A rice cytochrome P450 encoding gene OsRSb3 and its application

Abstract

The present invention discloses a rice cytochrome P450 encoding gene OsRSb3 and its application, and the nucleotide sequence of the gene is shown in SEQ ID No. 1. The present invention connects the cytochrome P450 encoding gene OsRSb3 gene with an overexpression vector by genetic engineering means, and then transforms it into rice, thereby increasing the expression of the gene in the transformed rice plant, and improving the resistance of the rice plant to sheath blight; in addition, the expression of the homologous gene of the gene in other plants can be overexpressed to enhance the disease resistance of other plants. In addition, the protein encoded by the OsRSb3 gene can be used as a bait protein to fish out the protein that interacts with the protein in rice or other plants, and clarify the regulatory mode of the gene in the process of resisting sheath blight.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a rice cytochrome P450 encoding gene OsRSb3 and an application thereof. Background Art

[0002] Rice is an important food crop, and its production directly affects the economic development of our country. Rice sheath blight is a soil-borne fungal disease caused by Rhizoctonia solani Kühn. It is known as one of the three major rice diseases along with rice blast and bacterial blight. In some areas with a high incidence of sheath blight, it can cause a 50% reduction in yield, and the situation has been getting worse year by year. It has become the main disease factor limiting rice high yield. In recent years, with climate change, the promotion and planting of high-yield, thick-stalked, multi-needle, and large-panicle rice germplasm, increased planting density, and excessive application of nitrogen fertilizer have created strong environmental conditions for sheath blight fungi to infect hosts.

[0003] At present, due to the lack of rice germplasm resources resistant to sheath blight, the wide host range of pathogens, strong stress resistance and genetic variation, there is still no systematic method for the prevention and control of sheath blight. In agricultural production, chemical agents are mainly used to prevent and control rice sheath blight, but problems such as the impact of chemical agents on the environment and the resistance of pathogens follow. In most rice-growing areas in the world, more than 90% of rice varieties show varying degrees of susceptibility. Although QTLs and genes related to resistance to rice sheath blight have been reported, the occurrence and prevalence of sheath blight in the field are affected by multiple factors such as rice growth, canopy density and field agronomic operations, and the identification of sheath blight resistance, such as inoculation methods, disease surveys and grading standards, are not uniform, resulting in the main effect genes and QTLs regulating sheath blight resistance have not been clearly resolved, and breeding for sheath blight resistance has progressed slowly. Therefore, the research on genes related to rice sheath blight resistance not only has important theoretical significance, but can also promote the exploration and genetic improvement of rice sheath blight-resistant germplasm resources, ultimately achieving effective prevention and control of sheath blight and increased agricultural production and income. Summary of the invention

[0004] The purpose of the present invention is to provide a rice cytochrome P450 encoding gene OsRSb3 and the application of the gene in resistance to rice sheath blight in order to provide a new method for breeding rice varieties resistant to sheath blight in view of the deficiencies of the above-mentioned prior art.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: to provide a cytochrome P450 protein OsRSb3 gene for positively regulating rice sheath blight resistance, the nucleotide sequence of which is shown in SEQ ID No. 1. After the gene was connected and recombined with a plant overexpression vector, it was transformed into the rice sheath blight susceptible variety Lement, and it was found that the expression level of the gene in the transgenic rice was increased, and the resistance to sheath blight was improved; after the gene was connected and recombined with a plant interference vector, it was transformed into the rice sheath blight resistant variety Teqing, and it was found that the expression level of the gene in the transgenic rice was reduced, and the resistance to sheath blight was reduced. By overexpressing the gene in rice, rice plants resistant to sheath blight can be obtained, which can be used for the breeding of rice varieties resistant to sheath blight.

[0006] The present invention also provides an overexpression vector containing the gene, and an Agrobacterium strain containing the overexpression vector.

