A mutated gene family, encoded proteins and uses thereof

Abstract

The present application relates to the field of plant disease and pest control, in particular to the field of crop gene editing biological breeding. Specifically, it relates to a mutant maize zinnia-glucoside-oxygen-methyltransferase gene family, a coding protein and a method and use for controlling hemipteran pests, especially controlling aphid damage to plants. For the first time, the present application simultaneously targets and edits multiple paralogous genes of the maize zinnia-glucoside-oxygen-methyltransferase gene family through multi-target gene editing technology, thereby enhancing the aphid resistance level of maize plants throughout the whole growth cycle and the whole plant. The technical solution of the present application solves the problems in the prior art such as poor effect of chemical control on aphids, serious environmental pollution, and invalidity of Bt insecticidal proteins against aphids; can widely create new anti-aphid maize germplasm resources, solve the problem of lack of aphid-resistant maize germplasm, and provide an efficient, environmentally friendly and low-cost aphid control solution for maize production, which has important production application value.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering, plant disease and pest control, and particularly to gene-edited crop breeding in agricultural biotechnology research. Specifically, this invention relates to a mutant zeatin-glucosinolate-oxy-methyltransferase gene family, its encoded protein, and methods and uses for controlling hemiptera pests, especially aphids, that damage plants. Background Technology

[0002] Insect pests are one of the major natural disasters threatening agricultural production. Aphids, belonging to the order Hemiptera, are herbivorous insects with piercing-sucking stylets. They use their sharp stylets to insert into the vascular system of plants and suck plant sap. At the insertion site of the stylet, not only are plant cells damaged, but the plant also loses the nutrients it needs for growth due to the aphids' feeding, thus reducing its growth capacity. Furthermore, aphids can transmit viral diseases, such as maize dwarf mosaic virus. Aphids have long been one of the major pests in maize production, causing significant yield reductions. Public statistics from the Plant Disease and Pest Prevention and Control Agency of the Ministry of Agriculture and Rural Affairs of China show that aphids damage approximately 100-150 million mu (6.67-6.67 million hectares) of maize planting area annually in my country, with more than 50 million mu (3.33 million hectares) suffering severe damage. Aphids reproduce multiple generations and adapt to a wide temperature range, but their presence is particularly severe during the flowering and pollination stage of corn. At this time, aphids mainly congregate on the male and female ears of corn, sucking the anthers of the male ears and the silks of the female ears. This causes pollen to clump together and prevents its release, or it can cause the silks of the female ears to fail to develop properly, preventing normal pollination and fertilization. This results in sterile ears, empty kernels, or even empty stalks and ears, leading to complete crop failure. Aphids also cause varying degrees of damage during the seedling and grain-filling stages of corn growth.

[0003] Currently, aphid control in corn production mainly relies on chemical methods. However, the control period is short, and the effect is unsatisfactory during the corn's silking and grain-filling stages, requiring large-scale use of pesticides and incurring high costs. Furthermore, the long-term and excessive use of pesticides also damages the environment, as pesticides kill aphids' natural enemies while controlling them, disrupting the ecological balance. Bacillus thuringiensis (Bt insecticidal protein) products can kill lepidopteran pests but are ineffective against aphids. Therefore, there is an urgent need in the corn planting and production sector for corn materials that can resist aphids to achieve efficient, environmentally friendly, and low-cost aphid control.

[0004] Maize (Zea mays L.) is a major food crop and feed source in many parts of the world, and it is also the world's highest-yielding food crop. Simultaneously, maize is widely used in agricultural processing, food processing, pharmaceuticals, and chemicals, showing broad application prospects. Biotechnology has been applied to maize to improve its agronomic traits and quality. Insect resistance is an important stress-resistance trait in maize production. Currently, mainstream biotechnology insect-resistant products worldwide focus on the control of lepidopteran pests, while products targeting hemiptera pests are relatively few, especially those specifically designed to improve resistance to maize aphids, which often have poor control effects. Therefore, using biotechnology to create new aphid-resistant maize germplasm, reduce the damage caused by aphids to maize production, and stabilize maize yields is urgent and of great practical significance.

[0005] Compared with existing technologies, this invention provides a mutated zeinib-glucosinolate-oxy-methyltransferase (Zmbx) gene family, its encoded protein, and its applications. By mutating the entire zeinib-glucosinolate-oxy-methyltransferase (ZmBx) gene family, or independently mutating any paralog gene or gene combination of the aforementioned gene family, the ability of plants to resist aphids can be enhanced, thereby creating new aphid-resistant maize germplasm and reducing the impact of aphids on maize production.

[0006] The main problems solved by the technical solution of this invention are:

[0007] (1) Existing technologies mainly use chemical measures to control hemiptera pests such as aphids. The control period is short and the control effect is poor. Moreover, the control of traditional pesticides requires a lot of manpower and material resources, which pollutes the environment and destroys the ecological balance.

[0008] (2) The problem that existing Bt insecticidal protein-related products are ineffective against aphids;

[0009] (3) Improve the problem of insufficient aphid resistance provided by single gene mutation by mutating the entire zein-glucosinolate-oxymethyltransferase (ZmBx) gene family;

[0010] (4) By combining paralogous genes of the mutant zeinbus-glucosinolate-oxy-methyltransferase (zmbx) gene family, a mutant homologous gene combination that provides better aphid resistance was found, which solved the problem of how to further improve plant aphid resistance.

[0011] (5) The urgent need in the field of corn planting and production is that it can effectively suppress the growth and / or reproduction rate of aphids in production practice, and can achieve aphid control throughout the entire growth cycle of corn, the whole plant and plant tissues, and solve the problem of the lack of new aphid-resistant corn germplasm. Summary of the Invention

[0012] The purpose of this invention is to provide a mutant zeinib-glucosinolate-oxy-methyltransferase gene family, its encoded protein, and its applications. This mutant gene family and its encoded protein can control hemiptera pests, especially aphids, which damage plants. Compared to existing technologies, this invention utilizes multi-target gene editing technology to simultaneously target and edit multiple paralogous genes of the zeinib-glucosinolate-oxy-methyltransferase gene family, enhancing aphid resistance throughout the entire growth cycle and across the entire maize plant. This can widely create novel aphid-resistant maize germplasm resources, solving the problem of aphid-resistant maize germplasm scarcity, and providing an efficient, environmentally friendly, and low-cost aphid control solution for maize production, possessing significant production and application value.

[0013] To achieve the above objectives, the present invention provides a mutant zeinib-glucosinolate-oxy-methyltransferase gene family, its encoded protein, and its applications.

[0014] To achieve the above objectives, this invention provides a mutant zeinib-glucosidase gene family and its encoded protein. Compared to the wild-type gene family and its encoded protein, specific conserved functional domain sequences of the zeinib-glucosidase are missing or partially missing, resulting in reduced enzyme activity or decreased gene expression levels. Mutating any homologous gene or gene combination of this gene family has the effect of regulating plant resistance to aphids. The wild-type zeinib-glucosidase gene family includes four homologous genes, namely ZmBx10, ZmBx11, ZmBx12, and ZmBx14. These four homologous genes exist in different maize ecotypes, and the amino acid sequences of each homologous gene exhibit sequence identity variations across different maize ecotypes.The amino acid sequences of the proteins encoded by the four homologous genes of the wild gene family are shown in SEQ ID NO.1 to SEQ ID NO.4 (based on the maize "B73" ecotype); the nucleotide sequences of the four homologous genes of the wild gene family are shown in SEQ ID NO.5 to SEQ ID NO.8 (based on the maize "B73" ecotype); the CDS sequences of the four homologous genes of the wild gene family are shown in SEQ ID NO.9 to SEQ ID NO.12 (based on the maize "B73" ecotype); the amino acid sequence of the ZmBx10 homologous gene has at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.2%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO.1; the amino acid sequence of the ZmBx11 homologous gene is similar to SEQ ID NO.1. Compared with SEQ ID NO. 2, it has at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 94.1%, 95%, 96%, 97%, 98%, or 99% sequence identity; the amino acid sequence of the ZmBx12 homologous gene compared with SEQ ID NO. 3 has at least 78%, 78.3%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity; the amino acid sequence of the ZmBx14 ...4%, 85%, 86%, 97%, 98%, or 99% sequence identity. Compared to NO.4, it has at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.6%, 97%, 98%, or 99% sequence identity.

[0015] To achieve the above objectives, the present invention provides a mutant zeinib-glucosinolate-oxy-methyltransferase gene family and its encoded protein, wherein the mutant zeinib-glucosinolate-oxy-methyltransferase gene family and its encoded protein have the function of regulating plant resistance to aphids.

[0016] To achieve the above objectives, the present invention provides a mutant zeinib-glucosidase-oxy-methyltransferase gene family and its encoded protein. The amino acid sequences of the mutant gene family and its encoded protein are shown in SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, and SEQ ID NO:75, or amino acid sequences with the same biological function or protein activity obtained by substitution, deletion, or addition of one or more amino acid residues in the shown amino acid sequences. The mutant gene family and its encoded protein have the function of regulating plant resistance to aphids.

[0017] To achieve the above objectives, the present invention provides a mutant zeinib-glucosidase-oxy-methyltransferase gene family and the protein it encodes. The nucleotide sequences of the mutant gene family are shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:73, and SEQ ID NO:76, or their complementary sequences, or different sequences encoding the same protein due to the degeneracy of the genetic code, or homologous gene sequences obtained by substitution, deletion, or addition of one or more nucleotide sequences; or the CDS sequences of the mutant gene family are shown in SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:74, and SEQ ID NO:77.

[0018] To achieve the above objectives, the present invention provides a method for mutating the zeinbad-glucosidase gene family and its encoded protein. The method includes: using gene editing to cause loss of function, reduced enzyme activity, or reduced expression of any homologous gene or gene combination of the wild-type zeinbad-glucosidase gene family; using gene silencing to downregulate any homologous gene or gene combination of the wild-type zeinbad-glucosidase gene family; using mutagenesis to cause loss of function, reduced enzyme activity, or reduced expression of any homologous gene or gene combination of the wild-type zeinbad-glucosidase gene family; or using homologous recombination to cause loss of function, reduced enzyme activity, or reduced expression of any homologous gene or gene combination of the wild-type zeinbad-glucosidase gene family; thereby obtaining the mutated zeinbad-glucosidase gene family and its encoded protein, wherein the mutated gene family and its encoded protein have the function of regulating plant resistance to aphids.

[0019] To achieve the above objectives, the present invention provides a target spacer sequence for multi-target gene editing, wherein the target spacer sequence is used to specifically target and edit the wild zeinib-glucosinolate-oxy-methyltransferase gene family, and to generate or obtain mutated zeinib-glucosinolate-oxy-methyltransferase gene family and its encoded protein, as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

[0020] To achieve the above objectives, the present invention provides a multi-target gene editing gRNA module, wherein the multi-target gene editing gRNA module comprises the above-mentioned multi-target spacer sequence, which is used to guide the multi-target spacer sequence to specifically target the wild-type zeinib-glucosinolate-oxy-methyltransferase gene family.

[0021] To achieve the above objectives, the present invention provides a gene editing system comprising a CRISPR / Cas protein and the aforementioned multi-target gene editing gRNA module, for gene editing of the wild-type zeinib-glucosinolate-oxymethyltransferase gene family.

[0022] To achieve the above objectives, the present invention provides a method for gene editing of wild-type zeinbus-glucosidase-oxy-methyltransferase gene family using the gene editing system described above, and the resulting gene editing target sequence is shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59.

[0023] To achieve the above objectives, the present invention provides a gene-editing expression cassette, which includes the aforementioned gene-editing system and is used for expression in monocotyledonous plants. Preferably, the monocotyledonous plant is maize, sorghum, wheat, barley, rye, millet, or oats.

[0024] To achieve the above objectives, the present invention provides a gene editing kit for creating aphid-resistant maize germplasm or aphid-resistant maize gene editing events, characterized in that the gene editing kit comprises the above-described gene editing system.

[0025] To achieve the above objectives, the present invention provides an aphid-resistant maize germplasm or an aphid-resistant maize gene editing event, wherein the aphid-resistant maize germplasm or the aphid-resistant maize gene editing event comprises the above-mentioned mutated maize zeinbus-glucosinolate-oxy-methyltransferase gene family, or any homologous gene or gene combination of the mutated gene family.

