CN118086242A - Rice methyltransferase SET36 and application of coding gene thereof in disease-resistant breeding - Google Patents
Rice methyltransferase SET36 and application of coding gene thereof in disease-resistant breeding Download PDFInfo
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Abstract
The invention discloses rice methyltransferase SET36 and application of a coding gene thereof in disease-resistant breeding, and belongs to the technical field of biology. Experiments prove that the SET36 gene has the function of improving the disease resistance of rice. The SET36 gene was knocked out in the ZH11 background, and the knocked-out plant SET36-KO was found to exhibit disease resistance higher than that of the control, compared to the wild-type ZH 11. The SET36 gene can be used as a target gene for molecular breeding for improving the disease resistance of rice or other plants, and lays a theoretical foundation for research on the disease resistance of plants.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to rice methyltransferase SET36 and application of a coding gene thereof in disease-resistant breeding.
Background
Fungal diseases such as rice blast cause large-scale yield reduction of crops, so that cultivation of disease-resistant crops is one of main targets of the planting industry. Several genes related to plant disease resistance have been reported, including effector genes and regulatory genes, which are derived from crops such as rice, wheat, corn, soybean, etc., and model plants such as Arabidopsis. Some of the genes are used as target genes for crop disease resistance genetic engineering, and disease resistance rice, wheat, corn, soybean and the like are successfully cultivated. However, due to the difference of genetic background, application of some genes is hindered, and thus new related genes are still needed to meet the breeding requirements.
The rice is one of the most important grain crops, improves the disease resistance of the grain crops, and has important theoretical and practical significance.
The SET (Su (var), enhancer ofzeste (E (z)), and Trithiorax domain gene family is a generic term for a group of proteins containing a conserved SET domain, involved in protein methylation, affecting chromosomal structure, and regulating gene expression, and plays an important role in plant development. Therefore, the research on SET has focused on the plant development in which SET participates, and no report on the application of SET36 gene to plant disease resistance is available.
Disclosure of Invention
The invention aims to provide application of rice methyltransferase SET36 and a coding gene thereof in disease-resistant breeding so as to solve the problems in the prior art. SET36 can negatively regulate rice disease resistance, and can improve rice disease resistance by knocking out SET36 in rice.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides the use of a SET36 protein in any one of the following:
(1) Application in improving disease resistance of plants;
(2) Application in preparing products for improving plant disease resistance;
(3) Application in cultivating high disease resistance plant variety;
(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;
the amino acid sequence of the SET36 protein is shown as SEQ ID No.2 or a protein sequence containing the amino acid sequence shown as SEQ ID No. 2.
The invention also provides application of the coding gene of the SET36 protein in any one of the following:
(1) Application in improving disease resistance of plants;
(2) Application in preparing products for improving plant disease resistance;
(3) Application in cultivating high disease resistance plant variety;
(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;
The amino acid sequence of the SET36 protein is shown as SEQ ID No.2 or a protein sequence containing the amino acid sequence shown as SEQ ID No. 2; the nucleotide sequence of the coding gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
The invention also provides the application of the recombinant vector containing the coding gene of the SET36 protein in any one of the following:
(1) Application in improving disease resistance of plants;
(2) Application in preparing products for improving plant disease resistance;
(3) Application in cultivating high disease resistance plant variety;
(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;
The amino acid sequence of the SET36 protein is shown as SEQ ID No.2 or a protein sequence containing the amino acid sequence shown as SEQ ID No. 2; the nucleotide sequence of the coding gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
The invention also provides the use of a host bacterium comprising a recombinant vector in any one of the following:
(1) Application in improving disease resistance of plants;
(2) Application in preparing products for improving plant disease resistance;
(3) Application in cultivating high disease resistance plant variety;
(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;
The recombinant vector is an expression vector integrating the SET36 gene, and the nucleotide sequence of the SET36 gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
Further, disease resistance of the plant is improved by knocking out the SET36 gene in the plant.
Further, the plant comprises rice.
The invention also provides a method for improving the disease resistance of plants, which improves the disease resistance of the plants by knocking out the SET36 gene in the plants;
the nucleotide sequence of the SET36 gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
Further, the plant comprises rice.
