Rice microtubulin OsTID1 and application of coding gene thereof in disease-resistant breeding

Abstract

The invention discloses application of rice microtubulin OsTID1 and a coding gene thereof in disease-resistant breeding, and belongs to the technical field of biological breeding. A rice tubulin family member participating in immunoreaction is identified and named as OsTID1, the amino acid sequence of the rice tubulin family member is as shown in SEQ ID NO.2, and the sequence of the coding gene is as shown in SEQ ID NO.1. A knockout vector and an overexpression vector are respectively constructed according to the sequence of the OsTID1 gene and transfected with Zhonghua 11 rice to obtain an OsTID1 gene knockout plant and an OsTID1 gene overexpression plant. Through a pyricularia grisea stabbing and grafting treatment experiment, an experiment result shows that compared with a control plant, the number of rice blast disease spots of the OsTID1 gene knockout plant is remarkably reduced, the length of the disease spots is remarkably shortened, and the rice blast resistance is remarkably improved. The OsTID1 can negatively regulate the disease resistance of plants, and has important significance for cultivating disease-resistant rice varieties.

Description

Technical Field

[0001] The invention relates to the technical field of biological breeding, and in particular to application of rice microtubule protein OsTID1 and its encoding gene in disease-resistant breeding. Background Art

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

[0003] As one of the most important food crops, improving rice's disease resistance has important theoretical and practical significance.

[0004] OsTID1 protein is a member of the microtubule protein family identified and named by the inventor's team in the study of rice immune response. Microtubule protein plays an important role in plant development, so previous research on OsTID1 protein focused on its regulation of plant development. Summary of the invention

[0005] The purpose of the present invention is to provide an application of rice microtubule protein OsTID1 and its encoding gene in disease resistance breeding to solve the problems existing in the above-mentioned prior art. Knocking out rice microtubule protein OsTID1 can effectively reduce the number of rice blast lesions, shorten the length of lesions, and improve the disease resistance of rice plants.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of rice tubulin OsTID1 in any of the following:

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

[0009] (2) Application in breeding transgenic rice with improved resistance to rice blast;

[0010] (3) Application in the preparation of products for improving rice resistance to rice blast;

[0011] The amino acid sequence of the rice tubulin OsTID1 is shown in SEQ ID NO.2.

[0012] The present invention provides the use of the gene encoding rice tubulin OsTID1 in any of the following:

[0013] (1) Application in regulating rice resistance to rice blast;

[0014] (2) Application in breeding transgenic rice with improved resistance to rice blast;

[0015] (3) Application in the preparation of products for improving rice resistance to rice blast;

[0016] The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

[0017] The present invention also provides the use of a recombinant vector comprising the encoding gene in any of the following:

[0018] (1) Application in regulating rice resistance to rice blast;

[0019] (2) Application in breeding transgenic rice with improved resistance to rice blast;

[0020] (3) Application in the preparation of products for improving the resistance of rice to rice blast.

[0021] The present invention also provides the use of an engineered bacterium comprising the recombinant vector in any of the following:

[0022] (1) Application in regulating rice resistance to rice blast;

[0023] (2) Application in breeding transgenic rice with improved resistance to rice blast;

[0024] (3) Application in the preparation of products for improving the resistance of rice to rice blast.

[0025] Preferably, the expression level of the rice microtubule protein OsTID1 or its encoding gene is down-regulated in rice to improve the resistance of the rice to rice blast.

[0026] Preferably, the method for down-regulating the expression level of the rice microtubule protein OsTID1 or its encoding gene in rice comprises the steps of constructing a knockout vector of the rice microtubule protein OsTID1 encoding gene, transforming the knockout vector into Escherichia coli, and then infecting rice plants.

[0027] The present invention also provides a method for improving the resistance of rice to rice blast, comprising the steps of down-regulating the expression level of a gene encoding rice microtubule protein OsTID1 in rice to improve the resistance of the rice to rice blast;

[0028] The nucleotide sequence of the gene encoding the rice microtubule protein OsTID1 is shown in SEQ ID NO.1.

