Application of rice receptor-like protein OsBIR1 in resistance to rice stripe virus

Abstract

The present application relates to a rice receptor class protein OsBIR1 coding gene and its use in breeding rice resistant to rice stripe virus.

Description

Technical Field

[0001] This invention relates to the fields of transgenic technology and plant virus disease control, specifically to the application of a rice receptor protein, OsBIR1, in plant resistance to rice stripe virus. Technical Background

[0002] Rice stripe virus (RSV) is one of the most serious viruses affecting rice production in my country. In its natural environment, rice is the primary host plant for RSV, but it can also infect various monocotyledonous crops such as wheat (Triticum aestivum), barley (Hordeum vulgare), and maize (Zea mays). The hemipteran insect, the brown planthopper (Laodelphax striatellus, SBPH), is an important vector for RSV. It is a destructive pest that feeds on the phloem of plants using its piercing-sucking stylet. Within the brown planthopper, RSV spreads through continuous and cyclical reproduction. After RSV infects rice, the first signs are mild stunting of the plant, followed by yellow-green stripes on the leaves. These stripes run parallel to the veins, with the spaces between the stripes remaining green, forming a typical striped mosaic pattern. In addition, the leaf tips of some leaves may become twisted, and the leaves may become stiff, wrinkled, or even distorted. The seed setting rate will decrease significantly, often producing empty grains, which seriously affects rice yield. RSV is a typical member of the genus *Schizoviravica*, a multispecific negative-sense single-stranded RNA virus. Its viral particles exhibit a filamentous structure under an electron microscope, with a diameter of approximately 3–8 nm, and lack an envelope. RSV consists of four single-stranded RNA strands, with a total length of approximately 17 kb, named RNA1, RNA2, RNA3, and RNA4 according to their molecular weight. These RNA strands have different coding schemes: RNA1 uses an antisense coding scheme, while RNA2, RNA3, and RNA4 employ a ambiguous coding strategy. These four RNA strands collectively encode seven viral proteins, including RdRp, P2, PC2, P3, PC3, P4, and PC4, each playing a different physiological role during RSV infection.

[0003] Plant immune responses and growth and development often rely on similar or overlapping cellular mechanisms for signal recognition and transduction via transmembrane receptors. Receptor proteins (RLPs), as an important class of cell surface receptors, have been found to be widely present in higher plants. Structurally, RLPs can be divided into the following seven distinct protein domains: signal peptide, cysteine-rich domain, eLRR domain, variable domain, acidic amino acid domain, transmembrane domain, and short cytoplasmic region. RLPs are important cell membrane receptors involved in plant growth, development, and immune responses. Because these proteins themselves lack kinase domains, they cannot independently transmit information after specifically recognizing external pathogens. They require the recruitment of RLKs to form signal transduction complexes, thereby activating downstream pathways and mediating plant responses to external signals. Existing research has extensively reported on the functions and roles of RLKs, but research on RLPs is relatively limited, especially on their role in disease resistance and defense. Summary of the Invention

[0004] Based on the deficiencies of the existing technology, the present invention relates to a rice receptor-like protein OsBIR1 gene and its application;

[0005] On the one hand, the OsBIR1 gene has the nucleotide sequence shown in SEQ ID NO.1, as follows:

[0006] In some embodiments, the OsBIR1 gene encodes a protein with the amino acid sequence shown in SEQ ID NO.2, as follows:

[0007] MSFDTGLLFWLLLLSSSSSLCFGSELDIQCLKSVKRSLIDPSGILRSSWKFSQDGTTNSICNF

[0008] MGVICWNPDENRILGLSLGSLGLQGQFPRGLEHCTSLVRLDLSNNSLSGPIPSGISWQLPDL

[0009] SSLNLSYNRFSGEIPVNISEMTYLYSIGLQHNKLTGSIPGKFALLSRLESFNVSDNLLSGPIPV

[0010] ALSKFSTSCFSGNQGLCGVPFDSCSTSYGDYSIGIIGAAVGFVVGFVGALYISHCLFFLRDAPALRLSHT.