The invention also provides a method for cultivating the transgenic plant with high disease resistance, which is to knock out the SET36 gene in the plant, reduce the expression quantity of the SET36 gene and obtain the transgenic plant with high disease resistance; the nucleotide sequence of the SET36 gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
Further, the plant comprises rice.
The invention discloses the following technical effects:
experiments prove that the SET36 gene has the function of improving the disease resistance of rice. The SET36 gene was knocked out in the ZH11 background, and the knocked-out plant SET36-KO was found to exhibit disease resistance higher than that of the control, compared to the wild-type ZH 11. The SET36 negative regulation of plant disease resistance is proved, and the SET36 protein and the coding gene thereof have important significance for cultivating disease-resistant rice varieties.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of the protein domain of SET36 and a knockout Target (Target);
FIG. 2 is a schematic diagram of a SET36 knockout carrier structure;
FIG. 3 shows the results of the stabbing of wild-type ZH11 (WT) and knockout lines SET36-KO1 and SET36-KO2, with a scale of 1cm and a significant difference (P < 0.05) indicated by the asterisks above the bar graph.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
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 invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 belongs. Although only 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 invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Flower 11 (o.sativa l. Spp. Japonica, var zhonghua, AA genome, ZH 11) of rice varieties is a subspecies, described in the following documents: ni Yuchong, a new variety of rice flower cultivation, namely Zhonghua No. 11, agricultural science and technology communication, 1989,07,35; is provided by the professor Chen Xuewei of the institute of genetic and developmental biology of the national academy of sciences.
SET36 knockout plants were completed by primary biotechnology company.
Plant binary expression vector pTCRISPR, provided by the professor Tang Yongyan, national emphasis laboratory at Sichuan university of agriculture.
Total RNA extraction kit: TRIzol available from Invitrogen, U.S. under the designation 15596026.
Reverse transcription kit: HISCRIPT III RT SuperMix for qPCR (+ GDNA WIPER) from Northenpran (Vanzyme) China under the designation R323-01.
Homologous recombination kit: clonExpress IIOne Step Cloning Kit from Northenan (Vanzyme) China, cat# C112-01.
Example 1 Rice protein SET36 and its cloning
Screening a protein SET36 in rice, and designing a primer according to a rice Nip genome reference gene sequence:
SET36F：ATGGCTGCTGCCATCCACCA；
SET36R：CTAGGATTCCCAGAAGATAA。
The primer is used for amplification by taking ZH11 total cDNA as a template to obtain 1488bp DNA bands. It was confirmed to be a SET36 gene belonging to the protein family after sequencing. Alignment of the LOC_Os09g24530 (SET 36) sequence in the rice genome reference sequence and the cloned sequence obtained from the ZH11 total cDNA amplification showed that the cloned sequence obtained in ZH11 was identical to the LOC_Os09g24530 (SET 36) sequence. The amino acid sequence of SET36 protein is shown in SEQ ID No.2, and the structural analysis is shown in FIG. 1. The CDS sequence of the coding chain of the gene SET36 encoding the SET36 protein is shown in SEQ ID No. 1.