[0007] The purpose of the present invention is:

[0008] (1) The present invention connects the gene to an overexpression vector through genetic engineering means and then transforms it into rice, thereby increasing the expression of the gene in the transformed rice plant, thereby improving the resistance of the rice plant to sheath blight. In addition, the gene can be used to overexpress the expression of its homologous genes in other plants, thereby enhancing the disease resistance of other plants.

[0009] (2) Used for analyzing the mechanism of rice resistance to sheath blight, such as connecting the gene sequence to any transformation vector containing a fluorescent protein gene, and using any transformation method to covalently introduce the gene and the fluorescent protein gene into rice or other plant cells. Fluorescence confocal transmission electron microscopy can be used to observe the migration and localization of the OsRSb3 protein expressed in rice or other plant cells in fusion with the fluorescent protein, thereby clarifying the site of action of the gene in the cell. The protein encoded by the OsRSb3 gene can also be used as a bait protein to fish out proteins in rice or other plants that interact with the protein, thereby clarifying the regulatory mode of the gene in the process of resistance to sheath blight.

[0010] The beneficial effect of the present invention is that the present invention helps to analyze the molecular mechanism of rice resistance to sheath blight by cloning the rice cytochrome P450 encoding gene OsRSb3 and analyzing its resistance to sheath blight. In practice, the gene can be used to cultivate sheath blight-resistant rice materials through transgenic means, and further through inter-variety hybridization, sheath blight-resistant rice varieties with stable traits can be obtained, effectively controlling the occurrence of sheath blight and increasing rice yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The expression pattern of OsRSb3 gene in resistant and susceptible rice varieties at different time points of sheath blight infection;

[0012] Figure 2 is the expression of OsRSb3 gene in overexpression plants;

[0013] Figure 3 is the expression of OsRSb3 gene in the interference plants;

[0014] Figure 4 Symptoms of rice sheath blight-susceptible material Lemont and plants overexpressing OsRSb3 gene with sheath blight-susceptible material Lemont as background after inoculation with sheath blight fungus for 72 hours;

[0015] Figure 5 Symptoms of rice sheath blight-resistant material Teqing and plants expressing OsRSb3 gene with sheath blight-resistant material Teqing as background 72 hours after inoculation with sheath blight fungus. DETAILED DESCRIPTION

[0016] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0017] Example 1: Cloning and sequence analysis of rice OsRSb3 gene cDNA

[0018] After germinating rice seeds at 37°C for 1 day, they were sown in a greenhouse and RNA was extracted from rice leaves at the three-leaf stage. The extraction method used the Trizol method, and the specific steps were as follows:

[0019] (1) Take 50-100 mg of fresh rice leaves, put them into a prepared mortar (soaked in DEPC water for 24 hours and sterilized at high temperature), add appropriate amount of liquid nitrogen and freeze grind them into powder;

[0020] (2) Transfer the ground powder into a 1.5 ml EP tube prepared without enzymes and immediately add 1 ml of Trizol reagent. Note that the total volume of the sample does not exceed 10% of the volume of Trizol.

[0021] (3) After placing the EP tube in an ice bath for 15 min, add 400 μL of chloroform, vortex for 1 min, and place in an ice bath for 15 min;

[0022] (4) Centrifuge at 12000 rpm for 15 min at 4°C and carefully pipette 450 μL of the supernatant into a 1.5 ml enzyme-free EP tube;

[0023] (5) Add 500 μL of isopropanol and mix well; place in a -20°C refrigerator and freeze-precipitate for at least 30 min;

[0024] (6) After the cryoprecipitation is completed, centrifuge at 13,000 rpm for 20 min at 4°C to obtain RNA;

[0025] (7) After centrifugation, pour off the upper liquid, being careful not to pour out the RNA. Add 1 ml of 75% ethanol (750 μL of anhydrous ethanol and 250 μL of DEPC water mixture), and wash the precipitate repeatedly 2 to 3 times at room temperature, each time for 5 to 10 minutes; after each wash, centrifuge at 7500 rpm for 5 minutes at 4°C;

[0026] (8) After washing, centrifuge at high speed and remove excess alcohol with a small gun tip;

