Target genes for improving genetic resistance to broad-spectrum diseases and insect pests in maize and their applications

Abstract

The present invention discloses a target gene for improving the genetic resistance of corn to a broad spectrum of diseases and insect pests and its application, and belongs to the technical field of molecular biological breeding. The present invention finds a gene editing target gene in corn that can improve its resistance to a broad spectrum of diseases and insect pests, and its nucleotide sequence is a nucleotide sequence including the nucleotide sequence shown in SEQ ID No.1; or the nucleotide sequence shown in SEQ ID No.2; or a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No.3 except the nucleotide sequence shown in SEQ ID No.2. The present invention not only enriches corn resistance genes, but also enables corn strains edited with target genes to have higher broad-spectrum resistance, produce stronger defensive physiological responses after physical damage, and show stronger resistance to corn pests and pathogenic microorganisms.

Description

Technical Field

[0001] The invention relates to a target gene for improving the genetic resistance of corn to a broad spectrum of diseases and insect pests and an application thereof, and belongs to the technical field of molecular biology breeding. Background Art

[0002] Crop pests and diseases pose a serious threat to crop yield and quality. According to statistics, 20%-40% of crops worldwide suffer yield losses due to pests and diseases. Corn is one of the world's important food crops, with a global total output of more than 1 billion tons. It can not only be used as food, but also as the main raw material for animal feed and bioenergy, and has a very wide range of uses in industrial production. Various pests and diseases are major risk factors for corn agricultural production. Common corn diseases include large and small leaf spot, gray leaf spot, gall smut, and silk smut. Common insect pests include corn borers, armyworms, fall armyworms, aphids, thrips, etc. These pests and diseases have a huge impact on the yield and quality of corn. Their large-scale outbreaks will cause a significant reduction in corn production. For the prevention and control of these pests and diseases, in addition to the use of chemical drugs, breeding high-resistant corn strains with genetic resistance to pests and diseases through genetic breeding is an indispensable part of the modern green pest and disease control system. At present, there are genes that can improve corn resistance to different diseases and insect pests in a wide range of corn germplasm resources. The protein products encoded by these genes include plant secondary metabolism synthases, signal transduction proteins, cell surface and intracellular receptors, etc. However, improving the long-lasting and broad-spectrum genetic resistance of corn requires a large number of resistance genes. Therefore, further enriching and accumulating corn resistance genes is an important method in this field to improve the prevention and control of corn diseases and insect pests. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a target gene for improving the genetic resistance of corn to a broad spectrum of diseases and insect pests and its application.

[0004] The first aspect of the present invention provides a target gene for improving the genetic resistance of crops to a broad spectrum of diseases and insect pests, wherein the nucleotide sequence of the target gene comprises or consists of the following sequence:

[0005] 1) the nucleotide sequence shown in SEQ ID No.1; or

[0006] 2) the nucleotide sequence shown in SEQ ID No. 2; or

[0007] 3) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 3 except 2).

[0008] In one embodiment of the present invention, the sequence of the target gene is shown as SEQ ID No.1, the nucleotide sequence of the protein in its protein coding region is shown as SEQ ID No.2, and the encoded amino acid sequence is shown as SEQ ID No.3.

[0009] The second aspect of the present invention provides a protein encoded by the above-mentioned target gene, wherein the amino acid sequence of the protein comprises or consists of the following sequence:

[0010] a) the amino acid sequence shown in SEQ ID No. 3; or

[0011] b) An amino acid sequence having the same protein activity as that of SEQ ID No. 3 by adding, deleting or replacing one or more amino acids.

[0012] In one embodiment of the present invention, the amino acid sequence of the protein encoded by the target gene is the amino acid sequence shown in SEQ ID No.3

[0013] In one embodiment of the present invention, the protein encoded by the target gene is an amino acid sequence in which one or more (for example, 1-10 or more, specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids are added, deleted, or replaced in the amino acid sequence shown in SEQ ID No. 3 and has the same protein activity.

[0014] The third aspect of the present invention provides the use of the above-mentioned target gene and the protein encoded by the target gene in at least one of the following:

[0015] i) Improve crop resistance to a broad spectrum of diseases and pests;

[0016] ii) Breeding crop varieties that are resistant to pests and diseases.

[0017] The fourth aspect of the present invention provides the use of sequences for specific knockout or silencing of target genes, recombinant knockout or silencing vectors or transformants in improving the broad-spectrum resistance of crops to pests and diseases or cultivating crop varieties with resistance to pests and diseases.

[0018] In one embodiment of the present invention, the sequences used to specifically knock out the target gene are shown as SEQ ID Nos. 4 to 23.