[0029] Preferably, the method for down-regulating the expression level of the gene encoding rice microtubule protein OsTID1 comprises knocking out the gene encoding rice microtubule protein OsTID1 in rice.

[0030] Preferably, the method for knocking out the gene encoding rice microtubule protein OsTID1 in rice comprises the following steps:

[0031] A knockout vector of the coding gene of the rice microtubule protein OsTID1 is constructed, and the knockout vector is transformed into Escherichia coli, and then infects rice plants.

[0032] The present invention also provides a method for breeding rice with improved resistance to rice blast, comprising the following steps:

[0033] Knocking out the gene encoding rice microtubule protein OsTID1 in rice cells, then cultivating the rice cells, and using the rice cells to regenerate rice, thereby obtaining the rice with improved resistance to rice blast;

[0034] The nucleotide sequence of the gene encoding the rice microtubule protein OsTID1 is shown in SEQ ID NO.1.

[0035] The present invention discloses the following technical effects:

[0036] The present invention identifies a member of the rice microtubule protein family that participates in immune response, named OsTID1, whose amino acid sequence is shown in SEQ ID NO.2, and the coding gene sequence is shown in SEQ ID NO.1. Knockout vectors and overexpression vectors were constructed according to the sequence of the OsTID1 gene, and the Zhonghua 11 rice was transfected to obtain OsTID1 gene knockout plants and OsTID1 gene overexpression plants. Through the experiment of treating with rice blast fungus by stabbing wounds, the experimental results showed that compared with the control plants, the number of rice blast lesions of the OsTID1 gene knockout plants was significantly reduced, the length of the lesions was significantly shortened, and the resistance to rice blast was significantly improved. OsTID1 negatively regulates the disease resistance of plants, which is of great significance for breeding disease-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1Figure 1 is a schematic diagram of the OsTID1 gene structure and the phenotype observation diagram of the OsTID1 gene knockout plant inoculated with rice blast fungus; A is a schematic diagram of the OsTID1 gene structure and its knockout target; B is the identification of OsTID1 knockout (KO) plants; C is a representative lesion image and lesion length (Lesion 20 mm) of the leaves of three-week-old Zhonghua 11 (ZH11), OsTID1-KO1, OsTID1-KO2 and OsTID1-KO3 plants 5 days after being pierced and inoculated with rice blast fungus (physiological subspecies zhong10-8-14). D is the representative lesion pictures and lesion number statistics of leaves of three-week-old Zhonghua11 (ZH11), OsTID1-KO1, OsTID1-KO2 and OsTID1-KO3 plants 5 days after spray inoculation with rice blast fungus (physiological subspecies zhong10-8-14); the scale bar is 1 cm, and * indicates significant difference (P<0.05);

[0039] Figure 2 Schematic diagram of the OsTID1 overexpression vector structure and the phenotype observation diagram of the OsTID1 gene overexpression plant inoculated with rice blast fungus; A is a schematic diagram of the OsTID1 overexpression vector structure; B is the expression level (Relative expression) detection of OsTID1 overexpression (OE) plants; C is a representative lesion picture and lesion length (Lesion 5 days after the leaves of three-week-old Zhonghua 11 (ZH11), OsTID1-OE1, OsTID1-OE2 and OsTID1-OE3 plants were inoculated with rice blast fungus (physiological subspecies zhong10-8-14) after being punctured. length); D is the representative lesion pictures and lesion number statistics of leaves of three-week-old Zhonghua11 (ZH11), OsTID1-OE1, OsTID1-OE2 and OsTID1-OE3 plants 5 days after spray inoculation with rice blast fungus (physiological subspecies zhong10-8-14); the scale bar is 1 cm, and * indicates significant difference (P<0.05). DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] The rice variety Zhonghua 11 (O. Sativa L. spp. japonica, var zhonghua11, AAgenome, ZH11) belongs to the japonica subspecies, as recorded in the following literature: Ni Yuchong, A new rice variety cultivated by flower culture—Zhonghua 11, Agricultural Science and Technology Newsletter, 1989, 07, 35; provided by Professor Chen Xuewei of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0046] OsTID1 overexpression and knockout transgenic rice were produced by Boyuan Biotechnology Company.

