Application of MYBR13 protein or related biological materials in regulation and control of insect resistance of plant seeds

Abstract

The invention discloses application of MYBR13 protein or related biological materials in regulation and control of insect resistance of plant seeds, and belongs to the technical field of biology. Experiments prove that when the expression quantity of the MYBR13 protein in the plant seeds is increased and the plant seeds are used for feeding tribolium castaneum, the growth of the tribolium castaneum is obviously inhibited; therefore, when the expression quantity of the MYBR13 protein in the plant seeds is increased, the insect resistance of the plant seeds is also correspondingly improved. The MYBR13 gene, the protein coded by the MYBR13 gene and related biological materials provided by the invention can be applied to plant insect resistance, and have great application and popularization values in the aspect of improving the biological resistance of plants such as corn.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of MYBR13 protein or related biological materials in regulating the insect resistance of plant seeds. Background Art

[0002] As one of the main pests in the growth of corn (Zea mays), the red flour beetle seriously affects the yield and quality of corn by damaging corn kernels. At present, the prevention and control of red flour beetles mainly relies on chemical pesticides. However, the long-term use of chemical pesticides will not only cause environmental pollution, but also produce pesticide-resistant insect species, reducing the control effect. Therefore, it is imperative to find a new and efficient method of insect control.

[0003] As an important regulatory factor, MYBR13 protein plays a key role in plant growth and development and resistance to adversity. Based on the important role of MYBR13 protein in plant resistance to adversity, its application in the field of corn grain insect resistance has important theoretical and practical significance. Through in-depth research on the expression and regulatory mechanism of MYBR13 protein in corn, it can provide theoretical basis and experimental foundation for the development of new insect-resistant corn varieties. Summary of the invention

[0004] The purpose of the present invention is to provide the application of MYBR13 protein or related biological materials in regulating the insect resistance of plant seeds to solve the problems existing in the above-mentioned prior art. The present invention confirms that the overexpression of MYBR13 gene significantly enhances the insect resistance of corn seeds, which can be used for plant insect resistance applications and has great application and promotion value in improving the biological resistance of plants such as corn.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of MYBR13 protein or related biological materials in regulating the insect resistance of plant seeds, thereby increasing the expression amount of the MYBR13 protein in the plant seeds, and the insect resistance of the plant seeds is also improved;

[0007] The amino acid sequence of the MYBR13 protein is as shown in SEQ ID No. 1, or has more than 80% identity with the amino acid sequence shown in SEQ ID No. 1 and has the same function;

[0008] The related biological materials include nucleic acid molecules encoding the MYBR13 protein or expression boxes containing the nucleic acid molecules, recombinant vectors, recombinant bacteria, transgenic cell lines, transgenic tissues or transgenic organs.

[0009] Furthermore, the nucleic acid molecule encoding the MYBR13 protein includes the coding gene of the MYBR13 protein and the CDS sequence of the MYBR13 protein, the nucleotide sequence of the coding gene is as shown in SEQ ID No.2, or has 90% or more identity with the nucleotide sequence shown in SEQ ID No.2; the CDS sequence is as shown in SEQ ID No.3, or has 90% or more identity with the nucleotide sequence shown in SEQ ID No.3.

[0010] Furthermore, the insect resistance includes resistance to Tribolium castaneum.

[0011] Furthermore, when the red flour beetle eats the plant seeds with increased expression level of the MYBR13 protein, its growth is inhibited.

[0012] The present invention also provides a method for improving the insect resistance of plant seeds, comprising the steps of introducing a nucleic acid molecule encoding the MYBR13 protein into the plant so that the seeds overexpress the MYBR13 protein;

[0013] The amino acid sequence of the MYBR13 protein is shown in SEQ ID No. 1;

[0014] The nucleic acid molecule encoding the MYBR13 protein includes the coding gene of the MYBR13 protein and the CDS sequence of the MYBR13 protein. The nucleotide sequence of the coding gene is shown as SEQ ID No.2, and the CDS sequence is shown as SEQ ID No.3.

