Application of OsAO gene for improving resistance of rice against rice stripe virus, rice black-streaked dwarf virus, or virus of same family

Abstract

The present application discloses an application of OsAO gene for improving rice resistance to rice stripe disease, rice black-streaked dwarf disease or other rice or corn virus diseases caused by homologous virus of rice black-streaked dwarf virus. The present application also provides an application of OsAO gene, the protein encoded by the gene or a recombinant vector containing the gene in regulating plant resistance to rice stripe disease, rice black-streaked dwarf disease or other rice and corn virus diseases caused by homologous virus of rice black-streaked dwarf virus, said protein has the amino acid sequence as shown in Seq 4. The experiment proved that the plant disease resistance is increased in rice overexpressing OsAO gene, indicating that the OsAO protein encoded by this gene plays an important role in rice resistance to rice stripe disease and rice black-streaked dwarf disease.

Claims

1. A method of improving plant resistance to rice stripe disease and rice black-streaked dwarf disease, comprising expressing a protein according to an amino acid sequence of SEQ ID NO: 4 in the plant, wherein expressing the protein in the plant comprises the following steps: a) importing a gene encoding the protein according to SEQ ID NO: 4 into the plant, and obtaining a transgenic plant; b) selecting transgenic plants with enhanced resistance to rice stripe disease, rice black-streak dwarf disease.

2. The method of claim 1, wherein the gene is any one of SEQ ID NO: 1 or SEQ ID NO: 2.

3. The method of claim 1, wherein the plant is rice.

4. The method of claim 1, wherein the plant expressing the protein according to an amino acid sequence of SEQ ID NO: 4 is resistant to a pathogen of rice stripe disease selected from rice stripe virus(RSV), a pathogen of rice black-streaked dwarf disease selected from rice black-streaked dwarf virus(RBSDV), wherein a viral mediator of both viruses is Laodelphax striatellus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is detection result of OsAO mRNA accumulations in RSV- and RBSDV-infected WT rice plants in the Nipponbare background. Realtime PCR analysis showed induced OsAO mRNA levels after inoculation with buffer (Mock), RSV and RBSDV. The expression levels were normalized using the signal from OsEF-1α, the relative values were calculated by comparison with the Mock (arbitrarily set to 1.0) wherein OsAO is SEQ ID NO: 1.

(2) FIG. 2 is detection result of OsAO protein (SEQ ID NO: 4) accumulations in mock-, RSV- and RBSDV-infected WT rice plants. Mock represents wild type rice Nipponbare not infected by RSV or RBSDV; RSV represents wild type rice Nipponbare infected by RSV; RBSDV represents wild type rice Nipponbare infected by RBSDV. Actin was detected and used as a control.

(3) FIG. 3 is detection result of relative enzyme activity of AO after mock, RSV or RBSDV infection. The results were based on the assumption that the relative enzyme activity of the Mock was 100%.

(4) FIG. 4 is detection result of OsAO mRNA expression levels in mock-(M) or RSV-inoculated (R) WT (Nipponbare, NPB) and pAO:AO and pAO:AO-Res transgenic rice lines. In the uninfected rice, the expression levels of OsAO mRNA were significantly increased in pAO:AO and pAO:AO-Res lines relative to NPB. In the RSV-infected rice plants, there was a marked rise in OsAO mRNA expression levels compared with the mock. OsEF-1α was served as a loading control, the expression levels in the Mock plants are set to a value of 1.0 and the expression levels in the other plants are relative to this reference value.

(5) FIG. 5 is detection result of relative enzyme activity of OsAO in mock-(M) or RSV-inoculated (R) WT(NPB) and pAO:AO and pAO:AO-Res transgenic rice lines. The enzyme activity of OsAO significantly increased in pAO:AO and pAO:AO-Res lines than NPB in the mock plants. And the enzyme activity of OsAO was dramatically higher after RSV infection.

(6) FIG. 6 is detection result of OsAO protein (SEQ ID NO: 4) levels in WT(NPB) and pAO: AO and pAO:AO-Res transgenic rice lines with (R) or without (M) RSV infection. The OsAO expression levels was greatly enhanced in pAO:AO and pAO:AO-Res lines when compared with NPB. In contrast with the uninfected, the OsAO expression levels in the RSV-inoculated plants increased significantly. α-Tubulin was probed and served as a loading control.

(7) FIG. 7 shows symptoms of mock- or RSV-inoculated WT(NPB) and transgenic plants expressing pAO:AO or pAO:AO-Res in the NPB background in example 3. The symptom of pAO:AO and pAO:AO-Res lines was apparently attenuated compared to NPB after RSV infection.

