Application of rice receptor-like protein OsBIR1 in resisting rice stripe virus

Abstract

The invention relates to a rice receptor-like protein OsBIR1 coding gene and application thereof in rice stripe virus resistant rice breeding.

Description

Technical Field

[0001] The invention relates to the field of transgenic technology and plant virus disease prevention and control, and in particular to 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 in rice production in my country. In the natural environment, the main host plant of RSV is rice. In addition, RSV can infect a variety of monocotyledonous crops such as wheat (Triticum aestivum), barley (Hordeum vulgare), and corn (Zea mays). The hemiptera insect Laodelphax striatellus (SBPH) is an important vector of RSV. It is a destructive pest that feeds on plant phloem through piercing and sucking mouth needles. In the body of the SBPH, RSV is transmitted in a continuous and cyclical way. After RSV infects rice, the diseased plants will first show a slight dwarfing phenomenon, and yellow-green stripes will appear on the leaves. These stripes are parallel to the veins, and the stripes remain green, forming typical striped mosaic symptoms. In addition, the tips of some leaves will be twisted, and the leaves may become stiff, wrinkled, or even twisted. The fruiting rate will drop significantly, and empty grains will often be produced, which seriously affects the yield of rice and has a significant impact on the growth, development and yield of rice. RSV is a typical member of the genus Tenuivirus, a multi-partite negative-sense single-stranded RNA virus. Its virus particles show a filamentous structure under an electron microscope, with a diameter of about 3 to 8 nm and no envelope. RSV consists of four single-stranded RNAs with a total length of about 17 kb, which are named RNA1, RNA2, RNA3 and RNA4 according to their molecular weight. These RNA chains are encoded in different ways, with RNA1 encoded in an antisense way, while RNA2, RNA3 and RNA4 use a double-sense coding strategy. These four RNA chains together encode seven viral proteins, including RdRp, P2, PC2, P3, PC3, P4 and PC4, which each have different physiological functions during RSV infection.

[0003] Plant immune responses and growth and development often rely on similar or overlapping cellular mechanisms to recognize and transduce signals through transmembrane receptors. Receptor-like 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 different 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 and development and immune responses. Since the protein itself lacks a kinase domain, it cannot independently transmit information after specifically recognizing external pathogens. It needs to recruit RLKs to form a signal transduction complex, thereby activating downstream pathways and mediating the plant's response to external signals. Existing studies have reported more on the functions and effects of RLKs, but there are fewer studies on RLPs, especially its research in the process of disease resistance. Summary of the invention

[0004] Based on the defects of the prior art, the present invention relates to a rice receptor protein OsBIR1 gene and its application;

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

[0006] In some embodiments, the amino acid sequence of the protein encoded by the OsBIR1 gene is shown in SEQ ID NO.2, which is as follows:

[0007] MSFDTGLLFWLLLLSSSSSLCFGSELDIQCLKSVKRSLIDPSGILRSSWKFSQDGTTNSICNF

[0008] MGVICWNPDENRILGLSLGSLGLQGQFPRGLEHCTSLVRLDLSNNSLSGPIPSGISWQLPDL

[0009] SSLNLSYNRFSGEIPVNISEMTYLYSIGLQHNKLTGSIPGKFALLSRLESFNVSDNLLSGPIPV

[0010] ALSKFSTSCFSGNQGLCGVPFDSCSTSYGDYSIGIIGAAVGFVVGFVGALYISHCLFFLRDAPALRLSHT.

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

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

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

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

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

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

[0017] 3) Identification of positive transgenic plants;

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

[0019] Primers with restriction sites were designed for the construction of the OsBIR1 binary expression vector PCV1300, and positive clones were selected for sequencing to confirm the successful construction of the PCV1300-OsBIR1 expression vector.

