WO2016065896A1 - OsAGO18蛋白或其编码基因在调控植物对水稻矮缩病毒或其同科病毒的抗性中的应用 - Google Patents

OsAGO18蛋白或其编码基因在调控植物对水稻矮缩病毒或其同科病毒的抗性中的应用 Download PDF

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WO2016065896A1
WO2016065896A1 PCT/CN2015/080183 CN2015080183W WO2016065896A1 WO 2016065896 A1 WO2016065896 A1 WO 2016065896A1 CN 2015080183 W CN2015080183 W CN 2015080183W WO 2016065896 A1 WO2016065896 A1 WO 2016065896A1
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rice
virus
protein
osago18
plant
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French (fr)
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李毅
戚益军
吴建国
杨志蕊
魏春红
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北京大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

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  • the invention belongs to the field of biotechnology, and particularly relates to the use of the OsAGO18 protein or a coding gene thereof or a recombinant vector containing the same for regulating the resistance of a plant to a rice dwarf virus or a homologous virus thereof.
  • RSV rice stripe virus
  • RDV rice dwarf virus
  • RBSDV rice black-streaked dwarf virus
  • SRBSDV Dwarf virus
  • RRSV rice serrated leaf dwarf virus
  • BYDV barley yellow dwarf virus
  • WYMV wheat yellow mosaic virus
  • RDV, RBSDV and RRSV belong to the rice reoviridae virus, in which RDV belongs to the reovirus genus, RBSDV belongs to the Fiji virus genus, and RRSV belongs to the rice genus.
  • the viral disease caused by Rice Dwarf Virus also known as rice common dwarf disease, Puxiao, Qingxiao, etc.
  • the disease is widely distributed in Japan, North Korea, Nepal, South Korea, the Philippines, as well as China's Zhejiang, Fujian, Yunnan, Guangxi, Jiangsu, Anhui, Jiangxi, Hunan, Hubei, Guangdong, Chongqing, Sichuan and Guizhou.
  • In Yunnan it is mainly distributed in Kunming, Yuxi, and Dehong and Xishuangbanna in the south of Yunnan. When an outbreak occurs, it can cause 30%-50% of production loss, even if the particles are not collected.
  • Rice infected with rice dwarf disease is generally characterized by dwarfing of the diseased plant, increased tillering, thick green leaves, stiff leaves, white spots on the leaves or sheaths, and dotted lines parallel to the veins. After the occurrence of dwarf disease in different rice varieties, the symptoms showed certain differences. Except that the dwarfing of the plants was a common feature, some varieties were obviously dark green and had no chlorotic streaks, while some varieties became yellowing complications. And there are obvious chlorotic streaks. Rice dwarf disease has slightly changed symptoms depending on the stage of the disease. When the seedling stage (Putian period) is infected, there is less tillering and more dead after transplanting.
  • the seedling stage is inoculated, resulting in dwarf and stiffness of the plant, severe dwarf, increased tillering, green leaves, short leaves, length less than 10cm, diseased plants can not head solid.
  • the young panicle the formation period of the differentiation phase
  • the flowering and fruiting period the mature stage of the waxy ripening period
  • chlorotic streaks in the leaves although the heading can be obtained, but the seed setting rate is low, and there are many glutinous grains.
  • rice stripe disease the obvious feature of rice dwarf disease is that the plant is dwarfed, and the diseased plants appear as stunted or random blocky distribution in the field.
  • Both the rice black-streaked dwarf virus (RBSDV) and the rice dwarf virus are reoviridae viruses, which are Fiji virus genus, and the virus has a total genome of 10 fragments.
  • the symptoms of rice black streak dwarf disease and corn rough disease are very similar. The sign is that the stems and leaves of the diseased leaves are shortened and stacked, the color is dark green, the texture is stiff, the flag leaves are short and wide and flat, and the waxy tears in the back, sheath or stem base of the leaves are waxy white, then become dark brown. Tillers in the tillering stage have dwarf clusters, heart leaf distortions and marginal curvatures ( ⁇ , Chen Shengxiang, Lin Ruifen, et al. Studies on rice black-streaked dwarf disease.
  • MRDV maiz rough dwarf virus
  • MRDV and RBSDV belong to the same family of reoviruses, Fiji virus genus, and the genome is 10 Double-stranded RNA, and is mainly transmitted by the same drug-transmitting mediator (Azuhata, F., Uyeda, I., Kimura, I. and Shikata, E. (1993). Close similarity between genome structures of rice black -streaked dwarf and maize rough dwarf viruses. J. Gen. Virol. 74, 1227-1232).
  • the rice dwarf virus (RDV) virion is a symmetrical icosahedral spherical structure with no lipoprotein outer membrane, no protrusions, and a double coat protein.
  • the complete particle diameter is 70 nm
  • the core is 53 nm
  • the thickness of the outer and inner shells are 17 nm and 7 nm, respectively.
  • the virus content is high, and the ultra-thin sections observe that the diseased leaf cells contain a large amount of virion and virus matrix.
  • the genome of RDV has a total length of 25,617 bp and consists of 12 double-stranded RNAs. According to its slow-to-fast mobility on polyacrylamide gel electrophoresis, it is named S1-S12.
  • the RDV virus itself has a deeper understanding, but little is known about the interaction between RDV and the host. This may be related to the nature of the RDV virions, and it is not possible to carry out effective invasive cloning.
  • the host of the virus is limited to gramineous plants and cannot infect tobacco and Arabidopsis, such as easy-to-operate Solanaceae and Cruciferae. plant. With the development and maturity of modern molecular biotechnology, research on the interaction between viruses and hosts has become a hot topic. In 2005, Zhu first revealed the dwarf mechanism of RDV pathogenesis.
  • Glycolate oxidase is a key protein in peroxisomes, and interaction with P8 may make P8 It also enters the peroxisome, which may be the site of replication of the RDV.
  • Transcriptional profiling of RDV-infected rice using rice genome-wide microarray technology revealed that RDV infects rice and causes a series of defense gene expression, including PR protein, WRKY transcription factor, etc., indicating rice RDV Infection initiated a defense response.
