Application of rice protein kinase OsTUPK and coding gene thereof in disease-resistant breeding

Abstract

The invention discloses application of rice protein kinase OsTUPK and a coding gene thereof in disease-resistant breeding, and belongs to the technical field of biology. The amino acid sequence of the rice protein kinase OsTUPK is as shown in SEQ ID NO. 2, and the nucleotide sequence of the gene for coding the OsTUPK protein is as shown in SEQ ID NO. 1. Experiments prove that the OsTUPK protein has the function of improving the disease resistance of rice. The Zhonghua 11 (ZH11) gene OsTUPK is over-expressed in ZH11, and the disease resistance of a transgenic plant is higher than that of a receptor; the OsTUPK in the rice is knocked out, the disease resistance of a transgenic plant is lower than that of a receptor, it is indicated that the OsTUPK positively regulates the disease resistance of the plant, and the OsTUPK protein and the coding gene thereof have important significance for breeding disease-resistant rice varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of rice protein kinase OsTUPK and its coding gene in disease resistance breeding. BACKGROUND

[0002] Fungal diseases such as rice blast cause large-scale yield reduction of crops, so breeding disease-resistant crops is one of the main goals of planting. Many genes related to plant disease resistance have been reported, including effector molecule genes and regulatory genes, which are derived from crops such as rice, wheat, corn, soybean, and model plants such as Arabidopsis. Some of them have been used as target genes for crop disease resistance genetic engineering, and disease-resistant rice, wheat, corn, soybean, etc. have been successfully bred. However, due to differences in genetic background, the application of some genes is hindered, so new related genes are still needed to meet the needs of breeding.

[0003] Rice is one of the most important food crops, and improving its disease resistance has important theoretical and practical significance.

[0004] In the study of rice immune response, the inventors identified a protein kinase, which was named OsTUPK, which can be used as a target gene for molecular breeding to improve plant disease resistance. SUMMARY

[0005] The purpose of the present application is to provide the application of rice protein kinase OsTUPK and its coding gene in disease resistance breeding, to solve the problems existing in the prior art. OsTUPK positively regulates the disease resistance of plants, and OsTUPK protein and its coding gene are of great significance for breeding disease-resistant rice varieties.

[0006] To achieve the above purpose, the present application provides the following solutions:

[0007] The present application provides the application of rice protein kinase OsTUPK in any of the following:

[0008] (1) in regulating rice blast resistance;

[0009] (2) in the preparation of products for regulating rice blast resistance;

[0010] (3) in the breeding of rice resistant to rice blast;

[0011] (4) in the breeding of rice resistant to rice blast;

[0012] The amino acid sequence of the rice protein kinase OsTUPK is shown in SEQ ID NO. 2.

[0013] The present application also provides the application of the coding gene OsTUPK of rice protein kinase OsTUPK in any of the following:

[0014] (1) in the application of regulating rice blast resistance;

[0015] (2) in the application of preparing products for regulating rice blast resistance;

[0016] (3) in the application of breeding rice resistant to blast;

[0017] (4) in the application of cultivating rice resistant to blast;

[0018] The nucleotide sequence of the coding gene OsTUPK is shown as SEQ ID NO. 1.

[0019] The present application also provides the application of a recombinant vector containing the coding gene OsTUPK in any of the following:

[0020] (1) in the application of regulating rice blast resistance;

[0021] (2) in the application of preparing products for regulating rice blast resistance;

[0022] (3) in the application of breeding rice resistant to blast;

[0023] (4) in the application of cultivating rice resistant to blast;

[0024] The nucleotide sequence of the coding gene OsTUPK is shown as SEQ ID NO. 1.

[0025] The present application also provides the application of a host bacterium containing a recombinant vector in any of the following:

[0026] (1) in the application of regulating rice blast resistance;

[0027] (2) in the application of preparing products for regulating rice blast resistance;

[0028] (3) in the application of breeding rice resistant to blast;

[0029] (4) in the application of cultivating rice resistant to blast;

[0030] The recombinant vector is a vector integrated with the coding gene OsTUPK in the genome, and the nucleotide sequence of the coding gene OsTUPK is shown as SEQ ID NO. 1.

[0031] Preferably, the regulation is positive regulation.