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

[0021] 1) Rice callus culture: Remove the shells of mature rice seeds, soak them in 75% alcohol for 10 minutes, wash them with sterile water three times, soak them in 30% sodium hypochlorite solution for 30 minutes, wash them with sterile water and soak them again for 30 minutes. Use sterile tweezers to place the seeds in mature embryo induction medium, culture them in a 28℃ light incubator for 3 weeks, transfer the grown callus to the subculture medium with sterile tweezers, and subculture them in a 28℃ light incubator for 1 week;

[0022] 2) Transformation and cultivation of Agrobacterium: Take the plasmid containing the target vector stored in a -80℃ refrigerator to transform Agrobacterium tumefaciens GV3101 (Shanghai Weidi, catalog number: AC1003S). The detailed operation steps are as follows: first, take 5μL of plasmid and add it to 100μL of competent cells, mix it by pipetting, add it to the electrode cup pre-cooled at 4℃, and perform electroporation transformation at a voltage of 220V. Add 600μL of antibiotic-free LB liquid culture medium, shake and culture it in a constant temperature shaker at 28℃ for about 3-4h, evenly spread it on LB solid culture medium containing 50μg / ml Kan and 50μg / ml Rif, and culture it in a 28℃ incubator for 3d;

[0023] 3) Agrobacterium infection: Prepare the infection solution, rinse the Agrobacterium on the plate with the infection solution, place the cultured callus in a 100ml sterile Erlenmeyer flask, add an appropriate amount of Agrobacterium suspension, and infect at room temperature for 20 minutes, shaking several times during the infection. Pour out the bacterial solution, place the callus on sterile filter paper to absorb the excess bacterial solution, and then transfer it to a solid co-culture medium covered with sterile filter paper, and culture it in the dark at 26℃ for 3 days;

[0024] 4) Screening and cultivation of resistant callus: Place callus tissue on a culture medium containing hygromycin B and screen after 30-45 days. Culture the screened callus tissue in a rooting medium until green plants with roots are produced. Culture for 2 weeks to obtain transgenic rice lines.

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

[0026] In some embodiments, the detection primer sequence is 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

[0032] On the other hand, the present invention also relates to a quantitative primer for detecting the OsBIR1 gene, and the nucleotide sequence of the quantitative primer is shown in SEQ ID NO.3-SEQ ID NO.4.

[0033] 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 primer is as follows:

[0034] qRSV-CP-F:AGGCAATCAATGACATCTCC SEQ ID NO.11

[0035] qRSV-CP-R:ATCTCTCAAAAGCCAGTGC SEQ ID NO.12

[0036] 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, and introducing it into an Agrobacterium strain by electroporation; and obtaining a transgenic plant overexpressing the OsBIR1 gene by a leaf disc method.

[0037] In some embodiments, the amino acid sequence of the OsBIR1 gene is as shown in SEQ ID NO.2.

[0038] In some embodiments, the nucleotide sequence of the OsBIR1 gene is as shown in SEQ ID NO.1.

[0039] In some embodiments, the plant binary expression vector is PCV1300.

[0040] In order to achieve the above-mentioned invention object, the present invention adopts the following technologies to achieve it:

[0041] The present invention takes the receptor protein OsBIR1 as the object, constructs an overexpression vector of pCV1300-OsBIR1, further transforms the constructed expression vector into mature rice Zhonghua 11 (ZH11) embryo using rice callus transfection technology, and finally induces callus formation. After obtaining T0 generation transgenic rice seeds, continue to expand and obtain T1 generation seeds, and detect the expression amount of OsBIR1 in rice by real-time fluorescence quantitative PCR technology, so as to obtain stable inheritance high-expression transgenic plants. Through artificial inoculation RSV experiment, the resistance of OsBIR1 transgenic plants is evaluated. The results show that compared with the wild type, the symptoms, morbidity and virus expression amount of the diseased plants of the OsBIR1 overexpression transgenic plants inoculated with RSV are significantly reduced. This shows that the OsBIR1 overexpression plant can significantly enhance the resistance of rice to RSV infection. The present invention has very important application value for us to cultivate new rice varieties, especially to cultivate anti-viral rice, and also has important application prospects in the field of plant disease prevention and control.

[0042] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0043] (1) The present invention overexpresses the OsBIR1 gene to obtain stably inherited transgenic rice, and analyzes the resistance of the OsBIR1 overexpressing plants to rice stripe disease. The research results further enrich the germplasm resource pool of rice stripe disease resistance.

[0044] (2) This study is the first to study the relationship between rice stripe virus and receptor protein OsBIR1. Previous studies have not reported in detail the role of receptor proteins in disease resistance. This study provides new genes and germplasm resources for rice genetic engineering disease resistance breeding, and also provides an important theoretical basis and new strategy for better prevention and control of rice virus diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Relative expression level of OsBIR1 in OsBIR1 overexpressing transgenic rice.

