Method for efficiently creating new corn germplasm with low mycotoxin content and high starch content by using gene editing technology and application of new corn germplasm

Abstract

The invention belongs to the technical field of genetic breeding, and particularly relates to a method for efficiently creating a low-mycotoxin and high-starch-content corn new germplasm by using a gene editing technology and application of the method. According to the method, a CRISPR / Cas9 polygene editing technology and a genetic transformation method are utilized, and in the genetic transformation process, an infection culture medium contains one or more of betaine and calcium chloride. On the basis of existing starch synthesis and grain rot related functional gene research, a high-throughput gene editing technology is utilized, numerous excellent sites are efficiently and randomly polymerized, and betaine and calcium chloride with a proper concentration are added in the genetic transformation process, so that the simultaneous editing quantity in a single transgenic event is more, the efficiency is better, and the yield is higher. According to the method, the variety degree is effectively improved, more excellent site combination modes are generated, numerous variant new germplasms with high starch content and resistance to diseases such as grain rot are efficiently generated on a large scale, and the method has good application value in gene editing, creation and breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic breeding, and specifically relates to a method for efficiently creating new corn germplasm with low mycophenolic acid and high starch content by using gene editing technology and its application. Background Art

[0002] Gene editing can cause point mutations in gene coding regions to weaken and delete gene functions. It can also delete fragments of promoter elements and UTR regions through a dual-target system to regulate gene expression and thus regulate gene function. In recent years, the CRISPR / Cas9 system has been widely used in gene editing of various species due to its high targeting and specificity, simple and fast operation, and has achieved large-scale creation of mutant resources in rice and soybean. However, these large-scale gene editing studies mainly use CRISPR / Cas9 as a substitute for traditional mutation methods. There is a lack of in-depth research on how to improve the high-throughput system from target design to mutant sequence detection for plant characteristics, reduce costs, and explore the laws of the impact of this emerging technology on the genome after application. Professor Yan Jianbing of Huazhong Agricultural University used high-throughput single-target gene editing technology to perform high-throughput gene editing on hundreds of gene coding regions, and obtained mutant plants with multiple phenotypes, including plant height, ear position, adversity, grains and other agronomic traits, which can verify the functions of multiple genes. However, its gene editing efficiency needs to be further improved. At the same time, it is mostly used for gene function verification and cannot achieve high-throughput material creation. Only by achieving high-throughput excellent material creation can we screen the required breeding materials and truly achieve genetic improvement of corn varieties.

[0003] High-throughput gene editing technology Gene editing is currently mostly used for gene function verification, and it is not possible to achieve high-throughput breeding material creation. Only by achieving high-throughput creation of excellent materials can we screen the best breeding materials and truly achieve genetic improvement of corn varieties. With the continuous optimization and improvement of corn genetic transformation efficiency, the number of verified functional genes continues to increase, and it becomes possible to achieve targeted shape improvement through gene editing technology. However, current gene editing is mostly used for the modification of single or several gene functions, and cannot achieve efficient aggregation of excellent functions on a large scale, resulting in a low rate of new germplasm innovation and low selectivity, which limits the development of germplasm innovation.

[0004] Therefore, seeking a method to use gene editing technology to efficiently create new corn germplasm is the key to achieving efficient and large-scale aggregation of excellent functional sites in corn and improving the innovation rate and selectivity of new germplasm. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method and application thereof for efficiently creating new corn germplasm with low mycotoxin and high starch content using gene editing technology. In the genetic transformation process, the present invention greatly increases the probability of site editing in a single transgenic event by adding an appropriate amount of betaine and calcium chloride to the infection medium, while achieving diversified and efficient polymerization of excellent sites with high starch content and low mycotoxin content (resistance to ear rot, etc.), greatly improving the speed and efficiency of creating excellent new germplasm, and is currently a fast and efficient method for creating new breeding materials. Based on the above research, this patent is completed.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for improving the efficiency of gene editing in corn, the method comprising utilizing CRISPR / Cas9 multi-gene editing technology and a genetic transformation method, wherein during the genetic transformation process, the infection culture medium contains one or more of betaine and calcium chloride.

