Application of OsMYB44 gene in regulating rice resistance to nematodes and breeding nematode-resistant rice varieties

Abstract

The present invention relates to the biological field, and in particular to the application of the OsMYB44 gene in regulating the nematode resistance of rice and cultivating nematode-resistant rice varieties. The present invention utilizes CRISPR / Cas9 gene editing technology, through an Agrobacterium-mediated transformation system, to site-directedly mutate the OsMYB44 gene in rice, and transforms rice explants to obtain rice mutant plants with OsMYB44 gene function loss, which can significantly enhance the resistance of rice to root-knot nematodes. The present invention first discovered that the rice transcription factor OsMYB44 has the function of regulating rice resistance to root-knot nematodes, which is of great significance for improving rice disease resistance and yield.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to the application of OsMYB44 gene in regulating the nematode resistance ability of rice and cultivating nematode-resistant rice varieties. Background Art

[0002] Rice (Oryza sativa L.), as one of the main food crops of the world's population, is often threatened by a variety of diseases and pests. Rice root-knot nematode disease has become an important pathogenic nematode in rice-growing areas in China and Southeast Asia, seriously affecting rice yield and quality. According to statistics, the annual rice yield loss is as high as 20%-80%. Production practice shows that exploring the transcription factors in rice that regulate resistance to rice root-knot nematodes, using gene editing technology to edit the transcription factor genes in rice, and then cultivating rice resistant varieties is the most effective and economical strategy for preventing and controlling rice root-knot nematode disease.

[0003] Transcription factors play a pivotal role in disease resistance signaling pathways. One transcription factor can often regulate the expression of multiple downstream genes related to similar traits. Compared with the traditional method of introducing or modifying a single gene to improve crop resistance to pathogens, it is more effective to improve crop disease resistance by exploring and modifying a key transcription factor. Exploring transcription factors that respond to nematode infection in resistant varieties, clarifying the upstream and downstream components of transcription factors and their relationships, and thus finding ideal transcription factor-related resources will provide strong technical support for breeding nematode-resistant plant varieties and studying special nematode molecular control strategies.

[0004] At present, CRISPR / Cas9 gene editing technology has been widely used in the study of transcription factor functions in rice, and has achieved the breeding of broad-spectrum disease-resistant rice varieties. For example, Zhu et al. used CRISPR / Cas9 technology to knock out the rice transcription factor OsEIL3, and found that it significantly enhanced the resistance of rice to sheath blight. Infection with rice blast or bacterial blight can enhance the binding of transcription factor OsEIL3 to the promoter of ERF transcription factor OsERF040, promote the biosynthesis and signal transduction of salicylic acid and jasmonic acid, and enhance the disease resistance of rice. However, there are few reports on methods for mutating key transcription factors in rice to improve rice resistance to root-knot nematode disease. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the OsMYB44 gene in regulating the nematode resistance of rice and cultivating nematode-resistant rice varieties to solve the problems existing in the above-mentioned prior art. The present invention confirms that the OsMYB44 gene is related to the nematode resistance of rice, and knocking out the OsMYB44 gene can improve the nematode resistance of rice.

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

[0007] The present invention provides the use of OsMYB44 protein in the following (1) or (2):

[0008] (1) Application in regulating rice's resistance to nematodes;

[0009] (2) Application in breeding transgenic rice resistant to nematodes;

[0010] Wherein, the amino acid sequence of the OsMYB44 protein is shown in SEQ ID NO.15.

[0011] The present invention provides the use of the OsMYB44 gene in the following (1) or (2):

[0012] (1) Application in regulating rice's resistance to nematodes;

[0013] (2) Application in breeding transgenic rice resistant to nematodes;

[0014] Wherein, the nucleotide sequence of the OsMYB44 gene is shown as SEQ ID NO.1.