[0011] On the other hand, this application relates to the use of a rice receptor protein OsBIR1 gene in rice breeding for resistance to rice stripe virus (RSV), the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] In some embodiments, this application also relates to the use of a rice receptor protein in rice breeding for resistance to rice stripe virus (RSV), said rice receptor protein being a protein encoded by the OsBIR1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] On the other hand, this application relates to a method for preparing rice resistant to rice stripe virus (RSV), the steps of which include: transferring a vector overexpressing the rice receptor protein OsBIR1 gene into the rice plant.

[0014] In some embodiments, the preparation method further includes:

[0015] 1) Construction of rice OsBIR1 gene overexpression vector;

[0016] 2) Rice genetic transformation, Agrobacterium-mediated transformation and callus induction culture

[0017] 3) Identification of positive transgenic plants;

[0018] In some embodiments, the construction steps of the rice OsBIR1 gene overexpression vector include:

[0019] Primers with restriction enzyme sites were designed for the construction of the OsBIR1 binary expression vector PCV1300. Positive clones were selected for sequencing, and the successful construction of the PCV1300-OsBIR1 expression vector was confirmed.

[0020] In some embodiments, the rice genetic transformation, Agrobacterium-mediated transformation, and callus induction culture steps further include:

[0021] 1) Rice callus culture: Mature rice seeds were dehulled, soaked in 75% alcohol for 10 minutes, and rinsed three times with sterile water; then soaked in 30% sodium hypochlorite solution for 30 minutes; rinsed with sterile water and soaked for another 30 minutes. Using sterile forceps, the seeds were placed in mature embryo induction medium and cultured at 28°C under light for 3 weeks. The resulting callus tissue was then transferred to subculture medium using sterile forceps and subcultured at 28°C under light for 1 week.

[0022] 2) Transformation and culture of Agrobacterium: Plasmid containing the target vector, stored at -80℃, was used to transform Agrobacterium rhizogenes GV3101 (Shanghai Weidi, catalog number: AC1003S). Detailed operating steps are as follows: First, 5 μL of plasmid was added to 100 μL of competent cells, mixed thoroughly by pipetting, and then added to an electrode cup pre-cooled to 4℃. Electroporation was performed at 220V. 600 μL of antibiotic-free LB liquid medium was added, and the mixture was incubated at 28℃ with shaking for approximately 3-4 hours. The mixture was then evenly spread onto LB solid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and incubated at 28℃ for 3 days.

[0023] 3) Agrobacterium infection: Prepare the infection solution. Rinse the Agrobacterium off the plate with the infection solution. Place the cultured callus tissue into a 100ml sterile Erlenmeyer flask, add an appropriate amount of Agrobacterium suspension, and incubate at room temperature for 20 minutes, shaking several times during the infection period. Discard the bacterial solution, place the callus tissue on sterile filter paper to absorb excess bacterial solution, and then transfer it to a solid co-culture medium lined with sterile filter paper. Incubate at 26℃ in the dark for 3 days.

[0024] 4) Screening and culture of resistant callus: The callus tissue was placed on a medium containing hygromycin B and screened after 30-45 days. The selected callus tissue was placed in a rooting medium and cultured until green plants with roots were produced. After 2 weeks of culture, transgenic rice lines were obtained.

[0025] On the other hand, the present invention also relates to a procedure for identifying rice resistant to rice stripe virus (RSV), comprising: extracting total RNA from positive transgenic plants and Zhonghua 11 rice using the TRIzol method, and reverse transcribing the RNA into cDNA using a reverse transcription kit. The quantitative primer used is qRT-OsBIR1, and the relative expression level of the OsBIR1 gene is measured to determine whether the plant is a positive transgenic plant.

[0026] In some implementations, the detection primer sequences are as follows:

[0027] qRT-OsBIR1-F:ATTCCAGGGAAATTCGCTCT SEQ ID NO.3

[0028] qRT-OsBIR1-R:TACAAGGCTCCCACAAAACC SEQ ID NO.4

[0029] qRT-OsUBQ5-F:ACCACTTCGACCGCCACTACT SEQ ID NO.5

[0030] qRT-OsUBQ5-R:ACGCCTAAGCCTGCTGGTT SEQ ID NO.6

[0031] On the other hand, the present invention also relates to a quantitative primer for detecting the OsBIR1 gene, the nucleotide sequence of which is shown in SEQ ID NO.3-SEQ ID NO.4.