[0032] The present application also provides a method for regulating rice blast resistance, comprising any of the following methods:

[0033] (1) reducing the resistance of the rice to rice blast by knocking out the OsTUPK gene in the rice;

[0034] (2) increasing the resistance of the rice to rice blast by overexpressing the OsTUPK gene in the rice;

[0035] The nucleotide sequence of the OsTUPK gene is shown as SEQ ID NO. 1.

[0036] The present application also provides a method for cultivating rice resistant to rice blast, comprising the steps of overexpressing the OsTUPK gene in the rice, increasing the expression amount of the OsTUPK, and obtaining rice resistant to rice blast.

[0037] The nucleotide sequence of the OsTUPK gene is shown as SEQ ID NO. 1.

[0038] The present application discloses the following technical effects:

[0039] The present application identifies a protein kinase, which is named as OsTUPK. The experiments of the present application prove that the OsTUPK protein has the function of improving the disease resistance of rice. Overexpressing the OsTUPK gene of Zhonghua 11 (ZH11) in ZH11, the transgenic plants show higher disease resistance than the receptor; knocking out the OsTUPK in rice, the transgenic plants show lower disease resistance than the receptor, which indicates that the OsTUPK positively regulates the disease resistance of plants, and the OsTUPK protein and the encoding gene have important significance for cultivating rice varieties resistant to diseases. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of 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 also be obtained by those skilled in the art without creative labor.

[0041] Figure 1 OsTUPK gene structure, knockout site, and identification of knockout plants and overexpression plants; A: schematic diagram of the gene structure of OsTUPK and the sgRNA designed at the knockout target site; B: identification of OsTUPK knockout (KO) plants; C: expression amount detection of OsTUPK overexpression (OE) plants; the * above the columnar chart indicates significant difference (P<0.05);

[0042] Figure 2Wounding and inoculation phenotype of wild type and OsTUPK overexpression plants; A: representative lesion pictures of three-week-old ZH11 (WT), OsTUPK-OE1 and OsTUPK-OE2 plant leaves 5 days after inoculation with Magnaporthe oryzae (physiological race zhong10-8-14); B: statistics of lesion length of three-week-old ZH11 (WT), OsTUPK-OE1 and OsTUPK-OE2 plant leaves 5 days after inoculation with Magnaporthe oryzae (physiological race zhong10-8-14); C: relative Magnaporthe oryzae DNA content of three-week-old ZH11 (WT), OsTUPK-OE1 and OsTUPK-OE2 plant leaves 5 days after inoculation with Magnaporthe oryzae (physiological race zhong10-8-14); ** above the column chart indicates significant difference (P < 0.01).

[0043] Figure 3 Wounding and inoculation phenotype of wild type and OsTUPK overexpression plants; A: representative lesion pictures of three-week-old ZH11 (WT), OsTUPK-OE1 and OsTUPK-OE2 plant leaves 5 days after inoculation with Magnaporthe oryzae (physiological race zhong10-8-14); B: statistics of lesion length of three-week-old ZH11 (WT), OsTUPK-OE1 and OsTUPK-OE2 plant leaves 5 days after inoculation with Magnaporthe oryzae (physiological race zhong10-8-14); C: relative Magnaporthe oryzae DNA content of three-week-old ZH11 (WT), OsTUPK-OE1 and OsTUPK-OE2 plant leaves 5 days after inoculation with Magnaporthe oryzae (physiological race zhong10-8-14); ** above the column chart indicates significant difference (P < 0.01). DETAILED DESCRIPTION

[0044] Various example embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is made in connection with the drawings, which are intended to illustrate certain aspects of the application, and should not be taken as a limitation to the scope of the application. It will be readily understood that the aspects of the present application, as generally described and illustrated in the figures, could be arranged, substituted, combined, separated, and designed in other different ways, and that some aspects of the application can be used independently or in combination with other aspects of the application.

[0045] 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 application. Additionally, the use of the term "about" in the description above and below means that the value is within 10% of the value being described. In addition, for numerical ranges expressed in the format "from X to Y," "X to Y," or "X / Y," it is meant that the numerical values include every number between X and Y, not just including the end points. Additionally, for numerical ranges expressed in the format "from X to Y," "X to Y," or "X / Y," it is meant that the numerical values include every number between X and Y, not just including the end points. Numerical ranges expressed in the format "from X to Y" are understood to include X and Y. Numerical ranges expressed in the format "X to Y" are understood to include X and Y. Numerical ranges expressed in the format "X / Y" are understood to include X and Y.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict between the content of the specification and that of any incorporated literature, the content of the specification controls.