(8) FIG. 8 shows the accumulation amount of RSV virus in mock- or RSV-inoculated WT(NPB) and transgenic plants expressing pAO:AO or pAO:AO-Res in the NPB background in example 3. Compared with NPB, the accumulation of four RNA strands of the RSV virus in pAO:AO and pAO:AO-Res lines reduced after RSV infection.

(9) FIG. 9 shows symptoms of mock- or RBSDV-inoculated DJ, NPB, mir528 and transgenic lines expressing pAO:AO-Res(pAO:AO-Res) or overexpressing miRNA528(miR5280E) in example 3. DJ and NPB are wild type rice, miR5280E is transgenic rice overexpressing miRNA528 in the NPB background, mir528 is the mutant rice of microRNA528 in the DJ background, pAO:AO-Res is the transgenic rice with NPB background.

(10) FIG. 10 shows the accumulation amount of RBSDV virus in mock- or RBSDV-inoculated DJ, NPB, mir528, and pAO:AO-Res or miR5280E transgenic lines in example 3. S1, S2, S6, and S10 are four representative viral chains of RBSDV. OsEF-la was probed and served as a loading control, the expression levels of each chain in the RBSDV-infected NPB plants are set to 1.0 and the expression levels in the other plants are relative to this reference value.

(11) FIG. 11 is detection result of OsAO mRNA expression levels in mock-(M) or RBSDV-inoculated(R) DJ, NPB, mir528, and pAO:AO-Res or miR5280E transgenic lines in example 3, wherein M represents uninfected rice, R represents RBSDV-inoculated rice.

(12) FIG. 12 is detection result of relative enzyme activity of OsAO in mock-(M) or RBSDV-inoculated(R) DJ, NPB, mir528, and pAO:AO-Res or miR5280E transgenic lines in example 3. In the uninfected rice, the enzyme activity of OsAO in pAO:AO-Res and miR5280E lines increased significantly relative to WT, while declined sharply in miR5280E line. And the enzyme activity of OsAO shows dramatic rise after RSV infection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(13) The experimental methods used in the following examples are conventional methods unless otherwise specified.

(14) The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

(15) Rice (Oryza sativa L.) cultivar Nipponbare: Oryza sativa L. subsp. japonica ‘Nipponbare’, reference: rice cultivar “Nipponbare”. Agricultural Science and Technology Newsletter, 1973, 02; available from Peking University.

(16) Rice (Oryza sativa L.) cultivar DJ: Dongjin rice (Oryza sativa L. subsp. japonica ‘Dongjin’) is a wild-type control of rice T-DNA insertion mutant used in the mutant study, from the mutant rice library in Korea. RISD (http://www.postech.ac.kr/life/pfg/risd/).

(17) Rice (Oryza sativa L.) cultivar Wuyujing 3: Susceptible to Rice Stripe Virus (RSV), reference: Wuyujing 3 soil-borne disease occurrence characteristics and control techniques, Jiangsu Agricultural Sciences, 1996 01; available from Peking University.

(18) Rice (Oryza sativa L.) cultivar Zhendao 88: Resistant to Rice Stripe Virus (RSV), described in the article “New Rice Cultivar—Zhendao 88, Agricultural Science and Technology Newsletter, 1998 05”, available from Peking University.

(19) Rice Stripe Virus (RSV): “Cai Xiaowei, Zhao Junling, Shao Ying, Gui Qingqing, Liu Fang. A review of the research on the transmission of rice stripe virus by Laodelphax striatellus. China Plant Protection Guide, 2011 09”, available from Peking University.

(20) pAO:AO and pAO:AO-Res transgenic rice were derived from Nipponbare background, recorded in Dr. Yang Rongxin's thesis Functional Research of Rice miRNA528, 2012 10. Dr. Yang was from Cao Xiaofeng's research group, in the Institute of Genetics and Developmental Biology (IGDB) of the Chinese Academy of Sciences (CAS); available from Peking University.

(21) Rice Black-Streaked Dwarf Virus (RBSDV): Chen Shengxiang, Zhang Qiaoyan. Research progress on rice black-streaked dwarf disease and maize rough disease in China. Journal of Plant Protection, Vol. 32, No. 1, 2005; available from Peking University.

(22) Agrobacterium EHA105: “Zhu et al., 2005. The Rice Dwarf Virus P2 Protein interacts with ent-Kaurene Oxidases in vivo, leading to reduced biosynthesis of Gibberellins and rice dwarf symptoms. Plant Physiology. 139: 1935-1945”; available from Peking University.

