Application of rice calmodulin OsCML15 in rice virus resistance

Abstract

The invention belongs to the field of plant genetic engineering technology and disease control, and relates to calmodulin OsCML15 and application thereof in rice virus disease resistance.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and disease control, and relates to a calmodulin-like protein OsCML15 and its application in rice resistance to viral diseases. Background Technology

[0002] Rice is one of my country's most important food crops, and its safe production is crucial to the national food security strategy and people's livelihood. During its growth cycle, rice faces threats from various viral diseases, among which the most serious is Rice Stripe Virus (RSV, a multispecific negative-sense RNA virus). When RSV infects rice, it causes stunted growth, leaf chlorosis and curling, and heart dieback, leading to reduced or no heading in infected plants and severe yield losses. RSV multiplies within the vector insect, the leafhopper (SBPH: Laodelphax striatellus Fallén), remaining a lifelong carrier and capable of transmission through eggs. More importantly, the use of pesticides such as imidacloprid in recent years has led to pesticide resistance in leafhoppers, resulting in increased insect populations in some areas and a higher risk of disease. Furthermore, long-term pesticide use causes serious environmental pollution, hindering the sustainable development of green agriculture and ecological balance. Therefore, identifying resistance genes and cultivating resistant varieties is beneficial for better control of rice viral diseases.

[0003] During the growth of rice, calmodulin-like proteins (CMLs) play an important role. OsCML is a class of proteins structurally similar to calmodulin (CaM) and dependent on calcium ions (Ca). 2+ Calcium signaling proteins are functional signaling proteins. Calcium signaling is an important signaling system for plants to sense changes in the external environment (such as stress and pathogen invasion) and internal physiological states. The core role of the CML family of proteins is to decode calcium signals and initiate downstream responses. The OsCML family is a key participant in calcium signaling, playing a multifunctional role in growth, development, and stress responses through a mechanism of "sensing calcium signals → activating target proteins → regulating physiological responses." Viral infection of rice can cause intracellular calcium... 2+ The concentration increases rapidly, thereby activating the calcium signaling pathway. Calmodulin may also resist viral infection by regulating downstream disease resistance pathways. In-depth research on the function of CML family genes will not only help reveal the molecular mechanisms of rice's environmental adaptation, but also provide a theoretical basis for improving rice stress resistance and increasing yield through gene editing and other technologies. However, the relationship between OsCML protein and rice virus resistance is not yet clearly documented in the literature. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention relates to an OsCML15 gene in rice and its application in rice antiviral activity;

[0005] On one hand, the present invention relates to an OsCML15 gene, the nucleotide sequence of which is shown in SEQ ID NO:1 and the amino acid sequence of which is shown in SEQ ID NO:2.

[0006] SEQ ID NO:1

[0007] ATGGGCAAGGTGAGGGCGTTCTTCTCGCGCAAGGGCAGGGGGAACAGCAGCGGCAGGTCCAGGTCGATGCGGGAGGCGGCGATGAACGTGGACTGGTCGCCGAGGCCGTCGGATCTCGCCGCCGCCGCCGCCGCGAAGCCCCGCCCGCCCG CGGCGGAGGACGAGACGGAGCGGGTGTTCAGGAAGTTCGACGCGAACGGCGACGGGCGGATCTCGCGGGCCGAGCTGGCGGCGCTGTTCCGCAGCGTGGGCCACGCCGTCACCGACGACGAGGTGGCGCGGATGATGCAGGAGGCCGACTCC GACGGCGACGGCTACATCTCGCTCGGCGAGTTCGCCGCCATCAGCGCCCCGCCGCCCGGCGACGCCGCCGCCGCCGAGGAGGACCTGCGCCACGCCTTCGGCGTGTTCGACGCCGACGGCAACGGCGTCATCACCCCCGCCGAGCTGGCCC GCGTCCTCCGCGGCATCGGCGAGGCCGCCACCGTCGCCCAGTGCCGCCGCATGATCGACGGCGTCGACCGCAACGGCGACGGCCTCATCAACTTCGAGGAGTTCAAGCTCATGATGGCCGCCGGCGCCGGCTTCGGCAGGATCGCTTCTTGA

