCTGTTGCACTGGCACCCACAGTTCCAGCTTTACCAAGGGATGCTTACTATGCCCCATCTGCTCAGCCGACCGAGACCATGCACCAGCAGTATCAAGCTCCGCCAGTTCCAGCCACAGGCACCTCCTGCACCACCTCAGCAGTACCCAATCCCAAACCCAGTTCCCTCAATATGCACAGCCACCTCAGCCTGCAA ATGTTAACCCTTCAACTCCCCATGTGCCCCATGCACCCCAGCAACCAGAGGAAACTATGCCTTATGCACCAGCTCAGAGCTATCCACCTAATGCAATCGCTGCACCCTTATATGCAACCACCTAGTGGACCTGCTCCTCCTTACTATGGGCAGCAAAACCCTAGCATGTATGAACCTCCTGCAGGCCGGGCTAACCCTGGGCCACCATCATCCTACGGTTCTGGT GGGTACGGGCCACAGGGTGGAGGTAGTTTCCTCTGAATCTTATGGTTACACTGGATCTCCTTCCCACCGTGGCAATGCTGGAATGAAGCAGTCTCACCTTTTGCTCAATCCTCTGGAGGAAGCGGCAGCTATGGCAGTGGCAAGCTCCTACTGCCCAGATGCTTCCAAGCAGTGCCGATCAGCTCCTCCAGCACCAGCGGTTCTTCTGGCAATA GAGTGCCACTTGACGATGTAGTGGAGAAGGTTGCTACGATGGGATTCTCAAGAGAGCAGGTGAGAGCAACCGTGCGGAGGCTGACTGAAAACGGGCAGAACGTGGACCTGAATGTGGTGCTCGACAAGCTGATGAACGGATGA; SEQ ID NO. 1.

The amino acid sequence of Zm00001d008708 protein is as follows:

MNASQFMDKQILGLAASASPSGGGAGGGGGVDLSDLMIPIPQEDAENRLGRRRSSTSVNGTADDMLPSYDFQPIRTSGGAAAAAAPQASWGSLDSKAPSASYNLKSAGILEPHVLKKVSHEEDKSNFPTVTIADIDRTMKKYSDNLLHALEGVSSRLSQMEGRTHQLENSVDELKLTIGNYNGSTDGKL RNLENMLREVQAGVQILRDKQEIVETQLHLAKLQTNKTDGQSSENSGSGQAGLQQQPVVPPQAAIQPQQVLTPSQPPALPALPAPNAPPPPTLQNQSSLQFPSHLQHSQVPSVALAPTVPALPRDAYYAPSAQPTETMHQQYQAPPVPQPQAPPPPQQYQSQTQFPQYAQPPQPAN VNPSTPHVPHAPQQPEETMPYAPAQSYPPNAIAIAAPYMQPPSGPAPPYYGQQNPSMYEPPAGRANPGPPSSYGSGGYGPQGGGSFSESYGYTGSPSHRGNAGMKQSSPFAQSSGGSGSYGSGKLPTAQMLPQAVPISSSSTSGSSGNRVPLDDVVEKVATMGFSREQVRATVRRLENGQNVDLNVVLDK LMNG; SEQ ID NO.2.

(2) For the gene Zm00001d008708, the nucleotide sequence of the sgRNA action site is:

5'-TCCAGCACCAGCGGTTCTTCTGG-3'; SEQ ID NO.3; (Target 1) and

5'-CCGTGCGGAGGCTGACTGAAAAC-3'; SEQ ID NO.4; (Target 2).

Primers were designed according to the selected editing site and the restriction site at the multiple cloning site of vectors pCBC-MT1T2 and pBUE411. The primer sequences are as follows:

MT1T2-F: 5'-AATAATGGTCTCAGGCGCCAGCACCAGCGGTTCTTC-3'; SEQ ID NO.5;

MT1T2-F0: 5'-GCCAGCACCAGCGGTTCTTCGTTTTAGGCTAGAAATAGC-3'; SEQ ID NO.6;

MT1T2-R0: 5'-TGCGGAGGCTGACTGAAAACGCTTCTTGGTGCC-3'; SEQ ID NO.7;

MT1T2-R: 5'-ATTATTGGTCTCTAAACTGCGGAGGCTGACTGAAAA-3'; SEQ ID NO.8;

(3) The target fragment was amplified by a round of PCR method, and the PCR reaction used two pairs of primers MT1T2-F, MT1T2-F0, MT2T2-R0, MT2T2-R, and the pCBC-MT1T2 plasmid was used as a template for amplification.