[0026] To achieve the above objectives, the present invention provides a method for detecting aphid-resistant plants, aphid-resistant maize germplasm, or aphid-resistant maize gene editing events, comprising using PCR primers to perform PCR amplification and sequencing detection on the aforementioned aphid-resistant plants, aphid-resistant maize germplasm, or aphid-resistant maize gene editing events, wherein the aphid-resistant plants, aphid-resistant maize germplasm, or aphid-resistant maize gene editing events comprise a zeatin-glucosidase gene family with effective mutations, or any homologous gene or gene combination of the mutated gene family.

[0027] To achieve the above objectives, the present invention provides a method for improving the resistance of maize plants to aphids. The method comprises effectively mutating any homologous gene or gene combination of the wild maize butyl glucoside-oxy-methyltransferase gene family or the mutant gene family to obtain aphid-resistant maize germplasm or aphid-resistant maize gene editing events. Aphids, by ingesting or coming into contact with plants containing the aforementioned aphid-resistant maize germplasm or aphid-resistant maize gene editing events, experience inhibited growth and / or reproductive capacity and / or die, thereby improving the resistance of maize plants to aphids.

[0028] To achieve the above objectives, the present invention provides a corn plant, plant cell, plant seed, plant tissue, plant part, or harvested agricultural product resistant to aphids, characterized in that the plant, plant cell, plant seed, plant tissue, plant part, or harvested agricultural product contains the above-mentioned mutated zeatin-glucosinolate-oxy-methyltransferase gene family or any homologous gene or gene combination of the gene family.

[0029] To achieve the above objectives, the present invention provides an application for improving plant aphid resistance, the application comprising introducing the aforementioned mutated zeatin-glucosidase gene family or any homologous gene or combination of the aforementioned gene family into plant cells, plant seeds, plant tissues, plant parts, or plants, for improving plant resistance to aphids. Preferably, the plant is corn, sorghum, wheat, barley, rye, millet, or oats.

[0030] To achieve the above objectives, the present invention provides a method for detecting aphid-resistant plants. The method utilizes PCR primers to perform PCR amplification and sequencing detection on plants, plant cells, plant seeds, plant tissues, or plant parts exhibiting aphid resistance traits. The plants, plant cells, plant seeds, plant tissues, or plant parts contain effectively mutated zeinib-glucosinolate-oxy-methyltransferase gene families, any homologous genes of gene families, or gene combinations thereof.

[0031] To achieve the above objectives, the present invention provides an application of a mutated zeinbaum-glucosinolate-oxy-methyltransferase gene family and its encoded protein. The application includes the creation of aphid-resistant maize germplasm resources, self-pollinating plants containing effective mutations of the zeinbaum-glucosinolate-oxy-methyltransferase gene family, or hybridizing a plant containing an effective mutation of the zeinbaum-glucosinolate-oxy-methyltransferase gene family as a first plant with a second plant, thereby producing maize plants containing the mutated zeinbaum-glucosinolate-oxy-methyltransferase gene family or any homologous gene or gene combination of the gene family.

[0032] The "Hemiptera" described in this invention is an order within the class Insecta, and one of the larger groups within that class. When at rest, the forewings of Hemiptera cover the back of their bodies, with the hindwings tucked beneath. In some groups, the base of the forewings becomes ossified and thickened, forming a "hemiewing" state, hence the name "Hemiptera." Hemiptera are hemimetabolous insects with piercing-sucking mouthparts, feeding on the sap of plants or other animals.

[0033] The term "aphid" as used in this invention is a general term for the superfamily Aphididae in the order Hemiptera, which includes all members of the superfamily Aphididae. As a specific embodiment of the present invention, the present invention discloses a mutant zeatin-glucosinolate-oxy-methyltransferase gene family, its encoded protein, and its application, which is also used to protect plants from any aphids, including the rice aphid (Rhopalosiphum padi), the corn leaf aphid (Rhopalosiphum maidis), the wheat aphid (Sitobion avenae), the wheat long-tubed aphid (Macrosiphum avenae), the wheat two-forked aphid (Schizaphis graminum), the peach aphid (Myzuspersicae), the bean aphid (Aphis fabae), the pea aphid (Acyrthosiphum pisun), the cabbage aphid (Brevicorynebrassicae), the wheat webless long-tubed aphid (Metopolophium dirhodum), the Russian wheat aphid (Diuraphis noxia), the carrot two-tailed aphid (Cavariella aegopodii), the potato aphid (Macrosiphum euphorbiae), the bean aphid (Aphis craccivora), and the cotton aphid (Aphis... The following aphids are listed: gossypii, black citrus aphid (Toxoptera aurantii), brown citrus aphid (Aphid Toxoptera ciidius), willow aphid (Cavariella spp.), willow leaf aphid (Chaitophorus spp.), black pine aphid (Cinara spp.), American sycamore aphid (Drepanosiphum platanoides), and spruce aphid (Elatobium spp.).), Spirea aphid (Aphis citricola), Turnip aphid (Lipaphis pserudobrassicae), Foxtail aphid (Aulacorthum solani), Asparagus aphid (Brachycorynella asparagi), Brown ragweed aphid (Uroleuconambrosiae), Rhamnaceae aphid (Aphis nasturtii), Corn root aphid (Aphis maidiradicis), Golden aphid (Dactynotus rudbeckiae), Honeysuckle aphid (Hyadaphis foeniculi), Plum aphid (Brachycaudushelichrysi), Gall aphid (Pemphigus bursarius), Pepper aphid (Ovatus crataegarius), Artichoke aphid (Capitophorus elaeagni), Onion aphid (Neotoxoptera formosana), Pea aphid (Macrosiphumpisi), Rust plum aphid (Hysteroneura setariae), Onion aphid (Myzus) The aphids include *Ascalonicus*, *Smynthurodes betae*, *Pemphigus betae*, *Dysaphistulipae*, *Aphis armoraciae*, and *Prociphilus erigeronensis*. In specific embodiments, the method of the present invention is used to control aphids that damage monocotyledonous plants, preferably aphids that damage corn or wheat, including *Rhopalosiphum padi*, *Rhopalosiphum maidis*, *Aphis maidiradicis*, *Sitobion avenae*, *Macrosiphum avenae*, *Schizaphis graminum*, *Metopolophium dirhodum*, and *Diuraphis noxia*.

[0034] Aphids are typically divided into three parts: head, thorax, and abdomen. Their body length ranges from 1.5 to 4.9 mm, with most around 2 mm. The body surface is smooth or covered with wax powder or filaments. The antennae have six segments, rarely five, and very rarely four. The sensory organs are circular, rarely elliptical, with the terminal segment often longer than the base. The eyes are large, with many small facets, and often have prominent three-facet tubercles. The terminal segment of the rostrum is short and blunt to long and pointed. The abdomen is larger than the sum of the head and thorax. The prothorax and abdominal segments often have marginal tubercles. The septa are usually tubular, often longer than wide, thick at the base, tapering towards the tip, sometimes swollen in the middle or at the tip, often with marginal projections at the apex. The surface is smooth or has imbricate patterns or reticulate markings at the tip; rarely do they have few or many hairs, and rarely are the septa annular or absent. The caudate plates are mostly semi-circular, tuberculate, conical, triangular, or pentagonal in shape and varying in length. The caudate plate ends in a rounded shape. The epidermis is smooth, reticulate, wrinkled, or mottled with micro-spines or granules. The tips of the body hairs are sharp or blunt, sometimes enlarged into a head-like or fan-shaped structure. Winged aphids typically have 6 antennae, with secondary sensory rings on the 3rd or 3rd and 4th or 3-5th segments. The median vein of the forewing usually branches into 3 branches, rarely into 2 branches. The hindwings usually have 2 cubital veins, rarely becoming smaller with reduced veins. The wing veins are sometimes edged with black. The body is translucent, mostly green or white.

[0035] The plant defense mechanisms against herbivorous insects described in this invention are divided into two types: constitutive defense mechanisms and inducible defense mechanisms. Constitutive defense mechanisms are inherent physical or chemical defense mechanisms of plants that can hinder insects. Inducible defense mechanisms refer to the indirect defense achieved by plants when they are attacked by insects, such as producing defensive proteins or secondary metabolites that are defensive to target insects. The DIMBOA described in this invention, chemically named 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, is mainly stored in vacuoles in plant cells as DIMBOA-Glc. When the plant is damaged, DIMBOA-Glc is converted into a defensive aglycone, DIMBOA, by hydrolytic enzymes.

[0036] In this invention, "contact" refers to insects and / or pests touching, staying on, and / or feeding on plants, plant organs, plant tissues, or plant cells. The plants, plant organs, plant tissues, or plant cells can be inside the plant or on the surface of the plant, plant organ, plant tissue, or plant cell.

[0037] The "control" and "prevention" of aphid resistance mentioned in this invention refers to the situation where aphids come into contact with plants carrying the effective mutation of the zein-glucosinolate-oxy-methyltransferase gene family, and the aphids' growth is inhibited and / or they die after contact.

[0038] The “enhancement” or “increase” of resistance to aphids as described in this invention can be characterized by one or more of the following: (1) a decrease in the survival or reproductive rate of insects capable of colonizing plants, such as aphids; (2) a decrease in the survival or reproductive rate of insects (such as aphids) that are capable of colonizing previously attacked plants to induce immunity; (3) no yield loss due to pests; (4) a decrease in the infection rate of insect-borne viruses; and / or (5) a reduction in virus-induced symptoms (e.g., yellowing of leaves and / or yield loss due to virus infection), any of which can be determined using standard techniques in the art.

[0039] The “gene editing” described in this invention refers to the process of modifying a specific target in the genome of an organism using gene editing technology. Specifically, it involves editing (directed modification) the target gene and its transcription products to add or delete specific DNA fragments, or to remove or replace specific DNA bases, thereby altering the sequence, expression level, or function of the target gene or regulatory element.

[0040] As a specific embodiment of the present invention, a gene editing system includes:

[0041] (1) Gene editing enzyme, wherein the gene editing enzyme includes nucleases of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nucleases), and ZFN (Zinc finger nuclease) editing tools. Preferably, the gene editing enzyme is a Cas protein, also known as a CRISPR enzyme or Cas effector protein, and its types include, but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, and FDK1 protein. In one embodiment of the present invention, the gene editing enzyme is a Cas12 protein, and the gene editing system further includes a direct repeat sequence (DR) that specifically binds to the Cas12 protein. The direct repeat sequence and the spacer are operably linked to form a guide RNA module;

[0042] (2) Regulatory elements, such as promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites and other expression control elements (e.g., transcription termination signals).

[0043] (3) Resistance genes, including hygromycin (hyg), glufosinate (bar), kanamycin (kana), rifamycin (rif), spectinomycin (spec), ampicillin (amp), etc., to facilitate the screening and editing of plants. The resistance genes and screening techniques are well known to those skilled in the art.

[0044] In this invention, the gene editing system is introduced into plants. Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, microemission bombardment, direct DNA uptake into protoplasts, electroporation, or whisker-silicon-mediated DNA introduction.

[0045] In this invention, "wild-type" or "wild-type gene" refers to a gene having the most common sequence or genotype in a particular plant species, or having a sequence or genotype that, relative to the most common sequence or genotype, has only natural variations, polymorphisms, or other silent mutations that do not significantly affect the expression and activity of the gene or allele. In fact, relative to the most common sequence or genotype, a "wild-type" gene or allele does not contain variations, polymorphisms, or any other type of mutation that substantially affect the normal function, activity, expression, or phenotypic outcome of the gene or allele.

[0046] In this invention, "mutation" refers to an individual that has undergone a mutation, possessing a sequence different from the wild type, which may lead to at least partial loss of function, such as changes in the sequence of conserved functional domains of a gene that will at least partially affect the expression of the coding sequence in an organism. The term "mutation" refers to any change in the sequence of a nucleic acid sequence that can be caused by deletion, addition, substitution, or rearrangement. As is well known to those skilled in the art, methods for creating "mutations" are not limited to the gene editing technology described in this invention, but also include RNA interference (RNAi) technology, induced mutagenesis technology, etc. Any mutations or equivalent mutations performed by those skilled in the art on the zeinib-glucosinolate-oxy-methyltransferase gene family described in this invention using different technical means are all within the spirit and scope of the technical solution of this invention.

[0047] As a specific embodiment of the present invention, the aphid-resistant maize germplasm or the aphid-resistant maize gene editing event refers to the deletion or partial deletion of specific conserved functional domain sequences of the maize butyl glucoside-oxy-methyltransferase gene family and its encoded protein, relative to the wild gene family and its encoded protein, resulting in reduced enzyme activity or reduced gene expression level.