(23) pCam1300 vector: “Jianguo Wu & Zhirui Yang et al., 2015. Viral-inducible Argonaute 18 confers broad-spectrum virus resistance in rice by sequestering a host microRNA. Elife. 2015 Feb. 17; 4”; available from Peking University.

Example 1. Infection of Rice Stripe Virus and Rice Black Streaked Dwarf Virus can Increase the Expression Level of OsAO mRNA

(24) The OsAO gene in this example was from rice (Oryza sativa L.). The genomic coding sequence of the OsAO gene is composed of 5472 nucleotides, as shown in SEQ ID NO: 1 in the sequence list. The protein encoded by SEQ ID NO: 1 consists of 633 amino acid residues, as shown in SEQ ID NO: 3. The coding sequence of the OsAO gene of the pAO:AO-Res transgenic line is shown in SEQ ID NO: 2, and the encoded protein sequence is shown in SEQ ID NO: 4.

(25) I. OsAO Gene Accumulates after RSV Infection at mRNA Level

(26) 0.5 g of leaves per sample from rice Nipponbare infected by RSV or RBSDV and rice not infected by virus (as control) is cut for testing and ground in liquid nitrogen, and total RNA is extracted according to Invitrogen's TRIzol Reagent instruction (Invitrogen Trizol Reagent, cat No. 15596-018). The total RNA concentration is measured, and then the rice genomic DNA in 10 μg of total RNA is digested according to the instructions of RQ1 Dnase (Promega, Cat. No. M610A). Digestion reaction system comprises: 10 μg of total RNA, 10 μl of 10×Dnase buffer, 10 μl of DNase, add DEPC water to 100 μl in total. The entire digestion reaction system is incubated at 37° C. for 35 min, 4 μl of RQ1 DNase stopping solution is added to the system to terminate the reaction, and incubated at 65° C. for 10 min to inactivate DNase.

(27) After digesting the genomic DNA, total RNA is extracted by chloroform, and the total RNA concentration is measured. Then 2 μg of RNA is reverse-transcribed with Invitrogen SuperScript II reverse transcriptase, the primer used is Oligod (T) primer of 16-nucleotide. For detailed protocol, please refer to invitrogen M-MLV Reverse Transcriptase (Cat. No. 28025-021). Using the rice cDNA obtained by reverse transcription as template, the transcriptional level of OsAO gene is detected by real-time quantitative fluorescent PCR according to TOYOBO SYBR® Green Realtime PCR Master Mix (Cat. No. QPK-201), the primers are:

(28) TABLE-US-00001 AO-qRT-F:  (SEQ ID NO: 7) 5′-CGAGAACGTGGAGACCTGCGTCGA-3′; AO-qRT-R:  (SEQ ID NO: 8) 5′-CCACCACCGTCATCTTGTGCCCTTG-3′.

(29) The internal control gene is rice EF, and the primers are:

(30) TABLE-US-00002 OsEF-la-F:  (SEQ ID NO: 9) 5′-GCACGCTCTTCTTGCTTTCACTCT-3′; OsEF-la-R:  (SEQ ID NO: 10) 5′-AAAGGTCACCACCATACCAGGCTT-3′.

(31) For the data processing method, refer to Bio-RAD CFX96 real-time quantitative fluorescence PCR instrument together with software CFX Manager™ Software (Version 2.1).

(32) The results are as shown in FIG. 1. The OsAO gene is significantly enriched after RSV and RBSDV infection in rice at the mRNA level, indicating that the infection of RSV and RBSDV can increase the expression of OsAO gene in rice, such that the transcription level of this gene is increased.

(33) II. OsAO Protein Accumulates in Rice Infected by RSV and RBSDV

(34) The leaves of Nipponbare infected by rice stripe virus (RSV) and rice black streaked dwarf virus (RBSDV) and control (not infected by virus) are used as experimental materials. Total protein is extracted by 2×SDS protein loading buffer, and the accumulation of OsAO protein in different samples is detected by Western blot. The details are as follows:

(35) (1) SDS-PAGE Electrophoresis Procedure and Precautions are According to “SDS-PAGE Gel Electrophoresis of Proteins”.

(36) After the electrophoresis, the gel is taken out and the glass plate is removed. The gel is cropped to target size and put in transfer buffer. A piece of ECL membrane slightly larger than the gel and two pieces of extra-thick filter paper are cut, and soaked in transfer buffer.

(37) The transfer sandwich is assembled on the semi-dry transfer cassette base by placing one piece of wet extra-thick filter paper on the bottom, then the ECL membrane, the protein gel, and finally, another piece of the wet filter paper on top. Be careful to remove the any air bubbles with a glass rod during the placement process. The cassette lid is placed and locked. The transfer process is carried out under a constant voltage of 15V for 90 minutes (the transfer time is determined according to the protein size).