The PCR amplification reaction system is: pCBC-MT1T2 plasmid 1 μl, MT1T2-F 0.5 μl, MT1T2-F0 0.5 μl, MT1T2-R0 0.5 μl, MT1T2-R 0.5 μl, 2×Mix 10 μl, ddH ₂ O 7 μl.

The PCR amplification reaction program was: 98°C for 3min; 98°C for 30s, 57°C for 30s, 72°C for 1min, 35 cycles; 72°C for 5min, 4°C∞.

The obtained target fragment was digested and ligated with the vector pBUE411, and the reaction system was as follows:

Target fragment (964bp) 2 μl, pBUE411 2 μl, 10xNEB T4 Buffer 1.5 μl, 10xBSA 1.5 μl, BsaI (NEB) 1 μl, T4 Ligase (NEB)/high concentration 1 μl, ddH ₂ O 6 μl, Total 15 μl.

Reaction conditions: 37°C for 5h, 50°C for 5min, 80°C for 10min.

The Zm00001d008708 gene editing vector pBUE411-2gR-Zm00001d008708 was constructed.

Example 2 Gene Editing Vector Transformed into Agrobacterium LBA4404

1) Preparation of Agrobacterium tumefaciens competent cells by CaCl ₂ method

(1) Pick a fresh LBA4404 single colony from the YEP plate (Rif ^R , Str ^R ) and inoculate it in YEP liquid medium containing 50 mg/L Str and 25 mg/L Rif, culture at 28°C and 220 rpm for 24-36 hours overnight with shaking ;

(2) Take 2ml of overnight activated bacterial solution in the logarithmic growth phase, inoculate it in 50mL of YEP liquid medium, and culture the bacterial solution at 20°C with an OD ₆₀₀ of about 0.4-0.6;

(3) Transfer the bacterial solution to a 50 mL sterile centrifuge tube pre-cooled with ice, bathe in ice for 30 min, centrifuge at 4,000×g for 10 min at 4°C to enrich the bacterial cells;

(4) Pre-cool the suspended cells with 0.05M CaCl ₂ in 10 mL of ice, bathe in ice for 30 min, centrifuge at 4,000×g for 10 min at 4°C to enrich the cells;

(5) Pre-cool 0.05M CaCl ₂ with 1mL of ice to resuspend the cells, store the prepared competent cells at 4°C, and use them within 24-48h for the highest transformation efficiency. You can also dispense 100μL per tube into sterile tubes 20% glycerol was added to the final concentration, and stored at -80°C after quick-freezing with liquid nitrogen.

2) Transformation of Agrobacterium tumefaciens competent cells by freeze-thaw method

(1) Take out the competent Agrobacterium (200 μL), put it on ice, add plasmid DNA (1 μg) when it is just thawed, put it in liquid nitrogen for 1 min, and then put it in a metal bath at 37 °C for 5 min;

(2) Take out the centrifuge tube, add 1mL YEB liquid medium (without antibiotics), place on a shaker, and incubate at 28°C, 180r/min for 35h;

(3) Centrifuge at 3000rpm for 1min, remove excess supernatant, retain 100μL, resuspend, pour onto YEB plate medium (kan, rif), spread evenly, and incubate in a constant temperature incubator at 28°C for 36-48h;

(4) Pick a single clone, detect it, and keep the positive colony. Using primers OsU3-FD3/TaU3-RD to carry out bacterial liquid PCR identification.

Among them, the OsU3-FD3/TaU3-RD primer sequence is as follows:

OsU3-FD3: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3'; SEQ ID NO.9;

TaU3-RD: 5'-CTCACAAATTATCAGCACGCTAGTC-3'; SEQ ID NO.10.

reaction system:

Bacteria solution 1μl, OsU3-FD3 1μl, TaU3-RD 1μl, 2×mix 10μl, ddH ₂ O 7μl, Total 20μl.

The PCR amplification reaction program was: 98°C for 3min; 98°C for 30s, 57°C for 45s, 72°C for 1min, 35 cycles; 72°C for 5min, 4°C∞.

The size of the colony PCR product was 831bp.

Embodiment 3 Maize genetic transformation

(1) The material for embryo extraction was the maize inbred line Zheng 58. The immature corn embryos were observed on the ninth day after pollination, and when they grew to about 1.5 mm, the ears were taken back to the laboratory for embryo extraction.