[0047] The plant binary expression vector pTCRISPR was provided by Associate Professor Tang Yongyan from the State Key Laboratory of Sichuan Agricultural University.

[0048] The plant binary expression vector pCAMBIA2300 was provided by Professor Wu Xianjun from the Rice Research Institute of Sichuan Agricultural University.

[0049] Total RNA extraction kit: TRIzol purchased from Invitrogen, USA, catalog number 15596026.

[0050] Reverse transcription kit: HiScript III RT SuperMix for qPCR (+gDNA wiper) purchased from Vanzyme, China, with the catalog number R323-01.

[0051] Fluorescence quantitative kit: AceQ qPCR SYBR Green MasterMix purchased from Vanzyme, China, with the catalog number Q111-02.

[0052] Homologous recombination kit: ClonExpress II One Step Cloning Kit purchased from Vanzyme, China, with the catalog number C112-01.

[0053] Example 1 OsTID1, a gene induced in rice immune response, and its cloning

[0054] In the process of studying the immune response of rice, the inventors found that the expression of OsTID1 was induced by rice blast fungus. After investigation, it was found that this gene encodes microtubule protein. According to the reference gene sequence of the rice genome, primers were designed:

[0055] OsTID1-F: ATGAGGGAGTGCATCTCGAT (SEQ ID NO.3);

[0056] OsTID1-R: GTACTCGTCACCCTCATCACCC (SEQ ID NO.4);

[0057] Using ZH11 total cDNA as a template, a DNA band of about 1.3Kb was amplified by the above primers. After sequencing, it was confirmed that it was the OsTID1 gene belonging to the tubulin family. The LOC_Os11g14220 sequence in the rice genome reference sequence and the clone sequence amplified from the ZH11 total cDNA were aligned, indicating that the clone sequence obtained in ZH11 was identical to the LOC_Os11g14220 sequence. The amino acid sequence of the OsTID1 protein is shown in SEQ ID NO.2. The CDS sequence of the coding chain of the gene LOC_Os11g14220 encoding the OsTID1 protein is a DNA molecule shown in SEQ ID NO.1.

[0058] SEQ ID NO.1:

[0059]

[0060] SEQ ID NO.2:

[0061] MRECISIHIGQAGIQVGNACWELYCLEHGIQPDGQMPGDKTVGGGDDAFNTFFSETGAGKHVPRAFVDLEPTVIDEVRTGDYRQLFHPEQLISGKEDAANNFARGHYTIGKE IVDLCLDRIRKLADNCTGLQGFLVFNAVGGGTGSGLGSLLLERLSVDYGKKSKLGFTVYPSPQVTSSVVEPYNSVLSTHSLLEHTDVAVLLDNEAIYDICRRSLDIERPTYTN LNRLVSQVISSLTASLRFDGALNVDVNEFQTNLVPYPRIHFMLSSYAPVISAEKAYHEQLSVAEITNSAFEPSSMMAKCDPRHGKYMACCLMYRGDVVPKDVNAAVATIKTKR TIQFVDWCPTGFKCGINYQPPSVVPGGDLAKVQRAVCMISNSTSVVEVFSRIDIKFDLMYSKRAFVHWYVGEGMEEGEFSEAREDLAALEKDYEEVGSEFDDGDEGDEGDEY;

[0062] Example 2 Construction of OsTID1 plant knockout vector

[0063] 1. Construction of OsTID1 knockout vector pTCRISPR-sgRNA

[0064] Using the genome of rice ZH11 as a template, the knockout target sequence was designed as follows:

[0065] GATCGACGAAGACAGCACGG(SEQ ID NO.5);

[0066] The following primers were synthesized:

[0067] KO-F: TGTG GATCGACGAAGACAGCACGG G (SEQ ID NO. 6);

[0068] KO-R:AAAAC CCGTGCTGTCTTCGTCGATC (SEQ ID NO.7);

[0069] After denaturation at 95°C, anneal to form a double strand. Digest the pTCRISPR vector with BsaI to recover the linearized vector. Mix the above DNA double strands and the linearized vector according to the following system, perform T4 DNA ligase ligation reaction and incubate at room temperature for 30 minutes:

[0070] Ligation reaction system: 3 μL double-stranded DNA fragment, 3 μL linearized vector, 2 μL T4 ligase, 2 μL double distilled water, total volume 10 μL.