[0027] (9) Air-dry naturally to evaporate the alcohol, add an appropriate amount of DEPC water according to the experimental requirements, and store in a -80℃ refrigerator for later use; obtain RNA and obtain cDNA by reverse transcription, and reverse transcription is performed using the ReverTra Ace qPCRRT Master Mix with gDNA Remover Kit of Y0Y0B0 Company; the specific operation is as follows: take 1 ug RNA and an appropriate amount of double distilled water and mix them evenly, with a total volume of 12ul, treat at 65℃ for 5min, quickly cool on ice, add 4ul of 4x DNase Mix, and treat at 37℃ for 5min. Add 4ul of 5x RT Master Mix, treat at 37℃ for 15min, then treat at 50℃ for 5min, and 98℃ for 5min to terminate the reaction.

[0028] Primer OsRS1 was designed using the Nipponbare genome as a template, and the sequence is as follows:

[0029] OsRS1-F:5'-ATGGAAGAACCAACCAGCTGCTAC-3' (SEQ ID No. 2);

[0030] OsRS1-R: 5'-CATGGAGTGGCACACGAACGACGG-3' (SEQ ID No. 3).

[0031] The cDNA obtained by reverse transcription was used as a template for amplification and the OsRSb3 gene of rice Teqing was cloned by RT-PCR. The specific reaction system was as follows: 2×Phanta Max Buffer 25μL, dNTP Mix (10mM each) 1μL, upstream primer (10μm) 2μL, downstream primer (10μm) 2μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, template cDNA 2μL, ddH2O up to 50μL; the reaction procedure was: 95℃ pre-denaturation for 2min; 95℃ denaturation for 20s, 62℃ annealing for 20s, 72℃ extension for 1min, 35 cycles; 72℃ extension for 10min. After the reaction, the obtained gene was sent to Qingke Xinye Biotechnology Co., Ltd. (Chengdu) for sequencing. The sequencing results are as follows:

[0032]

[0033] Example 2: Expression analysis of OsRSb3 gene in resistant and susceptible rice varieties

[0034] RNA was extracted from leaves of rice Teqing and Lemont at 0h, 12h, 24h, 48h and 72h of infection during the tillering period, and the expression levels of OsRSb3 gene at different infection time points in the two rice materials were analyzed.

[0035] The RNA extraction and reverse transcription methods of the two rice materials were the same as those in Example 1. The expression level of the OsRSb3 gene was analyzed by fluorescence quantitative PCR. The fluorescence quantitative PCR primer OsRS2 was designed based on the cloned OsRSb3 gene sequence, and the sequence is as follows:

[0036] OsRS2-F:5'-GATCGGGGACAGGTTCAAGAAG-3' (SEQ ID No. 4);

[0037] OsRS2-R: 5'-TCCATGAGCTCGAAGTTCTTG-3' (SEQ ID No. 5).

[0038] The RNA reverse transcription products of the leaves of two rice plants at different infection time points were used as templates. The reaction system was 20uL (2×AceQ qPCR SYBR Green Master Mix Primer1 (10μM) 10.0μl, Primer1 (10μM) 0.4μl, Primer2 (10μM) 0.4μl, 50×ROX Reference Dye 1 0.4μl, Template DNA / cDNA 2μl, ddH 2 O (20.0 μl), rice OsUBQ gene was used as the internal reference gene, and the primer sequences were as follows:

[0039] UBQ-F: 5'-CAAGATGATCTGCCGCAAATGC-3' (SEQ ID No. 6);

[0040] UBQ-R: 5'-TTTAACCAGTCCATGAACCCG-3' (SEQ ID No. 7).

[0041] The fluorescent quantitative PCR reaction was performed using the CFX Connect Real-Time PCR System fluorescent quantitative PCR instrument of Bio-Rad, USA. The reaction procedure was: pre-denaturation at 95℃ for 10min; denaturation at 95℃ for 15s, extension at 60℃ for 1min, and 40 cycles. The experimental results showed that the rice cytochrome P450 encoding gene OsRSb3 was highly expressed in the disease-resistant variety Teqing, and the expression level was upregulated at 12h of infection compared with the uninfected control; it was basically not expressed in the susceptible variety Lemont, such as Figure 1 shown.