[0015] The present invention provides the use of a recombinant vector for knocking out the OsMYB44 gene in the following (1) or (2):

[0016] (1) Application in regulating rice's resistance to nematodes;

[0017] (2) Application in breeding transgenic rice resistant to nematodes;

[0018] Wherein, the nucleotide sequence of the OsMYB44 gene is shown as SEQ ID NO.1.

[0019] The present invention provides the use of a recombinant bacterium comprising a recombinant vector for knocking out the OsMYB44 gene in the following (1) or (2):

[0020] (1) Application in regulating rice's resistance to nematodes;

[0021] (2) Application in breeding transgenic rice resistant to nematodes;

[0022] Wherein, the nucleotide sequence of the OsMYB44 gene is shown as SEQ ID NO.1.

[0023] Preferably, the nematode is a root-knot nematode.

[0024] The present invention provides a method for improving the nematode resistance of rice, comprising the steps of knocking out the OsMYB44 gene in rice to improve the nematode resistance of the rice; the nucleotide sequence of the OsMYB44 gene is shown in SEQ ID NO.1.

[0025] Preferably, the nematode is a root-knot nematode.

[0026] The present invention provides a method for cultivating nematode-resistant transgenic rice, comprising the steps of knocking out the OsMYB44 gene in rice to improve the nematode-resistant ability of the rice; the nucleotide sequence of the OsMYB44 gene is shown in SEQ ID NO.1.

[0027] Preferably, the nematode is a root-knot nematode.

[0028] The present invention discloses the following technical effects:

[0029] The present invention uses CRISPR / Cas9 gene editing technology to obtain Nipponbare rice OsMYB44 gene mutant plants, and verifies the regulatory ability of OsMYB44 gene in rice to resist root-knot nematodes through indoor inoculation. The research results show that knocking out the OsMYB44 gene can significantly improve the resistance of rice to root-knot nematodes, and will not affect the growth phenotype of rice plants. The present invention uses the transgenic rice cultivation method constructed by CRISPR / Cas9 gene editing technology to achieve the expansion of rice resistance resources, and can conveniently and efficiently apply fixed-point editing of rice transcription factor sites to obtain new root-knot nematode resistance rice lines with OsMYB44 gene function loss, which provides germplasm resources and creation methods for green prevention and control of rice root-knot nematodes and molecular breeding of resistant varieties, and is also of great significance to the improvement of rice resistance to nematodes and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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.

[0031] Figure 1 The OsMYB44 gene structure and guide RNA (gRNA) target sequence;

[0032] Figure 2 Schematic diagram of the T-DNA structure of the pYLCRISPR / Cas9-Pubi-H-OsMYB44-KO vector;

[0033] Figure 3 The target sequence mutation (a) and sequencing results of the target sequence homozygous mutation (b) and amino acid changes (c) of the OsMYB44 gene in mutant strains MYB44-KO-1 and MYB44-KO-15 are compared;

[0034] Figure 4 This is a schematic diagram of the subcellular localization of the OsMYB44 gene;

[0035] Figure 5 The statistical results of the number of nematode infection and root knots in the deletion mutant strains MYB44-KO-1, MYB44-KO-15 and Nipponbare plants inoculated with root-knot nematodes of the Poaceae family; a is the number of root knots of each plant; b is the number of nematode infection of each plant; c is a schematic diagram of nematode infection in rice roots; MYB-KO-1 is MYB44-KO-1, and MYB-KO-15 is MYB44-KO-15; the t-test showed significant differences (P<0.05). DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The inventors used DNA-pull down technology and subcellular localization analysis to find that the OsMYB44 gene is a nuclear transcription factor that regulates the OsThil gene ( Figure 4 ). In order to further analyze the function of the gene in the interaction between rice and root-knot nematodes, the present invention uses CRISPR / Cas9 gene editing technology to obtain Nipponbare rice OsMYB44 gene mutant plants, and obtains a transcription factor OsMYB44 gene related to root-knot nematode susceptibility in rice through indoor inoculation verification. This gene can mediate root-knot nematode infection of rice, and constructs a CRISPR / Cas9 plant expression vector containing the sgRNA sequence of the OsMYB44 gene. The target site of the OsMYB44 gene is mutated in rice through the Agrobacterium-mediated genetic transformation system to obtain a rice plant with a stable inheritance of the OsMYB44 gene function loss, and the resistance of the plant to root-knot nematodes is significantly improved. It shows that knocking out the OsMYB44 gene can significantly improve the resistance of rice to root-knot nematodes.