[0008] Furthermore, the method specifically comprises the following steps:

[0009] S1. Select target genes and design gRNA;

[0010] S2. Using the CRISPR / Cas9 system, a double-target knockout vector was obtained by enzyme ligation and gene recombination, and Agrobacterium was transformed separately and then mixed in equal amounts to prepare an Agrobacterium mixed pool;

[0011] S3, genetic transformation: the Agrobacterium mixed pool obtained in S2 is genetically transformed into corn transformants, and positive seedlings are obtained by herbicide resistance screening; wherein, during the genetic transformation process, the infection medium contains 80-120 mM betaine and / or 30-60 mM calcium chloride;

[0012] S4. After planting and screening homozygous edited plants, the editing efficiency was calculated and the phenotype was observed.

[0013] Furthermore, in S1, specific sgRNA sequences were designed for ear rot, low mycophenolic acid, and auxin content-related genes (predicted to be related to low mycophenolic acid) based on the maize genome sequence information;

[0014] or,

[0015] In step S2, the sgRNA prepared in step S1 is integrated into the CRISPR / Cas9 vector, and after sequencing is performed to verify that the construction is correct, multiple dual-target knockout vectors are formed;

[0016] The multiple dual-target knockout vectors are respectively transformed into Agrobacterium EHA105, and equal amounts of Agrobacterium are mixed to obtain a mixed pool of bacteria for subsequent genetic transformation.

[0017] Furthermore, the volume of the Agrobacterium EHA105 is 80-100uL.

[0018] Furthermore, in step S2, the CRISPR / Cas9 vector is a binary expression vector, preferably, the binary expression vector is pCXB053.

[0019] Furthermore, in step S3, the genetic transformation specifically comprises the following steps:

[0020] S3-1, placing corn embryos in 1.5-2 mL of suspension and treating for 25-35 min;

[0021] S3-2, remove the treatment solution, add the infection medium, and let it stand for infection for 3 to 8 minutes;

[0022] S3-3, transfer the infected maize embryos to the co-culture medium, culture them in the dark at 22-24°C for 3 days, then transfer the maize embryos to the resting medium, culture them in the dark at 27-29°C for 5-7 days;

[0023] S3-4, taking the immature corn embryos after the culture in S3-3, excising the radicles, transferring to a screening medium containing 5 mg / L Bialaphos and culturing for 14 days, and then transferring to a screening medium containing 8 mg / L Bialaphos and culturing for 14 days to obtain resistant callus;

[0024] S3-5. Transfer the resistant callus to differentiation medium and culture at 24-26°C, 5000 lx, light intensity for three weeks to obtain regenerated seedlings.

[0025] Furthermore, the infection medium includes 1 / 2MS, 68.5 g / L sucrose, 36 g / L glucose, 0.115 g / L L-proline, 200 mM acetosyringone, 200 mg / L cysteine, an Agrobacterium mixed solution with an OD600 of 0.5 to 1, 100 mM betaine and / or 50 mM calcium chloride.

[0026] Furthermore, the infection medium includes 1 / 2MS, 68.5 g / L sucrose, 36 g / L glucose, 0.115 g / L L-proline, 200 mM acetosyringone, 200 mg / L cysteine, an Agrobacterium mixture with an OD600 of 0.5 to 1, 100 mM betaine and 50 mM calcium chloride.

[0027] In a second aspect, the present invention provides application of the above method in creating new germplasm for plant breeding.

[0028] In a third aspect, the present invention provides application of the above method in creating new corn germplasm with low mycotoxin and high starch content.

[0029] In a fourth aspect, the present invention provides a method for creating a new corn germplasm with low mycophenolic acid and high starch content, which comprises editing the corn ZmGBSSI, Shrunken 2, Brittle 2, ZmDOF36, ZmTPS9, ZmAuxRP1, TAR4-1, TAR4-3, Yucca2, Yucca4, ZmSIZ1a, ZmSIZ1b, ZmFER1, ZmTSB2, ZmTSB1, ZmIGS, AUX2, TAR2, Yucca6, and AGPLS3 genes by the above-mentioned method and obtaining the new germplasm after screening.