[0071] Obtain pTCRISPR-sgRNA OsTID1 Knockout vector.

[0072] Transformed into DH5α competent cells, selected by kanamycin, single clones were picked for colony PCR identification, plasmids were extracted from positive colonies and sequenced to confirm pTCRISPR-sgRNA OsTID1 If the sequence of the target vector is correct, genetic transformation can be performed. Figure 1 As shown in A.

[0073] OsTID1 knockout transgenic rice was produced by Boyuan Biotechnology Company. After sequencing, the sequencing results of the relevant sites of the transgenic rice plants are as follows Figure 1 As shown in B, OsTID1 knockout transgenic rice was successfully constructed.

[0074] Example 3 Construction of OsTID1 plant expression vector

[0075] Construction of OsTID1 overexpression vector pCAMBIA2300-OsTID1

[0076] The cDNA obtained by reverse transcription of total RNA of rice ZH11 was used as template, and the cloning primers were:

[0077] OE-F: GGACAGGGTACCCGGGGATCC ATGAGGGAGTGCATCTCGAT(SEQ ID NO.8);

[0078] OE-R: GGTACTAGTGTCGACTCTAGA GTACTCGTCACCCTCATCACCC(SEQ ID NO.9);

[0079] The underlined sequence is the homologous recombination arm sequence.

[0080] The full-length cDNA of OsTID1 was obtained by PCR amplification, and the pCAMBIA2300-35S-eGFP-OCS vector was double-digested with BamHI and XbaI to recover the linearized vector. The full-length cDNA of OsTID1 recovered by PCR and the linearized vector were mixed according to the following system for homologous recombination.

[0081] Homologous recombination system: DNA fragment 5μL, linearized vector 5μL, 5×CE II Buffer 4μL, Exnase II 2μL, double distilled water 3μL, total volume 20μL.

[0082] Obtain the pCAMBIA2300-OsTID1 recombinant vector. The vector schematic diagram is shown in Figure 2 As shown in A.

[0083] The cells were transformed into DH5α competent cells and screened with kanamycin. Single clones were picked for colony PCR identification. Plasmids were extracted from positive colonies and sequenced to confirm that pCAMBIA2300-OsTID1 was correct, and genetic transformation could be performed.

[0084] OsTID1 overexpressing transgenic rice was constructed by Boyuan Biotechnology Company. The results of detecting the changes in OsTID1 expression levels in OsTID1 overexpressing rice plants are shown in Figure 2 As shown in B, transgenic rice overexpressing the OsTID1 gene was successfully constructed.

[0085] Example 4 OsTID1 is involved in regulating plant disease resistance

[0086] In plants, research on OsTID1 mainly focuses on development and abiotic stress regulation. The inventors first discovered that the microtubule protein OsTID1 is involved in regulating jasmonic acid synthesis in rice and is a regulatory factor in the jasmonic acid pathway. Jasmonic acid is known as a defense hormone and plays an important role in regulating plant disease resistance. Therefore, the effect of OsTID1 on rice disease resistance was tested.

[0087] Wounding and inoculation treatment at the seedling stage: The test materials were Zhonghua 11 (ZH11), OsTID1 knockout strain OsTID1-KO with ZH11 background and genetic stability, and OsTID1 overexpression strain OsTID1-OE with ZH11 background and genetic stability. Select seeds with full grains, put them in a conical bottle filled with tap water, and place them in a dark incubator at 37°C for germination, and change the water every day. After 2 days, select white seeds and put them in a 96-well seedling plate. The 96-well seedling plate was placed on a float and grown in Hoagland nutrient solution. After 21 days, the second-to-last rice leaf with the same growth and size was selected, and 5μL of a concentration of 5×10 5 mL -1 The 10-8-14 rice blast fungus spores were used and the length of the lesions was observed and counted after 5 days.