  • the expression of a large number of cell wall synthesis-related genes and chloroplast function-related genes was significantly decreased.
  • RNA silencing is a very important strategy for the regulation of eukaryotic gene expression and a method for plant resistance to viral infection (Hauptmann J and Gunter M, 2013; Li et al., 2013a; Maillard, et al., 2013; Meister G 2013; Aliyari and Ding 2009; Ding 2010; Cao et al., 2014; Wu et al., 2015).
  • Inhibition of RNA silencing and RNA silencing between viruses and hosts will play an important role in understanding the regulation of gene expression in eukaryotes.
  • the technology has been recognized by international research and applied to disease resistance genetic engineering breeding and functional gene research.
  • RNA silencing experiments performed on classical genetics experimental materials revealed several features of RNA silencing: specific, efficient, and diffusible.
  • RNA silencing or artificially constructed miRNAs have at least a 10-fold greater inhibitory effect on homologous gene expression than previous sense or antisense RNA technologies. How to construct the inverted repeat sequences of target genes quickly and efficiently has become a key step in the current use of RNA silencing mechanisms to obtain engineered plants immune to related viruses.
  • Dicer is a type III endonuclease that specifically recognizes double-stranded RNA in an organism or a double-stranded region formed by folding a single-stranded RNA to produce a small RNA of 20 to 25 nt in length. All Dicers have a homologous ribozyme domain: the RNaseIII domain. This domain can cleave double-stranded RNA to produce a duplex fragment, and the cleavage product typically has a 5' phosphate group and a 3' end with 2 base projections.
  • AGO protein an RNA-binding protein with a size of 90-130 kDa, is a core component of RNA silencing that binds to siRNA, miRNA or piRNA to form an active RISC (RNA-induced silencing complex). Studies have shown that AGO protein plays an important role in the plant antiviral process.
  • the model organism Arabidopsis contains 10 AGO proteins, of which the AtAGO1 protein participates in the process of plant resistance to various viruses by binding to virus-derived vsiRNA (Burgyan and Havelda, 2011; Zhang et al., 2006).
  • AGO2 and AGO7 also play different roles in antiviral responses (Carbonell et al., 2012; Ding, 2010).
  • Rice is currently known to have 19 AGO proteins, but little research has been done on the involvement of rice or other important crop AGO proteins in antiviral defense responses.
  • RDR RNA-dependent RNA polymerase (RDR) synthesizes dsRNA using RNA single strand as a template, and generates small RNAs under DCLs cleavage.
  • RNA silencing has become a conservative defense mechanism against plant infection.
  • VSR protein factor
  • Mutants are the basis for studying functional genomics and are important ways to isolate genes and identify gene functions. Since the 1970s, a series of mutant libraries have been established around the world. With the completion of rice genome-wide sequencing, rice mutants have been widely used to identify and regulate the linkage analysis of rice morphological and physiological traits and genes and the cloning and functional studies of related genes. Combined with rice expression profile data, screening of mutants of plant disease resistance-related genes and functional complementation analysis of transgenic plants is an important way to study the molecular genetic mechanisms of disease-resistant genes and their effects.
  • Solexa sequencing technology is one of the deep sequencing technologies and belongs to the second generation sequencing technology. Their rise and development provides a very good tool for genome and RNA research, which can high-throughput nucleotide sequence determination. This method is widely used not only in small RNA sequencing, but also in the study of genomic methylation, transgreen group and genome resequencing. Deep sequencing technology can reveal many of the information in the interaction between RDV and rice, such as the role of rice RNA silencing-related proteins in disease resistance. Before deep sequencing, it is necessary to construct a small RNA library and mRNA library. Firstly, extract total RNA from rice and healthy rice tissues infected with RDV, construct mRNA and small RNA libraries respectively, and connect the two ends to reverse transcription and PCR. The resulting cDNA was subjected to deep high-throughput sequencing.
  • A1 regulating the resistance of the plant to rice dwarf virus or its homologous virus
  • the OsAGO18 gene is the DNA molecule of any one of the following items 1) to 4):
  • the above stringent conditions may be that the solution is mixed with a solution of 6 x SSC, 0.5% SDS at 65 ° C, and then washed once with 2 x SSC, 0.1% SDS and 1 x SSC, 0.1% SDS.
  • sequence 1 consists of 3267 nucleotides, wherein positions 1-3267 are the coding sequence (ORF) of the OsAGO18 gene, encoding the protein represented by sequence 2 in the sequence listing, and sequence 2 consists of 1088 amino acid residues.
  • Sequence 3 consists of 3300 nucleotides, wherein positions 1-300300 are the coding sequence (ORF) encoding the fusion protein shown in SEQ ID NO: 4 in the sequence listing, and sequence 4 consists of 1099 amino acid residues.
  • all of the above-mentioned regulatory plants in a1) are plant diseases caused by rice dwarf poisoning or its homologous virus.
  • the resistance of toxic diseases such as rice dwarf disease and black streak dwarf disease are embodied in: pathogen-infecting plants can induce expression of the protein and its coding gene; and promoting expression of the protein or its coding gene, Plants are more resistant to rice dwarf disease.
  • the method for selecting a plant variety having enhanced resistance to rice dwarf disease as described in all of the above a2) may specifically include a step of hybridizing the plant or a plant having a higher expression level of the coding gene as a parent.
  • Another object of the present invention is to provide a method of cultivating a transgenic plant having enhanced resistance to rice dwarf poisoning or its isogenic virus.
  • the method for cultivating a transgenic plant having enhanced resistance to plant dwarf virus caused by rice dwarf poisoning or its homologous virus provided by the present invention may specifically include the following steps:
  • step b) obtaining a transgenic plant having enhanced resistance to rice dwarf poisoning or its isogenic virus as compared to the plant of interest obtained from the transgenic plant obtained in step a).