[0032] On the other hand, the present invention also relates to a method for detecting rice stripe virus in rice, wherein the nucleotide sequence of the detection primers is shown below:

[0033] qRSV-CP-F:AGGCAATCAATGACATCTCC SEQ ID NO.11

[0034] qRSV-CP-R:ATCTCTCAAAAGCCAGTGC SEQ ID NO.12

[0035] On the other hand, the present invention also relates to a method for obtaining transgenic plants, the method comprising: constructing the OsBIR1 gene into a plant binary expression vector, introducing it into an Agrobacterium strain by electroporation; and obtaining transgenic plants overexpressing the OsBIR1 gene by leaf disc method.

[0036] In some embodiments, the amino acid sequence of the OsBIR1 gene is as shown in SEQ ID NO.2.

[0037] In some embodiments, the nucleotide sequence of the OsBIR1 gene is as shown in SEQ ID NO.1.

[0038] In some embodiments, the plant binary expression vector is PCV1300.

[0039] To achieve the above-mentioned objectives, the present invention employs the following techniques:

[0040] This invention targets the receptor protein OsBIR1. An overexpression vector, pCV1300-OsBIR1, is constructed. This vector is then transformed into mature rice Zhonghua 11 (ZH11) embryos using rice callus transfection technology, ultimately inducing callus formation. After obtaining T0 generation transgenic rice seeds, propagation continues to yield T1 generation seeds. The expression level of OsBIR1 in rice is detected using real-time quantitative PCR, resulting in stably inherited high-expression transgenic plants. Resistance of the OsBIR1 transgenic plants is assessed through artificial RSV inoculation. Results show that, compared to the wild type, RSV-inoculated OsBIR1 overexpression transgenic plants exhibit significantly reduced symptoms, disease incidence, and viral RNA expression levels. This indicates that OsBIR1 overexpression significantly enhances rice resistance to RSV infection. This invention has significant application value in breeding new rice varieties, especially those resistant to viral diseases, and also holds important application prospects in the field of plant disease control.

[0041] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0042] (1) In this invention, the OsBIR1 gene is overexpressed to obtain transgenic rice with stable inheritance, and the resistance of OsBIR1 overexpressing plants to rice stripe virus disease is analyzed. The results further enrich the germplasm resource bank of rice stripe virus disease resistance.

[0043] (2) This invention is the first to study the relationship between rice stripe virus and the receptor protein OsBIR1. Previous studies have not reported in detail on the role of receptor proteins in the disease resistance process. This study provides new genes and germplasm resources for rice genetic engineering for disease resistance breeding, and at the same time provides important theoretical basis and new strategies for better control of rice viral diseases. Attached Figure Description

[0044] Figure 1 The relative expression level of OsBIR1 in transgenic rice overexpressing OsBIR1.

[0045] Figure 2 Comparison of disease symptoms in OsBIR1 overexpressing transgenic and control Zhonghua 11 30 days after RSV infection.

[0046] Figure 3 Comparison of the incidence rates of OsBIR1 overexpressing transgenic and control Zhonghua 11 30 days after RSV infection.

[0047] Figure 4 The virus content of OsBIR1 overexpressing transgene and control Zhonghua 11 was detected by qRT-PCR 30 days after RSV infection.

[0048] Figure 5 Figure 1: Viral protein detection results of OsBIR1 overexpressing transgene and control Zhonghua 11 30 days after RSV infection. Specific implementation methods

[0049] Example 1: Construction of rice OsBIR1 plant expression vector

[0050] (1) Cloning of the rice OsBIR1 gene

[0051] Primers OsBIR1-F and OsBIR1-R were designed based on the sequence of OsBIR1 (SEQ ID NO.1). The primer sequences used are as follows:

[0052] OsBIR1-F:ATGTCTTTTGACACCGGGCTT SEQ ID NO.7

[0053] OsBIR1-R:TAGACGTAGGGCCGGCCGTC SEQ ID NO.8

[0054] PCR amplification system: Total volume 50 μL, including 25 μL 2×PCR buffer, 1.5 μL forward and reverse primers (10 μM), 5 μL dNTP Mix (2.5 mM), 1 μL cDNA template, 1 μL KOD enzyme (5 U / μL), and 15 μL ddH2O.