[0046] Figure 2 : Comparison of disease symptoms of OsBIR1 overexpressing transgenic and control Zhonghua 11 30 days after RSV infection.

[0047] Figure 3 : Comparison of the incidence of Hua11 in the OsBIR1 overexpressing transgenic plant and the control 30 days after RSV infection.

[0048] Figure 4 : qRT-PCR detection of virus content in Hua11 of OsBIR1 overexpressing transgenic plants and controls 30 days after RSV infection.

[0049] Figure 5 : Virus protein detection results of OsBIR1 overexpressing transgenic and control Zhonghua 11 30 days after RSV infection. Specific implementation methods

[0051] Example 1: Construction of rice OsBIR1 plant expression vector

[0052] (1) Cloning of the rice OsBIR1 gene

[0053] 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:

[0054] OsBIR1-F:ATGTCTTTTGACACCGGGCTT SEQ ID NO.7

[0055] OsBIR1-R:TAGACGTAGGGCCGGCCGTC SEQ ID NO.8

[0056] PCR amplification system: total volume 50 μL, including 25 μL 2×PCR buffer, 1.5 μL upstream and downstream 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.

[0057] PCR program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min.

[0058] The PCR product was recovered, connected to the pMD18-T vector, cloned, and sent to Hangzhou Youkang Biological Company for sequencing to confirm that the correct pMD18-T-OsBIR1 recombinant plasmid was obtained.

[0059] (2) Construction of overexpression vector

[0060] Primers with restriction sites were designed for the construction of OsBIR1 binary expression vector PCV1300 (conventional expression vector), and positive clones were selected for sequencing to confirm the successful construction of PCV1300-OsBIR1 expression vector.

[0061] The primer sequences are as follows:

[0062] PCV-OsBIR1-F:GTTCCAGATTACGCTGGATCCATGTCTTTTGACACCGGGCTT

[0063] SEQ ID NO.9

[0064] PCV-OsBIR1-R:ATCGGGGAAATTCGAGCTCTAGACGTAGGGCCGGCGCGTC

[0065] SEQ ID NO.10

[0066] Example 2: Rice genetic transformation

[0067] 1) Rice callus culture: Remove the shells of mature rice seeds, soak them in 75% alcohol for 10 minutes, wash them with sterile water three times, soak them in 30% sodium hypochlorite solution for 30 minutes, wash them with sterile water and soak them again for 30 minutes. Use sterile tweezers to place the seeds in a mature embryo induction medium, culture them in a 28°C light incubator for 3 weeks, transfer the grown callus to a subculture medium with sterile tweezers, and subculture them in a 28°C light incubator for 1 week.

[0068] 2) Transformation and cultivation of Agrobacterium: Take the plasmid containing the target vector stored in a -80℃ refrigerator to transform Agrobacterium tumefaciens GV3101 (Shanghai Weidi, catalog number: AC1003S). The detailed operation steps are as follows: first, take 5μL of plasmid and add it to 100μL of competent cells, mix it by pipetting, add it to the electrode cup pre-cooled at 4℃, and perform electroporation transformation at a voltage of 220V. Add 600μL of antibiotic-free LB liquid culture medium, shake and culture it at a constant temperature shaker at 28℃ for about 3-4h, evenly spread it on LB solid culture medium containing 50μg / ml Kan and 50μg / ml Rif, and culture it in a 28℃ incubator for 3d.

[0069] 3) Agrobacterium infection: Prepare the infection solution, rinse the Agrobacterium on the plate with the infection solution, place the cultured callus in a 100 ml sterile Erlenmeyer flask, add an appropriate amount of Agrobacterium suspension, and infect at room temperature for 20 minutes, shaking several times during the infection. Pour out the bacterial solution, place the callus on sterile filter paper to absorb the excess bacterial solution, and then transfer it to a solid co-culture medium covered with sterile filter paper, and culture it in the dark at 26°C for 3 days.

[0070] 4) Screening and cultivation of resistant callus: The callus tissue was placed on a culture medium containing hygromycin B and screened after 30-45 days. The screened callus tissue was cultured in a rooting medium until green plants with roots were produced. After 2 weeks of cultivation, transgenic rice lines OsBIR1-4# and OsBIR-7# were obtained.