(2) Prepare the Agrobacterium infection solution. When the activated Agrobacterium is shaken to a specific concentration (OD ₅₅₀ =0.5) in the YEB liquid medium, the bacterial precipitate is collected by low-speed centrifugation, and then inf (composition per liter: N6 Salt and vitamin (sigma) 2g, sucrose 68.5g, glucose 36g, L-proline 0.7g, MES 0.5g, 1mg/ml 2,4-D 1.5ml)+AS(Acetosyringone, (100mM), 1ml)) The liquid medium was resuspended, and the bacteria were shaken at 75 r/min at 25°C for 24 hours until the concentration was OD ₅₅₀ =0.3-0.4.

(3) Wash the immature embryos taken out in (1) twice with inf+AS (same as above) liquid medium, then add Agrobacterium infection solution, and infect for 20min-30min.

(4) Transfer the immature embryos after infection to the co-cultivation medium (composition per liter: 4 grams of N6 salt and vitamins, 40 grams of sucrose, 30 grams of glucose, 0.7 grams of L-proline, 0.5 g of MES, 1 mg/ml 2 , 4-d 1.5ml, agarose (low EEO) 5g, 8.5mg/ml silver nitrate 0.1ml, 100mg/ml L-cysteine 0.4g, 0.5M/L DTT 0.154g), the shield of immature embryo With the sheet facing upwards, the hypocotyl was in contact with the surface of the culture medium, and the petri dish was sealed with parafilm, and cultured in the dark for 3 days in a 20°C incubator.

(5) Transfer the immature embryos from the co-cultivation medium to the resting medium (composition per liter: 4 grams of N6 salt and vitamins, 40 grams of sucrose, 30 grams of glucose, 0.7 grams of L-proline, 0.5 g of MES, 1 mg/ml 2,4-d 1.5ml, 8.5mg/ml silver nitrate 0.1ml, 100mg/ml L-cysteine 0.4g, 0.5M/L DTT 0.154g, Timentin (Timentin, Sigma) 100mg), use Seal the petri dish with a parafilm and culture it in the dark at 28°C for 7 days.

(6) Transfer all the immature embryos to selection medium I (composition per liter: 4 grams of N6 salt and vitamins, 40 grams of sucrose, 30 grams of glucose, 0.7 grams of L-proline, 0.5 g of MES, 1 mg/ml 2, 4-d 1.5ml, 8.5mg/ml silver nitrate 0.1ml, 100mg/ml L-cysteine 0.4g, 0.5M/L DTT 0.154g, Timentin 100mg, 3mg/ml Bialaphos 0.5ml), 28 ℃ dark Grow for two weeks.

(7) Transfer all immature embryos to selection medium II (composition per liter: 4 grams of N6 salt and vitamins, 40 grams of sucrose, 30 grams of glucose, 0.7 grams of L-proline, 0.5 g of MES, 1 mg/ml 2,4 -d 1.5ml, 8.5mg/ml silver nitrate 0.1ml, 100mg/ml L-cysteine 0.4g, 0.5M/L DTT 0.154g, Timentin 100mg, 3mg/ml Bialaphos1ml), you can choose at this time, Select brightly colored immature embryos and culture them in the dark at 28°C for two weeks.

(8) After two selections, start regeneration, in regeneration medium I (composition per liter: MS (Murashige and Skoog) salt (sigma) 4.3g, sucrose 60g, gel 2.5 grams, 2mg/ml glycine 1ml, Timentin 100mg) for germination and rooting, and transfer to regeneration medium II when obvious leaves and roots grow out (composition per liter: MS salts 2.9g, sucrose 30g, gel 2.5g, 2mg/ml glycine 1ml, Timentin 100mg) superior. From this step, light culture is carried out.

(9) When the regenerated seedlings grow 3-4 leaves, transfer them to the greenhouse for inspection, and keep the positive plants. After slowing down the seedlings for 2-3 days, transfer them to the soil, then carry out normal corn growth management, and flower for a week Then measure the plant height.

Example 4 Identification of edited transgenic maize plants

One week after being transferred to the soil, glufosinate-ammonium screening was carried out. After glufosinate-ammonium screening, DNA was extracted from the surviving corn leaves using the CTAB method, and PCR identification was carried out with Zm00001d008708 gene-specific primers, and the amplified products were electrophoresed on 1% agarose gel to test.

