Use of mybr13 protein or related biological material in regulating insect resistance of plant seeds

Abstract

The application discloses application of MYBR13 protein or related biological materials in regulation of plant grain insect resistance, and belongs to the technical field of biotechnology. The application proves that the expression amount of the MYBR13 protein in the plant grain is increased, and the growth of red flour beetles is significantly inhibited when the red flour beetles are fed with the plant grain; and it is indicated that the insect resistance of the plant grain is also increased when the expression amount of the MYBR13 protein in the plant grain is increased. The MYBR13 gene, the protein coded by the MYBR13 gene and the related biological materials provided by the application can be applied to plant insect resistance, and have great application and popularization value in increasing biological resistance of plants such as corn.

Description

TECHNICAL FIELD

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

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

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

[0004] The purpose of the present application is to provide the application of MYBR13 protein or related biological materials in regulating the insect resistance of plant seeds, in order to solve the problems existing in the prior art. The present application proves that overexpression of MYBR13 gene can significantly enhance the insect resistance of corn seeds, and can be used for plant insect resistance application, and has great application and popularization value in improving the biological resistance of plants such as corn.

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides the application of MYBR13 protein or related biological materials in regulating the insect resistance of plant seeds, improving the expression amount of MYBR13 protein in the plant seeds, and improving the insect resistance of the plant seeds;

[0007] The amino acid sequence of the MYBR13 protein is 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 cassettes, recombinant vectors, recombinant bacteria, transgenic cell lines, transgenic tissues or transgenic organs containing the nucleic acid molecules.

[0009] Further, the nucleic acid molecule encoding the MYBR13 protein comprises a coding gene of the MYBR13 protein and a CDS sequence of the MYBR13 protein, wherein the nucleotide sequence of the coding gene is shown as SEQ ID No. 2, or has 90% or above identity with the nucleotide sequence shown as SEQ ID No. 2; and the CDS sequence is shown as SEQ ID No. 3, or has 90% or above identity with the nucleotide sequence shown as SEQ ID No. 3.

[0010] Further, the insect resistance comprises the resistance to Tribolium castaneum.

[0011] Further, the plant seed with increased expression of the MYBR13 protein consumed by the Tribolium castaneum has its growth inhibited.

[0012] The present application also provides a method for improving the insect resistance of plant seeds, comprising the step 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 as SEQ ID No. 1.

[0014] The nucleic acid molecule encoding the MYBR13 protein comprises a coding gene of the MYBR13 protein and a CDS sequence of the MYBR13 protein, wherein 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] Further, the insect resistance comprises the resistance to Tribolium castaneum.

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

[0017] In the above biological material, the expression cassette refers to the DNA capable of expressing genes in host cells, which can include not only the promoter for initiating gene transcription, but also the terminator for terminating gene transcription.

[0018] Further, the expression cassette can also comprise an enhancer sequence.

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

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

[0021] d1) a dicotyledonous plant;

[0022] d2) a monocotyledonous plant;

[0023] d3) a grass plant;

[0024] d4) corn.

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

[0026] The present application proves that overexpression of the MYBR13 gene significantly enhances the insect resistance of corn kernels. Therefore, the MYBR13 gene, the protein encoded thereby and the related biological materials provided by the present application 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 DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0028] Figure 1 A schematic diagram for construction of the MYBR13 gene overexpression vector of Example 1;

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

[0030] Figure 3 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, and the scale bar is 1 cm; B represents the body length of the red flour beetle larvae. DETAILED DESCRIPTION

[0031] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0032] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, amounts, and the like, every intermediate value of that range is also specifically included. For example, a range from 1 to 50 includes every intermediate value such as 1, 4, 10, 14, 19, 25, 30, 35, 40, 45, and 50, etc. Likewise, a range from 6-9 includes every intermediate value such as 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, and 6.9. The same applies to every other similar

[0033] Unless defined otherwise, 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 application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0034] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0035] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0036] The experimental methods in the embodiments of the present application are all conventional methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0037] The data were processed using GraphPad Prism 8 statistical software, unless otherwise specified, and the experimental results were expressed as the mean value, which was tested by Student's t-test.

[0038] The overexpression vector pHB is described in Yao, D. S., et al. (2016). Maize opaque 10 encodes a cereal-specific protein that is essential for the proper distribution of zeins in endosperm protein bodies. PLoS Genetics, 12: e1006270.

[0039] W22 maize inbred line is described in Springer, N. M., et al. (2018). The maize W22 genome provides a foundation for functional genomics and transposon biology. Nature Genetics, 50: 1282-1288.

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

[0041] Maize Hi-II hybrid line is described in Frame, B. R., 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 the research group of Professor Song Rentao, China Agricultural University.

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

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

[0045] Red flour beetle feeding method: mature corn kernels are collected, and the endosperm is taken and ground into powder, which is dried for use. Wheat flour is used to feed red flour beetle adults, and the incubator is set to a temperature of 30°C and a humidity of 75%. After 2-3 days of culture, the red flour beetle eggs are screened using an 80-mesh sieve, and the corresponding kernel powder is added and transferred to the incubator for continued culture.