[0047] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. 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 given are exemplary only. It is to be understood that the application is not limited in scope by the specific embodiments described herein. Rather, the intent is to embrace all changes and modifications that will become apparent to those skilled in the art as a result of the practice of the application.

[0048] As used herein, the terms “comprises”, “comprising”, “includes”, “including”, “has”, “having” or the like are open-ended terms that are intended to mean including, but not limited to.

[0049] Oryza sativa L. spp. japonica, var. zhonghua 11, AA genome, ZH11, is a japonica subspecies and is described in the following document: Ni Y C, New Rice Variety Zhonghua 11, Agricultural Science and Technology Communication, 1989, 07, 35; provided by Professor Chen Xuewei of Sichuan Agricultural University.

[0050] OsTUPK knockout and overexpression transgenic rice were completed by Boyuan Biotechnology Co., Ltd.

[0051] Plant binary expression vector pTCRISPR was provided by Associate Professor Tang Yongyan of the State Key Laboratory of Sichuan Agricultural University.

[0052] Plant binary expression vector pCambia2300-35S:eGFP was provided by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University.

[0053] Total RNA extraction kit: TRIzol, Invitrogen, Cat No. 15596026.

[0054] Reverse transcription kit: HiScript III RT SuperMix for qPCR (+gDNA wiper), Vanzyme, Cat No. R323-01.

[0055] Homologous recombination kit: ClonExpress II One Step Cloning Kit, purchased from Vanzyme in China, with the product number C112-01.

[0056] Example 1 Rice protein kinase OsTUPK and cloning thereof

[0057] A protein kinase was screened in rice and named as OsTUPK. According to the rice ZH11 genome reference gene sequence, primers were designed as follows:

[0058] OsTUPK-F: 5'-ATGGAGGCGGAGGACGACGA-3' (SEQ ID NO. 3);

[0059] OsTUPK-R: 5'-CAGCGACATGCTGTGCTGCT-3' (SEQ ID NO. 4).

[0060] About 2.5 kb DNA band was obtained by amplification from the above primers with ZH11 total cDNA as a template. After sequencing, it was confirmed to be OsTUPK gene belonging to the protein kinase family. After comparing the sequence of LOC_Os06g06760 in the rice genome reference sequence and the cloned sequence obtained by amplification from the ZH11 total cDNA, it was shown that the cloned sequence obtained from ZH11 was the same as the sequence of LOC_Os06g06760. The amino acid sequence of OsTUPK protein is shown as SEQ ID NO. 2, and the CDS sequence of the coding strand of the gene OsTUPK encoding OsTUPK protein is shown as SEQ ID NO. 1.

[0061] SEQ ID NO. 1:

[0062]

[0063] SEQ ID NO. 2:

[0064] MEAEDDERAEAEAEARREKEAGNAAYRKLYLETAVRHYTRGALLDPRDISFLTNRAAAYLLMSKYKECVRDCDEAVEKGRELRADNKLVARALARKASALLKLAACAADYDPAIRALQQSLAEHYSEETLAKLGEAEEARKEIEERERLDQEAADHHRDRGNDFFKQKRYQEAAMHYTEAMKKNPKDPRVFSNRAQCHIYLGALPEGLEDADKCIALDPTFLKGYLRKAKVQLLMGNYEIALATYVEGLKCDPNNLEVLDGLRRCAACIKRANGGDSRAEDLREILGDLHLNDDLCNKLQKSMDEAAVLKKEASDERLKRIESERLARTLEDLYLSQVQQRKETEESLSRVQQEFEQLKIQQDEVTVELQRVNEQNENLLGQLSDSREHFEWLLSEHDQLLRERDNAVREVEELRQKRGQMLSVLVTAMHCEFSSSEVESATENFSNSLKIGEGGFGCVYKGILRNMTVAIKVLRPDSLQGQSQFEQEVSILSRVRHPHLVTLLGACSESSTLVYEFLPNGSLEDFLMCSDKRQTLTWQARIRIIAEICSALIFLHKNKPHPVVHGDLKPANILLGVNLVSKLSDFGISRLLIQSSTNNTTLYRTMHPVGTPLYMDPEFLSTGELTPQSDVYSFGIVVLRLLTGKPPVGIKNIVEDAMEKGDLNSVIDTSVGEWPHLHIEQLAYLALRCTELSRRCRPDLSGEVWAIVEAIRDAALSSPSSSRSAQDQNSPPSYFICPISQDIMDDPHIAADGFTYEAEAIRSWLCNGHDTSPMTNLLLEHEELIPNRALRSAIQEWLQQHSMSL.