(38) 1) After transfer process, the membrane is blocked in blocking buffer (5% milk formulated with TBST), and incubated for 1 hour at room temperature or overnight at 4° C.

(39) 2) After blocking, the membrane is incubated with a solution of primary antibody (or directly using the recovered primary antibody) diluted in fresh blocking buffer (5% milk formulated with TBST) under gentle agitation for typically 2 hours at room temperature, or overnight at 4° C.

(40) 3) Following incubation, the membrane is washed for 3-4 times with TBST wash buffer to remove unbound primary antibody, 5 min for each time, and the primary antibody is recovered.

(41) 4) After rinsing, the membrane is incubated with a solution of corresponding fresh secondary antibody diluted in blocking buffer for one hour at room temperature.

(42) 5) The membrane is washed for 3-4 times with TBST wash buffer, 5 min for each time.

(43) 6) The substrate kit (Immobilon™ Western: MILLIPORE, Shanghai Trading Co., Ltd., Cat. No. 1305701) corresponding to the enzyme coupled to the secondary antibody is developed and added to the membrane. After 1 minute of incubation reaction, the excess liquid on the membrane is exhausted. Try not to let the film dry, wrap it in plastic wrap as soon as possible, put it in a dark clip, expose it with X-ray film and develop, and finally scan the good film and save it.

(44) (2) Reagents Required for the Experiment:

(45) 1) 10 mL of 2×SDS protein loading buffer: 2 mL glycerol, 0.202 g bromophenol blue, 1 mL of 1 M Tris-HCl (pH 6.8), 0.14 mL β-mercaptoethanol, 4 mL 10% SDS, add H.sub.2O to 10 mL, stored at −20° C.

(46) 2) 1 L of Transfer buffer: 2.9 g of 39 mM glycine, 5.8 g Tris, 0.37 g of SDS, 200 mL methanol.

(47) 3) 100 mL of Alkaline phosphatase buffer: 100 mM NaCl, 5 mM MgCl.sub.2, 100 mM Tris-HCl, pH 9.5.

(48) 4) 1 L Tris-Buffered Saline with 0.1% Tween 20 (TBST): 2.42 g Tris, 8 g NaCl, 1 mL Tween 20, 900 mL water, pH is adjusted to a constant volume of 1 L, pH 7.6.

(49) 5) 100 mL Blocking buffer: 5 g non-fat dry milk in 100 mL TBST.

(50) Actin protein is used as the internal control. The primary antibody is Actin monoclonal antibody (from mouse, Sigma, Cat. No. T6793). The secondary antibody is anti-mouse (Promega, Cat. No.: 0000089661).

(51) The results are as shown in FIG. 2. The expression level of OsAO protein is low in the control rice (wild type Nipponbare not infected by RSV nor RBSDV), but is relatively high in wild type NPB infected by RSV or RBSDV. This result indicates that the infection of RSV and RBSDV can also increase the expression of OsAO protein in translational level.

(52) III. AO Enzyme Activity Increases after Infection by Rice Stripe Virus and Rice Black streaked dwarf virus.

(53) The leaves of wild type rice (Oryza sativa L.) cultivar Nipponbare infected by rice stripe virus (RSV) or rice black streaked dwarf virus (RBSDV) and control rice (NPB not infected by rice stripe virus nor rice black stripe) are sampled to detect the activity of AO as follows:

(54) 1) Preparation of Ascorbate Oxidase Extraction:

(55) 100 mg of plant stem and leaf tissue is ground to powder in liquid nitrogen, 1 mL of 10 mM PBS (pH 6.5) is added to dissolve and homogenized using vortex. The resulting material is placed on ice for 20 min, shaken every 5 min, and centrifuged at 4° C., 15000 g for 20 min; the supernatant is transferred to a new tube, which is the ascorbate oxidase extraction, and placed on ice and ready for use.

(56) 2) Detection of Ascorbate Oxidase Activity:

(57) The assay mixture consists of 10 μL of the extraction, 80 μL of 10 mM PBS (pH 5.6) and 10 μL of 2 mM L-AsA (L-ascorbic acid). The absorbance of A265 is measured with a Thermo full-wavelength microplate reader. Each sample have 3 to 5 replicates, and a mixture without the extraction is used as blank to compare the decrease in the absorbance of A265. The extinction coefficient (6) is 14.3 mM.sup.−1 cm.sup.−1.