[0042] In order to explain the above technical solution in more detail, a specific embodiment is further described below.

[0043] The gene sequences and primer sequences involved in the following examples are shown in Table 1 below.

[0044] Table 1 Gene and primer sequences

[0045]

[0046]

[0047] Example 1 Site-directed knockout of rice transcription factor OsMYB44 gene based on CRISPR / Cas9 gene editing technology

[0048] 1. Design of gRNA target sites

[0049] According to the sequence of OsMYB44 gene as shown in SEQ ID NO.1, a target site gRNA was designed on the exon of OsMYB44 gene, and its sequence is shown in SEQ ID NO.2 ( Figure 1 shown).

[0050] 2. Construction of plant expression vector pYLCRISPR / Cas9-Pubi-H-OsMYB44-KO

[0051] Using Overlapping PCR method:

[0052] The main purpose of the first round of PCR is to introduce gRNA into the downstream of OsU3 promoter and upstream of sgRNA sequence using pYLsgRNA-OsU3 as template. Reaction 1 is to introduce gRNA sequence into the downstream of OsU3 promoter after amplification using primer pair UF / OsMYB44-U3T1 (sequence as shown in SEQ ID NO.12); Reaction 2 is to introduce gRNA sequence into the upstream of sgRNA sequence after amplification using primer pair gR-R / OsMYB44-gRT1 (sequence as shown in SEQ ID NO.13).

[0053] The amplification reaction system is shown in Table 2 below.

[0054] Table 2 First round amplification reaction system

[0055] Reagents Dosage 2×PhantaMaxBuffer 7.5μL 10mmol / LdNTPsMix 0.25μL PhantaMaxPolymerase 0.2U YLgRNA-U3 4ng 10μmol / LU-F and OsMYB44-U3T1 (reaction 1) 0.3μL 10 μmol / L gR-R and OsMYB44-gRT1 (reaction 2) 0.3μL Sterile ultrapure water Make up to 15 μL

[0056] The reaction program was: 95°C for 5 min; 95°C for 10 s, 58°C for 15 s, 72°C for 15 s, 25 cycles; 72°C for 10 min.

[0057] Subsequently, the first round of PCR products (products of reaction 1 and reaction 2) were used as templates, and the promoter, target site and sgRNA backbone were constructed into a complete expression cassette using primer pair Pps / Pgs. The sequence after amplification is shown in SEQ ID NO.14.

[0058] The amplification reaction system is shown in Table 3 below.

[0059] Table 3 Second round amplification reaction system

[0060] Reagents Dosage 2×PhantaMaxBuffer 15μL 10mmol / LdNTPsMix 0.5μL PhantaMaxPolymerase 0.2U 10μmol / L mixed universal primers (Pps / Pgs) 0.5μL Reaction 1 + Reaction 2 dilution 0.5μL Sterile ultrapure water Make up to 30 μL

[0061] The reaction program was: 95°C for 5 min; 95°C for 10 s, 58°C for 15 s, 72°C for 20 s, 28 cycles; 72°C for 10 min.

[0062] Then, the gRNA expression cassette was cloned into pYLCRISPR / Cas9-Pubi-H to obtain the recombinant plant expression vector pYLCRISPR / Cas9-Pubi-H-OsMYB44-KO. The schematic diagram of its T-DNA structure is shown in Figure 2 .