[0065] Example 2 OsTUPK plant knock-out vector pTCRISPR-sgRNA OsTUPK Construction

[0066] The genome of rice ZH11 was used as a template to design a knockout target sequence: 5'-GCCGTGCGCCACTACACCCG-3'(sgRNA1, SEQ ID NO. 5) or 5'-CCGACAACAAGCTGGTCGCG-3'(sgRNA2, SEQ ID NO. 6, see Figure 1 The following primers were synthesized:

[0067] F: 5'-TGTGGCCGTGCGCCACTACACCCGG-3'(SEQ ID NO. 7);

[0068] R: 5'-AAAACGCCGTGCGCCACTACACCCG-3'(SEQ ID NO. 8).

[0069] After denaturation at 95°C, the double-stranded DNA was formed by annealing. The pTCRISPR vector was digested with Bsa I, and the linearized vector was recovered. The above DNA double-strand and linearized vector were mixed according to the system in Table 1, and T4 DNA ligase was used for ligation reaction at room temperature for 30 minutes. The pTCRISPR-sgRNA OsTUPK knockout vector.

[0070] Table 1 ligation system

[0071]

[0072]

[0073] The same effect can be achieved within the range shown in Table 1 above, and the specific embodiment adopted is 10 μL ligation system: 0.5 μL DNA double-strand fragment, 0.5 μL linearized vector, 2 μL T4 ligase, and ddH2O was supplemented to 10 μL.

[0074] The above pTCRISPR-sgRNA OsTUPK The knockout vector was transformed into DH5α competent cells, and after kanamycin screening, single colonies were picked for colony PCR identification. After the positive colonies were extracted for plasmid and sequencing was correct, the pTCRISPR-sgRNA OsTUPK was confirmed to be correct, and genetic transformation could be carried out.

[0075] OsTUPK knockout transgenic rice was completed by Boyuan Biotechnology Co., Ltd. Sequencing and screening of genetically stable knockout plants OsTUPK-KO1 and OsTUPK-KO2 showed that there were insertion or deletion of bases at the target sequence in the knockout plants, see Figure 1 in the middle of B.

[0076] Example 3 Construction of OsTUPK overexpression vector pCambia2300-35S:OsTUPK

[0077] The cDNA obtained by reverse transcription of total RNA of rice ZH11 was used as a template, and the cloning primer was:

[0078] F: 5'-TATCCAGATCCAGTGGGATCCATGGAGGCGGAGGACGACGA-3' (SEQ ID NO. 9);

[0079] R: 5'-CGCACTAGTAAGCTTGGTACCCAGCGACATGCTGTGCTGCT-3' (SEQ ID NO. 10).

[0080] The full-length cDNA of OsTUPK was obtained by PCR amplification, and the pCambia2300-35S:eGFP-nosT vector was digested with BamH I and Kpn I to obtain a linearized vector. The PCR recovery product OsTUPK full-length cDNA and the linearized vector were mixed according to the system in Table 2 to perform homologous recombination, and the pCambia2300-35S:OsTUPK recombinant vector was obtained.

[0081] Table 2 Homologous recombination reaction system

[0082]

[0083] The same effect can be achieved within the range shown in Table 2 above, and the specific embodiment adopted is a 20 μL homologous recombination reaction system: 1 μL of DNA double-stranded fragment, 1 μL of linearized vector, 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and ddH2O was supplemented to 20 μL.

[0084] The above-mentioned pCambia2300-35S:OsTUPK recombinant vector was transformed into DH5α competent cells, and after kanamycin screening, single colonies were picked for colony PCR identification. After the positive colonies were extracted for plasmid and sequencing was correct, it was confirmed that the pCambia2300-35S:OsTUPK was correct, and genetic transformation could be performed.