[0019] A fifth aspect of the present invention provides a method for improving the genetic resistance of crops to a broad spectrum of diseases and insect pests, which is achieved by knocking out or silencing the target gene as described above in the crops.

[0020] In one embodiment of the present invention, the target gene is knocked out or silenced by using CRISPR-Cas technology, T-DNA and transposon insertion, chemical mutagenesis or physical mutagenesis. The chemical mutagenesis here can be achieved by using ethyl methanesulfonate, and the physical mutagenesis can be achieved by using high-energy rays or plasma.

[0021] A sixth aspect of the present invention provides a method for breeding crops with resistance to pests and diseases, which is achieved by knocking out or silencing the above-mentioned target gene in the crop.

[0022] In one embodiment of the present invention, the target gene is knocked out or silenced by using CRISPR-Cas technology, T-DNA and transposon insertion, chemical mutagenesis or physical mutagenesis. The chemical mutagenesis here can be achieved by using ethyl methanesulfonate, and the physical mutagenesis can be achieved by using high-energy rays or plasma.

[0023] In one embodiment of the present invention, the crop is corn.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a target gene capable of improving the genetic resistance of corn to a broad spectrum of diseases and insect pests. By knocking out or silencing the target gene, the edited or regulated corn crop contains higher broad-spectrum resistance metabolites than the wild-type control. At the same time, the edited or regulated corn crop produces a stronger defense physiological response after the plant is physically damaged, and shows stronger resistance to corn pests and pathogenic microorganisms represented by fall armyworm, which has important significance and value for improving the genetic resistance of corn to a broad spectrum of diseases and insect pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the gene editing vector of Example 1 of the present invention.

[0027] Figure 2 This is a schematic diagram of the nucleotide sequence of the editing site where a frameshift mutation occurred in Example 2 of the present invention.

[0028] Figure 3 This is the measurement result of benzoxazine substances in Example 3 of the present invention.

[0029] Figure 4 The results of measuring plant hormones in Example 3 of the present invention are shown.

[0030] Figure 5 The results of testing the resistance of different corn strains to the fall armyworm in Example 4 of the present invention are shown. DETAILED DESCRIPTION

[0031] Below in conjunction with embodiment, technical scheme of the present invention is clearly and completely described, obviously, described embodiment is only a part of embodiment of the present invention, rather than all embodiments. Embodiment is carried out according to conventional experimental conditions or according to the condition suggested by manufacturer's specification. Unless otherwise defined, all professional terms used in the present invention have the same meaning as those skilled in the art are generally understood. In addition, any method and material similar or identical to the described content can be used in the inventive method, based on the embodiment in the present invention, all other embodiments obtained by those skilled in the art under the premise of not paying creative work, all belong to the scope of protection of the present invention.

[0032] The experimental methods used in the following examples were carried out according to conventional methods or those recommended by the manufacturer unless otherwise specified. The materials, reagents and consumables used were all commercially available unless otherwise specified.

[0033] Example 1 Construction of gene editing vector for target gene

[0034] Synthesize gRNA directed to the target gene, the sequence of gRNA is shown in SEQ ID No. 4 to 23, and then clone it into a cloning vector containing a kanamycin resistance gene, and then transfect it into an Escherichia coli strain. The Escherichia coli strain used in this embodiment is DH5α, and other suitable Escherichia coli strains can also be used in other embodiments. The transfected Escherichia coli is cultured on LB solid medium containing kanamycin (concentration of 50ug / mL), and the positive clones are selected and transferred to liquid LB medium for culture and expansion. Use a plasmid mini-extraction kit (TIANGEN: #DP103) to extract plasmid DNA, and then recombinant the target fragment containing gRNA into a plant binary expression vector containing the BASTA herbicide resistance gene (bar) and the Cas9 protein expression cassette. The schematic diagram of the gene editing vector is shown as follows Figure 1 As shown. The vector is transfected into an Agrobacterium strain. The Agrobacterium strain used in this embodiment is EHA105. In other embodiments, GV3101 or other suitable Agrobacterium strains may also be used. Then, LB medium containing rifampicin Rif and kanamycin (working concentrations are both 50ug / mL) is used to screen and culture the positive strains that have been successfully transfected and expand them for subsequent Agrobacterium transfection of explants.

[0035] Example 2 Target gene editing of corn plants

[0036] The transgenic Agrobacterium strain containing the plant binary expression vector constructed in Example 1 was co-cultured with the maize immature embryos after surface disinfection and slight friction injury under aseptic conditions on Murashige-Skoog medium. The bud tissue differentiated after co-cultivation was transferred to a new MS medium containing glyphosate (concentration of 4 mg / L) for resistance screening. The resistant positive differentiated buds obtained by screening were transferred to a root-promoting medium and transplanted to a sunlight greenhouse after confirming the root development and planting. T0 generation seeds were harvested after artificial cross-pollination with KN5585 wild-type pollen.

