Application of rice OsRING83 gene in improving rice blast resistance

Abstract

The application discloses application of a rice OsRING83 gene in improving rice blast resistance, and belongs to the field of genetic engineering. The application constructs transgenic rice by overexpressing the OsRING83 gene in the rice, and finds that overexpression of the OsRING83 gene significantly improves the resistance of the rice to the blast fungus through disease resistance detection. Therefore, the OsRING83 gene can be introduced into the rice as a target gene to improve the disease resistance of the rice and improve the rice variety. The application also proves that the protein expressed by the introduced OsRING83 gene effectively improves the defense ability of the rice by promoting the mRNA level of a defense-related gene OsPR10, and has important significance for rice improvement and breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the application of the rice OsRING83 gene in improving rice blast resistance. Background Technology

[0002] To defend against pathogen attacks, plants have evolved a highly complex immune system that requires strict regulation to initiate a powerful defense response while preventing autoimmunity. The ubiquitin-protease system, responsible for degrading over- or misfolded proteins, plays a crucial role in ensuring the initiation of a potent immune response. E3 ubiquitin ligases, as key components, play an important role in rice immunity by regulating the ubiquitination and degradation of downstream substrates involved in various immune signaling pathways.

[0003] Mounting evidence suggests that E3 ubiquitin ligase-mediated ubiquitination is involved in pattern-triggered immunity (PTI) in rice against multiple pathogen-associated molecular patterns. Most identified rice RING (Really Interesting New Gene) type E3 ubiquitin ligases are positive immunomodulators. OsRING113 positively regulates chitin-induced mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) bursts, and defense gene expression, promoting rice resistance to rice blast and bacterial blight. E3 ubiquitin ligases play an indispensable role in effector-triggered immunity (ETI) by binding to plant nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) or pathogen effector proteins. During bacterial blight infection, receptor-like protein kinase XA21 activates the E3 ubiquitin ligase XB3 via phosphorylation. XB3 promotes the accumulation of XA21 protein, thereby promoting XA21-mediated resistance to bacterial blight ETI. Although APIP10 positively regulates rice blast PTI, it negatively regulates Piz-t-mediated ETI by promoting the degradation of the NLR protein Piz-t. Knocking down APIP10 in the Piz-t background enhances rice resistance to the susceptible race RB22.

[0004] Based on current research findings, given that E3 ubiquitin ligases play a regulatory role in both immune and growth-related processes, various E3 ubiquitin ligases show great potential for fine-tuning rice growth and immunity in breeding programs. Furthermore, E3 ligases targeted by pathogen effectors are also potential targets for genetic engineering improvement in breeding programs. In the future, the identification of novel E3 ubiquitin ligases that simultaneously regulate rice PTI and ETI will provide entirely new genetic resources for breeding disease-resistant rice varieties through biotechnologies such as genome editing. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the rice OsRING83 gene in improving rice blast resistance, thereby solving the problems existing in the prior art. By overexpressing the OsRING83 gene in rice and increasing the expression level of the OsRING83 gene, rice blast resistance can be significantly improved, providing a new target for rice blast resistance and laying a theoretical foundation for breeding new varieties with high resistance to rice blast.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of the OsRING83 gene in any of the following:

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

[0009] (2) Application in the breeding of new rice varieties with high resistance to rice blast;

[0010] The nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.1.

[0011] This invention also provides the use of the protein encoded by the OsRING83 gene in any of the following:

[0012] (1) Application in improving rice blast resistance;

[0013] (2) Application in the breeding of new rice varieties with high resistance to rice blast;

[0014] The nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0015] The present invention also provides the use of a recombinant vector containing the OsRING83 gene in any of the following:

[0016] (1) Application in improving rice blast resistance;

[0017] (2) Application in the breeding of new rice varieties with high resistance to rice blast;

[0018] The recombinant vector was constructed by introducing the OsRING83 gene into an expression vector, and the nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.1.

[0019] The present invention also provides the use of recombinant engineered bacteria comprising the recombinant vector in any of the following:

[0020] (1) Application in improving rice blast resistance;

[0021] (2) Application in the cultivation of new rice varieties with high resistance to rice blast.

[0022] Preferably, the resistance of rice to rice blast is improved by overexpressing the OsRING83 gene in rice.

[0023] The present invention also provides a method for improving rice blast resistance, comprising overexpressing the OsRING83 gene in rice to increase the expression level of the OsRING83 gene, thereby improving the rice's resistance to rice blast; wherein the nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.1.