[0030] Beneficial technical effects of one or more of the above technical solutions:

[0031] (1) The present invention selects multiple functional genes related to starch synthesis and ear rot in order to improve the efficiency of the technology itself, including genes related to starch content and ear rot that have been studied: ZmGBSSI, Shrunken 2, AGPLS3, Brittle2, ZmDOF36, ZmTPS9, ZmAuxRP1, ZmSIZ1a, ZmSIZ1b, ZmFER1, and genes related by prediction: ZmTAR4-1, ZmTAR4-3, Yucca2, Yucca4, ZmTSB2, ZmTSB1, ZmIGS, ZmAUX2, ZmTAR2, ZmYUC6. By using high-throughput gene editing technology, many excellent sites are efficiently and randomly aggregated, and appropriate concentrations of betaine and calcium chloride are added during the genetic transformation process, so that more sites are edited simultaneously in a single transgenic event, the efficiency is better, the degree of diversification is effectively improved, more excellent site combination patterns are generated, and many new variant germplasms with high starch content and low mycotoxin content (resistance to diseases such as ear rot) are efficiently and large-scaled. This is an efficient application method for creating new germplasms required for breeding using gene editing. At the same time, the present invention can realize large-scale, systematic, and precise screening of excellent planting resources required for breeding by conducting multi-point and multi-repeated phenotypic identification in the field, and quickly and efficiently apply them to breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 is the average number of edits in a single event in the transformation events obtained by betaine and calcium chloride treatment in the embodiment of the present invention, wherein the betaine concentration is 100 mM and the calcium chloride concentration is 50 mM;

[0034] Figure 2The high starch content mutants and starch contents are identified and screened from the gene editing materials in the embodiments of the present invention, wherein zml-cr11 and zml-cr22 are selected from the transformation events obtained by betaine treatment; zml-cr42 and zml-cr56 are selected from the transformation events obtained by calcium chloride treatment; zml-cr74 and zml-cr92 are selected from the transformation events obtained by betaine + calcium chloride treatment. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0037] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0038] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0039] Glossary:

[0040] In the present invention, the multi-gene editing technology refers to the technology of using CRISPR / Ca9 gene editing technology to simultaneously achieve multiple site editing in a single transgenic event.

[0041] In the present invention, the gene editing efficiency is the aggregate editing efficiency, which is the number of vectors corresponding to multiple target site editing in one transgenic event using CRISPR / Cas9-mediated multi-gene editing technology.

[0042] In the present invention, the target site is the target.

[0043] Example 1

[0044] A method for efficiently creating new germplasm with low mycophenolic acid and high starch content using gene editing technology

[0045] 1. Experimental Materials

[0046] 1. Maize (Zea mays) inbred line KN5585 immature embryos;

[0047] The backbone vector used for gene editing target was pCXB053, and the Agrobacterium tumefaciens strain was EHA105 (purchased from Shanghai Weidi);

[0048] Plant seed starch content and mycotoxin content detection kit

[0049] Culture medium: YP medium, infection medium, co-cultivation medium, resting medium, screening medium 1, screening medium 2, differentiation medium, and rooting medium. The specific components are shown in Table 1.

[0050] Table 1 Specific components of different types of MS culture media

[0051]

[0052]

[0053] The above trace components are first prepared into mother liquor, and a large amount of solid components are weighed with a balance, and appropriate deionized water is added according to the target system to fully dissolve, and the pH value is adjusted, and the volume is fixed. Sterilize for 20 minutes under high temperature and high pressure conditions, and the temperature is reduced to about 50°C. Liquid components that are not resistant to high temperatures need to be filtered and sterilized with a bacteria filter, and then added to the culture medium with a disposable syringe and poured into a culture dish. Then store in a refrigerator at 4°C for future use.

[0054] 2. Experimental Methods

[0055] 1. Select target genes and design gRNA

[0056] 1.1 Gene selection: A total of 20 genes related to increasing starch content and low mycophenolic acid (ear rot related genes) were selected, including 6 starch related genes and 14 ear rot related genes; studies have shown that reducing the auxin content in the ear is beneficial to improving the resistance of corn to ear rot diseases, so the related genes affecting the auxin content were also selected as the pre-edited genes of the present invention, including Yucca2, Yucca4, Yucca6, TAR4-1, TAR4-3, AUX2, TAR2, ZmIGS, ZmTSB1, and ZmTSB2. The names and gene numbers of the genes are shown in Table 2.