(58) The results are as shown in FIG. 3. The OsAO protein has lower activity in the control rice (Nipponbare not infected by RSV nor RBSDV), but has higher enzymatic activity in NPB (Oryza sativa L.) infected by rice stripe virus (RSV) or rice black-streaked dwarf virus. This result indicates that the enzyme activity of OsAO protein can be induced by infection of rice stripe virus (RSV) or Rice black-streaked dwarf virus (RBSDV).

Example 2. Enhanced Rice Antiviral Defense Against Rice Stripe Virus of OsAO Overexpression Transgenic Rice Lines

(59) I. The Accumulation of AO in pAO: AO and AO-Res Transgenic Rice is Significantly Higher than that in Wild Type Rice Nipponbare (NPB)

(60) 1. The mRNA Levels of AO in pAO: AO and AO-Res Transgenic Rice are Significantly Higher than that in Wild Type.

(61) Some leaves of pAO: AO, AO-Res transgenic rice and wild type rice are ground in liquid nitrogen, total RNA is extracted, and real-time quantitative fluorescent PCR is performed to detect AO mRNA level. The method is carried out by referring to step 1 of example 1.

(62) Primer Sequences are Listed as Follows:

(63) TABLE-US-00003 AO-qRT-F: (SEQ ID NO: 11) 5′-CGAGAACGTGGAGACCTGCGTCGA-3′; AO-qRT-R: (SEQ ID NO: 12) 5′-CCACCACCGTCATCTTGTGCCCTTG-3′.

(64) The reference gene is OsEF gene in rice and the primers sequence are:

(65) TABLE-US-00004 OsEF-la-F: (SEQ ID NO: 13) 5′-GCACGCTCTTCTTGCTTTCACTCT-3′; OsEF-la-R: (SEQ ID NO: 14) 5′-AAAGGTCACCACCATACCAGGCTT-3′.

(66) From the results in FIG. 4, it can be seen that compared with the non-transgenic wild type rice (Oryza sativa L.) variety Nipponbare, the expression of OsAO gene in pAO:AO and AO-Res transgenic rice plants is higher, especially pAO: AO-Res transgenic line.

(67) 2. AO mRNA Levels are Significantly Increased in pAO: AO and AO-Res Transgenic Rice after Virus Infection.

(68) Non-transgenic wild-type rice cultivar Nipponbare, pAO:AO and pAO: AO-Res transgenic rice lines are infected with RSV or non-toxic Laodelphax striatellus in the same stage (tillering stage), in which the corresponding rice infected with non-toxic ash fly is used as a control group. The inoculated and control pAO:AO and AO-Res transgenic rice and wild type partial leaves are ground in liquid nitrogen to extract total RNA, and real-time quantitative fluorescent PCR is used to detect AO mRNA level. The method was carried out in accordance with step 1 of Example 1.

(69) The results are shown in FIG. 4. As can be seen from the figure, compared with the uninfected control rice, the mRNA levels of AO in all lines after infection are increased, especially in the pAO:AO and AO-Res transgenic rice.

(70) 3. The Relative Enzyme Activity of AO in pAO: AO and AO-Res Transgenic Rice is Significantly Higher than that of Wild Type Rice Nipponbare (NPB).

(71) For the specific operation, refer to step III in example 1.

(72) The results are shown in FIG. 5. As can be seen from the figure, compared with the non-transgenic wild type rice (Oryza sativa L.) variety Nipponbare, the relative enzyme activity of OsAO in pAO:AO and AO-Res transgenic rice plants is significantly increased, especially in pAO: AO-Res line. At the same time, the enzyme activity of OsAO is also increased significantly after virus infection.

(73) 4. The Accumulation of OsAO Protein in pAO: AO and AO-Res Transgenic Rice is Significantly Higher than Wild Type Rice Nipponbare (NPB).

(74) For the specific operation, refer to step III in example 1.

(75) The results are shown in FIG. 5. As can be seen from the figure, compared with the non-transgenic wild type rice (Oryza sativa L.) variety Nipponbare, the accumulation of OsAO protein in pAO:AO and AO-Res transgenic rice is increased significantly, especially in pAO: AO-Res line. At the same time, the protein accumulation of AO was also significantly up-regulated after virus infection.

(76) II. pAO: AO and AO-Res Transgenic Rice has Increased Resistance to RSV.

(77) 1. After RSV Infects pAO: AO and AO-Res Transgenic Rice, the Disease is Relieved, and the Incidence is Significantly Reduced.

(78) Non-transgenic wild-type rice cultivar Nipponbare, pAO:AO and pAO: AO-Res transgenic rice lines are infected with Laodelphax striatellus carrying RSV in the same stage (tillering stage), in which the corresponding rice infected with non-toxic ash fly is used as a control group.