[0024] Preferably, the overexpression includes the following steps:

[0025] The OsRING83 gene was ligated to an expression vector to construct a recombinant vector, which was then transformed into Agrobacterium to construct a recombinant engineered bacterium. The recombinant engineered bacterium was then inoculated into rice to significantly increase the expression level of the OsRING83 gene.

[0026] The present invention also provides a method for breeding transgenic rice with high resistance to rice blast, comprising overexpressing the OsRING83 gene in rice to increase the expression level of the OsRING83 gene and obtain transgenic rice with high resistance to rice blast; wherein the nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.1.

[0027] Preferably, the overexpression includes the following steps:

[0028] The OsRING83 gene was ligated to an expression vector to construct a recombinant vector, which was then transformed into Agrobacterium to construct a recombinant engineered bacterium. The recombinant engineered bacterium was then inoculated into rice to significantly increase the expression level of the OsRING83 gene.

[0029] The present invention discloses the following technical effects:

[0030] This invention significantly reduced the area of ​​lesions and the fungal biomass in lesion regions by overexpressing the rice E3 ubiquitin ligase gene OsRING83 in rice, thereby improving rice resistance to rice blast. Transgenic experiments demonstrated that overexpression of the OsRING83 gene enhanced rice resistance to rice blast fungus. Therefore, OsRING83 can be introduced into plants as a target gene to improve plant disease resistance and facilitate plant variety improvement. The introduced OsRING83 gene also effectively enhanced plant defense capabilities by increasing the expression level of the rice OsPR10 gene. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 Map of the pRHVcGFP expression vector;

[0033] Figure 2 This study analyzed the expression level of OsRING83 in transgenic rice; NPB represents the wild-type Nipponbare rice variety, and OsRING83-GFP-1-1 and OsRING83-GFP-3-1 represent two overexpression lines transformed with NPB as the background.

[0034] Figure 3 Phenotypic diagram showing the effect of OsRING83-GFP transgenic plants on resistance to blast fungus race RB22;

[0035] Figure 4 The results show the statistical results of fungal biomass in the lesion area of ​​OsRING83-GFP transgenic plants;

[0036] Figure 5 The expression level of OsPR10 in OsRING83-GFP transgenic plants. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] Example 1: Construction of OsRING83-GFP transgenic line

[0043] 1. Extraction of total RNA

[0044] The Nipponbare japonica rice variety was selected. When the rice seedlings were about two weeks old, leaves were immediately collected, frozen in liquid nitrogen, and stored at -80 ℃. A portion of the leaves was taken, crushed in a mortar, and transferred into a 1.5 mL EP tube containing Trizol lysis buffer. After thorough shaking, total RNA was extracted, and the quality of the total RNA was assessed by electrophoresis.

[0045] 2. Cloning of the rice E3 ubiquitin ligase gene OsRING83 and construction of its plant expression vector.

[0046] Design primers at both ends:

[0047] P1: 5'-GGATCCCCGGGTGAGCTCATGGAACTAACTGGTTGGCA-3' (SEQ ID NO.3);

[0048] P2: 5'-CGCACTAGTAAGCTTGCCGTGTCGGCGGCGC-3' (SEQ ID NO. 4).

[0049] The total RNA obtained above was reverse transcribed into cDNA first strand. Using this cDNA as a template, PCR amplification was performed with high-fidelity KOD enzyme. The PCR program was as follows: 96℃ pre-denaturation for 3 min, 96℃ denaturation for 30 s, 57℃ annealing for 30 s, 68℃ extension for 1 min, 35 cycles, followed by a final extension at 68℃ for 10 min. The target fragment was then recovered. Simultaneously, the pRHVcGFP vector was double-digested with restriction endonucleases SacI and HindIII. The digested vector fragment was then recovered, and OsRING83 was further cloned into the expression vector pRHVcGFP (… Figure 1 The ligation system consisted of: Exnase II 2 µL, 5×CE buffer 2 µL, target gene 2 µL, vector 2 µL, and ddH2O 2 µL; incubated at 37 ℃ for 30 min. The ligation product was then heat-shocked at 42 ℃ to transform competent *E. coli* cells JM109. The cells were plated on LB solid medium containing 50 µg / mL kanamycin and incubated upside down at 37 ℃ for approximately 12 h. Successfully transformed single clones were picked and cultured on LB liquid medium containing 50 µg / mL kanamycin for 10–12 h. Plasmids were extracted, and sequencing was performed to confirm the correct reading frame of the coding region in the expression vector. The recombinant vector pRHV-OsRING83-GFP was obtained and sequenced to confirm the correct reading frame of the coding region in the expression vector.