Zm00001d008708 gene-specific primer sequences are as follows:

Zm00001d008708-CRISPR-F1: 5'-CCACCATCATCCTACGGT-3'; SEQ ID NO.11;

Zm00001d008708-CRISPR-R1: 5'-CAGGCACAATCAGGTATGC-3'; SEQ ID NO.12;

The reaction system is:

DNA 1 μl, Zm00001d008708-CRISPR-F1 1 μl, Zm00001d008708-CRISPR-R1 1 μl, 2×mix 10 μl, ddH ₂ O 7 μl, Total 20 μl.

The reaction program was: 94°C for 5 min; 98°C for 30 s, 58°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 10 min, 4°C∞.

The primer pair (Zm00001d008708-CRISPR-F1/R1) is used to amplify the mutation of target 1 and target 2, and the product size is about 0.5Kb; the PCR product with the correct band size of the amplified product is sent for sequencing, and the sequencing result (Zm00001d008708MT ) and the wild type (Zm00001d008708WT), the results are shown in Figure 1 (only the mutation site is shown). One Zm00001d008708 gene-edited mutant line was screened (Fig. 2, A). The edited homozygous mutant material was significantly shorter than wild-type maize (Fig. 2, B) plants. This maize dwarf material has important breeding value.

The plant heights of the wild type (Zm00001d008708WT) and mutant (Zm00001d008708MT) lines were counted. The average plant height of the wild type plants was 158.2cm, and the average plant height of the mutant lines was 135.9cm. The variance analysis between the two showed significant differences.

The present invention provides a method for creating a corn dwarfing material. According to the above method, a target gene-edited corn plant is prepared, and then the gene-edited corn plant is subjected to hybridization, backcrossing, self-crossing or asexual reproduction, thereby creating a corn dwarfing material.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

sequence listing

<110> Jilin Academy of Agricultural Sciences

<120> A method for creating maize dwarf material based on Zm00001d008708 gene

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<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence

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atgaacgcgt cgcagttcat ggacaagcag atcctcggcc tggctgcctc cgcttccccc 60