[0085] OsTUPK overexpression transgenic rice was completed by Boyuan Biotechnology Co., Ltd., and the expression amount of the target gene was detected and the genetically stable overexpression plants OsTUPK-OE1 and OsTUPK-OE2 were screened out, and the results showed that the expression of the target gene in the overexpression plants was significantly increased compared with the expression of the target gene in the wild type (Fig. 3C). Figure 1

[0086] Example 4 OsTUPK participates in regulating the disease resistance of plants​

[0087] In plants, the OsTUPK is mainly focused on the heat tolerance of plants. The applicant found that the OsTUPK plays an important role in regulating the disease resistance of plants. Therefore, the effect of OsTUPK on the disease resistance of rice was tested.

[0088] Puncture inoculation treatment in seedling stage:

[0089] The tested materials were Zhonghua 11 (ZH11), ZH11 knockout lines OsTUPK-KO1 and OsTUPK-KO2 which were genetically stable and had ZH11 as background, and OsTUPK overexpression lines OsTUPK-OE1 and OsTUPK-OE2 which were genetically stable and had ZH11 as background.

[0090] Full seeds were selected and placed in conical bottles containing tap water, and then placed in a 37°C dark incubator for germination, and the water was changed every day. After 2 days, the seeds with white tips were selected and placed in 96-well seedling plates, and the 96-well seedling plates were placed on a float and grown in Hoagland nutrient solution. After 21 days, the penultimate leaves of rice with uniform growth and size were selected, punctured and inoculated with 5 μL of Zhong10-8-14 Magnaporthe grisea spore solution with a concentration of 5×10 5 ml -1 , and observed and counted the lesion length after 5 days.

[0091] The results showed that after puncture inoculation, compared with ZH11, the lesion length of OsTUPK-KO1 and OsTUPK-KO2 plants with ZH11 as background was extremely significantly increased, and the disease resistance was weakened ( Figure 2 ). Compared with ZH11, the lesion length of OsTUPK-OE1 and OsTUPK-OE2 plants with ZH11 as background was significantly reduced, and the disease resistance of the plants was significantly enhanced ( Figure 3 ). It is indicated that OsTUPK positively regulates the disease resistance of plants, and the encoded gene OsTUPK can be used as a target gene for molecular breeding to improve the disease resistance of plants.

[0092] The above-described embodiments are only preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. Use of rice protein kinase OsTUPK in any of the following: (1) Application in regulating rice blast resistance; (2) Application in the preparation of products for regulating rice blast resistance; (3) Application in blast-resistant rice breeding; (4) Application in breeding blast-resistant rice; The amino acid sequence of the rice protein kinase OsTUPK is shown in SEQ ID NO.

2.

2. Use of the gene encoding rice protein kinase OsTUPK in any of the following: (1) Application in regulating rice blast resistance; (2) Application in the preparation of products for regulating rice blast resistance; (3) Application in blast-resistant rice breeding; (4) Application in breeding blast-resistant rice; The nucleotide sequence encoding the gene OsTUPK is shown in SEQ ID NO.

1.

3. Use of a recombinant vector containing the OsTUPK encoding gene in any of the following: (1) Application in regulating rice blast resistance; (2) Application in the preparation of products for regulating rice blast resistance; (3) Application in blast-resistant rice breeding; (4) Application in breeding blast-resistant rice; The nucleotide sequence encoding the gene OsTUPK is shown in SEQ ID NO.

1.

4. Use of host bacteria containing recombinant vectors in any of the following: (1) Application in regulating rice blast resistance; (2) Application in the preparation of products for regulating rice blast resistance; (3) Application in blast-resistant rice breeding; (4) Application in breeding blast-resistant rice; The recombinant vector is a vector that integrates the coding gene OsTUPK into the genome, and the nucleotide sequence of the coding gene OsTUPK is shown in SEQ ID NO.

1.

5. The use according to any one of claims 1 to 4, characterized in that The regulation is positive regulation.

6. A method for regulating rice blast resistance, characterized in that: This includes any of the following methods: (1) Reducing the resistance of rice to rice blast by knocking out the OsTUPK gene in rice; (2) improving the resistance of rice to rice blast by overexpressing the OsTUPK gene in rice; The nucleotide sequence of the OsTUPK gene is shown in SEQ ID NO.

1.

7. A method for cultivating blast-resistant rice, characterized in that: The method comprises the steps of overexpressing the OsTUPK gene in rice, increasing the expression level of the OsTUPK gene, and obtaining rice blast-resistant rice; The nucleotide sequence of the OsTUPK gene is shown in SEQ ID NO.1.

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