[0008] SEQ ID NO:2

[0009] MGKVRAFFSRKGRGNSSGRSRSMREAAMNVDWSPRPSDLAAAAAAKPRPPAAEDETERVFRKFDANGDGRISRAELAALFRSVGHAVTDDEVARMMQEADSDGDGYISLGEFAAISAPPPGDAAAAEEDLRHAFGVFDADGNGVITPAELARVLRGIGEAATVAQCRRMIDGVDRNGDGLINFEEFKLMMAAGAGFGRIAS*

[0010] On the other hand, the present invention relates to the use of a rice calmodulin OsCML15 gene in rice breeding for resistance to rice stripe virus (RSV), wherein OsCML15 is a calmodulin-like gene derived from rice, and its nucleotide sequence is shown in SEQ ID NO:1 or has at least 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0011] In some embodiments, the protein sequence of the OsCML15 gene is as shown in SEQ ID NO:2 or has at least 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0012] In some implementations, the use is achieved by upregulating the expression of the OsCML15 gene, thereby enhancing the resistance of rice to rice stripe virus.

[0013] In some implementations, the up-adjustment of OsCML15 includes, but is not limited to:

[0014] (1) Transgenic overexpression;

[0015] (2) Promoter-enhanced expression;

[0016] (3) Knock out the negative regulatory factor of OsCML15 using the CRISPR / Cas system;

[0017] (4) Exogenous inducers promote the increase of OsCML15 transcription or translation.

[0018] In some implementations, the overexpression of OsCML15 reduces viral RNA accumulation, alleviates leaf yellowing and necrosis symptoms, and maintains high photosynthetic efficiency in rice after RSV inoculation.

[0019] In another aspect, the present invention relates to a method for improving rice resistance to rice stripe virus using the OsCML15 gene, comprising the following steps:

[0020] (1) Construct an expression vector containing the OsCML15 gene;

[0021] (2) Transformation of rice by Agrobacterium-mediated transformation;

[0022] (3) Screening to obtain plants with high expression of OsCML15.

[0023] In some implementations, the construction steps of the expression vector containing the OsCML15 gene are as follows:

[0024] (1) Using rice cDNA as a template, the open reading frame (ORF) of the OsCML15 gene was amplified using primers OsCML15-F and OsCML15-R.

[0025] (2) The PCR product was ligated into the pMD18-T vector, transformed into E. coli DH5α, positive clones were picked and verified by sequencing, and the pMD18-T-OsCML15 recombinant plasmid containing the OsCML15 insert fragment was obtained.

[0026] (3) The OsCML15 gene was amplified using primers PCV-OsCML15-F and PCV-OsCML15-R, which contain restriction ligation sites;

[0027] (4) After adding A / T ends to the PCR product, it is ligated with the plant binary expression vector pCV1300 treated with T ends, transformed into DH5α, screened and sequenced to verify, and the correct PCV1300-OsCML15 overexpression vector is obtained.

[0028] In some embodiments, the sequence of primer OsCML15-F is shown in SEQ ID NO:3, and the sequence of primer OsCML15-R is shown in SEQ ID NO:4.

[0029] In some embodiments, the sequence of the primer PCV-OsCML15-F is shown in SEQ ID NO:5, and the sequence of the primer PCV-OsCML15-R is shown in SEQ ID NO:6.

[0030] On the other hand, the present invention also relates to a recombinant overexpression vector containing the aforementioned OsCML15 gene and capable of driving the expression of the gene in plant cells.

[0031] This invention utilizes plant transgenic technology to transform the full-length OsCML15 gene into rice, and then selects transgenic lines with high expression levels. Experimental results show that after RSV infection, transgenic rice overexpressing OsCML15 exhibited significantly higher resistance to the virus than the control Zhonghua 11 (ZH11), indicating that OsCML15 plays a positive regulatory role in rice's antiviral process.