tccggcggcg gcgcgggggg cggtgggggt gtggatctca gcgatctgat gataccgatc 120

ccccaggagg acgccgagaa ccgcctcggt cgccggcgta gcagcaccag cgtcaacgga 180

accgcagacg acatgctacc cagttatgac ttccagccca tccgcactag tggcggcgcc 240

gcggccgccg ccgcgcctca ggcctcgtgg gggtcgctcg actccaaggc accctctgcc 300

tcatacaacc tcaagagtgc tggtatattg gagccgcatg tgctgaagaa agttagtcat 360

gaggaagaca agagtaactt tcctacagtt actattgcgg atattgatcg aaccatgaag 420

aagtactctg ataacctttt gcatgcactg gaaggtgtaa gctcaaggct ttcacagatg 480

gagggtagaa cacaccaact cgaaaactct gttgacgagt tgaagttaac aatcggtaac 540

tataatggta gcactgatgg aaaactgagg aaccttgaga acatgctcag ggaggtccaa 600

gcaggtgtgc agatttgcg agacaagcag gaaattgtcg aagacacagct ccaccttgcg 660

aagctccaga caaacaaaac cgatggccaa tcatcagaaa atagtgggtc tggacaggct 720

ggtttacagc agcagccggt ggttcctcca caagcagcca ttcagccaca acaagtccta 780

accccttcgc aaccacctgc acttcctgcc cttcctgctc caaatgcacc acctccacct 840

ccaacgcttc aaaaccaatc atcattacag tttccaagtc atttacaaca ttcacaggta 900

ccatctgtgc cttctgttgc actggcaccc acagttccag ctttaccaag ggatgcttac 960

tatgccccat ctgctcagcc gaccgagacc atgcaccagc agtatcaagc tccgccagtt 1020

ccacagccac aggcacctcc tgcaccacct cagcagtacc aatcccaaac ccagttccct 1080

caatatgcac agccacctca gcctgcaaat gttaacccctt caactcccca tgtgccccat 1140

gcaccccagc aaccagagga aactatgcct tatgcaccag ctcagagcta tccacctaat 1200

gcaatcgctg caccttatat gcaaccacct agtggacctg ctcctcctta ctatgggcag 1260

caaaacccta gcatgtatga acctcctgca ggccgggcta accctgggcc accatcatcc 1320

tacggttctg gtgggtacgg gccacagggt ggaggtagtt tctctgaatc ttatggttac 1380

actggatctc cttcccaccg tggcaatgct ggaatgaagc agtcttcacc ttttgctcaa 1440

tcctctggag gaagcggcag ctatggcagt ggcaagctcc ctactgccca gatgcttcca 1500

caagcagtgc cgatcagctc ctccagcacc agcggttctt ctggcaatag agtgccactt 1560

gacgatgtag tggagaaggt tgctacgatg ggattctcaa gagagcaggt gagagcaacc 1620

gtgcggaggc tgactgaaaa cgggcagaac gtggacctga atgtggtgct cgacaagctg 1680

atgaacggat ga 1692

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<213> Artificial Sequence

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Met Asn Ala Ser Gln Phe Met Asp Lys Gln Ile Leu Gly Leu Ala Ala