  • the above stringent conditions can be, for example, a solution of 6 x SSC, 0.5% SDS, hybridized at 65 ° C, and then washed once with 2 x SSC, 0.1% SDS and 1 x SSC, 0.1% SDS.
  • sequence 1 consists of 3267 nucleotides, wherein positions 1-3267 are the coding sequence (ORF) of the OsAGO18 gene, encoding the protein represented by sequence 2 in the sequence listing, and sequence 2 consists of 1088 amino acid residues.
  • Sequence 3 consists of 3300 nucleotides, wherein positions 1-300300 are the coding sequence (ORF) encoding the fusion protein shown in SEQ ID NO: 4 in the sequence listing, and sequence 4 consists of 1099 amino acid residues.
  • the protein consists of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing or the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing.
  • the protein consisting of the amino acid sequence shown in the sequence 2 in the sequence listing is named OsAGO18 protein (the coding gene is sequence 1, designated as OsAGO18 gene), and the protein consisting of the amino acid sequence shown in sequence 4 in the sequence listing is the OsAGO18 protein.
  • the function of the protein encoded by the gene can be affected by point mutation, addition or deletion of one or more bases in the gene sequence. Therefore, the present invention should be understood to include the above variation of the OsAGO18 gene.
  • the sequence of the OsAGO18 gene is not limited to the sequence 1 in the sequence listing, but also includes a DNA sequence which mutates any codon corresponding to an amino acid residue in the functional region of the protein. It is mainly to mutate functional amino acid residues into alanine which is generally considered to have no special function.
  • the gene encoding the protein is introduced into the plant of interest by a recombinant expression vector containing the gene encoding the protein.
  • the recombinant expression vector can be constructed using existing plant expression vectors.
  • the plant expression vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment, and the like, such as pCAMBIA3301, pCAMBIA2300, pCAMBIA2301, pCAMBIA1300, pCAMBIA 1301, pWM101, pGreen0029, pBI121, pBin19, pCAMBIA1301-UbiN, etc. or other derived plants.
  • Expression vector may further comprise a 3' untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression.
  • the polyadenylation signal directs the addition of polyadenylation to the 3' end of the mRNA precursor.
  • any enhanced, constitutive, tissue-specific or inducible promoter may be added before the transcription initiation nucleotide, for example, cauliflower mosaic virus (CAMV) 35S promoter a ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter Rd29A, etc., which can be used alone or in combination with other plant promoters; in addition, when the recombinant expression vector is constructed using the gene of the present invention, it can also be used.
  • CAMV cauliflower mosaic virus
  • pUbi ubiquitin gene Ubiquitin promoter
  • Rd29A stress-inducible promoter
  • Enhancers including translational enhancers or transcriptional enhancers, may be ATG start codons or contiguous region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the recombinant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound that can be expressed in plants, and a resistant antibiotic marker. Or anti-chemical reagents, etc. Transformed plants can also be screened directly under adverse conditions without any selectable marker genes.
  • the promoter that initiates transcription of the coding gene in the recombinant expression vector is specifically an Actin promoter.
  • the recombinant expression vector is a recombinant plasmid obtained by replacing the coding gene with a small fragment between the restriction sites Sma I and Sal I of the pCam23 ACT:OCS vector.
  • the promoter that initiates transcription of the coding gene is the Actin promoter.
  • the recombinant expression vector carrying the coding gene is introduced into the plant of interest, specifically: by Agrobacterium-mediated method, gene gun method, electroporation method, pollen tube introduction method, liposome fusion
  • the method and any other method for introducing a plasmid are transformed into a plant cell or tissue, and the transformed plant tissue is grown into a plant.
  • the plant may be a monocot or a dicot.
  • the monocot is a gramineous plant.
  • the plant is rice or corn.
  • rice varieties are preferably varieties that are sensitive to RDV, such as Zhonghua 11, Xiushui 11, Nipponbare, and the like.
  • the plant is a rice variety Nipponbare.
  • the pathogen of the rice dwarf disease is specifically Rice Dwarf Virus, Rice black-streaked dwarf virus, and Maize rough dwarf. Virus) or Rice stripe virus.
  • the above-mentioned homologous virus is preferably selected from Rice black-streaked dwarf virus, maiz rough dwarf virus or Rice stripe virus.
  • OsAGO18 is a disease-resistant gene that can be specifically induced by viruses, in rice dwarf virus (RDV), rice stripe virus (RSV), and rice black streaked dwarf virus (RBSDV) or maize rough virus. (Maiz rough dwarf virus) After infecting wild-type Japanese clear rice, the expression of OsAGO18 protein was significantly accumulated.
  • RDV rice dwarf virus
  • RSV rice stripe virus
  • RBSDV rice black streaked dwarf virus
  • Maize rough virus Maize rough virus
  • RDV infects overexpressing OsAGO18 transgenic rice, the accumulation of virions is significantly lower than that of wild-type rice, and the disease is also mild, and the incidence is decreased, indicating that OsAGO18 gene has the ability to improve rice antiviral.
  • rice black-streaked dwarf virus can infect corn at the same time, causing corn rough disease, and rice black-streaked dwarf virus can also induce high expression of OsAGO18 mRNA and protein levels after infecting rice, and ago18 mutant is resistant to the virus. It is weaker than wild type, indicating that AGO18 protein or its coding gene can enhance the ability of maize to resist rough corn disease caused by RDV, RBSDV, MRDV and the like.
  • Figure 1 shows the results of AGO18 protein accumulation after rice dwarf virus, rice black-streaked dwarf virus and rice stripe virus infecting wild-type Nipponbare rice.
  • Western blot analysis of AGO18 protein levels was induced after infection with various viruses.
  • Mock is the wild type rice variety Nipponbare infected by virus
  • RDV is the wild type rice variety Nipponbare infected by RDV
  • RBSDV is the wild type rice variety Nipponbare infected by RBSDV
  • RSV indicates the onset of RSV infection
  • Tubulin is a loading control.
  • FIG. 2 shows the results of OsAGO18 mRNA accumulation after infection by different viruses in rice.