[0032] The present invention mainly achieves the above-mentioned objectives by adopting the following solutions:

[0033] This invention focuses on the OsCML15 gene in rice. First, a recombinant expression vector for this gene was constructed and named pCV1300-OsCML15. Then, using Agrobacterium-mediated genetic transformation, the constructed expression vector was introduced into the embryo of the mature rice variety "Zhonghua 11" (ZH11), and callus formation was further induced. The Agrobacterium strain used throughout the transformation process was GV3101. After obtaining transgenic rice seedlings, transgenic plants were identified. The expression level of the OsCML15 gene in rice was detected using quantitative real-time PCR (qRT-PCR), and finally, stable T3 generation homozygous transgenic rice lines were successfully screened.

[0034] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0035] (1) The present invention successfully bred OsCML15 transgenic rice that is overexpressed and stably inherited, and then carried out resistance analysis on rice stripe virus for the transgenic rice.

[0036] (2) By exploring the role of the OsCML15 gene in the viral infection process, the interaction between plants and viruses was further revealed. These studies not only help to deepen the understanding of the relationship between rice viruses and plant host factors and enrich the knowledge of the molecular mechanism of rice-virus interaction, but also lay an important foundation for further in-depth research on disease resistance theory. Attached Figure Description

[0037] Figure 1 The relative expression level of OsCML15 in transgenic rice overexpressing OsCML15.

[0038] Figure 2 Symptoms of disease in RSV-infected OsCML15 overexpressing transgene and control wild-type.

[0039] Figure 3 Virus content of OsCML15 overexpressing transgene and control wild-type after RSV infection. Detailed Implementation

[0040] Example 1: Construction of rice OsCML15 overexpression vector

[0041] (1) Cloning of the rice OsCML15 gene

[0042] Primers OsCML15-F and OsCML15-R were designed based on the open reading frame (ORF) of OsCML15. The primer sequences are as follows:

[0043] OsCML15-F ATGGGCAAGG TGAGGCG (SEQ ID NO: 3);

[0044] OsCML15-R TCAAGAAGCG ATCCTGCCG (SEQ ID NO:4);

[0045] PCR amplification system: Total volume 40 μL, including 20 μL of 2×PCR Buffer for KOD FX, 1 μL each of forward and reverse primers (10 μM), 4 μL of dNTP Mix (2.5 mM), 1 μL of cDNA template, 0.8 μL of KOD FX, and 12.2 μL of ddH2O. PCR amplification program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ final extension for 10 min. PCR products were recovered, ligated into the pMD18-T vector, single clones were selected, colony PCR was performed, and sequencing was conducted. The samples were sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing, yielding the correct pMD18-T-OsCML15 recombinant plasmid.

[0046] (2) Construction of overexpression vector

[0047] Primers for ligating restriction enzyme sites were designed for the construction of the OsCML15 gene binary expression vector PCV1300. The primer sequences are as follows:

[0048] CGACGACAAGACCGTCACCATGGGCAAGGTGAGGGCG

[0049] PCV-OsCML15-F

[0050] (SEQ ID NO:5);

[0051] GAGGAGAAGAGCCGTCGAGAAGCGATCCTGCCG PCV-OsCML15-R

[0052] (SEQ ID NO:6);

[0053] The PCR products were recovered, and A bases were added to the ends of the recovered PCR products. The PCV1300 vector was end-capped with T bases. The A / T end addition system consisted of a total volume of 50 μL, including 38.5 μL of recovered PCR product, 1.5 μL of T4 DNA polymerase, 2.15 μL of 10×NEBBuffer, and 100 Mm dATP / dTTP. The A-terminal addition reaction program was: 37℃ for 20 min, then 75℃ for 20 min.

[0054] Ligation system: Total volume 10 μL, including 6 μL of the recovered product after addition of A and 4 μL of the T-terminated PCV1300 carrier. Ligation reaction program: 75℃ for 20 s, 37℃ for 30 min.