[0048] In this invention, "introduction" refers to the generation or acquisition of mutated zeinib-glucosinolate-oxy-methyltransferase gene family and their encoded proteins in plant cells, plant seeds, plant tissues, plant parts or plants by using genetic engineering methods such as gene editing, gene silencing, mutagenesis, homologous recombination, etc.

[0049] As is well known to those skilled in the art, DNA typically exists in a double-stranded form. In this arrangement, one strand is complementary to the other, and vice versa. Because DNA replicates in plants, other complementary strands of DNA are produced. Thus, this invention includes the use of the polynucleotides and their complementary strands as exemplified in the sequence listing. The term "coding strand" as commonly used in the art refers to the strand that binds to the antisense strand. To express a protein in vivo, typically one strand of DNA is transcribed into a complementary strand of mRNA, which serves as a template for protein translation. The mRNA is actually transcribed from the "antisense" strand of DNA. The "sense" or "coding" strand has a series of codons (codons are three nucleotides, and reading three at a time produces a specific amino acid), which can be read as an open reading frame (ORF) to form the target protein or peptide.

[0050] In this invention, nucleic acid molecules or fragments thereof hybridize under stringent conditions with any homologous gene or gene combination of the mutated zeinib-glucosinolate-oxy-methyltransferase gene family or said mutated gene family. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the mutated zeinib-glucosinolate-oxy-methyltransferase gene family or said mutated gene family. Nucleic acid molecules or fragments thereof can specifically hybridize with other nucleic acid molecules under certain conditions. In this invention, if two nucleic acid molecules can form antiparallel double-stranded nucleic acid structures, it can be said that these two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules exhibit complete complementarity, then one nucleic acid molecule is called a "complement" of the other nucleic acid molecule. In this invention, when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "complete complementarity". If two nucleic acid molecules can hybridize with sufficient stability to anneal and bind to each other under at least conventional "low-string" conditions, then the two nucleic acid molecules are called "minimally complementary". Similarly, if two nucleic acid molecules can hybridize with sufficient stability to anneal and bind to each other under conventional "highly stringent" conditions, then these two nucleic acid molecules are said to be "complementary." Deviations from perfect complementarity are permissible, as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. For a nucleic acid molecule to function as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the specific solvent and salt concentration used.

[0051] In this invention, the substantially homologous sequence is a nucleic acid molecule that, under highly stringent conditions, can specifically hybridize with the complementary strand of a matching nucleic acid molecule. Suitable stringent conditions that promote DNA hybridization, such as treatment with 6.0× sodium chloride / sodium citrate (SSC) at approximately 45°C followed by washing with 2.0× SSC at 50°C, are well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from approximately 2.0× SSC, 50°C under low-stringent conditions to approximately 0.2× SSC, 50°C under highly stringent conditions. Furthermore, the temperature conditions in the washing step can be increased from approximately 22°C (room temperature) under low-stringent conditions to approximately 65°C under highly stringent conditions. Both the temperature conditions and the salt concentration can be changed, or one can remain constant while the other is changed. Preferably, the stringent conditions described in this invention can be as follows: specific hybridization occurs at 65°C in a 6×SSC, 0.5% SDS solution, followed by washing the membrane once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0052] Therefore, sequences possessing anti-aphid activity and hybridizing under stringent conditions with the nucleotide sequences SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:70, SEQ ID NO:73; or the CDS sequences SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:71, SEQ ID NO:74 of the present invention are included in the present invention. These sequences are at least about 40%-50% homologous to the sequences of the present invention, about 60%, 65%, or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence homology.

[0053] The genes described in this invention include not only specific example sequences, but also portions and / or fragments (including deletions at the inside and / or ends compared to the full-length nucleotide, CDS, or amino acid sequence), variants, mutants, substitutes (proteins with substituted amino acids), chimeras, and fusion proteins having the aforementioned aphid-resistant activity characteristics. Due to the abundance of the genetic codon, many different DNA sequences can encode the same amino acid sequence. Alternative DNA sequences that generate these proteins encoding the same or substantially the same proteins are within the skill level of those skilled in the art. These different DNA sequences are included within the scope of this invention. Examples of conserved substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecule amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that typically do not alter specific activity are well known in the art and have been described, for example, by N. Neurath and R.R. Hill in *Protein*, published by Academic Press, New York, 1979. The most common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thu / Ser, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, as well as their opposites. It will be apparent to those skilled in the art that such substitutions can occur outside the regions where molecular function plays a crucial role, and still produce an active peptide. For the polypeptides of the present invention, the essential amino acid residues for their activity, and therefore selected as unsubstituted, can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, Cunningham and Wells, 1989, Science 244: 1081-1085). The latter technique involves introducing a mutation at each positively charged residue in the molecule and detecting the insecticidal activity of the resulting mutant molecule, thereby identifying the amino acid residues important for the activity of that molecule. The substrate-enzyme interaction sites can also be determined by analysis of their three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography, or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

[0054] In this invention, the nucleotide sequences of the mutated zeinib-glucosinolate-oxy-methyltransferase gene family or any homologous gene of the mutated gene family, including but not limited to amino acid sequences of SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:70, and SEQ ID NO:73 that have a certain degree of homology with the nucleotide sequences shown above, are also included in this invention. These sequences typically exhibit a similarity / identity greater than 78% with the sequences of this invention, preferably greater than 85%, more preferably greater than 90%, and even more preferably greater than 95%, and may be greater than 99%. Preferred nucleotide sequences of this invention may also be defined according to more specific ranges of similarity and / or identity. For example, sequences of the present invention have 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity.

[0055] In this invention, "insecticide" or "insect-resistant" refers to being toxic to crop pests, thereby achieving "control" and / or "prevention" of crop pests. Preferably, "insecticide" or "insect-resistant" refers to killing crop pests of the order Hemiptera. Specifically, the target insect is an aphid. More specifically, the target insect is an aphid that harms monocotyledonous plants, including the rice aphid (Rhopalosiphum padi), the corn aphid (Rhopalosiphum maidis), the wheat aphid (Sitobion avenae), the wheat long-tubed aphid (Macrosiphum avenae), and the wheat two-forked aphid (Schizaphis graminum).

[0056] In this invention, the mutated zeinib-glucosinolate-oxy-methyltransferase gene family, or any homologous gene or gene combination of the mutated gene family, has an inhibitory effect on aphid growth and / or causes death. Inhibition refers to lethality or sublethality. The plants of this invention, particularly maize, contain the mutated zeinib-glucosinolate-oxy-methyltransferase gene family or any homologous gene or gene combination of the mutated gene family in their genome. Aphids, through feeding on or contacting plant tissues, exhibit this inhibitory effect on their growth and / or reproductive capacity and / or cause death, essentially eliminating the need for chemical or biological pesticides. Furthermore, plants containing the mutated zeinib-glucosinolate-oxy-methyltransferase gene family or any homologous gene or gene combination of the mutated gene family in their genome should be morphologically and developmentally normal, and should not exhibit abnormal plant development or reduced yield due to carrying the mutated gene family or any homologous gene or gene combination of the mutated gene family.

[0057] The articles “a” and “an” used in this invention refer to one or more (i.e., at least one). For example, “an element” means one or more elements.

[0058] The terms “comprising,” “including,” or variations thereof, such as “including,” as used in this invention mean “including, but not limited to,” and should be understood to mean including one of the said elements, integers, or steps, or a group of elements, integers, or steps, but not excluding any other elements, integers, or steps, or groups of elements, integers, or steps.

[0059] The term "genetic material" as used in this invention includes all genes and nucleic acid molecules, such as DNA and RNA.

[0060] When used in the context of a particular nucleic acid, the term "encoding" or "coded" as used in this invention means that the nucleic acid contains the necessary information to guide the translation of a polynucleotide sequence or gene into a particular protein. The information used to encode the protein is detailed using codons. The nucleic acid encoding the protein may contain untranslated sequences (e.g., introns) within the translated region of the nucleic acid, or may lack such inserted untranslated sequences (e.g., in cDNA).

[0061] The terms “polypeptide,” “peptide,” and “protein” used in this invention are used interchangeably to refer to polymers of amino acid residues. These terms apply to polymers of amino acid residues, wherein one or more amino acid residues in the polymer are an artificial chemical analog of a corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers.

[0062] The term “pan-genome” used in this invention refers to all the genes of all ecotypes within the same species, including the core genome present in all individuals and the dispensable genome specific to individual ecotypes, which covers more genetic diversity than a single reference genome.

[0063] As used in this invention, the term "sequence identity" or "identity" refers to the same residues in two nucleic acid or polypeptide sequences when compared with maximum correspondence on a specified comparison window. To achieve optimal sequence alignment, the sequence portion in the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (which contains no additions or deletions). The percentage of sequence identity is calculated by determining the number of positions where the same nucleotide or amino acid residues appear in the sequence, dividing this number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100.

[0064] A non-limiting example of a mathematical algorithm for sequence comparison is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22: 4673-4680). ClustalW compares sequences and aligns them as a whole amino acid or DNA sequence, thus providing data on sequence conservation about the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After aligning amino acid sequences with ClustalW, the percentage of amino acid identity can be assessed. A non-limiting example of a software program that can be used for ClustalW alignment analysis is GeneDoc™. GeneDoc™ (Karl Nicholas) allows for the assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such algorithms are integrated into the ALIGN program (version 2.0), which is part of the GCG WisconsinGenetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA).

[0065] As used in this invention, the term "plant" refers to the whole plant, including all plants and plant populations, such as desired and unwanted wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants obtained through conventional breeding and optimization methods or through biotechnology and recombination methods, or a combination of these methods, including gene-edited plants.

[0066] The term "plant part" as used in this invention includes plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant clumps, and intact plant cells in a plant or plant part. Examples of plant parts include embryos, endosperm, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. It should be understood that parts of transgenic plants within the scope of this invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, endosperm, and flowers, stems, fruits, leaves, and roots derived from transgenic plants or their progeny that have been previously transformed with the DNA molecules of this invention and are therefore at least partially composed of transgenic cells. In one aspect, a plant part is a plant cell. In another aspect, a plant part is a non-regenerative or regenerative cell. In yet another aspect, a plant cell is a somatic cell. A non-regenerative cell is a cell that cannot be regenerated into a whole plant through in vitro culture. Non-regenerative cells can be in the plants or plant parts (e.g., leaves) of this invention. Non-regenerative cells can be cells in seeds or the seed coat of said seeds. Mature plant organs (including mature leaves, mature stems, or mature roots) contain at least one non-regenerating cell. On the other hand, plant cells are reproductive cells, such as ovules or cells that are part of pollen. In another aspect, pollen cells are vegetative (non-reproductive) cells, or sperm cells.

[0067] This invention can be applied to a variety of plants, including but not limited to corn, sorghum, wheat, barley, rye, millet, oats, or turfgrass; preferably, the monocotyledonous plants refer to corn, sorghum, wheat, barley, rye, millet, or oats.

[0068] This invention provides a commodity, product, or processed agricultural product obtained by processing a plant or harvest, which carries a mutated zeinib-glucosinolate-oxymethyltransferase gene family or any homologous gene or gene combination of said mutated gene family. The terms "commodity," "product," and "processed agricultural product" refer to any composition or product composed of material derived from plants, seeds, germplasm, plant cells, or plant parts containing the gene-editing events of this invention. Specifically, the terms "commodity," "product," and "processed agricultural product" include, but are not limited to, food or feed products, crude flour, starch, wheat flour, oil, crushed or whole kernels or seeds, protein concentrates, protein isolates, or biomass.

[0069] Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Definitions of common terms in molecular biology can be found in publications such as *Molecular Biology* (ISBN 9-78-703036853-9), Science Press, by Robert F. Weaver. Unless otherwise specified, all percentages are by weight, and all solvent mixture proportions are by volume. All temperatures are in degrees Celsius.

[0070] All references cited herein are incorporated herein by reference, and the extent of incorporation does not conflict with the explicit details disclosed herein. The references provided herein are solely for reference to their disclosures prior to the filing date of this application. Nothing herein should be construed as an admission that the inventor has no claim to prior disclosures due to prior invention.