[0015] Furthermore, the insect resistance includes the ability to resist Red Castanea castanea.

[0016] In the above scheme, the term "identity" refers to sequence similarity to a natural sequence. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0017] In the above biological materials, the expression cassette refers to a DNA capable of expressing a gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription, but also a terminator for terminating gene transcription.

[0018] Furthermore, the expression cassette may further include an enhancer sequence.

[0019] In the above scheme, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0020] In the above scheme, the plant is any one of the following d1) to d4):

[0021] d1) dicotyledonous plants;

[0022] d2) monocots;

[0023] d3) Gramineae;

[0024] d4) Corn.

[0025] The present invention discloses the following technical effects:

[0026] The present invention experiments prove that overexpression of MYBR13 gene significantly enhances the insect resistance of corn kernels. Therefore, the MYBR13 gene provided by the present invention, the protein encoded by it and the related biological materials can be used for plant insect resistance applications, and have great application and promotion value in improving the biological resistance of plants such as corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is a schematic diagram of the construction of the MYBR13 gene overexpression vector of Example 1;

[0029] Figure 2 Detection of MYBR13 protein content in the MYBR13 gene overexpression material of Example 1;

[0030] Figure 3 This is the insect resistance test of the MYBR13 gene overexpression material under the o2 background of Example 2, wherein A represents the growth state of the red flour beetle larvae, scale: bar=1 cm; B represents the body length of the red flour beetle larvae. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The experimental methods in the examples of the present invention are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0037] Unless otherwise specified, GraphPad Prism 8 statistical software was used to process the data, and the experimental results were expressed as mean values ​​and analyzed using Student's t-test.

[0038] The overexpression vector pHB is described in the following literature: Yao, DS, et al. (2016). Maize opaque10encodes acereal-specific protein that is essential for the proper distribution of zeins in endosperm protein bodies. PLoS Genetics, 12: e1006270.

[0039] The W22 maize inbred line is described in the following literature: Springer, NM, et al. (2018). The maizeW22 genome provides a foundation for functional genomics and transposonbiology. Nature Genetics, 50: 1282-1288.

[0040] The o2 maize mutant is described in the following literature: Li, CB, et al. (2015). Genome-wide characterization of cis-acting DNA targets reveals the transcriptionalregulatory framework of Opaque2 in maize. The Plant Cell, 27: 532-545.

[0041] The hybrid line Hi II is described in the following document: Frame, BR, et al. (2002). Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiology, 129: 13-22.

[0042] The above experimental materials were provided by Professor Song Rentao's research group at China Agricultural University.

[0043] The amino acid sequence of the MYBR13 protein described in the following examples is shown in SEQ ID No. 1, and the CDS sequence of the protein is shown in SEQ ID No. 3. The nucleotide sequence of the gene encoding the protein (MYBR13 gene) is shown in SEQ ID No. 2.

[0044] Protein detection method: Immature corn kernels were collected and their endosperms were used for total protein extraction. The content of MYBR13 protein was detected by immunoblotting, and ACTIN antibody (purchased from Thermo Fisher) was used as an internal reference.

[0045] Feeding method of red flour beetle: collect mature corn kernels, grind their endosperm into powder, dry and set aside. Feed red flour beetle adults with wheat flour, and set the temperature of the incubator to 30℃ and humidity to 75%. After 2-3 days of incubation, use an 80-mesh sieve to screen the red flour beetle eggs, add the powder of the corresponding kernels, and transfer to the incubator for further incubation.

[0046] Example 1 Overexpression of MYBR13 gene driven by 16kD γ-zein gene promoter

[0047] The promoter of the endosperm-specific gene 16kDγ-zein was used to drive overexpression of the MYBR13 gene (p16kD::MYBR13).

