Application of Rice Tubulin OsTID1 and Its Encoding Gene in Disease Resistance Breeding

Abstract

The present invention discloses the application of rice microtubule protein OsTID1 and its encoding gene in disease resistance breeding, belonging to the technical field of biological breeding. The present invention identifies a member of the rice microtubule protein family involved in immune response, named OsTID1, whose amino acid sequence is shown in SEQ ID NO.2, and the sequence of the encoding gene is shown in SEQ ID NO.1. According to the sequence of the OsTID1 gene, a knockout vector and an overexpression vector were constructed respectively, 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 puncture wound, 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 rice blast resistance was significantly improved. OsTID1 negatively regulates the disease resistance of plants, which is of great significance for breeding disease-resistant rice varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of biological breeding, 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 massive crop yield losses, making the development of disease-resistant crops a major goal of the agricultural industry. Numerous genes associated with plant disease resistance have been reported, including both effector and regulatory genes, from crops such as rice, wheat, maize, and soybean, as well as model plants such as Arabidopsis thaliana. Several of these have been used as target genes in crop disease resistance genetic engineering, resulting in the successful development of disease-resistant rice, wheat, maize, and soybean varieties. However, differences in genetic backgrounds hinder the application of some genes, necessitating the continued need for new relevant genes to meet breeding needs.

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

[0004] The OsTID1 protein is a member of the microtubule protein family identified and named by the inventor's team during their research on rice immune response. Microtubules play an important role in plant development, so previous research on the OsTID1 protein has focused on its regulation of plant development. Summary of the Invention

[0005] The purpose of the present invention is to provide the 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 the 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 tubulin OsTID1 or its encoding gene in rice comprises the steps of constructing a knockout vector of the rice tubulin 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 rice resistance 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 rice resistance to rice blast;

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

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

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

[0031] A knockout vector of the rice microtubule protein OsTID1 encoding gene is constructed, and the knockout vector is transformed into Escherichia coli, which is then used to infect 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 tubulin 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 tubulin OsTID1 is shown in SEQ ID NO.1.

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

[0036] The present invention identified a member of the rice microtubule protein family involved in immune response, named OsTID1, with its amino acid sequence shown in SEQ ID NO.2 and its encoding gene sequence shown in SEQ ID NO.1. A knockout vector and an overexpression vector were constructed based on the sequence of the OsTID1 gene, and the vectors were transfected into Zhonghua 11 rice to obtain OsTID1 gene knockout plants and OsTID1 gene overexpression plants. Experimental results of a wound inoculation experiment with rice blast fungus showed that compared with the control plants, the OsTID1 gene knockout plants had significantly fewer blast lesions, significantly shorter lesion lengths, and significantly improved blast resistance. OsTID1 negatively regulates plant disease resistance and 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0038] Figure 1Schematic diagram of the OsTID1 gene structure and the phenotype observation of OsTID1 gene knockout plants 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 10-8-14) of the leaves of three-week-old Zhonghua 11 (ZH11), OsTID1-KO1, OsTID1-KO2 and OsTID1-KO3 plants 5 days after being inoculated with rice blast fungus (physiological subspecies zhong10-8-14) D is the representative lesion images and lesion number statistics of leaves of three-week-old Zhonghua 11 (ZH11), OsTID1-KO1, OsTID1-KO2, and OsTID1-KO3 plants 5 days after spray inoculation with rice blast fungus (physiological subspecies zhong10-8-14); scale bar is 1 cm, * 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 relative expression detection of OsTID1 overexpression (OE) plants; C is a representative lesion image 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)). D is the representative lesion images and lesion number statistics of leaves of three-week-old Zhonghua 11 (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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative 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, AA genome, ZH11) belongs to the japonica subspecies and is described in the following literature: Ni Yuchong, "A New Rice Variety Under Flower Culture—Zhonghua 11," Agricultural Science and Technology Bulletin, July 35, 1989; 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 of 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, 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] While studying rice's immune response, the inventors discovered that OsTID1 expression is induced by the rice blast fungus. This gene encodes tubulin. Based on the reference gene sequence in 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, the aforementioned primers amplified a DNA band of approximately 1.3 kb. Sequencing confirmed that it was the OsTID1 gene, which belongs to the tubulin family. Alignment of the LOC_Os11g14220 sequence in the rice genome reference sequence and the cloned sequence amplified from ZH11 total cDNA revealed that the cloned sequence from ZH11 was identical to LOC_Os11g14220. The amino acid sequence of the OsTID1 protein is shown in SEQ ID NO. 2. The CDS sequence of the coding strand of the gene encoding the OsTID1 protein, LOC_Os11g14220, is 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 rice ZH11 genome as a template, the knockout target sequence was designed as follows:

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

[0066] And synthesize the following primers:

[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-stranded DNA. The pTCRISPR vector was digested with BsaI to recover the linearized vector. The double-stranded DNA and linearized vector were mixed according to the following system and ligated with T4 DNA ligase. The reaction was incubated 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 with kanamycin, single clones were picked for colony PCR identification, plasmids were extracted from positive colonies and sequenced to confirm the correct 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 completed 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 from rice ZH11 was used as the 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 OsTID1 cDNA was amplified by PCR, and the pCAMBIA2300-35S-eGFP-OCS vector was digested with BamHI and XbaI to recover the linearized vector. The OsTID1 full-length cDNA recovered by PCR and the linearized vector were mixed and homologous recombination was performed as follows.

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

[0082] Obtain the pCAMBIA2300-OsTID1 recombinant vector, the vector diagram is as follows Figure 2 As shown in A.

[0083] Transform into DH5α competent cells, select with kanamycin, pick single clones for colony PCR identification, extract plasmids from positive colonies and sequence them to confirm that pCAMBIA2300-OsTID1 is correct, and genetic transformation can be carried out.

[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 the figure below. 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 has primarily focused on its role in developmental and abiotic stress regulation. The inventors discovered for the first time that the microtubule protein OsTID1 is involved in regulating jasmonic acid (JA) synthesis in rice, acting as a regulator of the JA pathway. Jasmonic acid, known as a defense hormone, plays a crucial role in regulating plant disease resistance. Therefore, they investigated the impact of OsTID1 on rice disease resistance.

[0087] Seedling stage puncture inoculation treatment: the test materials are Zhonghua 11 (ZH11), the OsTID1 knockout strain OsTID1-KO with a ZH11 background and genetic stability, and the OsTID1 overexpression strain OsTID1-OE with a ZH11 background and genetic stability. Select seeds with full grains, put them in a conical flask 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 is 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 is selected, and 5μL of a concentration of 5×10 5 mL -1 The blast fungus spores of Zhong10-8-14 were inoculated, 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 with 20 plants of each type each time. Statistics showed that after puncture inoculation, 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of rice tubulin 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 encoding 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, thereby improving the resistance of the rice to rice blast.

6. 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 tubulin OsTID1 is shown in SEQ ID NO.

1.

7. The method according to claim 6, wherein 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.

8. 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 tubulin 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 tubulin OsTID1 is shown in SEQ ID NO.1.

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