[0055] PCR program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min.

[0056] The PCR product was recovered, ligated into the pMD18-T vector, clones were selected, and sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing to confirm that the correct pMD18-T-OsBIR1 recombinant plasmid was obtained.

[0057] (2) Construction of overexpression vectors

[0058] Primers with restriction enzyme sites were designed for the construction of the OsBIR1 binary expression vector PCV1300 (a conventional expression vector). Positive clones were selected for sequencing, and the successful construction of the PCV1300-OsBIR1 expression vector was confirmed.

[0059] The primer sequences are as follows:

[0060] PCV-OsBIR1-F:GTTCCAGATTACGCTGGATCCATGTCTTTTGACACCGGGCTT

[0061] SEQ ID NO.9

[0062] PCV-OsBIR1-R:ATCGGGGAAATTCGAGCTCTAGACGTAGGGCCGGCGCGTC

[0063] SEQ ID NO.10

[0064] Example 2: Genetic transformation of rice

[0065] 1) Rice callus culture: Mature rice seeds were dehulled, soaked in 75% alcohol for 10 minutes, and rinsed three times with sterile water; then soaked in 30% sodium hypochlorite solution for 30 minutes; rinsed with sterile water and soaked for another 30 minutes. Using sterile forceps, the seeds were placed in mature embryo induction medium and cultured at 28°C under light for 3 weeks. The resulting callus tissue was then transferred to subculture medium using sterile forceps and subcultured at 28°C under light for 1 week.

[0066] 2) Transformation and culture of Agrobacterium: Agrobacterium rhizogenes GV3101 (Shanghai Weidi, catalog number: AC1003S), containing the target vector and stored at -80℃, was transformed. Detailed operating steps are as follows: First, 5 μL of plasmid was added to 100 μL of competent cells, mixed thoroughly by pipetting, and then added to an electrode cup pre-cooled to 4℃. Electroporation was performed at 220V. 600 μL of antibiotic-free LB liquid medium was added, and the mixture was incubated at 28℃ with shaking for approximately 3-4 hours. The mixture was then evenly spread onto LB solid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and incubated at 28℃ for 3 days.

[0067] 3) Agrobacterium infection: Prepare the infection solution. Rinse the Agrobacterium off the plate with the infection solution. Place the cultured callus tissue into a 100ml sterile Erlenmeyer flask, add an appropriate amount of Agrobacterium suspension, and incubate at room temperature for 20 minutes, shaking several times during the infection period. Discard the bacterial solution, place the callus tissue on sterile filter paper to absorb excess bacterial solution, and then transfer it to a solid co-culture medium lined with sterile filter paper. Incubate at 26℃ in the dark for 3 days.

[0068] 4) Screening and culture of resistant callus: The callus tissue was placed on a medium containing hygromycin B and screened after 30-45 days. The screened callus tissue was placed in a rooting medium and cultured until green plants with roots were produced. After 2 weeks of culture, transgenic rice lines OsBIR1-4# and OsBIR-7# were obtained.

[0069] Example 3: Identification of positive results in transgenic rice

[0070] Total RNA was extracted from positive transgenic plants and Zhonghua 11 rice using the TRIzol method, and reverse transcription was performed using a 5* reverse transcription kit. III qRT Super Mix (manufacturer: Novizan, catalog number: R323-01) reverse transcribes RNA into cDNA. Quantitative primers use qRT-OsBIR1, and the relative expression level of the OsBIR1 gene is as follows: Figure 1 As shown.

[0071] qRT-OsBIR1-F:ATTCCAGGGAAATTCGCTCT SEQ ID NO.3

[0072] qRT-OsBIR1-R:TACAAGGCTCCCACAAAACC SEQ ID NO.4

[0073] qRT-OsUBQ5-F:ACCACTTCGACCGCCACTACT SEQ ID NO.5

[0074] qRT-OsUBQ5-R:ACGCCTAAGCCTGCTGGTT SEQ ID NO.6

[0075] Example 4: Transgenic rice inoculated with RSV

[0076] 1) After soaking and germinating the OsBIR1 transgenic and control Zhonghua 11 rice materials for 2-3 days, the seeds were sown in 1L beakers after they turned white, with about 20 seedlings per beaker. Three biological replicates were made and cultured at 30℃ under 16h light and 8h dark conditions.