[0071] This invention provides a mutated gene family, its encoded protein, and its applications, with the following advantages:

[0072] (1) This invention is the first to use multi-target gene editing technology to simultaneously target and edit four homologous genes (ZmBx10, ZmBx11, ZmBx12 and ZmBx14) of the zein-glucosinolate-oxy-methyltransferase gene family, and mutate any homologous gene or gene combination of the zein-glucosinolate-oxy-methyltransferase gene family. This not only enhances the aphid resistance level of the entire plant throughout its growth cycle, but also enables the editing and modification of the entire gene family at the same time, improving the efficiency of gene editing material creation and improving and avoiding the problem of insufficient resistance provided by single gene mutation.

[0073] (2) This invention simultaneously targets and edits four homologous genes (ZmBx10, ZmBx11, ZmBx12 and ZmBx14) of the zein-glucosinolate-oxy-methyltransferase gene family, creating a rich combination of mutant genes. Through the combination of different genotype mutations, it brings a rich variety of phenotypic variations to the resistance level of plants against aphid damage, greatly expands the gene pool of aphid-resistant maize, enriches the gene source for the creation of aphid-resistant maize germplasm materials, and provides a novel overall technical solution for solving the technical problems in the field of aphid resistance in maize.

[0074] (3) This invention creates a novel aphid-resistant maize germplasm resource, solving the global shortage of aphid-resistant maize germplasm. Compared with the maize germplasm commonly used in current maize planting and production, gene editing events or germplasm containing the mutant gene family and protein encoded by this invention unexpectedly show superior aphid resistance. In aphid survival tests during the seedling and young ear stages, the gene-edited maize plants showed significant improvement in aphid resistance, with a significant reduction in the number of surviving aphids, and the resistance level reached the "resistant" or "moderately resistant" level. The number of surviving aphids was only 26.3%~34.1% (seedling stage) and 33.3%~39.8% (young ear stage) of the aphid-susceptible control, and the aphid resistance effect of some gene editing events even exceeded that of known aphid-resistant maize germplasm materials. This invention discovers a combination of mutant homologous genes that can provide better aphid resistance, filling the technological gap in the field of plant aphid defense.

[0075] (4) This invention provides an efficient, environmentally friendly, and low-cost aphid control solution for corn planting and production. Compared with traditional chemical control methods, the technical solution of this invention reduces the use of pesticides, reduces environmental pollution, and lowers production costs, thus having significant production application value.

[0076] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0077] Figure 1 This is a schematic diagram of the DBN14234 expression cassette, a gene editing vector for specifically recognizing and cleaving target sequences according to the present invention.

[0078] Figure 2 The results show the relative expression levels of the ZmBx10, ZmBx11, ZmBx12 and ZmBx14 genes in the six maize inbred lines of this invention.

[0079] Figure 3 This is a diagram showing the seedling aphid inoculation test results of five maize inbred lines of this invention;

[0080] Figure 4 This is a diagram showing the effect of aphid inoculation experiment during the seedling stage of the gene editing event of the DBN567 maize inbred line germplasm and its genetic background.

[0081] Figure 5 A bar chart showing the statistical results of aphid infestation index from the seedling aphid inoculation experiment of the gene editing event of the DBN567 maize inbred line germplasm and its genetic background in this invention;

[0082] Figure 6 This is a diagram showing the effect of a female ear aphid inoculation experiment on the gene editing event of the DBN567 maize inbred line germplasm and its genetic background in this invention;

[0083] Figure 7 This is a bar chart showing the statistical results of the aphid infestation index of the female ear aphid inoculation experiment of the gene editing event of the DBN567 maize inbred line germplasm and its genetic background. Detailed Implementation

[0084] The following specific embodiments further illustrate the technical solution of the present invention regarding a mutated gene family, its encoded protein, and its application. The following description is merely an illustration of preferred embodiments of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0085] First embodiment: Obtaining the zeatinib-glucosinolate-oxymethyltransferase (ZmBx) gene family and its paralogous genes.

[0086] By searching the MaizeGDB database using the keyword "DIMBOA-glucoside O-methyltransferase", you can obtain an overview of the zimboside-glucoside-O-methyltransferase (ZmBx) gene family. The data can be obtained at the following URL: https: / / maizegdb.org / data_center / gene_product?id=2804560.

[0087] Maize possesses four paralogous genes for butyl-glucosidase-oxy-methyltransferases, including ZmBx10, ZmBx11, ZmBx12, and ZmBx14, which are present in different maize ecotypes. Pan-genome amino acid sequence analysis of ZmBx10, ZmBx11, ZmBx12, and ZmBx14 provided by the MaizeGDB database showed that there are amino acid sequence identity variations across different maize ecotypes for each gene. The ranges of sequence identity variations were: 95.2%–100% for ZmBx10, 94.1%–100% for ZmBx11, 78.3%–100% for ZmBx12, and 96.6%–100% for ZmBx14.

[0088] According to the conserved functional domain data of the four types of butyl-glucosinolate-oxy-methyltransferase genes provided by the MaizeGDB database, the URLs for obtaining the conserved functional domain data are as follows:

[0089] (1) Conserved functional domain of oxygen-methyltransferase in ZmBx10: https: / / maizegdb.org / gene_center / gene / 9024004

[0090] (2) Conserved functional domain of oxygen-methyltransferase in ZmBx11: https: / / maizegdb.org / gene_center / gene / 9024005

[0091] (3) The conserved functional domain of oxygen-methyltransferase in ZmBx12:

[0092] https: / / maizegdb.org / gene_center / gene / 9024006

[0093] (4) Conserved functional domain of oxygen-methyltransferase in ZmBx14: https: / / maizegdb.org / gene_center / gene / 1199963

[0094] Data on conserved functional domains indicate that although different maize ecotypes of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes exhibit amino acid sequence identity variations at the pan-genome level, the amino acid sequences of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes in any ecotype all possess a specific and consistent conserved O-methyltransferase domain. The database accession number for the conserved functional domains of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes is Accession ID: PF00891.

[0095] Therefore, researchers selected the core ecotype genome sequences based on the maize "B73" ecotype as the base sequences for the paralog genes of the four types of butyl-glucosidases, namely ZmBx10 (Gene Model ID: Zm00001d029359), ZmBx11 (Gene Model ID: Zm00001d029356), ZmBx12 (Gene Model ID: Zm00001d029353), and ZmBx14 (Gene Model ID: Zm00001d004921). Their amino acid sequences are shown in SEQ ID NO:1 to SEQ ID NO:4 in the sequence listing, their nucleotide sequences are shown in SEQ ID NO:5 to SEQ ID NO:8 in the sequence listing, and their CDS sequences are shown in SEQ ID NO:9 to SEQ ID NO:12 in the sequence listing. Those skilled in the art can mutate or make equivalent mutations in the zeinib-glucosinolate-oxy-methyltransferase gene family described in this invention by using sequences of different ecotypes, all of which fall within the spirit and scope of the technical solution of this invention.

[0096] The second embodiment involved the determination of the relative expression levels of maize bismuth subglucosidase (ZmBx) paralogous genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) and the seedling aphid survival test of maize inbred lines.

[0097] Researchers determined the relative expression levels of four types of maize dimbroside-glucosidase genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) in six maize inbred lines: DBN567, DBN319, DBN317, DBN316, DBN623, and DBN625. Simultaneously, seedling aphid survival tests were conducted on five of these maize inbred lines (DBN567, DBN319, DBN317, DBN623, and DBN625) to clarify the relationship between the expression levels of dimbroside-glucosidase genes and aphid reproduction rates. This provides data support for further selection of specific aphid-susceptible maize inbred lines for mutation manipulation of the dimbroside-glucosidase gene family and for improving aphid resistance traits in these inbred lines.

[0098] 1. Determination of relative expression levels

[0099] Approximately 100 mg of leaves from six maize inbred lines (DBN567, DBN319, DBN317, DBN316, DBN623, and DBN625) were collected as samples. Total RNA was extracted using Magen's HiPure Plant RNA Mini Kit, and then reverse transcribed into cDNA using TransGen's First-Strand cDNA Synthesis SuperMix. The relative expression levels of ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes were determined by real-time quantitative PCR (2^-ΔΔCt method). Simultaneously, DBN567 plants served as the control, and DBN319, DBN317, DBN316, DBN623, and DBN625 plants served as experimental groups, with analysis performed using the same method. The experiment was conducted in triplicate, and the average value was used.

[0100] The specific methods for determining the relative expression levels of ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes are as follows:

[0101] Step 1: Take 100 mg of maize plant leaves from five maize inbred lines DBN567, DBN319, DBN317, DBN316, DBN623 and DBN625 respectively, grind them into homogenates in a mortar with liquid nitrogen, and take 3 replicates for each sample.

[0102] Step 2: Use Magen's HiPure Plant RNA Mini Kit to extract total RNA from the above samples. Refer to the product instructions for specific methods.

[0103] Step 3: Use TransGen's First-Strand cDNA Synthesis SuperMix to reverse transcribe the total RNA of the above samples into cDNA. Refer to the product instructions for specific methods.

[0104] Step 4: Determine the cDNA concentration of the above samples using NanoDrop 2000 (Thermo Scientific);

[0105] Step 5: Adjust the cDNA concentration of the above samples to the same concentration value, wherein the concentration value ranges from 1 to 10 ng / μl;

[0106] Step 6: The relative gene expression levels in the samples were determined using real-time quantitative PCR (2^-ΔΔCt method). The CUL gene was used as an internal reference gene for the real-time quantitative PCR reaction, and samples from the maize inbred line DBN567 were used as controls. Each sample was tested in triplicate, and the average value was taken. The primers for the real-time quantitative PCR were:

[0107] The following primers were used to determine the relative expression levels of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes:

[0108] Primers 1: Bx10 F, Bx10 R, used to determine the relative expression level of the ZmBx10 gene. The forward and reverse primers are shown in SEQ ID NO:13 and SEQ ID NO:78 in the sequence listing.

[0109] Primers 2: Bx11 F and Bx11 R are used to determine the relative expression level of the ZmBx11 gene. The forward and reverse primers are shown in SEQ ID NO:14 and SEQ ID NO:79 in the sequence listing.

[0110] Primers 3: Bx12F and Bx12R are used to determine the relative expression level of the ZmBx12 gene. The forward and reverse primers are shown in SEQ ID NO:15 and SEQ ID NO:80 in the sequence listing.

[0111] Primers 4: Bx14F and Bx14R are used to determine the relative expression level of the ZmBx14 gene. The forward and reverse primers are shown in SEQ ID NO:16 and SEQ ID NO:81 in the sequence listing.

[0112] Primers 5: CUL F and CUL R are used for the amplification of the CUL internal reference gene by real-time PCR. The forward and reverse primers are shown in SEQ ID NO:17 and SEQ ID NO:82 in the sequence listing.

[0113] The PCR reaction system is as follows:

[0114] PowerUp™ SYBR™ Green Master Mix (2X) 10 μl

[0115] 2 μl of primer mixture

[0116] cDNA 2 μl

[0117] Water (ddH2O) 6 μl

[0118] The primer mixture contains 10 μM of F-terminal and R-terminal primers.

[0119] PCR reaction conditions are as follows:

[0120] Step temperature time

[0121] Step 7: UDG activation at 50℃ for 2 minutes

[0122] Step 8: Activate with Dual-Lock™ DNA polymerase at 95°C for 2 minutes.

[0123] Step 9: Denaturation at 95℃ for 15 seconds

[0124] Step 10: Annealing and extending at 60°C for 1 minute

[0125] Return to steps 9-10 of the denaturation-annealing and extension process, and repeat 40 times.

[0126] During PCR amplification, fluorescence signals were monitored in real time, and the Ct value of each reaction tube was recorded. Data were analyzed using SDS 2.3 software (Applied Biosystems). The methods for analyzing the relative expression levels of ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes are as follows:

[0127] Step 11: Calculate the ΔCt value of the target gene and internal reference gene for each sample. The formula is: ΔCt = Ct (target gene) - Ct (internal reference gene).

[0128] Step 12: Calculate the ΔΔCt values ​​for the experimental group and the control group using the formula: ΔΔCt = ΔCt (experimental group) - ΔCt (control group);

[0129] Step 13: Calculate the relative expression level of the target gene using the formula 2^-ΔΔCt. The formula is: relative expression level = 2^-ΔΔCt.

[0130] Step 14: Result Judgment

[0131] (1) If the relative expression level is greater than 1, it means that the expression level of the target gene in the experimental group is higher than that in the control group;

[0132] (2) If the relative expression level is less than 1, it means that the expression level of the target gene in the experimental group is lower than that in the control group.