(79) After 2 weeks, the incidence of rice is detected (the identification of rice incidence refers to the literature “Chapter 1.1 of the Identification and Control of Major Virus Diseases in Rice and Wheat”), and the incidence rates of the experimental group and the control group are statistically calculated. Simultaneously, the difference of the phenotypes after the onset of rice among different experimental groups is observed.

(80) In the experimental group, the statistical results of the incidence of each rice line are shown in Table 1. It can be seen from the table that the incidence of pAO:AO and pAO:AO-Res transgenic rice infected by RSV was significantly lower than that of wild-type rice cultivar Nipponbare. Further, the symptoms of each rice line inoculated with RSV is shown in FIG. 7. After RSV infects pAO:AO and pAO:AO-Res transgenic rice lines, the disease is lighter than wild type rice, and the green streak is reduced. The incidence of the leaves is also reduced.

(81) TABLE-US-00005 TABLE 1 Statistics of incidence of OsAO and OsAO-Res overexpressing rice after RSV infection. Rice line N.sup.a D.sup.b P.sup.c NPB 73 41 56.16% pAO:AO 75 29 38.67% pAO:AO-Res #1 73 24 32.87% pAO:AO-Res #2 74 27 36.47% pAO:AO-Res #3 75 29 38.66% Note: .sup.athe total number of rice plants observed; .sup.bthe number of rice plants with disease phenotype after two weeks of infection; .sup.cthe proportion of diseased rice relative to all infected rice; .sup.dcontrol without the preference of insect mediators to rice lines.

(82) The above experimental results show that the overexpression of OsAO gene in pAO:AO and AO-Res transgenic rice lines enhances the disease resistance of plants, making rice less susceptible to RSV infection.

(83) 2. Northern Blot Analysis about Accumulation of RNA Strands of RSV Genome in Diseased Rice

(84) The wild-type rice variety Nipponbare, diseased pAO:AO and pAO:AO-Res transgenic rice, undiseased wild-type rice variety Nipponbare, undiseased pAO:AO and pAO:AO-Res transgenic rice obtained in step 1 are used as an experimental material. 2 g of each rice leaf is taken and ground into powder in liquid nitrogen, and total RNA is extracted according to the Invitrogen TRIzol Reagent specification (Invitrogen Trizol Reagent, cat No. 15596-018), and the resulting samples are ready for use after measuring the concentration.

(85) A. 120 mL of 1.2% agarose formaldehyde denatured gel is prepared in a ventilated cabinet: 1.44 g agarose is added to 87.6 mL DEPC water, heated in a microwave to melt the agarose, then cooled to a temperature of about 60° C. Thereto 12 ml 10×mops stock solution and 20.4 mL of formaldehyde is added. After shaking and mixing well, the resulting mixture is quickly poured into the gel groove and insert the comb.

(86) B. RNA loading buffer is added to 10-20 μg RNA sample, heated at 100° C. for 10 minutes, and then placed on ice for 2-3 min, and centrifuged for 1-2 min before loading.

(87) C. the denatured sample is added by pipette to the cooled agarose formaldehyde denaturing gel. The electrophoresis solution is 1×mops solution, and at voltage of 100V, electrophoresis is carried out for 3-4 hours. The gel is cut and placed in a 20×SSC solution for 10-20 min.

(88) D. After electrophoresis is completed, two methods, i.e., vacuum transfer and capillary transfer are used for transmembrane. The basic method of capillary transfer comprises: 20×SSC solutions is poured into the culture dish, and 2-3 layers of filter paper are placed on the glass plate to form a paper bridge. The gel is put on the paper bridge, the PDVF film is placed on the top of the gel, and thereto 3 layers of filter paper and 10-25 cm thick of absorbent paper are put. A weight is pressed thereon again and transferring (transmembrane) is conducted for 24-36 hours. The principle of vacuum transfer is the same as that of capillary transfer, using a vacuum pump to speed up the transfer of the solution.

(89) E. UV crosslinking: the film is crosslinked at the energy of 1800. The film can then be baked at 80° C. for 30 min. The treated film can be stained with methylene blue, and tested about the presence or absence of degradation of the RNA in the previous step and whether the amount of sample loading is consistent. The band of the dyed rRNA can be used as a control.

(90) F. The film is placed in a hybridization flask containing a pre-mixed liquid (Sigma, serial number SLBG7228V), and pre-mixed at 65° C. for 1-2 hours.