  • Realtime PCR analysis of OsAGO18 mRNA was induced after infection with different rice viruses.
  • Mock indicates that the wild-type rice variety Nipponbare is not infected by the corresponding virus;
  • RDV, RBSDV, RRSV, and RGSV indicate the wild-type rice variety Nipponbare, which is infected by the corresponding virus.
  • the expression level of EF in the internal reference gene rice was 1.
  • Figure 3 shows the insertion position information and identification results of the ago18 mutant T-DNA.
  • A is an RT18-PCR method to identify the ago18 mutant (NF6013)
  • LP+RP is the result of amplification using the primer pair 6013_LP/6013_RP
  • LB+RP is the result of amplification using the primer pair tos17_tail6(LB)/6013_RP.
  • AGO18 indicates the result of amplification of OsAGO18-F/OsAGO18-R using primers
  • Actin indicates the result of amplification using Actin-RT-F/Actin-RT-R with primers
  • B is a schematic diagram of insertion site of Tos17.
  • Fig. 4 is a diagram showing the condition of each rice line in Example 2 after inoculation of RDV.
  • WT-mock indicates that the wild type rice variety Nipponbare is not infected by RDV
  • WT-RDV indicates the wild type rice variety Nipponbare which is infected by RDV
  • ago18-mock indicates that the ago18 mutant homozygous is not infected by RDV
  • ago18-RDV indicates homozygous for the ago18 mutant infected by RDV.
  • Fig. 5 is a diagram showing the condition of each rice line in Example 2 after inoculation of RBSDV.
  • Mock indicates wild type rice which is not infected by virus
  • WT-RBSDV indicates wild type rice which was infected by RBSDV
  • ago18-RBSDV indicates ago18 mutant rice which was infected by RBSDV.
  • Figure 6 shows the results of RDV genomic RNA accumulation in susceptible ago18 mutant rice.
  • WT-mock indicates that the wild type rice variety Nipponbare is not infected by RDV
  • WT-RDV indicates the wild type rice variety Nipponbare which is infected by RDV
  • ago18-mock indicates that the ago18 mutant homozygous is not infected by RDV
  • ago18-RDV indicates homozygous for the ago18 mutant infected by RDV.
  • Figure 7 shows the results of identification of OsAGO18 overexpressing transgenic rice.
  • A is the result of RT-PCR identification;
  • B is the result of Western blot.
  • Fig. 8 is a diagram showing the condition of each rice line in Example 3 after inoculation of RDV virus.
  • Mock indicates that the wild type rice variety Nipponbare is not infected by RDV; WT-RDV indicates the wild type rice variety Nipponbare infected by RDV; Vector-RDV indicates the transgenic vector rice infected by RDV; ago18-RDV shows The homolog of the ago18 mutant infected by RDV; AGO18OE-5-RDV indicates transgenic rice line 5# infected by RDV; AGO18OE-2-RDV indicates transgenic rice line 2# infected by RDV; AGO18OE-1 -RDV indicates transgenic rice line 1# infested with RDV.
  • Rice Dwarf Virus Recorded in Zhang Zhongkai, Fang Qi, Wei Chunhong, Ding Ming, Dong Jiahong, Yu Li, Li Yi. cytopathological comparison of rice plant dwarf virus Yunnan and Zhejiang isolates infecting rice plants Research. Journal of Electron Microscopy, Vol. 25, No. 2, 2006, available to the public from Peking University.
  • Rice Stripe Virus Recorded in "Cai Xiaowei, Zhao Junling, Shao Ying, Gui Qingqing, Liu Fang. A review of the research on the propagation of rice dwarf virus by Laodelphax striatellus. China Plant Protection Guide, 2011, 09" In the article, the public can get it from Peking University.
  • Agrobacterium tumefaciens EHA105 described in "Zhu et al., 2005.
  • the Rice Dwarf Virus P2Protein interacts with ent-Kaurene Oxidases in vivo, leading to reduced biosynthesis of Gibberellins and rice dwarf symptoms. Plant Physiology. 139: 1935-1945.” The public can get it from Peking University.
  • pEASY-simple-T1 carrier Beijing Quanjin Biotechnology Co., Ltd., its catalog number is CT111-01.
  • pCam23ACT OCS vector: described in "Liang Wu et al., 2009. Rice Mr croRNA Effector Complexes and Targets. Plant Cell. 21 (11): 3421-3435.” The public is available from Peking University.
  • Example 1 It was found that the OsAGO18 gene can be induced by various viruses in rice.
  • the OsAGO18 gene involved in the present example is derived from rice (Oryza. sativa L.), the cDNA sequence of the OsAGO18 gene is shown in SEQ ID NO: 1 in the sequence listing, and the sequence 1 is composed of 3267 nucleotides, of which 1-3267 Is the coding sequence (ORF); sequence 1 encodes the protein shown in SEQ ID NO: 2 in the sequence listing, and sequence 2 consists of 1088 amino acid residues.
  • OsAGO18 protein accumulates after rice dwarf virus, rice black streaked dwarf virus and rice stripe virus infection
  • Wild type rice (Oryza.sativa L.) cultivar Nipponbare and control rice (not rice) infected by rice dwarf virus (RDV), rice black streaked dwarf virus (RBSDV) and rice stripe virus (RSV) Dwarf virus infection
  • RDV rice dwarf virus
  • RBSDV rice black streaked dwarf virus
  • RSV rice stripe virus
  • PBS-T 1L pH 7.5 1 M sodium dihydrogen phosphate 31.6 ml; 1 M disodium hydrogen phosphate 68.4 ml Tween 20 0.1% 1 ml; water 900 ml; adjusted to a pH of 1 L.
  • TBS-T 1L pH 7.6 Tris 2.42 g; NaCl 8 g; Tween 201 ml; water 900 ml; adjusted to a volume of 1 L.
  • Blocking solution 100 ml 100 ml of TBS-T was added with 5 g of skim milk powder.