[0133] Tables 1-4 present the relative expression levels of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes in six maize inbred lines: DBN567, DBN319, DBN317, DBN316, DBN623, and DBN625. The relative expression levels are further presented in bar charts. Figure 2 middle.

[0134] Table 1. Relative expression levels of the ZmBx10 gene in six maize inbred lines.

[0135]

[0136] Table 2. Relative expression levels of the ZmBx11 gene in six maize inbred lines.

[0137]

[0138] Table 3. Relative expression levels of the ZmBx12 gene in six maize inbred lines.

[0139]

[0140] Table 4. Relative expression levels of the ZmBx14 gene in six maize inbred lines.

[0141]

[0142] Data on the relative expression levels of dimbro-glucosinolate-oxy-methyltransferase genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) in six maize inbred lines DBN567, DBN319, DBN317, DBN316, DBN623, and DBN625 showed that the relative expression levels of DBN623 and DBN625 were significantly lower than those of DBN567, DBN319, DBN317, and DBN316, indicating that the relative expression levels of dimbro-glucosinolate-oxy-methyltransferase genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) were higher in the DBN567, DBN319, DBN317, and DBN316 germplasms. Maize inbred lines DBN567, DBN319, DBN317, and DBN316 are potential maize inbred lines highly susceptible to aphids and are candidate germplasm resources for aphid resistance improvement.

[0143] 2. Seedling stage aphid survival test

[0144] Researchers further selected five maize inbred lines—DBN567, DBN319, DBN317, DBN623, and DBN625—for aphid survival testing during the seedling stage (V3-V4). Six healthy, uniformly shaped maize seedlings from each inbred line were selected for inoculation. Ten vigorous Rhopalosiphum padi aphids were inoculated at the base of each seedling stem. Ten days after inoculation, the number of surviving aphids on each seedling was counted. The results showed that DBN623 and DBN625 had the lowest aphid population, averaging less than 100 aphids per seedling; DBN567 and DBN317 had an average of 190 aphids per seedling; and DBN319 had the highest population, averaging 290 aphids per seedling. The seedling aphid inoculation test results of the five maize inbred lines are shown in the figure below. Figure 3 As shown.

[0145] The results of aphid life tests during the maize seedling stage showed that the DBN623 and DBN625 maize inbred lines had the lowest aphid reproduction rates and could provide aphid resistance for maize. In contrast, the DBN567, DBN319, and DBN317 maize inbred lines were aphid-sensitive, with low aphid resistance levels and high susceptibility to aphids.

[0146] Further analysis of the experimental data from "1. Determination of Relative Expression Levels" in this embodiment demonstrates that the relative expression levels of the dimbro-glucosinolate-oxy-methyltransferase genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) affected the aphid resistance levels of different maize inbred lines. The DBN623 and DBN625 maize inbred lines exhibited lower dimbro-glucosinolate-oxy-methyltransferase gene expression levels, while the DBN567, DBN319, and DBN317 maize inbred lines showed higher dimbro-glucosinolate-oxy-methyltransferase gene expression levels. Systematically altering the relative expression levels of the dimbro-glucosinolate-oxy-methyltransferase gene through innovative biotechnological methods can improve the plant's resistance to biological stress throughout its entire growth cycle, reduce aphid damage and losses, and has significant practical value in agricultural applications.

[0147] Third embodiment: Design of target gene sites and construction of gene editing vectors

[0148] 1. Target design of the target gene

[0149] This embodiment aims to design gene editing targets for four butyl glucoside-oxy-methyltransferase genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) in the butyl glucoside-oxy-methyltransferase gene family of maize. Using the Cas12 protein and gRNA targeting the first exon sequence of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes, the four genes are simultaneously edited in maize inbred lines DBN567 and DBN319.

[0150] The specific methods for designing gene editing targets ZmBx10, ZmBx11, ZmBx12, and ZmBx14 are as follows:

[0151] Step 15: Obtaining the target gene sequence

[0152] The zein butyl glucoside-oxy-methyltransferase gene family and its paralogous genes ZmBx10, ZmBx11, ZmBx12 and ZmBx14 were obtained using the method of Example 1 of the present invention.

[0153] Step 16: Target design for the CRISPR / Cas12 gene editing system

[0154] The CRISPR / Cas12 gene editing system guides the Cas12 protein via gRNA to recognize and cleave target DNA. The target sequence should contain the PAM sequence recognized by the Cas12 protein. In this embodiment, the recognition PAM sequence for the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes is TTTG.

[0155] Step 17: Screening of target spacer sequences

[0156] To design target spacer sequences, use the online tool CRISPR-GE (http: / / skl.scau.edu.cn / ). Input the target gene sequence and search for potential target spacer sequences that meet the PAM sequence requirements. Ensure that the target spacer sequences are specific. Spacer sequences are an important component of the CRISPR gene editing system, used to accurately identify target DNA sequences.

[0157] The target spacer sequences spacer1, spacer2, and spacer3 targeting the first exon sequences of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes are designed as follows. Spacer1 can simultaneously target either ZmBx10 or ZmBx11, spacer2 targets ZmBx12, and spacer3 targets ZmBx14. The four 5' nucleotides TTTG at the end of the three spacer sequences are used to recognize the PAM sequence.

[0158] (1) The target spacer sequence targeting the ZmBx10 gene or the ZmBx11 gene is as follows:

[0159] 5'-TGAGTCTGGCGCTCCAGCCTA-3' (SEQ ID NO:19)

[0160] (2) The target spacer sequence targeting the ZmBx12 gene is as follows:

[0161] 5'-GCGAAGCACAAGGAATGGTGC-3' (SEQ ID NO: 20)

[0162] (3) The target spacer sequence targeting the ZmBx14 gene is as follows:

[0163] 5'-GCGAAGCACAGGCAGTGGTGG-3' (SEQ ID NO: 21)

[0164] Step 18: Design of a multi-target gRNA tandem module

[0165] A multi-target gRNA tandem module was designed based on the selected target spacer sequences. In this embodiment, to achieve simultaneous editing of four target genes, an expression cassette containing three gRNA tandem modules was constructed: gRNA1 (SEQ ID NO:22), gRNA2 (SEQ ID NO:23), and gRNA3 (SEQ ID NO:24), which respectively guide the spacer sequences to target the ZmBx10 and ZmBx11, ZmBx12, and ZmBx14 genes. The sequence of the multi-target gRNA tandem module is shown in SEQ ID NO:25 of the sequence listing.

[0166] 2. Construction of gene editing vectors

[0167] The multi-target gRNA tandem module disclosed in this embodiment is assembled into an expression cassette containing a CRISPR / Cas12 gene editing system to form a multi-target gene editing expression cassette "14234" (SEQ ID NO:26) containing three gRNA modules tandemly connected. A schematic diagram of the gene editing expression cassette is shown below. Figure 1 As shown in the diagram. RB represents the right boundary; prPvUbi1 (SEQ ID NO:27) is the Panicum virgatum L. Ubi promoter; CaMV poly(A) (SEQ ID NO:28) is the 3' tailing signal for terminating transcription; prUbi1 (SEQ ID NO:18) is the Zea mays L. Ubi promoter; Cas12 is the RNA-guided editing enzyme of the CRISPR gene editing system; NOS (SEQ ID NO:29) is the t-Nos terminator for ending transcription; and LB represents the left boundary.

[0168] The specific procedures for constructing gene-editing vectors can be performed according to conventional molecular biology experimental methods in this field. A brief description of the method is provided in this embodiment:

[0169] Step 19: The pUC57-14234 vector with the "14234" expression cassette was synthesized by Nanjing GenScript Co., Ltd., and the target fragment was obtained by double digestion with HindIII and SpeI.

[0170] Step 20: The backbone vector DBNBC-01 was double-digested with HindIII and SpeI to obtain a backbone with sticky ends of restriction endonucleases.

[0171] Step 21: Connect the expression box "14234" to the skeleton carrier DBNBC-01 to construct the final carrier DBN14234;

[0172] Step 22: The ligation product was transformed into *E. coli* competent cells Trans-T1 and plated on LB agar plates containing 100 mg / L kanamycin (Kan). Eight colonies were selected and incubated at 28°C and 200 rpm for 2 hours. PCR amplification was performed on the colonies, and the band lengths of the PCR products were checked by agarose gel electrophoresis. Two single clones with correct amplified bands were selected for further sequencing to verify the accuracy of the sequences. The forward and reverse primer sequences for the bacterial culture PCR are shown in SEQ ID NO:83 and SEQ ID NO:84 in the sequence listing.

[0173] Step 23: Select the correctly sequenced single clones for propagation, preservation, and plasmid extraction. Transform them into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) using the heat shock method. Select 5 single colonies for culture. After PCR detection and identification, preserve the bacteria in a -80℃ freezer for later use.

[0174] Fourth Example: Obtaining Gene-Edited Positive Plants

[0175] Following conventional Agrobacterium-mediated plant genetic transformation methods, aseptically cultured maize inbred lines DBN567 and DBN319 embryos were co-cultured with the Agrobacterium used in "2. Construction of Gene Editing Vector" in the third embodiment to transfer the expression cassette from the gene editing vector DBN14234 constructed in the third embodiment into the maize chromosome, resulting in maize plants with the nucleotide sequence of the multi-target gene editing expression cassette "14234" containing three gRNA modules (gRNA1 module, gRNA2 module, and gRNA3 module) tandemly.

[0176] For maize plants transformed with the multi-target gene editing expression cassette, leaves were taken when the plants reached a height of 5-6 cm for sequencing analysis of the target genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14). Transformed seedlings showing editing at any one of the target genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) in the first-generation sequencing results were selected and transplanted to a greenhouse for seed production. Sequencing data showed that the multi-target gene editing expression cassette "14234" can simultaneously target and edit four homologous genes (ZmBx10, ZmBx11, ZmBx12, and ZmBx14) in the maize butyl glucoside-oxymethyltransferase gene family, improving the efficiency of creating new aphid-resistant maize germplasm using gene editing technology.

[0177] Among them, 51 gene-editing events were positive in maize inbred line DBN567. The gene-editing event numbers are as follows: EC0012.1, EC0012.2, EC0012.3, EC0012.4, EC0012.5, EC0012.7, EC0012.8, EC0012.9, EC0012.10, EC0012.11, EC0012.13, EC0012.14, EC0012.15, EC0012.16, EC0012.17, EC0012.18, EC0012.19, EC0012.10, EC0012.11, EC0012.12 ... 0012.20, EC0012.21, EC0012.22, EC0012.23, EC0012.24, EC0012.28, EC0012.30, EC0012.31, EC0012.32, EC0012.34, EC00 12.35, EC0012.36, EC0012.37, EC0012.38, EC0012.39, EC0012.40, EC0012.41, EC0012.42, EC0012.44, EC0012.45, EC0012. 46, EC0012.47, EC0012.48, EC0012.49, EC0012.50, EC0012.52, EC0012.53, EC0012.54, EC0012.55, EC0012.57, EC0012.58, EC0012.59, EC0012.60, EC0012.61, EC0012.62; A total of 19 gene-editing events were obtained from maize inbred line DBN319 with positive gene-editing event numbers EC0021.1, EC002, EC002, and EC002, respectively. 1.2, EC0021.3, EC0021.6, EC0021.7, EC0021.10, EC0021.11, EC0021.12, EC0021.13, EC0021.14, EC0021.15, EC0021.17, EC0021.18, EC0021.19, EC0021.20, EC0021.21, EC0021.23, EC0021.24, EC0021.25; plants from wild-type maize inbred lines DBN567 and DBN319 were used as controls.

[0178] Using the methods described above, researchers created gene-editing-positive plants with maize inbred lines DBN567 and DBN319 as genetic background materials. Each independent gene-editing-positive plant is referred to as a "gene-editing event." Those skilled in the art can obtain the sequence of the gene-editing target using conventional PCR amplification product sequencing methods, which can be used to detect whether an effective mutation has occurred in the gene-editing event and to determine the mutated sequence form of the target.

[0179] Effective mutations in gene editing events refer to the deletion, partial deletion, reduced enzyme activity, or decreased expression of specific conserved functional domain sequences of the zeinib-glucosidase gene family and its encoded proteins, relative to the wild-type gene family and its encoded proteins.