[0057] Table 2 Gene names and gene numbers

[0058]

[0059]

[0060] 1.2 Batch target synthesis

[0061] Batch target synthesis is performed using a dual-target method for each vector. Genes are divided into two types: negative regulation and positive regulation. For a total of 13 genes for negative regulation, frameshift mutations are performed by designing targets in the coding region. Two targets are designed for each gene, and a total of 13 dual-target vectors are constructed in order to obtain variant germplasms with partial or complete loss of gene function; 3'UTR (3'Untranslated Region) is the untranslated region at the tail end of the gene after transcription, which plays an important role in the regulation process after gene transcription, and has an important influence on the regulation of gene expression, mRNA stability, positioning, and post-transcriptional modification. In the present invention, for a total of 7 genes for positive regulation, a method of fragment knockout is adopted in the 3'UTR region dual target, 2 pairs (4 targets) are designed for each gene, and 2 dual vectors are constructed for each gene, a total of 14 vectors, in order to obtain variant germplasms with upregulated expression of gene protein. Finally, a total of 27 dual-target vectors are required, and it is expected to create new germplasm materials with high starch and low mycotoxin content. Specific primers and sequences are shown in Table 3.

[0062] Table 3 Specific primers and their sequences

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] 1.3 High-throughput vector construction

[0069] CRISPR-P 2.0 software was used to design and screen specific sgRNA sequences for target genes. After adding the restriction site BsaI, they were used to synthesize primers and then used to prepare dual-target vectors. The vector construction method was referred to [Zhang Maolin, Wang Juan, Yan Jiali, et al. Using CRISPR / Cas9 gene editing fragment knockout technology to create diversified allelic variation in maize [J]. Shandong Agricultural Science, 2024, 56(09): 1-5. DOI: 10.14083 / j.issn.1001-4942.2024.09.001.]. The backbone vector used for the target was pCXB053 (specific reference doi: 10.1105 / tpc.19.00934.), and the dual-target vector was used for standby after sequencing was correct.

[0070] 1.4 Cultivation of Agrobacterium

[0071] The constructed plasmids were transformed into 100uL of Agrobacterium EHA105 respectively, and the successfully transformed Agrobacterium was activated in YP liquid culture medium respectively. When OD600 was the same value between 0.5 and 1 (OD600 used in this experiment was 0.6), 100uL of bacterial solution was taken and mixed to make Agrobacterium mixed pool for the next step of genetic transformation.

[0072] 1.5 High-throughput genetic transformation

[0073] The plasmid-transformed mixed pool Agrobacterium was transformed into the recipient inbred line KN5585. During the genetic transformation process, the infection medium was treated with betaine and / or calcium chloride, treatment 1: 100mM betaine, treatment 2: 50mM calcium chloride, treatment 3: 100mM betaine + 50mM calcium chloride, and a normal AS suspension without betaine and calcium chloride was used as a control. As shown in Table 4:

[0074] Table 4 Infection medium treatment method

[0075]

[0076] Among them, the suspension is: 1 / 2MS + sucrose 68.5g / L + glucose 36g / L + L-proline 0.115g / L;

[0077] The AS-containing suspension is: 1 / 2MS + sucrose 68.5 g / L + glucose 36 g / L + L-proline 0.115 g / L + acetosyringone (AS) 200 mM + cysteine ​​200 mg / L.

[0078] Specifically, freshly peeled immature embryos of corn inbred line KN5585 (inbred line bred by Weimi Biotechnology (Jiangsu) Co., Ltd.) of about 1 mm were used as materials, and the peeled corn embryos were placed in a 2 mL plastic centrifuge tube containing 1.8 mL of suspension or treatment solution (see the table below), and the osmotic pressure of the immature embryos was maintained. About 150 immature embryos were treated within 30 minutes; the suspension was sucked off, and 1.0 mL of infection medium containing Agrobacterium was added to the remaining corn embryos in the tube, and placed for 5 minutes. The bacterial solution concentration in the infection medium was diluted to an OD600 of between 0.5 and 1, and the concentration used in this experiment was 0.6.

[0079] After suspending the young embryos in the centrifuge tube, pour them into the co-culture medium, and use a pipette to remove the excess Agrobacterium liquid on the surface, and co-culture them at 23°C in the dark for 3 days. After co-culture, transfer the young embryos to the resting culture medium, culture them at 28°C in the dark for 6 days, and then place them on the screening culture medium 1 containing 5 mg / L Bialaphos (a kind of herbicide), start screening culture for 2 weeks, and then transfer them to the screening culture medium 2 containing 8 mg / L Bialaphos for 2 weeks to obtain resistant callus.