  • Anti-diluent 100 ml 100 ml PBS-T was added with 0.25 g of BSA.
  • OsAGO18 protein accumulates in the infected rice material, indicating that different viral infections can induce the accumulation of OsAGO18 protein.
  • OsAGO18 protein may be involved in rice defense against multiple viruses.
  • OsAGO18 gene mRNA accumulates after infection by rice dwarf virus, rice black-streaked dwarf virus, rice stripe virus and rice sawtooth dwarf virus
  • Wild type rice (Oryza.sativa L.) infected by rice dwarf virus (RDV), rice black streaked dwarf virus (RBSDV), rice stripe virus (RSV) and rice serrated dwarf virus (RRSV)
  • RDV rice dwarf virus
  • RSV rice stripe virus
  • RRSV rice serrated dwarf virus
  • the cultivar Nipponbare and the control rice were each 0.5g, and after grinding in liquid nitrogen, according to Invitrogen's TRIzol Reagent specification (Invitrogen Trizol Reagent, cat No. 15596- 018) Total RNA was extracted.
  • RNA concentration 10 ⁇ g was taken, and the rice genomic DNA in total RNA was digested according to the instructions of RQ1Dnase (Promega, Cat. No. M610A).
  • Digestion reaction system 10 ⁇ g of total RNA, 10 ⁇ l of 10 ⁇ Dnase buffer, 10 ⁇ l of DNase, and 100 ⁇ l of DEPC water.
  • the entire digestion reaction system was incubated at 37 ° C for 35 min. After the incubation, 4 ⁇ l of RQ1DNase was added to the system to terminate the reaction solution, and incubation was carried out at 65 ° C for 10 min to inactivate DNase.
  • RNA concentration was determined. 2 ⁇ g of RNA was reverse-transcribed with Invitrogen SuperScript II reverse transcriptase, and the primer used was a 16-nucleotide Oligod (T) primer.
  • T 16-nucleotide Oligod
  • the rice cDNA was obtained by reverse transcription as a template, and the transcription level of OsAGO18 gene was detected by realtime PCR. The experimental method was based on TOYOBO. Realtime PCR Master Mix (Cat. No. QPK-201) instructions, the primers are:
  • OsAGO18-F 5'-TGTTCGTCCAGGCACAGTAG-3' (positions 2901-2920 of sequence 1);
  • OsAGO18-R 5'-GCGGTGAAGTTGTTGTCGTC-3' (reverse complement of positions 3022-3041 of SEQ ID NO: 1).
  • the internal reference gene is rice EF, and the primers are:
  • OsEF-1a-F 5'-GCACGCTCTTCTTGCTTTCACTCT-3';
  • OsEF-1a-R 5'-AAAGGTCACCACCATACCAGGCTT-3'.
  • the ago18 mutant seed (NF6013) was purchased in the Tos17 database.
  • the T-DNA insertion sequence is:
  • the insertion site is shown as B in Figure 3.
  • the 6013_LP/6013_RP primer pair, the tos17_tail6(LB)/6013_RP primer pair, and the OsAGO18-F/OsAGO18-R primer pair were subjected to semi-quantitative RT-PCR reaction.
  • wild type rice (Oryza.sativa L.) variety Nipponbare was set as a control.
  • the primer sequences are as follows:
  • 6013_LP 5'-GATCGAGGGAACTCGACAAG-3';
  • 6013_RP 5'-CAAGATCAACTCCACGCAAA-3'.
  • the tos17_tail6(LB), OsAGO18-F and OsAGO18-R sequences are described above.
  • Actin-RT-R 5'-GAGAAACAAGCAGGAGGACGG-3'.
  • the wild type rice (Oryza.sativa L.) variety Nipponbare uses the 6013_LP/6013_RP primer pair and the OsAGO18-F/OsAGO18-R primer pair to amplify the corresponding target band, but using tos17_tail6(LB)/6013_RP The primer pair did not amplify the corresponding target band. Consistent with the expected results.
  • OsAGO18 gene is involved in rice anti-RDV defense response
  • the wild type rice (Oryza. sativa L.) variety Nipponbare and the first 18 mutant homozygous obtained in the first step (experimental group) were infested with the leafhopper of RDV virus.
  • the corresponding rice was infested with avirulence leafhopper as a control group.
  • a the total number of rice plants observed
  • b the number of rice plants with disease phenotype after two weeks of infection
  • c the proportion of rice that is affected by all infected rice.
  • the wild type rice variety Nipponbare, the pathogenic ago18 mutant rice obtained in the first step, the uninfected wild type rice variety Nipponbare, and the uninfected ago18 mutant rice were used as experimental materials. 2 g of each rice material leaf was taken, ground into powder in liquid nitrogen, and total RNA was extracted according to the Invitrogen TRIzol Reagent instruction (Invitrogen Trizol Reagent, cat No. 15596-018), and the concentration was measured and used.
  • RNA loading buffer 10-20 ⁇ g RNA sample, heat at 100 °C for 10 minutes, then place on ice for 2-3 min, and centrifuge for 1-2 min before loading.
  • the denatured sample is added to the cooled agarose formaldehyde denaturing gel.
  • the electrophoresis solution is 1 ⁇ mops solution, voltage is 100V, and electrophoresis is carried out for 3-4 hours.
  • the gel was cut and placed in a 20 x SSC solution for 10-20 min.
  • Electrophoresis is completed by two methods: vacuum transfer method and capillary transfer.
  • the basic method of capillary transfer pour 20 ⁇ SSC solution into the culture dish, and put 2-3 layers of filter paper on the glass plate to form a paper bridge. Place the glue on the paper bridge, place the PDVF film on top of the glue, and place 3 layers of filter paper and absorbent paper after 10-25cm. Press the weight again and transfer the film 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.
  • UV crosslinking The energy is 1800 to crosslink the film. After that, the film can be baked at 80 ° C for 30 min. The treated membrane can be stained with methylene blue, and the presence or absence of degradation of the RNA in the previous step and the amount of sample loading are consistent. The band of the dyed rRNA can be used as a control.