[0180] Researchers further clustered the target editing patterns of the four gene mutation genotypes (zmbx10, zmbx11, zmbx12, and zmbx14) in all 70 gene editing events, classifying the "zmbx10-zmbx11-zmbx12-zmbx14 mutant gene combinations" into five types:

[0181] (1) Type A: Gene editing events in which the ZmBx12 gene is mutated but the ZmBx10, ZmBx11, and ZmBx14 genes are not mutated, i.e., the gene combination type of “ZmBx10- ZmBx11-zmbx12- ZmBx14”.

[0182] (2) Type B: Gene editing events in which the ZmBx14 gene is mutated but the ZmBx10, ZmBx11, and ZmBx12 genes are not mutated, i.e., the gene combination type of “ZmBx10- ZmBx11- ZmBx12- zmbx14”.

[0183] (3) Type C: Gene editing events in which the ZmBx12 and ZmBx14 genes are mutated but the ZmBx10 and ZmBx11 genes are not mutated, i.e., the gene combination type of “ZmBx10- ZmBx11- zmbx12-zmbx14”.

[0184] (4) Type D: Gene editing events in which the ZmBx11, ZmBx12, and ZmBx14 genes are mutated, but the ZmBx10 gene is not mutated, i.e., the gene combination type “ZmBx10- zmbx11- zmbx12-zmbx14”.

[0185] (5) Type E: Gene editing events in which the ZmBx10, ZmBx12, and ZmBx14 genes are mutated, but the ZmBx11 gene is not mutated, i.e., the gene combination type of “zmbx10- ZmBx11- zmbx12-zmbx14”.

[0186] The following shows the target editing patterns of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes under any gene editing event in the DBN567 genetic background. Bold characters represent PAM sequences, and underlined characters represent spacer sequences. The target sequence of the wild-type maize inbred line DBN567 is used as a control.

[0187] DBN567WT (wild-type control)

[0188] ZmBX10: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCTGGG

[0189] ZmBX11: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCTGGG

[0190] ZmBX12: TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0191] ZmBX14: TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0192] EC0012.1 Type B

[0193] ZmBX10: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCTGGG

[0194] ZmBX11: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCTGGG

[0195] ZmBX12: TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0196] zmbx14:TGCTCGAAGCGCACGACGAGCT (-11bp) GCCTGTGCTTCGC CAAA

[0197] EC0012.2 Type C

[0198] ZmBX10: TTTGTGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0199] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0200] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC (-1bp) TTCCTTGTGCTTCGC CAAA

[0201] zmbx14:TGCTCGAAGCGCACGACGAGCT(-11bp) GCCTGTGCTTCGC CAAA

[0202] EC0012.3C

[0203] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0204] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0205] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC (-1bp) TTCCTTGTGCTTCGC CAAA

[0206] zmbx14:TGCTCGAAGCGCACGACGAGCTCTT CCAC (-3bp) TGCCTGTGCTTCGCC CAAA

[0207] EC0012.4 cylinderC

[0208] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0209] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0210] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GC (-3bp) TATCCTTGTGCTTCGC CAAA

[0211] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-7bp) ACTGCCTGTGCTTCGC CAAA

[0212] EC0012.5 cylinderC

[0213] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0214] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0215] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC (-4bp) CTTGTGCTTCGC CAAA

[0216] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-7bp) ACTGCCTGTGCTTCGC CAAA

[0217] EC0012.7C

[0218] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0219] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0220] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC (-4bp) CTTGTGCTTCGC CAAA

[0221] zmbx14:TGCTCGAAGCGCACGACGAGCTCTT CC (-9bp) TGTGCTTCGC CAAA

[0222] EC0012.8C

[0223] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0224] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0225] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACCAT (-1bp) CCTTGTGCTTCGC CAAA

[0226] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-6bp) CACTGCCTGTGCTTCGC CAAA

[0227] EC0012.9C

[0228] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0229] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0230] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-10bp) CCTTGTGCTTCGC CAAA

[0231] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-6bp) CACTGCCTGTGCTTCGC CAAA

[0232] EC0012.10C

[0233] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0234] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0235] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT G (+2bpTG) CACCATTCCTTGTGCTTCGC CAAA

[0236] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-6bp) CACTGCCTGTGCTTCGC CAAA

[0237] EC0012.11C

[0238] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0239] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0240] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT G (+2bpTG) CACCATTCCTTGTGCTTCGC CAAA

[0241] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-13bp) GTGCTTCGC CAAA

[0242] EC0012.13C

[0243] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0244] ZmBX11:TTTG [[ID= TCGCTGCCTGTCCGCACGCCCCTGGG

[0245] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT ​ (-7bp) ​ CAAA

[0246] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-6bp) ​ CAAA

[0247] EC0012.14C

[0248] ZmBX10:TTTG ​ TCGCTGCCTGTCCGCACGCCCCTGGG

[0249] ZmBX11:TTTG ​ TCGCTGCCTGTCCGCACGCCCCTGGG

[0250] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT ​ (-7bp) ​ CAAA

[0251] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-9bp) ​ CAAA

[0252] EC0012.15C

[0253] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0254] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0255] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0256] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-6bp) ACTGCCTGTGCTTCGC CAAA

[0257] EC0012.16C

[0258] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0259] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0260] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GC (-7bp) CTTGTGCTTCGC CAAA

[0261] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-10bp) GCCTGTGCTTCGC CAAA

[0262] EC0012.17C

[0263] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0264] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0265] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0266] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-10bp) GCCTGTGCTTCGC CAAA

[0267] EC0012.18th B

[0268] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0269] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0270] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0271] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-7bp) CTGCCTGTGCTTCGC CAAA

[0272] EC0012.20C

[0273] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0274] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0275] zmbx12:TGCTCCAAGCTCACGACGAGC(-13bp) CTTGTGCTTCGC CAAA

[0276] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-7bp) CTGCCTGTGCTTCGC CAAA

[0277] EC0012.21stC

[0278] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0279] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0280] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC (-1bp) TTCCTTGTGCTTCGC CAAA

[0281] zmbx14:TGCTCGAAGCGCACGACGAGC(-12bp) GCCTGTGCTTCGC CAAA

[0282] EC0012.22C

[0283] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0284] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0285] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-11bp) CTTGTGCTTCGC CAAA

[0286] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-9bp) GCCTGTGCTTCGC CAAA

[0287] EC0012.23C

[0288] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0289] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0290] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-11bp) CTTGTGCTTCGC CAAA

[0291] zmbx14:TGCTCGAAGCGCACGACGAGC(-12bp) GCCTGTGCTTCGC CAAA

[0292] EC0012.24C

[0293] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0294] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0295] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC (-1bp) TTCCTTGTGCTTCGC CAAA

[0296] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-9bp) GCCTGTGCTTCGC CAAA

[0297] EC0012.28th A

[0298] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0299] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0300] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-7bp) TCCTTGTGCTTCGC CAAA

[0301] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0302] EC0012.30C

[0303] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0304] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0305] zmbx12:TGCTCCAAGCTCACGACGA(-29bp)AA

[0306] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0307] EC0012.31 SECTIONB

[0308] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0309] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0310] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0311] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0312] EC0012.32 SECTIONB

[0313] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0314] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0315] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0316] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-7bp) ACTGCCTGTGCTTCGC CAAA

[0317] EC0012.34C

[0318] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0319] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0320] zmbx12:TGCTCCAA(-41bp)A

[0321] zmbx14:TGCTCGAAGCGCACGACGAGCTCTTCCACC(-3bp) GCCTGTGCTTCGC CAAA

[0322] EC0012.35 SECTIONA

[0323] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0324] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0325] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0326] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0327] EC0012.36 SECTIONB

[0328] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0329] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0330] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0331] zmbx14:TGCTCGAAGCGCACGACGAGCTCTT CC (-4bp) CTGCCTGTGCTTCGC CAAA

[0332] EC0012.37C

[0333] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0334] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0335] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0336] zmbx14:TGCTCGAAGCGCACGACGAGCTCTT CC (-4bp) CTGCCTGTGCTTCGC CAAA

[0337] EC0012.38 B.C

[0338] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0339] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0340] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0341] zmbx14:TGCTCGAAGCGCACGACGAGCT(-11bp) GCCTGTGCTTCGC CAAA

[0342] EC0012.39C

[0343] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0344] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0345] zmbx12:TGCTCCAAGCTCACGA(-17bp&+2bpTG) CCTTGTGCTTCGC CAAA

[0346] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-7bp) CTGCCTGTGCTTCGC CAAA

[0347] EC0012.40 cylinderB

[0348] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0349] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0350] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0351] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0352] EC0012.41C

[0353] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0354] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0355] zmbx12:TGCTCCAAGCTCACGA(-17bp&+2bpTG) CCTTGTGCTTCGC CAAA

[0356] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0357] EC0012.42 SECTIONA

[0358] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0359] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0360] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-7bp) ATTCCTTGTGCTTCGC CAAA

[0361] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0362] EC0012.44 SECTIOND

[0363] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0364] zmbx11:TTTG TGAGTCTGGCGCTCC (-10bp)TGCCTGTCCGCACGCCCCTGGG

[0365] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GC (-11bp) TGCTTCGC CAAA

[0366] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-9bp) GCCTGTGCTTCGC CAAA

[0367] EC0012.45 SECTIOND

[0368] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0369] zmbx11:TTTG TGAGTCTGGCGCTCC (-10bp)TGCCTGTCCGCACGCCCCTGGG

[0370] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-11bp) CTTGTGCTTCGC CAAA

[0371] zmbx14:GGAGAGCAGCCAGGACTT(-78bp)CCGCATCCCCGACGCGATC

[0372] EC0012.46C

[0373] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0374] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0375] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-11bp) CTTGTGCTTCGC CAAA

[0376] zmbx14:GGAGAGCAGCCAGGACTT(-78bp)CCGCATCCCCGACGCGATC

[0377] EC0012.47 SECTIONE

[0378] zmbx10:TTTG TGAGTCTGGCGCTCCAG (-4bp)TCGCTGCCTGTCCGCACGCCTGGG

[0379] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0380] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT G (-6bp) TCCTTGTGCTTCGC CAAA

[0381] zmbx14:TGCTCGAAGCGCACGACGAG(-11bp) CTGCCTGTGCTTCGC CAAA

[0382] EC0012.48 sq.D

[0383] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0384] zmbx11:TTTG TGAGTCTGGCGCTCC (-8bp)GCTGCCTGTCCGCACGCCCCTGGG

[0385] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-7bp) ATTCCTTGTGCTTCGC CAAA

[0386] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-10bp) CCTGTGCTTCGC CAAA

[0387] EC0012.49 SEC D

[0388] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0389] zmbx11:TTTG TGAGTCTGGCGCTCC (-8bp)GCTGCCTGTCCGCACGCCCCTGGG

[0390] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-7bp) ATTCCTTGTGCTTCGC CAAA

[0391] zmbx14:TGCTCGAAGCGCACGACGAG(-11bp) CTGCCTGTGCTTCGC CAAA

[0392] EC0012.50 SECTIONE

[0393] zmbx10:TTTG TGAGTCTGGCGCTCCAG (-4bp)TCGCTGCCTGTCCGCACGCCTGGG

[0394] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0395] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT G (-6bp) TCCTTGTGCTTCGC CAAA

[0396] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-10bp) CCTGTGCTTCGC CAAA

[0397] EC0012.52C

[0398] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0399] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0400] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GC (-7bp) CTTGTGCTTCGC CAAA

[0401] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-10bp) GCCTGTGCTTCGC CAAA

[0402] EC0012.53C

[0403] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0404] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0405] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0406] zmbx14:TGCTCGAAGCGCACGACGAGCT(-8bp) ACTGCCTGTGCTTCGC CAAA

[0407] EC0012.54 SECTIONE

[0408] zmbx10:TTTG TGAGTCTGGCGCT (-5bp) CTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0409] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0410] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC (-3bp) CCTTGTGCTTCGC CAAA

[0411] zmbx14:TGCTCGAAGCGCACGACGAGCT(-9bp) CTGCCTGTGCTTCGC CAAA

[0412] EC0012.55C

[0413] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0414] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0415] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-7bp) TCCTTGTGCTTCGC CAAA

[0416] zmbx14:TGCTCGAAGCGCACGACGAGCT(-9bp) CTGCCTGTGCTTCGC CAAA

[0417] EC0012.57C

[0418] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0419] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0420] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-5bp) ATTCCTTGTGCTTCGC CAAA

[0421] zmbx14:TGCTCGAAGCGCACGACGAGCT(-8bp) ACTGCCTGTGCTTCGC CAAA

[0422] EC0012.58C

[0423] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0424] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0425] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-5bp) ATTCCTTGTGCTTCGC CAAA

[0426] zmbx14:TGCTCGAAGCGCACGACGAGCTCTT(-3bp) CCACTGCCTGTGCTTCGC CAAA

[0427] EC0012.59 SECTIONA

[0428] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0429] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0430] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT G (+2bpTG) CACCATTCCTTGTGCTTCGC CAAA

[0431] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0432] EC0012.60 cylinderB

[0433] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0434] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0435] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0436] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-10bp) GCCTGTGCTTCGC CAAA

[0437] EC0012.61 Type C

[0438] ZmBX10: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCTGGG

[0439] ZmBX11: TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCTGGG

[0440] zmbx12: TGCTCCAAGCTCACGACGAGCTC (-7bp) ATTCCTTGTGCTTCGC CAAA

[0441] zmbx14:TGCTCGAAGCGCACGACGAG (-11bp) CTGCCTGTGCTTCGC CAAA

[0442] EC0012.62 Type B

[0443] ZmBX10: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCTGGG

[0444] ZmBX11: TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCTGGG

[0445] ZmBX12: TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0446] zmbx14:TGCTCGAAGCGCACGACGAG (-11bp) CTGCCTGTGCTTCGC CAAA

[0447] The following shows the target editing patterns of the ZmBx10, ZmBx11, ZmBx12, and ZmBx14 genes under any gene editing event in the DBN319 genetic background. Bold characters represent PAM sequences, and underlined characters represent spacer sequences. The target sequence of the wild-type maize inbred line DBN319 is used as a control.