[0042] Example 3: Obtaining transgenic rice plants overexpressing the OsRSb3 gene

[0043] In order to identify the function of OsRSb3 gene in resistance to sheath blight, the present invention cloned the sequence fragment obtained in Example 1 into the plant expression vector pBWA(V)KS to construct an overexpression vector of OsRSb3 gene. The constructed OsRSb3 gene overexpression vector was transformed into Agrobacterium GV3101 by electroporation transformation method, and then transformed into the susceptible variety Lemont. The specific operation was completed by Wuhan Boyuan Biotechnology Co., Ltd. The transformed plants were tested and showed that the expression of OsRSb3 gene in the overexpressed transformed plants was significantly higher than that in the wild type, such as Figure 2 shown.

[0044] Example 4: Obtaining OsRSb3 gene interference transgenic rice plants

[0045] To further verify the function of OsRSb3 gene in sheath blight resistance, the present invention constructed an interference vector of OsRSb3 gene and transformed the rice sheath blight resistant variety Teqing. The specific method is as follows: Based on the CDS sequence of OsRSb3 gene, a specific primer OsRSb3i was designed, and the sequence is as follows:

[0046] OsRSb3i-F:5'-AGAGTGATGAAGAGA-3' (SEQ ID No. 8);

[0047] OsRSb3i-R:5'-TGCGCGAACATTATAACCA-3' (SEQ ID No. 9).

[0048] The cloned OsRSb3 gene was used as a template and the high-fidelity enzyme TransStart FastPfu DNA Polymerase (TRNAsGen) was used to PCR amplify the OsRSb3 gene sequence. The amplified sequence was connected to the pBWA(V)HS vector to obtain the interference vector of the OsRSb3 gene. The constructed OsRSb3 gene interference vector was transformed into Agrobacterium GV3101 by electroporation and transformed into the disease-resistant variety Teqing. The specific operation was completed by Wuhan Boyuan Biotechnology Co., Ltd. The transformed plants were tested and showed that the expression of the OsRSb3 gene in the interference transformed plants was significantly lower than that in the wild type. Figure 3 shown.

[0049] Example 5: Identification of resistance to sheath blight in rice plants by overexpression and interference

[0050] The resistance of overexpressed and knocked-out rice plants to sheath blight was identified by inoculating detached rice leaves with sheath blight pathogens. The specific method is as follows:

[0051] (1) Use a hole puncher (about 5 mm in diameter) to punch holes on the edge of the PDA culture medium with hyphae of Rhizoctonia solani to take small pieces of uniform diameter, inoculate them onto a fresh PDA culture medium plate, place it upside down in a 28°C incubator, activate and culture for 2 to 3 days, and take it out after the surface of the culture medium is covered with white hyphae;

[0052] (2) Cut the second leaf of rice at the tillering stage and place it on a 40×30×3 cm white porcelain plate covered with filter paper. Soak the filter paper with sterile water and keep the back of the leaf close to the filter paper. Punch holes evenly on the edge of the culture dish of fresh sheath blight bacteria to ensure that each agar block contains a roughly uniform amount of hyphae. Place the bacterial block in the middle of the detached leaf, with the side with hyphae close to the leaf surface. To maintain the high humidity required for pathogen infection, wrap the white porcelain plate with plastic wrap and place it in a 28°C light incubator.

[0053] (3) After 72 hours, the disease progression was observed and the length of the lesions was measured.

[0054] The experimental results showed that rice plants with OsRSb3 gene interference were significantly more susceptible to disease than wild type, and rice plants with OsRSb3 gene overexpression were significantly more resistant to disease than wild type. Figure 4 and Figure 5 This further indicates that the OsRSb3 gene has the function of positively regulating rice sheath blight resistance.