[0050] 3. Obtaining transgenic plants

[0051] The recombinant vector pRHV-OsRING83-GFP obtained above was transformed into Agrobacterium strain EHA105, and further transformed into the japonica rice variety Nipponbare (NPB). After qRT-PCR verification of the obtained transgenic plants OsRING83-GFP, the transgenic rice was confirmed. The confirmation steps were as follows:

[0052] Young leaves of transgenic rice were collected, RNA was extracted and cDNA was reverse transcribed, and qRT-PCR was performed using the following primers:

[0053] OsRING83-F1: 5'-GAGTTGTCGGCTTCTGCTCT-3' (SEQ ID NO.5);

[0054] OsRING83-F2: 5'-GTGGTACGTAGCCAAGACGG-3' (SEQ ID NO. 6).

[0055] The expression level of the OsRING83 gene was detected by qRT-PCR. The qRT-PCR reaction system consisted of 1 µL each of primers OsRING83-F1 and OsRING83-F2, 10 µL of 2×SYBR qPCR Mix, and 8 µL of diluted cDNA. The reaction program was as follows: 95 ℃ pre-denaturation for 30 s, 95 ℃ for 10 s, 60 ℃ for 30 s, for 40 cycles, followed by melting curve generation at 95 ℃ for 15 s, 60 ℃ for 60 s, and 95 ℃ for 15 s. The obtained OsRING83 overexpressing plants were subcultured for disease resistance testing in rice. The overexpressing plants OsRING83-GFP-1-1 and OsRING83-GFP-3-1 were detected. Figure 2 ).

[0056] Example 2: Identification of disease resistance in OsRING83-GFP transgenic lines

[0057] Inoculation by punching holes: When the OsRING83-GFP transgenic rice reaches 6-8 weeks of leaf age, select the second leaf from the top and inoculate it with rice blast fungus (rice blast fungus physiological race RB22) by punching holes. Then, place the rice in a plant growth chamber and culture it in the dark for 24 hours. Then, transfer it to a 12-hour light / 12-hour dark condition for growth. After 10-14 days, observe the expansion of lesions, investigate the disease, and count the lesion area and biomass. Transgenic rice plants that show obvious resistance compared with the control are the transgenic plants that are resistant to rice blast.

[0058] The results showed that, compared with wild-type NPB, OsRING83 overexpression transgenic plants exhibited enhanced resistance to blast fungus race RB22. Specifically, the fungal biomass in the lesion area of ​​OsRING83 overexpression transgenic plants was significantly lower than that of the control NPB. Figures 3-4 Simultaneously, qRT-PCR was used to detect the expression of the defense-related gene OsPR10 in OsRING83 overexpressing plants. Primer design:

[0059] OsPR10 - F1: 5'-CCCTGCCGAATACGCCTAA-3' (SEQ ID NO.7);

[0060] OsPR10 - F2: 5'-CTCAAACGCCACGAGAATTTG-3' (SEQ ID NO. 8);

[0061] The expression level of the defense-related gene OsPR10 in OsRING83 overexpressing plants was detected by qRT-PCR. The qRT-PCR reaction system consisted of 1 µL each of primers OsPR10-F1 and OsPR10-F2, 10 µL of 2×SYBR qPCR Mix, and 8 µL of diluted cDNA. The reaction program was as follows: 40 cycles of pre-denaturation at 95 °C for 30 s, 95 °C for 10 s, and 60 °C for 30 s, followed by melting curve generation at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s.

[0062] qRT-PCR results showed that the expression level of the defense-related gene OsPR10 was significantly increased in OsRING83 overexpressing plants. Figure 5 The above results indicate that overexpression of the OsRING83 gene in rice enhances the resistance of rice to rice blast fungus.