[0063] The vectors pYLsgRNA-OsU3 and pYLCRISPR / Cas9-Pubi-H are derived from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, and have been disclosed in the document "Xingliang Ma, Qunyu Zhang, Qinlong Zhu, et al. 2015, A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant, 8(8): 1274-1284".

[0064] 3. Heat shock method for the introduction of recombinant vector into Agrobacterium tumefaciens EHA105

[0065] Referring to the EHA105 competent transformation method, the recombinant plant expression vector pYLCRISPR / Cas9-Pub-H-OsMYB44-KO constructed above was transformed into the Agrobacterium tumefaciens EHA105 strain by heat shock method. After transformation, a single colony was picked for PCR detection, and the positive clone was named EHA105-OsMYB44-CRISPR / Cas9-KO.

[0066] 4. Agrobacterium-mediated rice callus transformation

[0067] The specific method of Agrobacterium-mediated rice callus transformation is as follows:

[0068] S1. After surface disinfection, rice seeds are inoculated into an induction medium and cultured in a dark place at 30°C for about 1 month. The induced callus tissue is inoculated into a new subculture medium and can be used as material for Agrobacterium transformation after 1 week.

[0069] S2. Using the recombinant bacteria prepared above, take out the EHA105-OsMYB44-CRISPR / Cas9-KO original tube bacterial liquid from the -80℃ refrigerator, streak culture on an LB plate containing 50μg / mL rifampicin and 50μg / mL kanamycin sulfate, and culture in the dark at 28℃ for 2 days. Then, use a pipette to draw 1mL of japonica rice suspension medium, wash the Agrobacterium on the plate, add it to a triangular flask containing 50mL of suspension liquid, place it on a constant temperature shaker at 28℃ for 1.5h, adjust the OD value to about 1.0, and place it on a clean bench for use;

[0070] S3. Select callus tissue with light yellow surface, dry structure and compact structure, soak it in the bacterial suspension obtained in step S2 for 30 minutes, and then transfer it to the solid co-culture medium and co-culture it at 25°C in the dark for 3 days;

[0071] S4. Wash the co-cultured callus tissue with sterile water three times, then dry it with sterile filter paper and transfer it to the screening medium. Culture it in the dark at 30°C. After 20 days, transfer the resistant callus to a new screening medium for a second screening.

[0072] S5. The selected resistant callus was transferred to differentiation medium and cultured in a light incubator at 28°C, 16h light / 8h dark, and a light intensity of about 12000LX. After differentiation for about one and a half months, genetically transformed plants (MYB44-KO-1 and MYB44-KO-15) were obtained.

[0073] In the above-mentioned Agrobacterium tumefaciens-mediated genetic transformation of rice mature embryos, the culture medium and formulation components required are shown in Table 4 below.

[0074] Table 4 Culture medium

[0075]

[0076] Example 2 Identification of rice mutants expressing OsMYB44 gene by CRISPR / Cas9 gene editing

[0077] 1. Identification of rice mutants expressing OsMYB44 gene by CRISPR / Cas9 gene editing

[0078] Rice leaf DNA was extracted using the CTAB method, and the CRISPR / Cas9 gene-edited OsMYB44 gene rice mutant was amplified by PCR using primer pairs (MYB44 KO-F and MYB44 KO-R), and the amplified fragments were sequenced and verified (the results are shown in Figure 3 shown).

[0079] The nucleotide sequences of the primer pairs are as follows:

[0080] MYB44 KO-F:5'-ACAACTCTCGCTCATCCACA-3', SEQ ID NO.16;

[0081] MYB44 KO-R: 5'-AGCATTGGTCCTCGGTTCA-3', SEQ ID NO. 17.

[0082] The amplification reaction system is shown in Table 5 below.