[0080] The resistant callus tissue is transferred to a differentiation medium and cultured at 25°C, 5000lx, and light intensity for 3 weeks; the differentiated seedlings are transferred to a rooting medium and cultured at 25°C, 5000lx, and light intensity until they take root; the seedlings are transferred to small pots for growth, and transplanted to a greenhouse after a certain growth stage, and the offspring seeds are harvested after 3-4 months.

[0081] 1.6 The positive seedlings obtained were taken to the modern greenhouse in Xishuangbanna, where they were carefully cared for and managed for harvest. Then, the transgenic system was removed by separation to obtain homozygous transformation events, with no less than 80 homozygous transformation events obtained for each treatment.

[0082] 1.7 Take leaves of experimental materials in the field and extract DNA. At the same time, design 23 pairs of primers according to the target editing position to identify the gene editing type. The primers used are shown in Table 5.

[0083] Table 5 Identification primer sequences

[0084]

[0085]

[0086] 30 gene-edited materials obtained from each treatment were randomly selected (a total of 120) for editing type identification. Each single plant was amplified by PCR with 23 pairs of primers and then sequenced by Sanger to obtain the accurate editing type. The number of edits at the statistical sites was calculated according to the following statistical rules: each vector has two targets, one of the two target sites is edited (base deletion, insertion, substitution, etc.), both are edited, or the corresponding fragment between the two targets is edited (fragment deletion, substitution, etc.). In any of the above cases, the vector is considered to have played a role in editing and is counted as 1 time, that is, the number of edits counted refers to the number of vectors that play an editing role in a single transformation event ( Figure 1 ).

[0087] The results are as follows Figure 1 As shown, whether betaine or calcium chloride was added alone or both were added, the number of edits in a single transformation event was significantly higher than the control, and the number of editing sites added with both was the largest. According to statistics, compared with the control, the editing efficiency increased by 22.6% after treatment with betaine alone, and by 15.1% after treatment with calcium chloride alone. Betaine and calcium chloride synergistically promoted the gene editing efficiency, which was 81% higher than the control group. Therefore, betaine and calcium chloride can effectively promote the efficiency of target site aggregation editing and improve the gene editing efficiency, which provides new ideas for the innovation of corn germplasm.

[0088] 1.8 The seeds of homozygous gene-edited materials were planted in the field (Shandong) for multi-point and multi-repeated field phenotypic identification and screening. At the same time, the starch content and mycotoxin content of the grains were tested after pollination and harvest. Here, the well-known and relatively important aflatoxin B1 content was used to represent the mycotoxin content. New germplasm materials with good comprehensive performance such as high starch and low mycotoxin were screened ( Figure 2, Table 6). Six gene-edited materials with relatively high starch content were identified and screened, of which two were selected from transformation events obtained by betaine treatment, two were from calcium chloride treatment, and two were from betaine + calcium chloride treatment. Shandong belongs to the Huanghuaihai region, where the climatic conditions are changeable. Therefore, experimental points with more serious ear and grain rot were selected from multiple repeated environments to determine the mycotoxin content of grains. From these, seven germplasms with very low mycotoxin content were selected (because it is not easy to determine below 3ug / kg, it is expressed as <3ug / kg) (Table 6), of which two were from transformation events obtained by betaine treatment, two were from calcium chloride treatment, and three were from betaine + calcium chloride treatment. Due to the limitation of sample size, we have not yet screened and identified enough gene-edited materials with high starch and low mycotoxin content, so we cannot statistically compare which treatment has obtained more germplasms with high starch and low mycotoxin content. However, it is certain that after treatment with betaine, calcium chloride, and betaine + calcium chloride, the average number of edits in a single transformation event was effectively increased, providing new ideas for the efficient, targeted and precise creation of new germplasm materials that are beneficial to breeding.

[0089] Table 6 Identification and screening of 6 gene-edited materials with relatively high starch content

[0090] name Mycotoxin content (ug / KG) deal with KN5585 6.2 Comparison zml-cr14 <3 Betaine 100mM zml-cr16 <3 Betaine 100mM zml-cr19 <3 Calcium chloride 50mM zml-cr44 <3 Calcium chloride 50mM zml-cr48 <3 Betaine 100mM + Calcium Chloride 50mM zml-cr76 <3 Betaine 100mM + Calcium Chloride 50mM zml-cr96 <3 Betaine 100mM + Calcium Chloride 50mM

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for improving gene editing efficiency in corn, characterized in that: It includes the use of CRISPR / Cas9 multi-gene editing technology and genetic transformation methods. During the genetic transformation process, the infection culture medium contains one or more of betaine and calcium chloride.