SEQ ID No.1：
ATGGCTGCTGCCATCCACCACCACCACCTCCTCCCTCCTCGCCTCCTCTCCGTCCACCCGCAGCCTCCCCGCCTCCGCCTCCGGCGCCCGCTCCCCCGCCGCGCGGCCGCGTCCGGCGCAGCGGCCGGGACGTCGTCGTCGACCGCGGCCGCGCCGCCGCCCACGGACGCCGCGCTGCAGGAGTTCAGGCGCTGGGTGTCGTCCCACGGCGCGGACGCGGGGGCCGGCGCGGCGGCGCCCGCCGCCGTCCCCGAGGGTGGCCTCGGCCTCGTCGCGGCGCGGGACCTGCCCCGCGGGGAGGTGCTCGCCGAGGTGCCCAAGAAGCTCTGGCTGGACGCCGACGCCGTCGCGGCCTCCGACCTCGGCGGCGCCGTCGGCCGCGGGGGGCTCAGGCCCTGGGTCGCCGTCGCGCTGCTTCTCCTCCGCGAGGCCGCCCGCGGCGCCGGCTCGCCGTGGGCGCCCTACCTCGCCATCCTCCCGCGCCAGACCGACTCCACCATCTTCTGGTCAGAAGAAGAGCTCTTGGAGATACAAGGAACACAGTTACTGAGCACAACAATGGGTGTGAAGGAGTATGTGCAGAGTGAATTTGAGAGTGTTGAAGCTGAGATCATAAGCGAGAACAGGGAGCTCTTTCCTGGTACTGTAACATTCAATGATTTCCTATGGGCATTCGGCATACTCAGATCACGGGTGTTTGCGGAGCTCCGTGGAGATAAGCTTGCTCTCATACCATTTGCTGATCTTGTAAATCACAGCGATGATATAACCTCAAAAGAGTCCAGTTGGGAGATCAAAGGAAAGGGTCTTTTTGGTAGGGATGTTGTGTTTTCTTTGCGAACACCGGTGAATGTTAAATCTGGAGAACAGATATATATTCAGTATGATTTGGACAAGAGCAACGCAGAATTAGCGCTTGATTATGGTTTCACCGAATCAAATTCATCAAGGGATGCATATACTCTGACCTTGGAGATATCTGAATCTGATCCATTTTATGATGACAAGCTTGACATTGCAGAGCTAAATGGGATGGGGGAGACTGCATACTTTGATATTGTCCTTGGTGAATCTCTTCCTCCTCAAATGCTACCTTACCTGCGATTACTCTGCCTTGGGGGAACAGATGCATTTCTCTTGGAAGCACTCTTCAGAAATGCTGTTTGGGGCCACCTTGAACTGCCAGTGAGTCAAGATAATGAAGAAGCGATATGTCAGGTCATCCGAAATGCCTGCAAATCTGCCCTTGGTGCTTACCACACTACCATAGAAGAGGACGAAGAACTGTTGGGAAGTGAAAATCTTCAGCCAAGGCTTCAAATTGCCGTTGAAGTCAGGGCCGGTGAGAAGAAAGTGCTACAGCAGATTGATGACATTTTCAAGCAGAGGGAGGAGGAACTGGATGGCCTAGAGTACTACCAAGAACGGAGACTCAAGGATATTGGTTTAGTTGGCGACAATGGTGAAATTATCTTCTGGGAATCCTAG.
SEQ ID No.2：
MAAAIHHHHLLPPRLLSVHPQPPRLRLRRPLPRRAAASGAAAGTSSSTAAAPPPTDAALQEFRRWVSSHGADAGAGAAAPAAVPEGGLGLVAARDLPRGEVLAEVPKKLWLDADAVAASDLGGAVGRGGLRPWVAVALLLLREAARGAGSPWAPYLAILPRQTDSTIFWSEEELLEIQGTQLLSTTMGVKEYVQSEFESVEAEIISENRELFPGTVTFNDFLWAFGILRSRVFAELRGDKLALIPFADLVNHSDDITSKESSWEIKGKGLFGRDVVFSLRTPVNVKSGEQIYIQYDLDKSNAELALDYGFTESNSSRDAYTLTLEISESDPFYDDKLDIAELNGMGETAYFDIVLGESLPPQMLPYLRLLCLGGTDAFLLEALFRNAVWGHLELPVSQDNEEAICQVIRNACKSALGAYHTTIEEDEELLGSENLQPRLQIAVEVRAGEKKVLQQIDDIFKQREEELDGLEYYQERRLKDIGLVGDNGEIIFWES.
EXAMPLE 2 construction of SET36 plant knockout vector
(1) Construction of SET36 knockout vector pTCRISPR-sgRNA SET36
Taking a genome of rice Nip as a template, and designing a knockout target sequence as follows: 5'-AATGATTTCCTATGGGCATT-3'.
And the following primers were synthesized:
F：5’-TGTGAATGATTTCCTATGGGCATTG-3’；
R：5’-AAAACAATGCCCATAGGAAATCATT-3’。
After denaturation at 95℃a double strand is formed by annealing. And (3) carrying out BsaI digestion on pTCRISPR vectors, and recovering to obtain linearization vectors. The above DNA double strand and linearized vector were mixed in the system shown in Table 1 below, and T4 DNALIGASE ligation was performed and incubated at room temperature for 30 minutes.
TABLE 1
The pTCRISPR-sgRNA SET36 knockout vector was obtained after ligation (see FIG. 2).