[0037] For the different gRNA sequences used in Example 1, the corresponding upstream and downstream amplification nucleotide primers were designed and synthesized, and the oligonucleotide amplification fragments containing the editing target site were obtained by PCR amplification. The obtained fragments were sequenced to verify whether the editing target had a frameshift mutation (i.e., a nucleotide addition or deletion that was not an integer multiple of 3 occurred). Figure 2 This is an example of the nucleotide sequence of the editing site where a frameshift mutation occurred. The gene-edited strain with a frameshift mutation at the editing target site is determined as the desired positive strain, and the homozygous gene-edited plants without T-DNA insertion events can be screened by artificial self-pollination for subsequent physiological and pathological index determination.

[0038] Example 3 Identification of antibacterial and insect-resistant small molecule metabolites in corn

[0039] Leaves were sampled after physical wounding and insect defense induction treatments, and small molecule metabolites and phytohormones were quantitatively determined.

[0040] Determination of benzoxazine substances: 150±2.5 mg of frozen ground leaf samples were taken for the extraction of benzoxazine compounds. The specific extraction method is: the leaf sample was suspended in a 2 mL centrifuge tube with MeOH / HO (50:50, v / v; 0.5% formic acid) and vortexed for 15-20 min. After the sample was centrifuged for 15 min, 400 μL of the supernatant was transferred to a glass vial and analyzed on HPLC MS / MS (LCMS-8040, Shimadzu). The analysis method can refer to the method described in the literature Qi J. et al. (2016) Oral secretions from Mythimna separata insects specifically induce defense responses in maize as revealed by high-dimensional biological data. Plant Cell & Environ. 39 (8): 1749-66. The determination results are as follows Figure 3 As shown, Figure 3The wild type in the above is the corn line that has not been successfully gene-edited, the edited line is the corn line that has been successfully gene-edited, and the overexpression line is the corn line in which the target gene is overexpressed. Figure 3 It can be seen that the corn strains after the target gene was knocked out contain higher broad-spectrum resistance metabolites than the wild-type corn strains.

[0041] Determination of plant hormones: 150-200 mg of leaf samples were fully ground in liquid nitrogen and ethyl acetate containing 20 ng D6-JA and 5 ng 13C6-JA-Ile was added. After centrifugation at room temperature for 10 min, the supernatant was removed and transferred to a new centrifuge tube. The precipitate was extracted again with 0.5 mL ethyl acetate and centrifuged. The supernatants were combined and then evaporated to dryness on a vacuum concentrator. The residue was resuspended in 0.5 mL 70% methanol and analyzed by HPLC MS / MS (LCMS-8040, Shimadzu). The analysis method can be referred to the method described in the literature Wu JQ.et al. (2007) Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata. Plant Cell 19(3): 1096-122. The measurement results are as follows. Figure 4 As shown, the edited corn lines produced significantly stronger defensive physiological responses after physical damage. This shows that knocking out or silencing the target gene makes corn crops more defensive after physical damage.

[0042] Example 4: Bioassay of corn insect resistance

[0043] The newly hatched larvae of fall armyworm were inoculated on different corn strains of about 2 weeks old, and transparent plastic boxes were placed on the plants to prevent them from escaping. After 7 days, the fall armyworm larvae were weighed to test the resistance of different corn strains to fall armyworm. Figure 5 As shown by Figure 5 It can be seen that the editing of the target gene significantly inhibited the growth of the fall armyworm larvae that fed on the leaves, making the edited corn lines show stronger resistance to pests.

[0044] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. Use of a nucleic acid molecule, a recombinant knockout vector or a transformant for specifically knocking out a target gene in improving the broad-spectrum disease and insect pest resistance of corn or cultivating a corn variety with disease and insect pest resistance, characterized in that: The nucleotide sequence of the target gene is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The sequences of the nucleic acid molecules used to specifically knock out the target gene are shown in SEQ ID No. 4-5.

3. A method for improving the broad-spectrum disease and insect pest resistance of corn, characterized by: The target gene in corn is knocked out, and the nucleotide sequence of the target gene is shown as SEQ ID No.

2.

4. The method according to claim 3, characterized in that: The target gene is knocked out using CRISPR-Cas technology.

5. A method for breeding corn with resistance to pests and diseases, characterized in that: The target gene in corn is knocked out, and the nucleotide sequence of the target gene is shown as SEQ ID No.

2.

6. The method according to claim 5, characterized in that: The target gene is knocked out using CRISPR-Cas technology.

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