[0071] Example 3: Identification of positive transgenic rice

[0072] The total RNA of positive transgenic plants and Zhonghua 11 rice was extracted by TRIzol method and reverse transcription kit 5* The RNA was reverse transcribed into cDNA using III qRT Super Mix (manufacturer: Novozymes, catalog number: R323-01). The quantitative primer used was qRT-OsBIR1, and the relative expression level of the OsBIR1 gene was as follows: Figure 1 shown.

[0073] qRT-OsBIR1-F:ATTCCAGGGAAATTCGCTCT SEQ ID NO.3

[0074] qRT-OsBIR1-R:TACAAGGCTCCCACAAAACC SEQ ID NO.4

[0075] qRT-OsUBQ5-F:ACCACTTCGACCGCCACTACT SEQ ID NO.5

[0076] qRT-OsUBQ5-R:ACGCCTAAGCCTGCTGGTT SEQ ID NO.6

[0077] Example 4: RSV inoculation of transgenic rice

[0078] 1) After soaking and germinating the seeds of OsBIR1 transgenic and control Zhonghua 11 rice materials for 2-3 days, the seeds were sown in 1L beakers after turning white, with about 20 seedlings per beaker, and three biological replicates. The seeds were cultured at 30°C, 16h light and 8h dark.

[0079] 2) The newly hatched 1-2-year-old non-virus-carrying gray leafhoppers were inoculated on RSV-infected rice seedlings for 3 days, and the insects were swept out on healthy rice seedlings for 10 days to obtain virulent gray leafhoppers. An artificial inoculation experiment was conducted, with 3 insects per seedling. The virus-carrying / non-virus-carrying gray leafhoppers were inoculated on rice seedlings at the three- to four-leaf stage, and the insects were swept out after feeding for 3 days.

[0080] 3) After 30 days, observe the symptoms of diseased rice and determine the virus status of rice by qRT-PCR.

[0081] Example 5: Resistance analysis of rice after RSV inoculation

[0082] 1) Statistical incidence: Figure 3 As shown, the disease incidence of two OsBIR1 overexpressing transgenic lines (OsBIR1-4# and OsBIR-7#) was 26% and 14%, while the disease incidence of the control was 38%. The above results show that overexpression of OsBIR1 significantly reduces the disease incidence of transgenics.

[0083] 2) qRT-PCR detection of virus content: 30 days after transplanting rice, RSV-infected rice showed symptoms of heart blight. Compared with the control Zhonghua 11, the two OsBIR1-overexpressing transgenic lines showed obvious resistance to RSV, such as Figure 2 As shown. The total RNA of rice material was extracted as shown in Example 3, and reverse transcribed into cDNA, with the OsUBQ5 gene of rice as an internal reference. The expression of RSV CP gene was further detected by qRT-PCR, as shown. Figure 4 As shown in Figure 1, the expression level of the RSV CP gene in the transgenic strain 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 showed that the resistance of transgenic plants to RSV infection was affected by the OsBIR1 gene, and overexpression of OsBIR1 in rice could significantly enhance the resistance of rice to RSV infection.

[0084] The sequences of the quantitative primers are as follows:

[0085] qRSV-CP-F:AGGCAATCAATGACATCTCC SEQ ID NO.11

[0086] qRSV-CP-R:ATCTCTCAAAAGCCAGTGC SEQ ID NO.12

[0087] qRT-OsUBQ5-F:ACCACTTCGACCGCCACTACT SEQ ID NO.5

[0088] qRT-OsUBQ5-R:ACGCCTAAGCCTGCTGGTT SEQ ID NO.6.

Claims

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

1.

2. The use according to claim 1, wherein the anti-Tenuivirus virus is preferably Rice stripe virus (RSV).

3. A method for obtaining a transgenic plant, the method comprising: The OsBIR1 gene was constructed into a plant binary expression vector and introduced into Agrobacterium strains by electroporation; The transgenic plants overexpressing OsBIR1 gene were obtained by leaf disc method.

4. The method according to 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 to 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 to 5, wherein the plant binary expression vector is PCV1300.

7. A method for identifying rice resistant to rice stripe virus (RSV), comprising the steps of: The total RNA of the tested plants and Zhonghua 11 rice was extracted using the TRIzol method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit; The quantitative primer is qRT-OsBIR1, and the control primer is qRT-OsUBQ5. The expression level of the OsBIR1 gene relative to the control gene is measured to determine whether the rice plant is resistant to rice stripe virus. The sequences of the quantitative primer and the control primer 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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