(91) G. The labeled probe (primer sequences for amplifying the four RNA strand probes is shown below) was denatured at 100° C. for 10 min and then placed on ice for 3 min to cool. This is added to the pre-mixed solution and hybridized overnight (over 24 hours) at 65° C. Labeled Probes Random Primer Reaction System refers to the method provided by TAKARA Probe Labeling Kit (Cat. No. D6045):

(92) TABLE-US-00006 ddH.sub.2O added to 50 μl volume 29 μl Labeling 5× buffer 10 μl unlabeled dNTPs mixture  2 μl Denatured RNA template (30-50 ng)  1 μl BSA  2 μl α-32P dCTP (50 μCi, 3000 Ci/mmol)  5 μl DNA Polymerase I Klenow Large  1 μl Fragment (5 U)

(93) The primers used to amplify the four RNA strand probes are shown below (5′-3′)

(94) TABLE-US-00007 RSV-RNAl-F: (SEQ ID NO: 15) 5′-GCACCCAATAGGTATCTCCTTGAT-3′; RSV-RNAl-R: (SEQ ID NO: 16) 5′-CAAATGACCCTACTAGATGGACGA-3′. RSV-RNA2-F: (SEQ ID NO: 17) 5′-CAACCACCCTTATCACAAACTTCA-3′; RSV-RNA2-R: (SEQ ID NO: 18) 5′-CACCAATACCTTTCCCTGACACCC-3′. RSV-RNA3-F: (SEQ ID NO: 19) 5′-TATATGGGCACCAACAAGCCAGCC-3′; RSV-RNA3-R: (SEQ ID NO: 20) 5′-TATGACTTAGGGAGTGAGTTGTGCAGT-3′. RSV-RNA4-F: (SEQ ID NO: 21) 5′-GCTTCACCACACCGAACTCCTTCT-3′; RSV-RNA4-R: (SEQ ID NO: 22) 5′-GTTACGATTGACCAAGCTGCCACA-3′.

(95) H. After the hybridization is completed, the film is washed twice with a 2× is washing solution (2×SSC, SDS is added to a final concentration of 1 g/L) at 65° C. for 20 minutes each time. Then, a 0.1× washing solution (0.1×SSC, SDS is added to a final concentration of 1 g/L), and the film is washed once at 65° C. for about 20 minutes.

(96) I. The film is air-dried and wrapped with plastic wrap to measure the radiation intensity. Tableting (X-ray film or phosphor screen) is carried out, and the length of the tableting time is determined according to the radiation intensity.

(97) rRNA is used as a control in the experiment.

(98) J. The experimental results are shown in FIG. 8, the enrichment of the four RNA strands of the RSV genome in the pAO:AO and pAO:AO-Res transgenic rice lines is lower than that of the wild type rice Nipponbare, which further indicates that the amount of replication of RSV in pAO:AO and pAO:AO-Res transgenic rice lines is relatively small. This is consistent with the results measured in step 1: the pAO:AO and pAO:AO-Res transgenic rice lines are more resistant to disease than the wild type rice variety Nipponbare.

(99) K. The above results all proved that the OsAO gene is involved in rice defense against rice stripe virus. After overexpressing the gene in rice, the plant is more resistant to disease, the disease is weakened, and the amount of virion replication is reduced.

Example 3: Enhanced Ability of Transgenic Rice Overexpressing OsAO Against Rice Black Streak Dwarf Disease

(100) I. pAO: AO-Res Transgenic Lines and Mir528 Mutant Rice have Enhanced Resistance to RBSDV

(101) 1. After RBSDV Infects pAO: AO-Res Transgenic Lines and Mir528 Mutant Rice, the Disease is Alleviated and the Incidence is Significantly Reduced.

(102) Non-transgenic wild-type rice cultivar Nipponbare, pAO: AO-Res transgenic rice line, miR5280E rice, and mir528 mutant rice and wild type DJ are infected with Laodelphax striatellus carrying RBSDV virus in the same stage (Tiller period). The corresponding rice is infected with non-toxic Laodelphax striatellus (gray planthopper) as a control group.

(103) After 2 weeks, the incidence of rice was detected (the identification of rice incidence refers to Chapter 1.3 of the book Identification and Control of Major Virus Diseases in Rice and Wheat), and the incidence rates of the experimental group and the control group are statistically analyzed. Simultaneously, the difference of the phenotypes after the onset of rice among different experimental groups is observed.

(104) In the experimental group, the statistical results of the incidence of each rice line are shown in Table 2. It can be seen from the table that the incidence of RBSDV-infected pAO:AO-Res transgenic rice lines and mir528 mutants with higher AO expression is significantly lower than that of RBSDV-infected wild-type rice varieties Nipponbare and DJ. Further, the symptoms of each rice line inoculated with RBSDV virus is shown in FIG. 9. After RBSDV infects the line with higher AO expression, i.e., mir528 mutant and pAO: AO-Res transgenic rice line, the disease is significantly lighter than wild-type rice, especially in pAO: AO-Res transgenic lines, plant dwarfing is weakened or even no dwarfing.