1 5 10 15

Ser Ala Ser Pro Ser Gly Gly Gly Ala Gly Gly Gly Gly Gly Val Asp

20 25 30

Leu Ser Asp Leu Met Ile Pro Ile Pro Gln Glu Asp Ala Glu Asn Arg

35 40 45

Leu Gly Arg Arg Arg Ser Ser Thr Ser Val Asn Gly Thr Ala Asp Asp

50 55 60

Met Leu Pro Ser Tyr Asp Phe Gln Pro Ile Arg Thr Ser Gly Gly Ala

65 70 75 80

Ala Ala Ala Ala Ala Pro Gln Ala Ser Trp Gly Ser Leu Asp Ser Lys

85 90 95

Ala Pro Ser Ala Ser Tyr Asn Leu Lys Ser Ala Gly Ile Leu Glu Pro

100 105 110

His Val Leu Lys Lys Val Ser His Glu Glu Asp Lys Ser Asn Phe Pro

115 120 125

Thr Val Thr Ile Ala Asp Ile Asp Arg Thr Met Lys Lys Tyr Ser Asp

130 135 140

Asn Leu Leu His Ala Leu Glu Gly Val Ser Ser Arg Leu Ser Gln Met

145 150 155 160

Glu Gly Arg Thr His Gln Leu Glu Asn Ser Val Asp Glu Leu Lys Leu

165 170 175

Thr Ile Gly Asn Tyr Asn Gly Ser Thr Asp Gly Lys Leu Arg Asn Leu

180 185 190

Glu Asn Met Leu Arg Glu Val Gln Ala Gly Val Gln Ile Leu Arg Asp

195 200 205

Lys Gln Glu Ile Val Glu Thr Gln Leu His Leu Ala Lys Leu Gln Thr

210 215 220

Asn Lys Thr Asp Gly Gln Ser Ser Glu Asn Ser Gly Ser Gly Gln Ala

225 230 235 240

Gly Leu Gln Gln Gln Pro Val Val Pro Pro Gln Ala Ala Ile Gln Pro

245 250 255

Gln Gln Val Leu Thr Pro Ser Gln Pro Pro Ala Leu Pro Ala Leu Pro

260 265 270

Ala Pro Asn Ala Pro Pro Pro Pro Pro Pro Thr Leu Gln Asn Gln Ser Ser

275 280 285

Leu Gln Phe Pro Ser His Leu Gln His Ser Gln Val Pro Ser Val Pro

290 295 300

Ser Val Ala Leu Ala Pro Thr Val Pro Ala Leu Pro Arg Asp Ala Tyr

305 310 315 320

Tyr Ala Pro Ser Ala Gln Pro Thr Glu Thr Met His Gln Gln Tyr Gln

325 330 335

Ala Pro Pro Val Pro Gln Pro Gln Ala Pro Pro Ala Pro Pro Gln Gln

340 345 350

Tyr Gln Ser Gln Thr Gln Phe Pro Gln Tyr Ala Gln Pro Pro Gln Pro

355 360 365

Ala Asn Val Asn Pro Ser Thr Pro His Val Pro His Ala Pro Gln Gln

370 375 380

Pro Glu Glu Thr Met Pro Tyr Ala Pro Ala Gln Ser Tyr Pro Pro Asn

385 390 395 400

Ala Ile Ala Ala Pro Tyr Met Gln Pro Pro Ser Gly Pro Ala Pro Pro

405 410 415

Tyr Tyr Gly Gln Gln Asn Pro Ser Met Tyr Glu Pro Pro Ala Gly Arg

420 425 430

Ala Asn Pro Gly Pro Pro Ser Ser Tyr Gly Ser Gly Gly Tyr Gly Pro

435 440 445

Gln Gly Gly Gly Ser Phe Ser Glu Ser Tyr Gly Tyr Thr Gly Ser Pro

450 455 460

Ser His Arg Gly Asn Ala Gly Met Lys Gln Ser Ser Pro Phe Ala Gln

465 470 475 480

Ser Ser Gly Gly Ser Gly Ser Tyr Gly Ser Gly Lys Leu Pro Thr Ala

485 490 495

Gln Met Leu Pro Gln Ala Val Pro Ile Ser Ser Ser Ser Thr Ser Gly

500 505 510

Ser Ser Gly Asn Arg Val Pro Leu Asp Asp Val Val Glu Lys Val Ala

515 520 525

Thr Met Gly Phe Ser Arg Glu Gln Val Arg Ala Thr Val Arg Arg Leu

530 535 540

Thr Glu Asn Gly Gln Asn Val Asp Leu Asn Val Val Leu Asp Lys Leu

545 550 555 560

Met Asn Gly

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 3

tccagcacca gcggttcttc tgg 23

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 4

ccgtgcggag gctgactgaa aac 23

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 5

aataatggtc tcaggcgcca gcaccagcgg ttcttc 36

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 6

gccagcacca gcggttcttc gttttagagc tagaaatagc 40

<210> 7

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 7

tgcggaggct gactgaaaac gcttcttggt gcc 33

<210> 8

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 8

attattggtc tctaaactgc ggaggctgac tgaaaa 36

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

gacaggcgtc ttctactggt gctac 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 10

ctcacaaatt atcagcacgc tagtc 25

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 11

ccaccatcat cctacggt 18

<210> 12

<211> 19

<212>DNA

<213> Artificial Sequence

<400> 12

caggcacaat caggtatgc 19

Claims (2)

1. A method for creating corn dwarfing material based on Zm00001d008708 gene, is characterized in that, concrete steps are as follows: (1) Design the sgRNA action site based on CRISPR/Cas9 for the gene Zm00001d008708; The nucleotide sequence of the sgRNA action site is: 5'-TCCAGCACCAGCGGTTCTTCTGG-3'; SEQ ID NO. 3; and 5'-CCGTGCGGAGGCTGACTGAAAAC-3'; SEQ ID NO.4; (2) Using pCBC-MT1T2 plasmid as template, MT1T2-F, MT1T2-F0, MT2T2-R0, MT2T2-R as primers, PCR amplifies the target fragment; The primer sequences are as follows: MT1T2-F: 5'-AATAATGGTCTCAGGCGCCAGCACCAGCGGTTCTTC-3'; SEQ ID NO.5; MT1T2-F0: 5'-GCCAGCACCAGCGGTTCTTCGTTTTAGGCTAGAAATAGC-3'; SEQ ID NO.6; MT1T2-R0: 5'-TGCGGAGGCTGACTGAAAACGCTTCTTGGTGCC-3'; SEQ ID NO.7; MT1T2-R: 5'-ATTATTGGTCTCTAAACTGCGGAGGCTGACTGAAAA-3'; SEQ ID NO.8; (3) Ligate the target fragment obtained in step (2) with the vector pBUE411 to construct the Zm00001d008708 gene editing vector pBUE411-2gR-Zm00001d008708; The enzyme digestion ligation reaction system is as follows: target fragment 2 μL, pBUE411 2 μL, 10xNEB T4 Buffer 1.5 μL, 10xBSA 1.5 μL, BsaI 1 μL, T4 Ligase 1 μL, ddH ₂ O 6 μL, Total 15 μL; (4) The gene editing vector pBUE411-2gR-Zm00001d008708 obtained in step (3) was transformed into Agrobacterium LBA4404 for genetic transformation of maize, and the dwarf maize material was obtained through screening and identification. 2. A method for creating dwarf corn materials based on the Zm00001d008708 gene according to claim 1, wherein the PCR reaction system in step (2) is: 1 μL of pCBC-MT1T2 plasmid, 0.5 μL of MT1T2-F, 0.5 μL of MT1T2 -F00.5μL, MT1T2-R0 0.5μL, MT1T2-R 0.5μL, 2×Mix 10μL, ddH ₂ O 7μL; PCR amplification reaction program: 98℃ for 3min; 98℃ for 30s, 57℃ for 30s, 72℃ for 1min, 35 cycles; 72°C for 5min, 4°C∞.

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