  • the membrane was placed in a hybridization flask containing a pre-mixed liquid (Sigma, serial number SLBG7228V), and pre-mixed at 65 ° C for 1-2 hours.
  • a pre-mixed liquid Sigma, serial number SLBG7228V
  • the labeled probe (primer sequence for the three RNA fragment probes used to amplify RDV is shown below) was denatured at 100 ° C for 10 min and then placed on ice for 3 min to cool. It was added to the pre-mixed solution and hybridized overnight (over 24 hours) at 65 °C.
  • Labeled Probes Random Primer Reaction System Refer to the method provided by TAKARA Probe Labeling Kit (Cat. No. D6045):
  • RDV-S2-F 5'-CCGGGACGTGCCAAACCGTGAAC-3';
  • RDV-S2-R 5'-ACGCCATCAACAGAGCAGAATCCATTA-3'.
  • RDV-S8-F 5'-TCAATAGCGATACCAAGCCTACCGTTTC-3';
  • RDV-S8-R 5'-ATCGATGTCGGGGTCTGAGTTATCGAGTTCAAT-3'.
  • RDV-S11-F 5'-CATGCCATGGCAATGAGTGGAACATTACCCTTG-3';
  • RDV-S11-R 5'-AGAATGCGGCCGCTTACTTACGCTTTGATTTGCGAG-3'.
  • the membrane was washed twice with a 2 ⁇ washing solution (2 ⁇ SSC, SDS was 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 was added to a final concentration of 1 g/L), and the membrane was washed once at 65 ° C for about 20 minutes.
  • 2 ⁇ washing solution 2 ⁇ SSC, SDS was added to a final concentration of 1 g/L
  • a 0.1 ⁇ washing solution 0.1 ⁇ SSC, SDS was added to a final concentration of 1 g/L
  • the film is air-dried and wrapped with plastic wrap to measure the radiation intensity. Tableting (X-ray film or phosphor screen), the time depends on the radiation intensity to determine the length of the tableting time.
  • the experiment also used rRNA as a control.
  • the experimental results are shown in B of Fig. 6.
  • the enrichment of the three RNA fragments of the RDV genome in the ago18 mutant rice was higher than that of the wild type rice Nipponbare, which further indicates that the RDV has an increased replication amount in the ago18 mutant.
  • the ago18 mutant measured in step 1 is more susceptible to disease and weaker disease resistance than the wild type rice variety Nipponbare.
  • OsAGO18 gene is involved in rice anti-RBSDV defense response
  • the wild type rice (Oryza. sativa L.) variety Nipponbare and the first 18 mutant homozygous obtained in the first step (experimental group) were infested with the RBSDV virus.
  • the corresponding rice was infested with avirulence leafhopper as a control group.
  • RNA of Oryza sativa L. japonica. cv. Nipponbare was extracted with TRIzol Reagent of Invitrogen according to the instructions, and reverse transcription was carried out using the company's SuperScript II reverse transcriptase.
  • the primer used for reverse transcription was a 16-nucleotide Oligod (T) primer, and finally a rice cDNA template obtained by reverse transcription was obtained.
  • PCR amplification was carried out using the following primer pair.
  • AGO18cds-F 5'-ATAATGGCGAGCCGAGGAGGAGGC-3' (positions 4-24 of the sequence are position 1-21 of sequence 1, and ATA of the first three positions are protective bases);
  • AGO18cds-R 5'-GACCTAGCAAAAGAACATGGACTTTTTC-3' (positions 4-28 of this sequence are the reverse complement of positions 3243-3267 of sequence 1, and the ATA of the first three positions are protected bases).
  • the full-length sequence was ligated to the pEASY-simple-T1 vector, transformed into E. coli, and the plasmid was subjected to sequencing.
  • the recombinant plasmid which was sequenced and inserted into the DNA fragment shown by "ATA+SEQ ID NO: 1 + GTC" in the pEASY-simple-T1 vector was designated as pEASY-OsAGO18.
  • the recombinant plasmid pEASY-OsAGO18 obtained in the step 2 was used as a template, and PCR amplification was carried out using the following primers.
  • AGO18cds-Sma 1-MYC-F 5'-ATA cccggg ATG GAG CAG AAG CTG ATC TCA GAG GAG GAC CTG -ATG GCG AGC CGA GGA GGC-3' (underlined lowercase letters are Sma I recognition sequences, underlined uppercase The letter part is the MYC tag sequence, and the -sequence is the 1st bit of sequence 1);
  • AGO18cds-Sal 1-R 5'-GAC gtcgac CTAGCAAAAGAACATGGACTTTTTC-3' (the underlined lowercase portion is the recognition sequence of Sal I, and the subsequent sequence is the reverse complement of positions 3243-3267 of sequence 1).
  • the PCR product was digested with SmaI and SalI (products of NEB, product numbers: R0141 and R0138, respectively), and the gel was recovered and ligated to the large fragment of the pCam23ACT: OCS vector which was subjected to the same double digestion to obtain a recombinant plasmid.
  • the correct recombinant plasmid (a band of about 10300 bp and 3300 bp in size) was initially identified by double digestion with SmaI and SalI.
  • the recombinant plasmid which was sequenced to replace the small fragment between the cleavage sites Sma I and Sal I of the pCam23 ACT:OCS vector with the DNA fragment shown in SEQ ID NO:3 in the sequence listing was named pCambia2300-Actin-MYC-OsAGO18.
  • Sequence 3 has a MYC tag sequence at the 5' end of the OsAGO18 gene shown in SEQ ID NO: 1 compared to SEQ ID NO: 1.
  • Sequence 3 encodes the protein shown in SEQ ID NO: 4 in the sequence listing (a fusion protein of MYC tag and OsAGO18).
  • the promoter for transcription of the DNA fragment shown in the promoter sequence 3 is the Actin promoter.