(105) TABLE-US-00008 TABLE 2 Statistics of incidence after RBDSV infection of OsAO and OsAO-Res overexpression rice Rice line N.sup.a D.sup.b P.sup.c NPB 75 55 73.33% DJ 73 52 71.93% miR528OE#1 73 62 84.93% miR528OE#2 73 60 82.19% mir528 75 39   52% pAO:AO-Res#1 75 31 41.33% pAO:AO-Res#2 75 31 41.33%

(106) 2. After RBSDV Infects pAO: AO-Res Transgenic Lines and Mir528 Mutant Rice, the Virus Accumulation is Less.

(107) The diseased wild type rice varieties Nipponbare and DJ, diseased pAO: AO-Res transgenic rice, diseased mir528 mutant and the miR5280E line and undiseased lines obtained in the step 1 are used as experimental materials. 2 g of each rice leaf material is ground into powder in liquid nitrogen. Total RNA is extracted according to Invitrogen TRIzol Reagent instructions (Invitrogen Trizol Reagent, cat No. 15596-018) for further detection of RBSDV four genomic RNA strands (S1,S2,S6,S10) by qRT-PCR. The specific experimental steps refer to step I of the example 1.

(108) The primer sequences used in the experiment are as follows (5′-3′)

(109) TABLE-US-00009 RBSDV-S1-F: (SEQ ID NO: 23) 5′-AACCCAGTCAAGACGCTCA-3′; RBSDV-S1-R: (SEQ ID NO: 24) 5′-CGACATCAAATGAAGCACCT-3′. RBSDV-S2-F:  (SEQ ID NO: 25) 5′-TGTGATAACAGAATGACGGC-3′; RBSDV-S2-R: (SEQ ID NO: 26) 5′-CTTCGGTCGGACAATACAC-3′. RBSDV-S6-F:  (SEQ ID NO: 27) 5′-TCAGCAGTCTTGGGTTGAT-3′; RBSDV-S6-R: (SEQ ID NO: 28) 5′-CAGTTTCAGCAGAGTAACGC-3′. RBSDV-S10-F: (SEQ ID NO: 29) 5′-ATTGGCGAAGTGTTGAGC-3′; RBSDV-S10-R: (SEQ ID NO: 30) 5′-CGGGTGCTAAATGAAATGC-3′.

(110) The results are shown in FIG. 10. In the pAO:AO-Res transgenic rice and mir528 mutants with relatively high AO expression, the accumulation of viral RNA strands is less than that of the wild type, while the accumulation of the four RNA strands of the virus in the miR5280E rice with relatively low AO expression is higher than that of the wild type, which is consistent with the phenotype observed in FIG. 9.

(111) II. The Accumulation of OsAO and the Enzyme Activity of OsAO Protein in Rice are Up-Regulated after RBSDV Infection.

(112) 1. After RBSDV Infects Different Rice Lines, the mRNA Level of OsAO is Up-Regulated.

(113) The diseased wild type rice varieties Nipponbare and DJ, the pathogenic pAO: AO-Res transgenic rice, diseased mir528 mutant and miR5280E line and undiseased lines obtained in step I are used as experimental materials. 2 g of each rice leaf material is taken and ground into powder in liquid nitrogen. Total RNA is extracted according to Invitrogen's TRIzol Reagent instruction (Invitrogen Trizol Reagent, cat No. 15596-018) for further detection of OsAO mRNA accumulation by qRT-PCR. The specific experimental steps refer to step I of Example 1.

(114) The results are shown in FIG. 11. The mRNA level of OsAO is significantly up-regulated after RBSDV infection, indicating that the expression of OsAO is induced in plants infected with RBSDV.

(115) 2. The Relative Enzyme Activity of OsAO is Enhanced after RBSDV Infects Rice of Different Lines.

(116) The diseased wild type rice varieties Nipponbare and DJ, diseased pAO: AO-Res transgenic rice, diseased mir528 mutant and the miR5280E line and undiseased lines obtained in step I are used as experimental materials to detect the relative enzyme activity of OsAO of each material. The specific experimental steps refer to step I of example 1.

(117) The results are shown in FIG. 12. The enzyme activity of OsAO is significantly enhanced after RBSDV infection, indicating that OsAO has function during the infection of RBSDV.

(118) Based on the above experimental results, it is shown that OsAO plays a role in plant anti-RBSDV process and can enhance the resistance of plants to rice black-streaked dwarf virus.