2. The method for improving gene editing efficiency in corn according to claim 1, characterized in that: The specific steps include: S1. Select target genes and design gRNA; S2. Using the CRISPR / Cas9 system, a dual-target knockout vector was obtained by enzyme ligation and gene recombination, and Agrobacterium was transformed individually and then mixed in equal amounts to prepare a mixed pool of Agrobacterium; S3, genetic transformation: the mixed Agrobacterium obtained in S2 is genetically transformed into corn transformants, and positive seedlings are obtained by herbicide resistance screening; wherein, during the genetic transformation process, the infection medium contains 80-120 mM betaine and / or 30-60 mM calcium chloride; S4. After planting and screening homozygous edited plants, the editing efficiency was calculated and the phenotype was observed.

3. The method for improving gene editing efficiency in corn according to claim 2, characterized in that: In the step S1, specific sgRNA sequences are designed for genes related to ear rot, low mycophenolic acid and auxin content according to the maize genome sequence information; or, In step S2, the sgRNA prepared in step S1 is integrated into the CRISPR / Cas9 vector, and after sequencing is performed to verify that the construction is correct, multiple dual-target knockout vectors are formed; The multiple dual-target knockout vectors are respectively transformed into Agrobacterium EHA105, and equal amounts of Agrobacterium are mixed to obtain a mixed pool of bacteria for subsequent genetic transformation.

4. The method for improving gene editing efficiency in corn according to claim 3, characterized in that: In step S2, the CRISPR / Cas9 vector is a binary expression vector, preferably, the binary expression vector is pCXB053.

5. The method for improving gene editing efficiency in corn according to claim 2, characterized in that: In step S3, genetic transformation specifically includes the following steps: S3-1, placing corn embryos in 1.5-2 mL of suspension and treating for 25-35 min; S3-2, remove the treatment solution, add the infection medium, and let it stand for infection for 3 to 8 minutes; S3-3, transfer the infected maize embryos to the co-culture medium, culture them in the dark at 22-24°C for 3 days, then transfer the maize embryos to the resting medium, culture them in the dark at 27-29°C for 5-7 days; S3-4, taking the immature corn embryos after the culture in S3-3, excising the radicles, transferring to a screening medium containing 5 mg / L Bialaphos and culturing for 14 days, and then transferring to a screening medium containing 8 mg / L Bialaphos and culturing for 14 days to obtain resistant callus; S3-5. Transfer the resistant callus to differentiation medium and culture at 24-26°C, 5000 lx, light intensity for 3 weeks to obtain regenerated seedlings.

6. The method for improving gene editing efficiency in corn according to claim 5, characterized in that: The infection culture medium comprises 1 / 2MS, 68.5 g / L sucrose, 36 g / L glucose, 0.115 g / L L-proline, 200 mM acetosyringone, 200 mg / L cysteine, an Agrobacterium mixed solution with an OD600 of 0.5 to 1, 100 mM betaine and / or 50 mM calcium chloride.

7. The method for improving gene editing efficiency in corn according to claim 6, characterized in that: The infection culture medium comprises 1 / 2MS, 68.5 g / L sucrose, 36 g / L glucose, 0.115 g / L L-proline, 200 mM acetosyringone, 200 mg / L cysteine, an Agrobacterium mixed solution with an OD600 of 0.5 to 1, 100 mM betaine and 50 mM calcium chloride.

8. Use of the method according to claim 1 in creating new germplasm for plant breeding.

9. Use of the method according to claim 1 in creating new corn germplasm with low mycotoxin content and high starch content.

10. A method for creating a new corn germplasm with low mycophenolic acid and high starch content, characterized in that: The method of claim 1 is used to edit maize ZmGBSSI, Shrunken 2, Brittle 2, ZmDOF36, ZmTPS9, ZmAuxRP1, TAR4-1, TAR4-3, Yucca2, Yucca4, ZmSIZ1a, ZmSIZ1b, ZmFER1, ZmTSB2, ZmTSB1, ZmIGS, AUX2, TAR2, Yucca6, and AGPLS3 genes, and the obtained product is obtained after screening.

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