  • the seeds of Nippon rice (Oryza sativa L. japonica.cv. Nipponbare, hereinafter referred to as wild type rice) are dehulled, first soaked with 70% (volume fraction) ethanol for 10 min, and then soaked with 0.1% (volume fraction) of mercury for 30 min; get on Surface sterilization.
  • the solution on the surface of the seed is washed with a large amount of sterile water, and the moisture on the surface of the seed is absorbed by a sterile filter paper.
  • the seeds were placed on a mature embryo callus induction medium (see below for the formulation of the medium), and the edges of the plates were closed with a Parafilm membrane and incubated in a 26 ° C incubator in the dark.
  • the grown callus was carefully removed and transferred to mature embryo subculture medium (see below for the formulation of the medium), and the culture was continued under the same conditions. Subculture is required every two weeks. For transformation, it is necessary to select a pale yellow callus after subculture for about 5 days.
  • the recombinant expression vector pCambia2300-Actin-MYC-OsAGO18 obtained in the first step was electrotransferred into Agrobacterium EHA105, and the selection was able to grow on the LB plate supplemented with the antibiotic rifampicin and kanamycin, and the primer AGO18-PCR-F was used.
  • PCR amplification with AGO18-PCR-R gave a positive result of a 793 bp PCR product, which was named EHA105/pCambia2300-Actin-MYC-OsAGO18.
  • AGO18-PCR-F 5'-CCCAACTATTATATTTGGTGCTGAT-3' (positions 2475-2499 of SEQ ID NO: 1);
  • AGO18-PCR-R 5'-CTAGCAAAAGAACATGGACTTTTTC-3' (reverse complement of positions 3243-3267 of SEQ ID NO: 1).
  • Recombinant Agrobacterium tumefaciens EHA105/pCambia2300-Actin-MYC-OsAGO18 was streaked on LB plates containing antibiotics (50 mg/L Kan, 50 mg/L Rif) and cultured at 28 °C for 2 days. Single colonies were picked into liquid LB medium, shake cultured at 28 ° C to an OD 600 of about 0.5, and acetosyringone was added to a final concentration of 100 mM to obtain an Agrobacterium suspension for transforming rice callus.
  • the subcultured callus prepared in step 1 is placed in a sterilized conical flask, and the Agrobacterium suspension obtained in the step 2 is poured into the callus. Leave at room temperature for 20 min, and occasionally shake gently to make the callus fully contact with the bacterial liquid. Gently remove the callus with sterile forceps, place on sterile filter paper and remove excess bacterial solution, and transfer to a plate of co-culture medium (see formula below) with a layer of sterile filter paper. Dark culture at 28 ° C for 2-3 days gave co-cultured calli.
  • the co-cultured callus was washed with an appropriate amount of sterile water to remove the residual Agrobacterium on the surface, placed in a screening medium (see the formula below), and screened at 26 ° C in the dark, and transferred to new after two weeks. Continue screening for two weeks on the screening medium.
  • the callus with good condition after two rounds of screening was selected and transferred to a differentiation medium (see the medium formula below) for the plate, first protected from light for 3 days, and then transferred to a light incubator (15h/day). ) Light culture.
  • the differentiated seedlings can be seen after one month.
  • the differentiated seedlings When the differentiated seedlings grew to about 2 cm, they were transferred to a rooting medium (see below for the formulation of the medium) in an Erlenmeyer flask and continued to be cultured for about two weeks. After selecting growing well developed root system of seedlings, roots washed with tap water medium transplanted into the soil, seed charge, to give T 0 generation of gene transfer OsAGO18 rice seeds.
  • a rooting medium see below for the formulation of the medium
  • the medium required for the formulation is as follows:
  • the pCam23ACT:OCS empty vector was transferred into wild type rice, and the transgenic vector rice was obtained as a control.
  • the OsAGO18 gene overexpressed rice has enhanced anti-RDV ability.
  • T 0 obtained in step two
  • Substitute OsAGO18 transgenic rice seeds were sown to obtain T 1 generation of rice seedlings, from six randomly selected, numbered # 1, # 2, # 3, # 4, # 5 and # 6. Part of the leaves were taken and ground in liquid nitrogen, and some were semi-quantitative RT-PCR, and some were identified by Western blot.
  • the primer sequences are as follows:
  • OsAGO18-F 5'-TGTTCGTCCAGGCACAGTAG-3' (positions 2901-2920 of sequence 1);
  • OsAGO18-R 5'-GCGGTGAAGTTGTTGTCGTC-3' (reverse complement of positions 3022-3041 of SEQ ID NO: 1).
  • Actin-RT-R 5'-GAGAAACAAGCAGGAGGACGG-3'.
  • the strains in which OsAGO18 mRNA was significantly accumulated in the RT-PCR assay were used as RT-PCR positive vaccines.
  • results shown in FIG. 7 B can be seen, compared to the wild-type non-transgenic rice (Oryza.sativa L.) cultivar Nipponbare, 6 T 1 generation of transgenic rice plants transfected OsAGO18 higher protein expression OsAGO18 .
  • the expression level of OsAGO18 protein in transgenic rice plants was basically the same as that of untransgenic wild type rice (Oryza.sativa L.) cultivar Nipponbare (low content, no detectable expression of OsAGO18), no statistical significance. .
  • each rice line Seen from the table, RDV virus infection OsAGO18 Substitute T 1 transgenic rice lines # 1, # 2 and wild-type rice varieties of rice plants were significantly lower than the incidence RDV virus # 5 after infection of non-transgenic rice plants after Japan Clear Incidence. Further, the disease pattern of each rice line inoculated with RDV virus is shown in Fig. 8. After the RDV infects the OsAGO18 transgenic positive line, the disease is lighter than that of the wild type rice, and the white spots distributed along the veins are reduced, and the dwarf is weakened. The blades are reduced. Correspondingly, after RDV infested the ago18 mutant rice, the white spots distributed along the veins increased significantly, the leaves were dark green, and the plants were dwarfed.
  • a the total number of rice plants observed
  • b the number of rice plants with disease phenotype after two weeks of infection
  • c the proportion of rice affected by all infected rice
  • AGO18OE-5#, AGO18OE-2# and AGO18OE -1# indicates the transgenic rice lines 5#, 2# and 1# infected by RDV, respectively.