[0077] 2) Newly hatched 1st-2nd instar non-toxic planthoppers were infected with RSV on rice seedlings for 3 days. After the insects were removed from healthy rice seedlings and allowed to circulate for 10 days, usable infected planthoppers were obtained. Artificial inoculation experiments were conducted, with 3 insects per seedling. Infected / non-toxic planthoppers were inoculated on rice seedlings at the three- to four-leaf stage. After feeding for 3 days, the insects were removed.

[0078] 3) After 30 days, observe the symptoms of diseased rice and determine the virus-carrying status of rice by qRT-PCR.

[0079] Example 5: Resistance analysis of rice after RSV inoculation

[0080] 1) Statistics on incidence rates: such as Figure 3 As shown, the incidence rates in the two OsBIR1 overexpression transgenic lines (OsBIR1-4# and OsBIR-7#) were 26% and 14%, respectively, while the incidence rate in the control was 38%. These results indicate that overexpression of OsBIR1 significantly reduces the incidence rate of transgenic diseases.

[0081] 2) qRT-PCR detection of virus content: 30 days after transplanting, RSV-infected rice showed symptoms of heart rot. Compared with the control Zhonghua 11, the two transgenic lines overexpressing OsBIR1 showed significant resistance to RSV. Figure 2 As shown in Example 3, total RNA was extracted from rice materials and reverse transcribed into cDNA, using the rice OsUBQ5 gene as an internal control. The expression level of the RSV CP gene was further detected by qRT-PCR. Figure 4 As shown, the expression level of the CP gene in the transgenic lines RSV was significantly lower than that in ZH11; Figure 5 As shown, its protein expression level was also lower than that of ZH11; the above results indicate that the resistance of transgenic plants to RSV infection is affected by the OsBIR1 gene, and overexpression of OsBIR1 in rice can significantly enhance the resistance of rice to RSV infection.

[0082] The quantitative primer sequences are as follows:

[0083] qRSV-CP-F:AGGCAATCAATGACATCTCC SEQ ID NO.11

[0084] qRSV-CP-R:ATCTCTCAAAAGCCAGTGC SEQ ID NO.12

[0085] qRT-OsUBQ5-F:ACCACTTCGACCGCCACTACT SEQ ID NO.5

[0086] qRT-OsUBQ5-R:ACGCCTAAGCCTGCTGGTT SEQ ID NO.6.

Claims

1. The use of a rice OsBIR1 gene in rice breeding resistant to Tenuivirus, wherein the nucleotide sequence of the OsBIR1 gene is shown in SEQ ID NO.

1.

2. The use as described in claim 1, wherein the anti-tenuivirus is rice stripe virus (RSV).

3. A method for obtaining a transgenic plant, the method comprising: The OsBIR1 gene as described in claim 1 was constructed into a plant binary expression vector and introduced into Agrobacterium strain by electroporation; Transgenic plants overexpressing the OsBIR1 gene were obtained using the leaf disc method.

4. The method of claim 3, wherein the amino acid sequence of the OsBIR1 gene is as shown in SEQ ID NO.

2.

5. The method according to any one of claims 3-4, wherein the nucleotide sequence of the OsBIR1 gene is as shown in SEQ ID NO.

1.

6. The method according to any one of claims 3-5, wherein the plant binary expression vector is PCV1300.

7. A method for identifying rice resistant to rice stripe virus (RSV), comprising the following steps: Total RNA was extracted from the test plants and Zhonghua 11 rice using the TRIzol method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. The quantitative primer used was qRT-OsBIR1, and the control primer used was qRT-OsUBQ5. The expression level of the OsBIR1 gene relative to the control gene was measured to determine whether the rice plant was resistant to rice stripe virus. The sequences of the quantitative and control primers are as follows: qRT-OsBIR1-F:ATTCCAGGGAAATTCGCTCT SEQ ID NO.3 qRT-OsBIR1-R:TACAAGGCTCCCACAAAACC SEQ ID NO.4 qRT-OsUBQ5-F:ACCACTTCGACCGCCACTACT SEQ ID NO.5 qRT-OsUBQ5-R:ACGCCTAAGCCTGCTGGTT SEQ ID NO.6.

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