[0055] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent. Sequence Listing <110> Sichuan Agricultural University <120> A Rice Cytochrome P450 Encoding Gene OsRSb3 and Its Application <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1560 <212> DNA <213> Artificial Sequence <400> 1 atggaagaac caaccagctg ctacggttac taccactacc tcgccctcgc cgtggccgtg 60 ctggtgctcg tccgggtcac gaggacgcgc ggcggcggca gcgatggcgt caggctgccg 120 ccggggccgt ggcggctgcc ggtgatcggc agcctgcacc acctcgccgg gaagccgctg 180 gtgcaccgcg cgctggcgga cctcgcgcgg cggatggacg cgccgctcat gtacctccgg 240 ctcggcgagg tccccgtggt ggtggccacg tcgccgggcg ccgcgcgcga ggtgatgcgg 300 acgcacgacg tcgcgttcgc gacgcggccg gtgaacccca ccgtgaggat catgacggcc 360 gacggggagg gcctcgtgtt cgcgccctac ggcgcgctgt ggcgacagct ccgccgcatc 420 gccatcctgg agctcctctc cgcgcgccgc gtccagtcgt tccgccgcgt cagggaggag 480 gaggccgcgc gcctcgccgc cgccgtcgcg gcggcggcgc cccacggcga ggcggccgtc 540 aacgtcagcg agcggatcgc cgtgctcatc gccgactcgg cggtgcgcgc catgatcggg 600 gacaggttca agaagcggga cgagttcttg gaggccctcg ccgaggggct caagctcgtc 660 tccgggttca gcctcgccga cctgttcccg tcgtcgtggc tcgccagctt cgtcaccggc 720 gcggcgcggc gggcgcagga gaaccaccgc aagaacttcg agctcatgga ccgcgccatc 780 gagcagcacc aggagcggcg cgcggcggca gcggcggcga gcggcgacgt cgtggaagac 840 gacgatctgg tggacgtcct cttgaggatt cagaagggag gtggcctcga cgtgcctctc 900 accatgggga tcatcaaagc agtaatcctt gaccttttta gcgccggaag tgagacatcg 960 gcgactacaa tccaatgggc catgtcggag ctcatgagga acccaagagt gatgaagaga 1020 gcgcaagctg agttacgcga caatctccaa gggaagccaa aggtgaccga ggaagacttg 1080 gccgacctaa attacctaa gctcattatc aaagagacgc tgaggttgca tctgccagcg 1140 cctttgctcc ttcctaggga gtcgagggag tcatgcaaaa tctttggata tgatgtgcca 1200 aaaggtacca ctgtgtttgt taacgcttgg gcaattggta gagatcctaa gtattgggat 1260 gaccccgaag agttcaagcc tgagcgattt gaggatagca agattgactt taagggtttg 1320 gactttgagt tcttgccatt tgggtctgga cggagaatgt gtcctggtat aatgttcgcg 1380 cagccgaaca tagaactcgc cctcgccacc cttctctacc acttcgattg gagcctccct 1440 gctggggtga agccaagtga gctagatatg accgaggaga tgggcatcac tgttaggagg 1500 aagaatgacc tatacctaca tgccgtcgtt cgtgtgccac tccatgcgac gacaccctag 1560 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <400> 2 atggaagaac caaccagctg ctac 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 catggagtgg cacacgaacg acgg 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 gatcggggac aggttcaaga ag 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 tccatgagct cgaagttctt g 21 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 caagatgatc tgccgcaaat gc 22 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 tttaaccagt ccatgaaccc g 21 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <400> 8 agagtgatga agaga 15 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <400> 9 tgcgcgaaca ttatacca 18

Claims

1. A rice cytochrome P450 encoding gene OsRSb3, whose nucleotide sequence is shown in SEQ ID No. 1, and overexpression of the encoding gene OsRSb3 has an effect of resisting plant sheath blight.

2. An overexpression vector containing the rice cytochrome P450 encoding gene OsRSb3 according to claim 1.

3. An Agrobacterium strain containing the overexpression vector according to claim 2.

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