[0048] 1. Construction of p16kD::MYBR13 overexpression vector

[0049] (1) EcoRI and HindIII were used to excise the double 35S promoter of the overexpression vector pHB;

[0050] (2) amplifying a fragment of about 2 kb upstream of the ATG of the 16 kD γ-zein gene (SEQ ID No. 4) as its promoter;

[0051] (3) amplifying the coding sequence of the MYBR13 gene, as shown in SEQ ID No. 3;

[0052] (4) The promoter sequence of the 16kD γ-zein gene and the coding sequence of the MYBR13 gene were sequentially connected to the pHB vector of the Wushuang 35S promoter to generate the p16kD::MYBR13 overexpression vector ( Figure 1 ). The promoter sequence of the 16kDγ-zein gene is the sequence shown in SEQ ID No. 4. The p16kD::MYBR13 overexpression vector is a vector obtained by replacing the promoter sequence of the 16kDγ-zein gene and the coding region sequence of the MYBR13 gene with a small fragment between the EcoRI and HindIII restriction sites in the pHB vector of the Wushuang 35S promoter, while keeping the other nucleotides of the pHB vector of the Wushuang 35S promoter unchanged.

[0053] 2. Transformation of maize embryos with p16kD::MYBR13 overexpression vector

[0054] The overexpression vector was transformed into Agrobacterium EHA105, and then the vector was transformed into the immature embryos of maize Hi-II hybrid using the Agrobacterium-mediated method to obtain transgenic T0 generation plants.

[0055] The genomic DNA of the leaves of the T0 generation plants at the seedling stage was extracted and PCR amplified and identified using Bar-F / R primers. The primer sequences are as follows:

[0056] Bar-F: GAAGTCCAGCTGCCAGAAAC (SEQ ID No. 5); Bar-R: GCACCATCGTCAA CCACTAC (SEQ ID No. 6).

[0057] The reaction system and reaction procedure for PCR amplification are shown in Table 1-2.

[0058] Table 1 PCR reaction system

[0059] Components volume 2×TaqPCRMix 10μL 10 μM Bar-F 0.5μL 10 μM Bar-R 0.5μL gDNA 1μL <![CDATA[ddH 2 The]]> Up to 20 μL

[0060] Table 2 PCR reaction program

[0061]

[0062] The T0 generation plants that can amplify the 440 bp fragment are the plants that have successfully been loaded with the p16kD::MYBR13 overexpression vector and are named T0 generation positive plants.

[0063] 3. Detection of MYBR13 protein content in p16kD::MYBR13 plants

[0064] The T0 positive plants were crossed with the W22 inbred line, and the grains were taken from the separated ears produced. The wild-type WT grains without transgenic elements were used as the control group (WT), and the grains containing transgenic elements were used as the experimental group with p16kD::MYBR13 overexpression (p16kD::MYBR13) by using Bar-F / R primers for PCR amplification and identification. The total protein of the two groups of grains was extracted, and the content of MYBR13 protein was detected.

[0065] At the protein level, the accumulation of MYBR13 protein in p16kD::MYBR13 was significantly higher than that in WT ( Figure 2 ). The above results indicate that the promoter of 16kDγ-zein gene can successfully drive the overexpression of MYBR13 gene.

[0066] Example 2. Insect resistance test of MYBR13 gene overexpression under o2 background

[0067] The manifestations of plant insect resistance mainly include non-selectivity, antibiotics, tolerance and compensation. Among them, antibiotics refer to the ability of plants to produce certain chemical substances, which have a direct killing effect on pests and can inhibit the growth or development of pests. Therefore, the present invention analyzes its insect resistance effect by studying the antibiotics of MYBR13 gene overexpression on red flour beetle.

[0068] The p16kD::MYBR13 transgenic material was planted in the field and hybridized with o2 to obtain MYBR13 overexpressing material in the o2 background. The mature grain powder was used to feed the larvae of Tribolium castaneum, and the body length was measured. The larvae of Tribolium castaneum fed with o2 were used as the control group. The results of the observation of the growth status of the larvae of Tribolium castaneum showed that compared with the control group, the larvae of Tribolium castaneum fed with p16kD::MYBR13 were smaller in size ( Figure 3 A). The results of body length measurement showed that the body length of Tribolium castaneum fed with p16kD::MYBR13 was significantly reduced, indicating that its growth was inhibited ( Figure 3 B). This indicates that overexpression of the MYBR13 gene has a certain resistance to Tribolium castaneum.