[0448] DBN319 WT (wild-type control)

[0449] ZmBX10: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCTGGG

[0450] ZmBX11: TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0451] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0452] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0453] EC0021.1 SectionC

[0454] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0455] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0456] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0457] zmbx14:TGCTCGAAGCGCACGACGA(+5bpCGAAC&-14bp)GCCTGTGCTTCGCCAAA

[0458] EC0021.2 SectionC

[0459] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0460] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0461] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0462] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-7bp) CTGCCTGTGCTTCGC CAAA

[0463] EC0021.3 SECTIONA

[0464] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0465] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0466] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0467] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0468] EC0021.6 SectionB

[0469] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0470] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0471] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0472] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0473] EC0021.7 SectionA

[0474] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0475] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0476] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACCAT(-5bp)GTGCTTCGC CAAA

[0477] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0478] EC0021.10 BlockC

[0479] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0480] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0481] zmbx12: TGCTCCAAGCTCACGACGAGCTCTT GCACC(-2bp)TCCTTGTGCTTCGC CAAA

[0482] zmbx14:TGCTCGAAGCGCACGACGAGCTCTT(-18bp) CGC CAAA

[0483] EC0021.11 BlockC

[0484] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0485] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0486] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-10bp) CCTTGTGCTTCGC CAAA

[0487] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0488] EC0021.12 BlockC

[0489] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0490] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0491] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-11bp) CTTGTGCTTCGC CAAA

[0492] zmbx14:GCAGGAGAGCAGCCAG(+4bpCACT&-33bp)CACTGCCTGTGCTTCGCCAAA

[0493] EC0021.13 SectionC

[0494] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0495] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0496] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-10bp) CCTTGTGCTTCGC CAAA

[0497] zmbx14:GCAGGAGAGCAGCCAG(+4bpCACT&-33bp)CACTGCCTGTGCTTCGCCAAA

[0498] EC0021.14 BlockC

[0499] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0500] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0501] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-11bp) CTTGTGCTTCGC CAAA

[0502] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0503] EC0021.15 BlockC

[0504] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0505] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0506] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-39bp)ACCGTGGCGCTGGA

[0507] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0508] EC0021.17 ScreenB

[0509] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0510] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0511] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0512] zmbx14:TGCTCGAAGCGCACGACGAGCTC(-8bp) CTGCCTGTGCTTCGC CAAA

[0513] EC0021.18 ScreenB

[0514] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0515] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0516] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0517] zmbx14:TGCTCGAAGCGCACGACGAGCT(-11bp) GCCTGTGCTTCGC CAAA

[0518] EC0021.19 BlockC

[0519] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0520] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0521] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-39bp)ACCGTGGCGCTGGA

[0522] zmbx14:TGCTCGAAGCGCACGACGA(-11bp) ACTGCCTGTGCTTCGC CAAA

[0523] EC0021.20 Block B

[0524] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0525] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0526] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0527] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-7bp) CTGCCTGTGCTTCGC CAAA

[0528] EC0021.2 SectionB

[0529] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0530] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTA TCGCTGCCTGTCCGCACGCCCCTGGG

[0531] ZmBX12:TGCTCCAAGCTCACGACGAGCTCTT GCACCATTCCTTGTGCTTCGC CAAA

[0532] zmbx14:TGCTCGAAGCGCACGACGAGC(-10bp) CTGCCTGTGCTTCGC CAAA

[0533] EC0021.23 SectionA

[0534] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCCTGGG

[0535] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCCTGGG

[0536] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGCCAAA

[0537] ZmBX14:TGCTCGAAGCGCACGACGAGC(-12bp) GCCTGTGCTTCGC CAAA

[0538] EC0021.24 BlockC

[0539] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCCTGGG

[0540] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCCTGGG

[0541] zmbx12:TGCTCCAAGCTCACGACGAGCTCTT(-8bp) CCTTGTGCTTCGC CAAA

[0542] zmbx14:TGCTCGAAGCGCACGACGAGCTCT(-7bp)CTGCCTGTGCTTCGCCAAA

[0543] EC0021.25 SectionA

[0544] ZmBX10:TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCCTGGG

[0545] ZmBX11:TTTG TGAGTCTGGCGCTCCAGCCTAT CGCTGCCTGTCCGCACGCCCCTGGG

[0546] zmbx12:TGCTCCAAGCTCACGACGAGCTC(-10bp) CCTTGTGCTTCGC CAAA

[0547] ZmBX14:TGCTCGAAGCGCACGACGAGCTCTT CCACCACTGCCTGTGCTTCGC CAAA

[0548] The 70 maize lines with positive gene-editing events obtained above were grown in a greenhouse for multiple generations. These lines underwent a first-generation backcross (T0 generation) followed by a second-generation self-pollination (T1 generation) to achieve Mendelian segregation of the zmbx10, zmbx11, zmbx12, and zmbx14 mutant genes, while ensuring sufficient seed quantity for subsequent experiments. Seeds produced through self-pollination were harvested and continued to be grown in the greenhouse (T2 generation). Leaves were collected during the seedling stage (V3-V4) for PCR testing and genotype screening. The forward and reverse primers used for PCR detection of gene editing events are the ZmBx10 gene PCR detection primers (SEQ ID NO:85, SEQ ID NO:89), the ZmBx11 gene PCR detection primers (SEQ ID NO:86, SEQ ID NO:90), the ZmBx12 gene PCR detection primers (SEQ ID NO:87, SEQ ID NO:91), and the ZmBx14 gene PCR detection primers (SEQ ID NO:88, SEQ ID NO:92).

[0549] Plants with all homozygous alleles after Mendelian genetic segregation of the four mutant genes zmbx10, zmbx11, zmbx12, and zmbx14 were selected and further self-pollinated (T3 generation) for aphid life test in the fifth embodiment and aphid reproduction test in the sixth embodiment.

[0550] Fifth Example: Seedling aphid physiology test and female ear aphid physiology test of DBN567 genetic background gene-edited positive plants.

[0551] Researchers selected six positive gene editing events from the T3 generation with representative genotypes (all target alleles were homozygous according to PCR detection) based on DBN567. The gene editing event numbers were EC0012.17, EC0012.35, EC0012.49, EC0012.58, EC0012.60, and EC0012.61, respectively. These events were used for greenhouse aphid survival tests at the maize seedling stage (V3 stage) and field aphid survival tests at the maize young ear (female ear). The maize inbred line DBN567 was used as the aphid-susceptible control, and the moderately aphid-resistant maize inbred line DBN625 was used as the aphid-resistant control.

[0552] Table 5 shows the editing target sequence number, amino acid sequence number of the protein encoded by the mutant gene, nucleotide sequence number of the mutant gene, and CDS sequence number of the mutant gene for the six gene editing events with DBN567 as the genetic background in this embodiment.

[0553] Table 5. Gene editing event mutation sequence numbers in the genetic background of maize inbred line DBN567

[0554]

[0555] Based on the research by Li Yuan et al. on the population growth and aphid resistance traits of maize aphids ([D]. Henan Agricultural University, 2012.DOI:10.7666), the aphid index (or aphid ratio) was used as the resistance evaluation index for aphid life tests. Simultaneously, based on the aphid index, the aphid resistance level of maize germplasm was divided into five levels: highly resistant (0-0.25), resistant (0.26-0.50), moderately resistant (0.51-0.75), moderately susceptible (0.76-1.25), and highly susceptible (>1.25).

[0556] The formula for calculating the aphid infestation index of the evaluated material is:

[0557] Aphid Infestation Index = Aphid Quantity of a Certain Maize Material / Average Aphid Quantity of All Observed Maize Materials

[0558] 1. Aphid life test during the corn seedling stage

[0559] Using the aforementioned gene-editing event with maize DBN567 as the genetic background, a life test of aphid infestation in maize seedlings was conducted under greenhouse conditions. Six to ten healthy maize seedlings with similar appearance from each gene-editing event were selected for inoculation, including the gene-edited event, the aphid-susceptible control DBN567, and the aphid-resistant control DBN625. Ten vigorous Rhopalosiphum padi aphids were inoculated at the base of the stem of each maize seedling. Ten days after inoculation, the total number of surviving aphids on each maize seedling was counted.

[0560] The results of the seedling aphid life test showed that after aphid inoculation at the seedling stage, the maize inbred lines DBN567, which was highly susceptible to aphids, and DBN625, which was moderately resistant to aphids, all exhibited aphid resistance at the "resistant" or "moderately resistant" level when carrying gene editing events with different "zmbx10-zmbx11-zmbx12-zmbx14 mutant gene combinations". The results of the seedling aphid life test are shown in Table 6.

[0561] Table 6. Results of seedling aphid biotests using gene editing events with DBN567 as the genetic background.

[0562]

[0563] Compared to the highly susceptible aphid-prone seedlings of the maize inbred line DBN567 (aphid-susceptible control CK), the gene-editing events all demonstrated a significant increase in aphid resistance, with the number of surviving aphids on the plants only reaching 26.3%–34.1% of the number in the susceptible control. Figure 4 Among the gene-editing events, EC0012.49 (zmbx11-zmbx12-zmbx14 multi-gene mutation) showed the best resistance to aphids, with an average aphid index of 0.571, lower than the average aphid index of 0.646 for the moderately aphid-resistant maize inbred line DBN625 (resistance control CK). This indicates that the aphid resistance level of gene-editing event EC0012.49 is superior to that of known aphid-resistant maize germplasm. Other gene-editing events in the biotesting experiments, such as EC0012.58, EC0012.60, EC0012.61, and EC0012.17, also achieved aphid resistance levels consistent with those of DBN625 (resistance control CK), indicating that these gene-editing events can provide aphid resistance for maize planting and production. After effective mutation of the dimbutase-glucosinolate-oxy-methyltransferase gene family in the aphid-susceptible maize inbred line DBN567, aphid resistance was significantly enhanced.

[0564] The data results from the aforementioned gene-editing event and aphid life test are further presented in a bar chart. Figure 5 middle.

[0565] 2. Field aphid life test on young maize ears (female ears)

[0566] The gene-editing events with the aforementioned maize DBN567 genetic background were selected, and a life test of aphids on maize young ears (female ears) was conducted under field conditions. Three to four healthy maize plants with similar appearance from each gene-editing event were selected for inoculation, including the gene-edited event, the aphid-susceptible control DBN567, and the aphid-resistant control DBN625. Ten vigorous Rhopalosiphum padi aphids were inoculated at the husk of each maize female ear. After inoculation, each inoculated female ear was spatially isolated and covered with insect-proof netting to prevent interference from the open field environment on the experimental data. Ten days after aphid inoculation, the total number of surviving aphids on each maize young ear (female ear) was counted.

[0567] The field aphid life test results of maize young ears (female ears) showed that after inoculation with aphids, maize inbred lines DBN567 were highly susceptible to aphids, DBN625 were moderately resistant to aphids, and gene editing events with different "zmbx10-zmbx11-zmbx12-zmbx14 mutant gene combinations" all showed aphid resistance at the "moderate resistance" level. The field aphid life test results of maize young ears (female ears) are shown in Table 7.