[0055] After ligation, 10 μL of the ligation product was transformed into DH5α, incubated on ice for 30 min, and then heat-shocked at 42℃ for 45 s. After heat shock, the plate was immediately placed on ice. 500 μL of antibiotic-free LB liquid medium was added to a clean bench and the plate was placed in a shaker at 37℃. After incubation at 200 rpm for 1 h, the plate was centrifuged at 5000 rpm for 1 min. Part of the supernatant was discarded, and the remaining precipitate was evenly spread on LB plates containing kanamycin sulfate resistance. The plate was incubated overnight at 37℃. Single clones were picked, colony PCR was performed, and sequencing was performed to obtain the correct recombinant plasmid PCV1300-OsCML15.

[0056] Example 2: Genetic transformation of rice

[0057] (1) Agrobacterium transformation and callus induction culture: The plasmid containing the target vector was transformed into Agrobacterium GV3101. Procedure: 2 μL of plasmid was added to 50 μL of competent cells and mixed well. The mixture was then added to a sterilized electrode cup and transformed by electroporation at 2200 V. 500 μL of antibiotic-free LB was added, and the mixture was cultured at 28℃ and 200 rpm for 2-3 h. After centrifugation at 5000 rpm for 1 min, part of the supernatant was discarded, and the remaining precipitate was spread on Rif plates containing 50 μg / ml Kan and 50 μg / ml, and cultured at 28℃ for 3 days. Mature rice seeds were dehulled, soaked in 75% alcohol for 5 min, rinsed several times with ddH2O, soaked in 30% sodium hypochlorite solution for 30 min, and then soaked in ddH2O for 30 min. Use tweezers to place the seeds into the induction medium and incubate them in a 28°C light incubator for 3 weeks. Then, use pre-sterilized tweezers to transfer the callus tissue into the subculture medium and subculture it in a 28°C light incubator for 1 week.

[0058] (2) Agrobacterium-mediated callus transfection and screening and culture of resistant callus: Single colonies of GV3101 were picked and inoculated into LB liquid medium and cultured to OD 600= Approximately 0.6. The induced callus tissue was immersed in Agrobacterium suspension culture for 5 minutes. The callus tissue was then removed and placed in a co-culture medium, incubated at 26°C under light for 2.5 days. The callus tissue was then grown on a medium containing hygromycin for 30-45 days. The selected callus tissue was then placed in rooting medium and cultured under light to produce green plantlets with roots. After 2 weeks of culture, transgenic plantlets were obtained, namely OsCML15#1 and OsCML15#12.

[0059] Example 3: Positive identification of transgenic plants

[0060] Total RNA was extracted from positive transgenic plants and reverse transcribed into cDNA, using the rice OsUBQ5 gene as an internal control. Quantitative primers are shown in SEQ ID NO:6-9. The relative expression levels of the transgenic lines OsCML15#1 and OsCML15, which overexpress the OsCML15 gene, were significantly higher than the control. Figure 1 ).

[0061] qRTOsCML15F AGGAAGTTCG ACGCGAACG (SEQ ID NO:7);

[0062] qRTOsCML15RATGTAGCCGTCGCCGTCG(SEQ ID NO:8);

[0063] OsUBQ5-F ACCACTTCGACCGCCACTACT(SEQ ID NO:9);

[0064] OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 10);

[0065] Example 4: Artificial inoculation of RSV

[0066] OsCML15 transgenic and control rice seeds were soaked in a 37℃ incubator for 2-3 days until they turned white. Then, they were sown in 1L glass beakers with 30-35 seedlings per beaker, with 3 biological replicates. These beakers were then cultured in an artificial climate chamber at 25℃ with 16 hours of light and 8 hours of darkness. First- and second-instar non-toxic planthoppers acquired RSV infection on RSV-infected rice seedlings for 3-5 days, and were then transferred to healthy rice seedlings for a 10-12 day cycle. The virus-carrying rate of the insects was measured, and the number of insects inoculated per seedling was calculated based on the rate. Virus-carrying / non-virulent planthoppers were inoculated onto three- to four-leaf stage (approximately 15 days old) rice seedlings. Three days after inoculation, all insects were removed, and the seedlings were transplanted to the field to await natural disease development. Around 30 days later, the symptoms of disease were observed, and the disease incidence was statistically analyzed to determine the overall disease situation in the rice. The two overexpressing transgenic lines of OsCML15, OsCML15#1 and OsCML15#12, showed resistance to RSV, such as Figure 2 As shown. Further analysis of viral load revealed that the viral load in both OsCML15 overexpression transgenic lines was significantly lower than that in the wild type, such as... Figure 3 As shown.