[0083] Table 5 Reaction system

[0084] Reagents Dosage PrimeSTARMaxPremix (2×) 12.5μL MYB44KO-F (10 μM) 0.5μL MYB44KO-R (10 μM) 0.5μL DNA template <200ng Sterile ultrapure water Make up to 25 μL

[0085] The amplification reaction program was: 98°C for 5 min; 98°C for 10 s, 58°C for 15 s, 72°C for 10 s, 35 cycles; 72°C for 10 min.

[0086] Example 3 Resistance evaluation of rice mutants with OsMYB44 gene edited by CRISPR / Cas9

[0087] The roots of rice seedlings cultured in indoor pot experiments were used for inoculation of root-knot nematodes of the Poaceae family 14 days after sowing. Before inoculation, it was necessary to stop watering with rice complete nutrient solution (manufacturer: Yoshida, item number: HZ1040-2L) 2 days in advance to reduce the moisture in the sand and absorbent resin matrix, so as to facilitate the nematode to infect the rice root system. During inoculation, a blue gun tip was used to insert holes in the sand near the rice stems, and then a rubber-tipped dropper was used to absorb 1 mL of a suspension of about 300 second-instar larvae of the Poaceae root-knot nematodes, and then slowly dripped into the sand and absorbent resin matrix along the holes close to the rice roots. Stop watering with rice complete nutrient solution 3 days after inoculation to avoid affecting the infection of the root-knot nematodes of the Poaceae family. 14 days after the inoculation of the Poaceae root-knot nematodes, the number of nematode infections and development were counted by acid fuchsin staining, and finally the average value of multiple rice plants was taken for T-test difference analysis (P<0.05).

[0088] The results are as follows Figure 5 As shown, compared with Nipponbare rice, using CRISPR / Cas9 to edit the OsMYB44 gene in the susceptible variety Nipponbare significantly enhanced the resistance of rice to root-knot nematodes.

[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. Use of OsMYB44 protein or OsMYB44 gene in the following (1) or (2): (1) Application in improving the nematode resistance of rice; improving the nematode resistance of rice by knocking out the OsMYB44 gene in rice; (2) Application in breeding nematode-resistant transgenic rice; breeding nematode-resistant transgenic rice by knocking out the OsMYB44 gene in rice; in, The amino acid sequence of the OsMYB44 protein is shown in SEQ ID NO.15; the nucleotide sequence of the OsMYB44 gene is shown in SEQ ID NO.1; and the nematode is a root-knot nematode.

2. Use of the recombinant vector for knocking out the OsMYB44 gene in the following (1) or (2): (1) Application in improving the nematode resistance of rice; improving the nematode resistance of rice by knocking out the OsMYB44 gene in rice; (2) Application in breeding nematode-resistant transgenic rice; breeding nematode-resistant transgenic rice by knocking out the OsMYB44 gene in rice; in, The nucleotide sequence of the OsMYB44 gene is shown in SEQ ID NO.1; the nematode is a root-knot nematode.

3. Use of a recombinant bacterium containing a recombinant vector for knocking out the OsMYB44 gene in the following (1) or (2): (1) Application in improving the nematode resistance of rice; improving the nematode resistance of rice by knocking out the OsMYB44 gene in rice; (2) Application in breeding nematode-resistant transgenic rice; breeding nematode-resistant transgenic rice by knocking out the OsMYB44 gene in rice; in, The nucleotide sequence of the OsMYB44 gene is shown in SEQ ID NO.1; the nematode is a root-knot nematode.

4. A method for improving the nematode resistance of rice, characterized in that: The method comprises the steps of knocking out the OsMYB44 gene in rice to improve the nematode resistance of the rice; the nucleotide sequence of the OsMYB44 gene is shown in SEQ ID NO.1; and the nematode is a root-knot nematode.

5. A method for cultivating nematode-resistant transgenic rice, characterized in that: The method comprises the steps of knocking out the OsMYB44 gene in rice to improve the nematode resistance of the rice; the nucleotide sequence of the OsMYB44 gene is shown in SEQ ID NO.1; and the nematode is a root-knot nematode.

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