[0063] The nucleotide sequence of the rice OsRING83 gene is shown in SEQ ID NO.1:

[0064] ATGGAACTAACTGGTTGGCATCTTCCAGAACCACCGTCTTCCACCGTCCGATCTGTGATCGACGGCGGCGGTGCTCACCACCCACGTTTGATAAGCAGCCAGGTAGGCCAGCCAGCGAGGCCTACCTCGCCATCACCATTGCCACCGCGCCATCGCCACCGCCACATTCCAAAAGCAGAAGCAGTCCAAGGCGAGAGACAGAGTCCGCGACATGGTTCGATGGAGTTGTCGGCTTCTGCTCTGGGTTTCGGTCTTTGTCTTGACCCTCACCCAGTTGGGGGCCTTCGTCGTCGTCTTCGGCGAGGGGGAGGACGCCATACCTCGCGTTCTGATTGCCGCCGCCGTCTTGGCTACGTACCACGTCCTGCTCACTTTCCGGTGAGCGGAGTCGGCGGCGGCTTGCGCGGGTTCCTCCACCGCGCGCCGCCGGGTTCTGCCCGGAACGACAAGGGCTGCGCGAGGCGCCACAAGACGGCGTCGAGTTCGGCGACGAGCGCGGAACCCATGGACGAGGGCGAGGCCGACCTCCTCCAGTTTCTCTTCGTCACCTCCGGTGTCTCCTGGGGAGGCATCGTGGGGGTCGTCGTCGCCGTGAACGCCTCCGTACCGGCTGCGCGGGTGTGGATGCTCCCCGGCGTCACCACGCTGGACCGGGAGCTGGGCGGCGACGACTGCTCGATGTGCCAGTACGACAAGGATGCCGGAGCCGTCGTCCGCACGCTCTCCTGCGACCACGTCTTCCACAAGGCGTGCATCGACGTGTGGCTCCGCGAGCACGGCATGGCCTGCCGACTTTGCCGCCGTACCGCTAGCTGCGTGCTGCCCTGGAAAACCGGAGGGCGCCGCCGACACGGCTGA。

[0065] The amino acid sequence encoded by the rice OsRING83 gene is shown in SEQ ID NO.2:

[0066] MELTGWHLPEPPSSTVRSVIDGGGAHHPRLISSQVGQPARPTSPSPLPPRHRHRHIPKAEAVQGERQSPRHGSMELSASALGFGLCLDPHPVGGLRRRLRRGGGRHTSRSDCRRRLGYVPRPAHFPVSGVGGGLRGFLHRAPP GSARNDKGCARRHKTASSSATSAEPMDEGEADLLQFLFVTSGVSWGGIVGVVVAVNASVPAARVWMLPGVTTLDRELGGDDCSMCQYDKDAGAVVRTLSCDHVFHKACIDVWLREHGMACRLCRRTASCVLPWKTGGRRRHG.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of the OsRING83 gene in any of the following: (1) Application in improving rice blast resistance; (2) Application in the breeding of new rice varieties with high resistance to rice blast; The nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.

1.

2. The application of the protein encoded by the OsRING83 gene in any of the following: (1) Application in improving rice blast resistance; (2) Application in the breeding of new rice varieties with high resistance to rice blast; The nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Application of recombinant vectors containing the OsRING83 gene in any of the following: (1) Application in improving rice blast resistance; (2) Application in the breeding of new rice varieties with high resistance to rice blast; The recombinant vector was constructed by introducing the OsRING83 gene into an expression vector, and the nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.

1.

4. The use of the recombinant engineered bacteria comprising the recombinant vector of claim 3 in any of the following: (1) Application in improving rice blast resistance; (2) Application in the cultivation of new rice varieties with high resistance to rice blast.

5. The application as described in any one of claims 1-4, characterized in that, The resistance of rice to rice blast was improved by overexpressing the OsRING83 gene in rice.

6. A method for improving rice blast resistance, characterized in that, The method includes overexpressing the OsRING83 gene in rice to increase the expression level of the OsRING83 gene, thereby improving the resistance of rice to rice blast; wherein the nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.

1.

7. The method as described in claim 6, characterized in that, The overexpression includes the following steps: The OsRING83 gene was ligated to an expression vector to construct a recombinant vector, which was then transformed into Agrobacterium to construct a recombinant engineered bacterium. The recombinant engineered bacterium was then inoculated into rice to significantly increase the expression level of the OsRING83 gene.

8. A method for cultivating transgenic rice with high resistance to rice blast, characterized in that, The invention includes overexpressing the OsRING83 gene in rice to increase the expression level of the OsRING83 gene and obtain transgenic rice with high resistance to rice blast; wherein the nucleotide sequence of the OsRING83 gene is shown in SEQ ID NO.

1.

9. The method as described in claim 8, characterized in that, The overexpression includes the following steps: The OsRING83 gene was ligated to an expression vector to construct a recombinant vector, which was then transformed into Agrobacterium to construct a recombinant engineered bacterium. The recombinant engineered bacterium was then inoculated into rice to significantly increase the expression level of the OsRING83 gene.

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