[0067] In summary, to evaluate the impact of the OsCML15 gene on the virus resistance of rice, the inventors artificially inoculated OsCML15-overexpressing transgenic rice and wild-type ZH11 rice with rice stripe virus (RSV) using the vector insect, the planthopper. Approximately 30 days after inoculation and natural disease development in the field, phenotypic observation revealed that, compared to wild-type plants, the OsCML15-overexpressing transgenic rice showed significantly reduced symptoms under RSV infection, with significantly lower viral transcription and protein levels. This demonstrates that overexpression of the OsCML15 gene can effectively enhance rice resistance to RSV and also affect the infection efficiency of RSV in rice. These findings not only provide crucial evidence for elucidating the interaction mechanism between rice and viruses but also lay the foundation for subsequent research in related fields.

Claims

1. The use of the OsCML15 gene in rice breeding for resistance to rice stripe virus (RSV), characterized in that, The OsCML15 gene is a calmodulin-like protein derived from rice, and its nucleotide sequence is shown in SEQ ID NO:1 or has at least 95%, 96%, 97%, 98%, or 99% sequence identity with it.

2. The use according to claim 1, wherein the protein sequence of the OsCML15 gene is as shown in SEQ ID NO:2 or has at least 95%, 96%, 97%, 98%, or 99% sequence identity with it.

3. The use according to any one of claims 1-2, which enhances the resistance of rice to rice stripe virus by upregulating the expression level of the OsCML15 gene.

4. The use according to any one of claims 1-3, wherein the up-adjustment of OsCML15 includes, but is not limited to: (1) Transgenic overexpression; (2) Promoter-enhanced expression; (3) Knock out the negative regulatory factor of OsCML15 using the CRISPR / Cas system; (4) Exogenous inducers promote the increase of OsCML15 transcription or translation.

5. The use according to any one of claims 1-3, wherein the overexpression of OsCML15 causes rice to exhibit reduced viral RNA accumulation, alleviated leaf yellowing and necrosis symptoms, and maintain high photosynthetic efficiency after RSV inoculation.

6. A method for improving rice resistance to rice stripe virus using the OsCML15 gene, characterized in that, Includes the following steps: (1) Construct an expression vector containing the OsCML15 gene; (2) Transformation of rice by Agrobacterium-mediated transformation; (3) Screening to obtain plants with high expression of OsCML15.

7. The method of claim 6, wherein the construction steps of the expression vector containing the OsCML15 gene are as follows: (1) Using rice cDNA as a template, the open reading frame (ORF) of the OsCML15 gene was amplified using primers OsCML15-F and OsCML15-R. (2) The PCR product was ligated into the pMD18-T vector, transformed into E. coli DH5α, positive clones were picked and verified by sequencing, and the pMD18-T-OsCML15 recombinant plasmid containing the OsCML15 insert fragment was obtained. (3) The OsCML15 gene was amplified using primers PCV-OsCML15-F and PCV-OsCML15-R, which contain restriction ligation sites; (4) After adding A / T ends to the PCR product, it is ligated with the plant binary expression vector pCV1300 treated with T ends, transformed into DH5α, screened and sequenced to verify, and the correct PCV1300-OsCML15 overexpression vector is obtained.

8. The method according to any one of claims 6-7, wherein the sequence of primer OsCML15-F is shown in SEQ ID NO:3, and the sequence of primer OsCML15-R is shown in SEQ ID NO:

4.

9. The method according to any one of claims 6-7, wherein the sequence of the primer PCV-OsCML15-F is shown in SEQ ID NO:5, and the sequence of the PCV-OsCML15-R is shown in SEQ ID NO:6.

Images