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

[0047] The overexpression of the MYBR13 gene is driven by the promoter of the endosperm-specific expressed gene 16kD γ-zein (p16kD::MYBR13).

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

[0049] (1) The double 35S promoter of the overexpression vector pHB is excised using EcoRI and HindIII;

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

[0051] (3) The coding sequence of the MYBR13 gene is amplified, 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 are sequentially connected to the pHB vector without the double 35S promoter, generating the p16kD::MYBR13 overexpression vector Figure 1 ). The promoter sequence of the 16kD γ-zein gene is shown in SEQ ID No. 4. The p16kD::MYBR13 overexpression vector is a vector obtained by replacing the small fragment between the EcoRI and HindIII enzyme cutting sites in the pHB vector without the double 35S promoter with the promoter sequence of the 16kD γ-zein gene and the coding region sequence of the MYBR13 gene, and keeping the other nucleotides of the pHB vector without the double 35S promoter unchanged.

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

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

[0055] Genomic DNA of the seedling leaves of the T0 generation plants above was extracted, and PCR amplification and identification were performed using Bar-F / R primers. The primer sequences above are as follows:

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

[0057] The reaction system and the reaction procedure of PCR amplification are shown in Tables 1-2.

[0058] Table 1 PCR reaction system

[0059] Component Volume 2 x Taq PCR Mix 10 μL 10 μM Bar-F 0.5 μL 10 μM Bar-R 0.5 μL gDNA 1 μL ddH2O up to 20 μL

[0060] Table 2 PCR reaction procedure

[0061]

[0062] The T0 generation plants that can amplify a 440 bp fragment are plants in which the p16kD::MYBR13 overexpression vector is successfully introduced, and are named as T0 generation positive plants.

[0063] 3. Content detection of MYBR13 protein in p16kD::MYBR13 plants

[0064] The T0 generation positive plants above are crossed with a W22 self-crossing line, and kernels are taken from the separated ears generated therefrom. By using Bar-F / R primers for PCR amplification and identification, wild type WT kernels without transgenic elements are used as a control group (WT), and kernels containing transgenic elements are used as an experimental group of p16kD::MYBR13 overexpression (p16kD::MYBR13). Total proteins of the kernels in the two groups are extracted, and the content of MYBR13 protein is detected.

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

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

[0067] The expression of plant insect resistance mainly includes non-selectivity, anti-natalism, damage tolerance and compensation. Among them, anti-natalism refers to that plants can produce certain chemical substances, which have a direct killing effect on pests and can inhibit the growth or development of pests. Therefore, the insect resistance effect of the present application is analyzed by studying the anti-natalism of Tribolium castaneum of MYBR13 gene overexpression.

[0068] The p16kD::MYBR13 transgenic material was planted in the field and crossed with o2 to obtain MYBR13 overexpression material in the o2 background. The red flour beetle larvae were fed with mature grain powder, and the body length was measured. The o2 fed red flour beetle larvae were used as the control group. The observation results of the growth state of the red flour beetle larvae showed that, compared with the control group, the volume of the p16kD::MYBR13 fed red flour beetle larvae was smaller Figure 3 A) of FIG. 6. Through the body length measurement, it was found that the body length of the p16kD::MYBR13 fed red flour beetle was significantly reduced, indicating that its growth was inhibited Figure 3 B) of FIG. 6. It is shown that MYBR13 gene overexpression produces certain resistance to the red flour beetle.

[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 above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art based on the above-described embodiments should fall within the scope of the present application defined by the claims.

Claims

1. The application of MYBR13 protein or related biological materials in regulating insect resistance in plant seeds, characterized in that, Increasing the expression level of the MYBR13 protein in the plant seeds also enhances the insect resistance of the plant seeds. The amino acid sequence of the MYBR13 protein is shown in SEQ ID No. 1; The relevant biological materials include nucleic acid molecules encoding the MYBR13 protein or expression cassettes containing the nucleic acid molecules, recombinant vectors, recombinant bacteria, transgenic cell lines, transgenic tissues, or transgenic organs; The insect resistance mentioned refers to resistance to the red flour beetle; The plant in question is corn.

2. The application according to claim 1, characterized in that, The nucleic acid molecule encoding the MYBR13 protein includes the CDS sequence of the MYBR13 protein, as shown in SEQ ID No.

3.

3. The application according to claim 1, characterized in that, The growth of the red flour beetle is inhibited when it consumes plant seeds with increased expression of the MYBR13 protein.

4. A method for improving the insect resistance of plant seeds, characterized in that, The method includes the step of introducing a nucleic acid molecule encoding the MYBR13 protein into the plant, causing its seeds to 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 CDS sequence of the MYBR13 protein, as shown in SEQ ID No. 3; The plant in question is corn; The insect resistance mentioned refers to the resistance to the red flour beetle.

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