(2) Transformation
The knock-out vector constructed above is transformed into DH5 alpha competence, after kanamycin screening, monoclonal is selected for colony PCR identification, positive colony extracts plasmid and confirms pTCRISPR-sgRNA SET36 without error after sequencing correctly, and genetic transformation can be carried out.
(3) Construction of knockout transgenic Rice
The SET36 knockout of transgenic rice was accomplished by Boer Biotechnology.
Example 3 detection of the involvement of SET36 in the modulation of plant disease resistance
And (3) stabbing and inoculation treatment in a seedling stage: the test materials were Zhonghua (ZH 11), the ZH11 knockout lines SET36-KO1 and SET36-KO2, which are genetically stable against ZH11 as background. Selecting full seeds, placing the seeds into a conical flask filled with tap water, placing the conical flask into a dark incubator at 37 ℃ for germination, and changing water every day. After 2 days, the white seeds are selected and placed in a 96-hole seedling raising plate, and the 96 Kong Yomiao plate is placed on a buoy to grow in Hoagland nutrient solution. After 21 days, selecting the penultimate rice leaf with consistent growth vigor and size, inoculating 5 mu L of Zhong10-8-14 rice blast spores with the concentration of 5X 10 5 pieces per ml -1 after stabbing, and observing and counting the lesion length after 5 days.
Statistics show that after stab inoculation, the lesion length of SET36-KO1 and SET36-KO2 plants in the ZH11 background was significantly reduced and disease resistance of the plants was significantly increased compared to ZH11 (see fig. 3). The SET36 negative regulation of plant disease resistance is illustrated, and the coding gene SET36 can be used as a target gene for molecular breeding for improving plant disease resistance.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
- Use of a set36 protein in any one of the following:(1) Application in improving disease resistance of plants;(2) Application in preparing products for improving plant disease resistance;(3) Application in cultivating high disease resistance plant variety;(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;the amino acid sequence of the SET36 protein is shown as SEQ ID No.2 or a protein sequence containing the amino acid sequence shown as SEQ ID No. 2.
- Use of a gene encoding a set36 protein in any one of:(1) Application in improving disease resistance of plants;(2) Application in preparing products for improving plant disease resistance;(3) Application in cultivating high disease resistance plant variety;(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;The amino acid sequence of the SET36 protein is shown as SEQ ID No.2 or a protein sequence containing the amino acid sequence shown as SEQ ID No. 2; the nucleotide sequence of the coding gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
- 3. Use of a recombinant vector comprising a gene encoding a SET36 protein in any one of:(1) Application in improving disease resistance of plants;(2) Application in preparing products for improving plant disease resistance;(3) Application in cultivating high disease resistance plant variety;(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;The amino acid sequence of the SET36 protein is shown as SEQ ID No.2 or a protein sequence containing the amino acid sequence shown as SEQ ID No. 2; the nucleotide sequence of the coding gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
- 4. Use of a host bacterium comprising a recombinant vector in any one of the following:(1) Application in improving disease resistance of plants;(2) Application in preparing products for improving plant disease resistance;(3) Application in cultivating high disease resistance plant variety;(4) The application of the plant variety with high disease resistance in preparing the product for cultivating the plant variety with high disease resistance;The recombinant vector is an expression vector integrating the SET36 gene, and the nucleotide sequence of the SET36 gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
- 5. The use of any one of claims 1-4, wherein disease resistance of the plant is increased by knocking out the SET36 gene in the plant.
- 6. The use according to any one of claims 1 to 4, wherein the plant comprises rice.
- 7. A method for improving disease resistance of a plant, characterized in that disease resistance of the plant is improved by knocking out the SET36 gene in the plant;the nucleotide sequence of the SET36 gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
- 8. The method of claim 7, wherein the plant comprises rice.
- 9. A method for cultivating a transgenic plant with high disease resistance, which is characterized in that the expression level of SET36 gene is reduced by knocking out the SET36 gene in the plant, so as to obtain the transgenic plant with high disease resistance;The nucleotide sequence of the SET36 gene is shown as SEQ ID No.1 or comprises the nucleotide sequence shown as SEQ ID No. 1.
- 10. The method of claim 9, wherein the plant comprises rice.
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