[0088] The results showed that there was no significant difference in the growth of ZH11, OsTID1-KO and OsTID1-OE under hydroponic conditions. The experiment was repeated three times. Each time, 20 plants were planted. Statistics showed that after puncture inoculation, compared with ZH11, the length of lesions in OsTID1-KO plants under the ZH11 background was significantly reduced, the number of lesions was significantly reduced, and the disease resistance of the knockout plants was significantly increased ( Figure 1 C and D), while in ZH11, the lesion length and number of plants overexpressing OsTID1 were significantly increased, and the overexpressing plants were more sensitive to pathogens ( Figure 2 C and D). This indicates that OsTID1 negatively regulates plant disease resistance and its encoding gene OsTID1 can be used as a target gene for molecular breeding to improve plant disease resistance.

[0089] 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 rice microtubule protein OsTID1 in any of the following: (1) Application in regulating rice resistance to rice blast; (2) Application in breeding transgenic rice with improved resistance to rice blast; (3) Application in the preparation of products for improving rice resistance to rice blast; The amino acid sequence of the rice tubulin OsTID1 is shown in SEQ ID NO.

2.

2. Use of the gene encoding rice tubulin OsTID1 as claimed in claim 1 in any of the following: (1) Application in regulating rice resistance to rice blast; (2) Application in breeding transgenic rice with improved resistance to rice blast; (3) Application in the preparation of products for improving rice resistance to rice blast; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

3. Use of the recombinant vector comprising the coding gene according to claim 2 in any of the following: (1) Application in regulating rice resistance to rice blast; (2) Application in breeding transgenic rice with improved resistance to rice blast; (3) Application in the preparation of products for improving the resistance of rice to rice blast.

4. Use of the engineered bacteria comprising the recombinant vector according to claim 3 in any of the following: (1) Application in regulating rice resistance to rice blast; (2) Application in breeding transgenic rice with improved resistance to rice blast; (3) Application in the preparation of products for improving the resistance of rice to rice blast.

5. The use according to any one of claims 1 to 4, characterized in that: The expression level of the rice microtubule protein OsTID1 or its encoding gene is down-regulated in rice to improve the resistance of the rice to rice blast.

6. The use according to claim 5, characterized in that The method for down-regulating the expression level of the rice microtubule protein OsTID1 or its encoding gene in rice comprises the steps of constructing a knockout vector of the encoding gene of the rice microtubule protein OsTID1, transforming the knockout vector into Escherichia coli, and then infecting rice plants.

7. A method for improving rice resistance to rice blast, characterized in that: The method comprises the steps of down-regulating the expression level of a gene encoding rice microtubule protein OsTID1 in rice to improve the resistance of the rice to rice blast; The nucleotide sequence of the gene encoding the rice microtubule protein OsTID1 is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that The method for down-regulating the expression level of the gene encoding rice microtubule protein OsTID1 comprises knocking out the gene encoding rice microtubule protein OsTID1 in rice.

9. The method according to claim 8, characterized in that The method for knocking out the gene encoding rice microtubule protein OsTID1 in rice comprises the following steps: A knockout vector of the coding gene of the rice microtubule protein OsTID1 is constructed, and the knockout vector is transformed into Escherichia coli, and then infects rice plants.

10. A method for breeding rice with improved resistance to rice blast, characterized in that: The following steps are involved: Knocking out the gene encoding rice microtubule protein OsTID1 in rice cells, then cultivating the rice cells, and using the rice cells to regenerate rice, thereby obtaining the rice with improved resistance to rice blast; The nucleotide sequence of the gene encoding the rice microtubule protein OsTID1 is shown in SEQ ID NO.1.

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