[0069] SEQ ID No.1:

[0070] MAFYYGVGGQSSAWAAPGGSRPWTKAEDKVFEGALVMFPEHLPNRWALVASRLYDRTPQEAWDHYQALVTDVDLIERGMVEAPDSWDDDAAAAGGGRSGRGRGGGSGSGEERRRGVPWTEDEHRLFLEGLEKYGRGDWRNISRWSVKTRTPTQVASHAQKYFIRQASAASRGDSKRKSIHDITTP.

[0071] SEQ ID No.2:

[0072]

[0073] SEQ ID No.3:

[0074] ATGGCCTTCTACTACGGCGTGGGCGGACAGTCGTCGGCGTGGGCGGCGCCGGGCGGCAGCAGGCCATGGACCAAGGCGGAGGACAAGGTGTTCGAGGGCGCGCTGGTGATGTTCCCGGAGCACCTGCCGAACCGGTGGGCGCTGGTGGCGTCCCGGCTGTACGACCGCACGCCGCAGGAGGCCTGGGACCACTACCAGGCGCTGGTGACCGACGTCGACCTCATCGAGCGCGGCATGGTGGAGGCCCCGGACTCCTGGGACGACGACGCGGCTGCCGCCGGAGGGGGACGCTCGGGCCGCGGCCGCGGGGGCGGCAGCGGCAGCGGAGAGGAGCGCCGCCGCGGGGTGCCGTGGACCGAGGACGAGCACAGGCTGTTTCTGGAGGGGCTTGAGAAGTACGGGCGCGGCGACTGGCGGAACATCTCGCGCTGGTCGGTGAAGACGCGGACGCCCACGCAGGTGGCCAGCCACGCGCAGAAGTACTTCATCCGCCAGGCCAGCGCCGCCAGCCGCGGCGACAGCAAGCGCAAGAGCATCCACGACATCACCACCCCGTGA。

[0075] SEQ ID No.4:

[0076]

[0077] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of MYBR13 protein or related biological materials in regulating insect resistance of plant seeds, characterized in that: The expression level of the MYBR13 protein in the plant seeds is increased, and the insect resistance of the plant seeds is also improved; The amino acid sequence of the MYBR13 protein is shown in SEQ ID No. 1; The related biological materials include nucleic acid molecules encoding the MYBR13 protein or expression boxes containing the nucleic acid molecules, recombinant vectors, recombinant bacteria, transgenic cell lines, transgenic tissues or transgenic organs.

2. The use according to claim 1, characterized in that: The nucleic acid molecule encoding the MYBR13 protein includes the coding gene of the MYBR13 protein and the CDS sequence of the MYBR13 protein. The nucleotide sequence of the coding gene is shown in SEQ ID No.2, and the CDS sequence is shown in SEQ ID No.

3.

3. The use according to claim 1, characterized in that: The plants include corn.

4. The use according to claim 1, characterized in that: The insect resistance includes resistance to Tribolium castaneum.

5. The use according to claim 4, characterized in that: The red flour beetle eats the plant seeds with increased expression of the MYBR13 protein, and its growth is inhibited.

6. A method for improving the insect resistance of plant seeds, characterized in that: The method comprises the steps of introducing a nucleic acid molecule encoding the MYBR13 protein into the plant so that the plant's grains overexpress the MYBR13 protein; The amino acid sequence of the MYBR13 protein is shown in SEQ ID No. 1; The nucleic acid molecule encoding the MYBR13 protein includes the coding gene of the MYBR13 protein and the CDS sequence of the MYBR13 protein. The nucleotide sequence of the coding gene is shown in SEQ ID No.2, and the CDS sequence is shown in SEQ ID No.

3.

7. The method according to claim 6, characterized in that The plants include corn.

8. The method according to claim 6, characterized in that The insect resistance includes the ability to resist Red Castanea castanea.

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