[0568] Table 7. Field aphid biotest results of maize young ears (female ears) in the gene-editing event with DBN567 as the genetic background.

[0569]

[0570] Compared to the highly susceptible young ears (female ears) of the maize inbred line DBN567 (aphid-susceptible control CK), the gene editing events all demonstrated a significant increase in aphid resistance, with the number of surviving aphids on the young ears only reaching 33.3%–39.8% of that in the susceptible control. Figure 6 In the gene editing event described in the biotest, the aphid resistance level reached the same level as that of DBN625 (resistance control CK), indicating that the gene editing event can provide aphid resistance for maize planting and production. After effective mutation of the dimbu-glucosinolate-oxy-methyltransferase gene family in the aphid-susceptible maize inbred line DBN567, aphid resistance in the maize ear region was also significantly improved.

[0571] The data results from the field aphid life test on maize young ears (female ears) following the aforementioned gene-editing event are further presented in a bar chart. Figure 7 middle.

[0572] Sixth Example: Aphid Reproduction Test in DBN319 Genetic Background Gene-Edited Positive Plants

[0573] For the T3 generation gene editing event of the "zmbx10-zmbx11-zmbx12-zmbx14 mutant gene combination" (all target alleles were homozygous according to PCR detection) from the genetic background of the highly aphid-susceptible maize inbred line DBN319, researchers selected 6 positive gene editing events to test the reproductive capacity of greenhouse aphids during the seedling stage (V3-V4 stage) under greenhouse conditions, and counted the number of aphids reproduced on the plants. The gene editing event numbers were EC0021.11, EC0021.17, EC0021.20, EC0021.23, EC0021.24, and EC0021.25, respectively.

[0574] For each gene-editing event, 6-8 healthy corn seedlings with similar appearance were selected for inoculation. Ten vigorous Rhopalosiphum padi aphids were inoculated at the base of the stem of each corn seedling. Ten days after inoculation, the total number of surviving aphids on each corn seedling was counted.

[0575] Table 8 shows the editing target sequence number, amino acid sequence number of the protein encoded by the mutant gene, nucleotide sequence number of the mutant gene, and CDS sequence number of the mutant gene for the six gene editing events with DBN319 as the genetic background in this embodiment.

[0576] Table 8. Gene editing event mutation sequence numbers in the genetic background of maize inbred line DBN319

[0577]

[0578] Statistical results from aphid reproduction testing experiments showed that different gene editing events involving the "zmbx10-zmbx11-zmbx12-zmbx14 mutant gene combinations" significantly increased aphid resistance and reduced aphid reproduction compared to the highly susceptible DBN319 (aphid-susceptible control CK) aphids with an average reproduction rate of 289 aphids per plant. Table 9 shows the aphid reproduction data for gene editing events with DBN319 as the genetic background.

[0579] Table 9. Results of aphid reproduction testing using the DBN319 gene editing event as the genetic background.

[0580]

[0581] Based on the data from aphid reproduction tests, the number of surviving aphids after gene editing events was only 24.9%–37.4% of that of the susceptible control DBN319, and was basically consistent with, or even better than, the aphid reproduction level of DBN625 (resistant control CK). For example, the gene editing event EC0021.11 (zmbx12-zmbx14 multi-gene mutation) resulted in a 17.2% reduction in aphid reproduction compared to the average of 87 aphids per plant in DBN625 (resistant control CK), with an average of only 72 aphids per plant. These results demonstrate the superior aphid resistance of gene editing events, indicating that these gene editing events can provide aphid resistance for maize cultivation. After effective mutation of the dimbut-glucosinolate-oxy-methyltransferase gene family in the highly aphid-susceptible maize inbred line DBN319, aphid resistance was significantly improved.

[0582] In summary, by systematically mutating the butyl-glucosidase gene family and altering the relative expression level of the butyl-glucosidase gene through innovative biotechnological methods, it is possible to improve the plant's resistance to biological stress throughout its entire growth cycle and throughout the entire plant, thereby reducing the damage and losses caused by hemiptera insects, especially aphids. The technological innovations and superior technical effects brought about by this invention include, but are not limited to:

[0583] (1) The multi-target gene editing expression cassette “14234” can simultaneously target and edit four homologous genes (ZmBx10, ZmBx11, ZmBx12 and ZmBx14) of the maize butyl glucoside-oxy-methyltransferase gene family, which improves the efficiency of creating new maize aphid-resistant germplasm materials using gene editing technology.

[0584] (2) By mutating the zein-glucosidase gene family, the resistance level of plants to aphids can be improved, and the resistance level even exceeds that of existing aphid-resistant maize germplasm in nature, which is not obvious and difficult to predict.

[0585] (3) Different “zmbx10-zmbx11-zmbx12-zmbx14 mutant gene combinations” of the maize butyl glucoside-oxy-methyltransferase gene family can bring rich phenotypic variations to the resistance level of plants against aphid damage, providing a novel technical solution to solve the technical problems and urgent needs in the field of maize aphid resistance, and can bring huge improvement space for further improving and optimizing the aphid resistance level of plants, and achieve efficient, environmentally friendly and low-cost aphid control;

[0586] (4) It greatly expands the global gene pool of aphid-resistant maize, enriches the source of aphid-resistant maize germplasm materials, enables the creation of new aphid-resistant maize germplasm resources, solves the problem of aphid-resistant maize germplasm scarcity, brings hope to global maize production and aphid control, and has important production and application value.

[0587] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A mutant zeinib-glucosinolate-oxymethyltransferase gene family and its encoded protein, characterized in that, The mutated zeinib-glucosidase gene family and its encoded proteins, compared to the wild-type gene family and its encoded proteins, exhibit the loss or partial loss of specific conserved functional domain sequences of oxygen-methyltransferases, reduced enzyme activity, or decreased gene expression levels. Mutations in any homologous gene or gene combination of this gene family have a regulatory effect on plant resistance to aphids. The wild-type zeinib-glucosidase gene family includes four homologous genes: ZmBx10, ZmBx11, ZmBx12, and ZmBx14. These four homologous genes exist in different maize ecotypes, and the amino acid sequences of each homologous gene exhibit sequence identity variations across different maize ecotypes. (1) The amino acid sequence of the ZmBx10 homologous gene has at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.2%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:1; (2) The amino acid sequence of the ZmBx11 homologous gene has at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 94.1%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:2; (3) The amino acid sequence of the ZmBx12 homologous gene has at least 78%, 78.3%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:3; (4) The amino acid sequence of the ZmBx14 homologous gene has at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96.6%, 97%, 98% or 99% sequence identity with SEQ ID NO:

4.

2. The mutated zeinib-glucosinolate-oxymethyltransferase gene family and its encoded protein according to claim 1, characterized in that, The amino acid sequences of the mutant gene family and its encoded proteins are shown in SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:72, and SEQ ID NO:75, or amino acid sequences with the same biological function or the same protein activity obtained by substituting, deleting, or adding one or more amino acid residues in the shown amino acid sequences. The mutant gene family and its encoded proteins have the function of regulating plant resistance to aphids.

3. The mutated zeinib-glucosinolate-oxymethyltransferase gene family and its encoded protein according to claim 1 or 2, characterized in that, (1) The nucleotide sequences of the mutant gene family are as shown in SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, or their complementary sequences, or different sequences encoding the same protein due to the degeneracy of the genetic code, or homologous gene sequences obtained by substitution, deletion, or addition of one or more nucleotide sequences; or (2) The CDS sequences of the mutant gene family are shown in SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:71, SEQ ID NO:74, and SEQ ID NO:

77.

4. A method for mutating the zeinib-glucosinolate-oxymethyltransferase gene family and its encoded protein, characterized in that, The method includes: (1) Using gene editing methods to cause the loss of function, reduction of enzyme activity or reduction of expression of any homologous gene or gene combination of the wild zein-glucosinolate-oxy-methyltransferase gene family as described in claim 1. (2) Down-regulate any homologous gene or gene combination of the wild zein butyl glucoside-oxymethyltransferase gene family as described in claim 1 by gene silencing method; (3) To induce a loss of function, reduced enzyme activity, or reduced expression of any homologous gene or gene combination of the wild-type zeinib-glucosinolate-oxymethyltransferase gene family as described in claim 1 by mutagenesis; or (4) Using homologous recombination, the function of any homologous gene or gene combination of the wild zein butyl glucoside-oxy-methyltransferase gene family described in claim 1 is lost, the enzyme activity is reduced, or the expression level is reduced. Thus, the mutated zeinib-glucosinolate-oxy-methyltransferase gene family and its encoded protein as described in claim 1 are obtained, and the mutated gene family and its encoded protein have the function of regulating plant resistance to aphids.

5. A target spacer sequence for multi-target gene editing, characterized in that, The target spacer sequence of the multi-target gene editing is used for specific targeted editing of the wild zeinib-glucosinolate-oxy-methyltransferase gene family of claim 1, to generate or obtain the mutated zeinib-glucosinolate-oxy-methyltransferase gene family of claim 1 and its encoded protein, and the target spacer sequence is shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:

21.

6. A gRNA module for multi-target gene editing, characterized in that, The multi-target gene editing gRNA module includes the multi-target spacer sequence of claim 5, used to guide the target spacer sequence of claim 5 to specifically target the wild-type zeinib-glucosinolate-oxy-methyltransferase gene family of claim 1.

7. A gene editing system, characterized in that, The gene editing system comprises a CRISPR / Cas protein and the multi-target gene editing gRNA module of claim 6, for gene editing of the wild-type zeinib-glucosinolate-oxy-methyltransferase gene family of claim 1.

8. The gene editing system according to claim 7, characterized in that, The gene editing system is used to perform gene editing on the wild-type zeinib-glucosidase-oxy-methyltransferase gene family as described in claim 1, and the resulting gene editing target sequence is shown in SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:

59.

9. A gene editing expression cassette, characterized in that, The gene editing expression cassette comprises the gene editing system of claim 7 for expression in maize.

10. A gene editing kit for creating aphid-resistant maize germplasm or aphid-resistant maize gene editing events, characterized in that, The gene editing kit comprises the gene editing system of claim 7.

11. A type of aphid-resistant maize germplasm or aphid-resistant maize gene editing event, characterized in that, The aphid-resistant maize germplasm or aphid-resistant maize gene editing event includes the mutated zeinib-glucosinolate-oxymethyltransferase gene family and its encoded protein as described in claim 2.

12. A method for improving the resistance of maize plants to aphids, characterized in that, The method includes effectively mutating the wild maize butyl-glucosinolate-oxy-methyltransferase gene family or any homologous gene or gene combination of the gene family as described in claim 1 to obtain aphid-resistant maize germplasm or aphid-resistant maize gene editing events; the aphids, by ingesting or contacting plants or plant parts containing the aphid-resistant maize germplasm or aphid-resistant maize gene editing events, have their growth and / or reproductive capacity inhibited and / or die, thereby improving the aphid resistance of maize plants.

13. The application of a mutant zeinib-glucosinolate-oxymethyltransferase gene family and its encoded protein, characterized in that, The application includes the creation of aphid-resistant maize germplasm resources, self-pollinating plants obtained by the method of claim 12, or hybridizing plants obtained by the method of claim 12 as first plants with second plants, thereby producing maize plants containing the maize butyl-glucosinolate-oxy-methyltransferase gene family, any homologous gene or gene combination of the gene family containing the mutations described in any one of claims 1-3.

14. An application for improving plant aphid resistance, characterized in that, The application includes introducing the mutated zeinib-glucosinolate-oxy-methyltransferase gene family or any homologous gene or gene combination of the mutated gene family as described in any one of claims 1-3 into plant cells, plant seeds, plant tissues, plant parts or plants to improve plant resistance to aphids.

15. The application for improving plant aphid resistance according to claim 14, characterized in that, The plants mentioned are corn, sorghum, wheat, barley, rye, millet, or oats.

16. A method for detecting aphid-resistant plants, characterized in that, Using PCR primers, PCR amplification and sequencing detection are performed on the plant, plant cell, plant seed, plant tissue or plant part as described in claim 14 or 15, wherein the plant, plant cell, plant seed, plant tissue or plant part contains an effectively mutated zeinib-glucosinolate-oxy-methyltransferase gene family, any homologous gene or gene combination of the gene family.

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