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Abstract

提供了一种OsAGO18蛋白或其编码基因或含有所述编码基因的重组载体在提高植物对水稻矮缩病毒或其同科病毒引发的病毒病害抗性中的应用；所述OsAGO18蛋白的氨基酸序列具体为序列表中的序列2或序列4。在OsAGO18基因过表达植物抗病性增强，相反，ago18突变体的抗病性减弱，表明该基因编码的OsAGO18蛋白在植物抵抗水稻矮缩病毒或其同科病毒引发的病毒病害中发挥着重要的作用。

Description

OsAGO18蛋白或其编码基因在调控植物对水稻矮缩病毒或其同科病毒的抗性中的应用 技术领域
本发明属于生物技术领域，具体涉及OsAGO18蛋白或其编码基因或含有该编码基因的重组载体在调控植物对水稻矮缩病毒或其同科病毒的抗性中的应用。
背景技术
我国稻、麦生产中发生严重和间歇暴发流行的这些病毒病害，主要由水稻条纹叶枯病毒(RSV)、水稻矮缩病毒(RDV)、水稻黑条矮缩病毒(RBSDV)、南方水稻黑条矮缩病毒(SRBSDV)、水稻锯齿叶矮缩病毒(RRSV)、大麦黄矮病毒(BYDV)和小麦黄花叶病毒(WYMV)所引起，多数病毒除侵染稻、小麦外还可侵染玉米等禾本科作物，玉米对该病毒尤其敏感，例如RBSDV侵染玉米后造成的玉米粗缩病已经成为玉米的主要病毒病害，导致玉米大规模减产。RDV、RBSDV以及RRSV同属于水稻呼肠孤病毒科病毒，其中RDV属于呼肠孤病毒属，RBSDV属于斐济病毒属，RRSV属于水稻病毒属。
由水稻矮缩病毒(Rice Dwarf Virus，RDV)引起的病毒病，又称水稻普通矮缩病、普矮、青矮等，靠昆虫介体叶蝉传播。该病广泛分布于日本、朝鲜、尼泊尔、韩国、菲律宾，以及我国浙江、福建、云南、广西、江苏、安徽、江西、湖南、湖北、广东、重庆、四川和贵州等稻区。在云南主要分布在滇中的昆明、玉溪和滇西滇南的德宏和西双版纳等地区。暴发流行时，可导致30％-50％的产量损失，甚至颗粒无收。感染水稻矮缩病的水稻普遍表现为病株矮缩，分蘖增多，叶色浓绿，叶片僵硬，在叶片或叶鞘上出现白色斑点，与叶脉平行排列成虚线状。不同水稻品种发生矮缩病后，症状表现出一定的差异，除植株的矮化为共同特征外，有的品种明显浓绿，无褪绿条斑，而有的品种则成黄化并发症，并有明显的褪绿条斑。水稻矮缩病根据发病时期的不同，其症状略有变化。幼苗期(秧田期)受侵染时，分蘖少，移栽后多枯死。秧苗分蘖期(返青期有效分蘖期无效分蘖期)苗期发病后，导致植株的矮缩僵硬，严重矮化，分蘖增多，叶色浓绿，叶片较短，长度在10cm以下，病株不能抽穗结实。幼穗发育期(分化期形成期完成期)和开花结实期(乳熟期蜡熟期完熟期)发病，叶部有褪绿条斑，虽能抽穗，但结实率低，多秕谷。与水稻条纹叶枯病比较，水稻矮缩病的明显特征是植株矮化，病株在田间表现为成片矮化或随机块状分布。由水稻黑条矮缩病毒(Rice Black-Streaked Dwarf Virus，RBSDV)引起的水稻黑条矮缩病和玉米粗缩病，靠传毒介体灰飞虱传播，两者在我国均于20世纪60年代初发现，前者最早于1963年浙江省余姚县的早稻上发现，同时在上海市嘉定和奉贤县、江苏省的苏州和镇江等水稻专区有局部危害，后者分别于70年代和90年代在华北和西北等地区的玉米上局部流行成灾。
水稻黑条矮缩病毒(RBSDV)与水稻矮缩病毒均是呼肠孤病毒科病毒，该病毒为斐济病毒属，病毒全基因组共10个片段。水稻黑条矮缩病和玉米粗缩病的发病症状十分相似，其特 征是始病叶以上茎叶短缩而叠层、色泽浓绿，质地僵硬、剑叶短宽而平展，在叶片背部、叶鞘或茎基部生蜡泪状脉肿，先是蜡白色，而后变成黑褐色。分蘖期病株的分蘖矮化丛生、心叶扭曲以及边缘有曲刻(阮义理，陈声祥，林瑞芬，等.水稻黑条矮缩病的研究.浙江农业科学，1984，(4)：185—187；27杨本荣，马巧月.玉米粗缩病的寄主范围研究.植物病理学报，1983，13(3)：1—8)。在欧洲、南美洲、东南亚以及澳洲侵染玉米引起玉米严重矮化的病毒有玉米粗缩病毒(maiz rough dwarf virus,MRDV)，MRDV与RBSDV同属呼肠孤病毒科，斐济病毒属，基因组为10条双链RNA，并主要由相同的传毒介体灰飞虱传播(Azuhata,F.,Uyeda,I.,Kimura,I.and Shikata,E.(1993).Close similarity between genome structures of rice black-streaked dwarf and maize rough dwarf viruses.J.Gen.Virol.74,1227-1232)。
水稻矮缩病毒(RDV)病毒粒子是一个呈正二十面体对称的球形结构，无脂蛋白外膜，无突起，含有双层外壳蛋白。完整粒子直径为70nm，核心为53nm,外壳和内壳的厚度分别为17nm和7nm。外壳层三角形剖分数为T＝13。在感染的水稻病株中，病毒含量很高，经超薄切片观察，病叶细胞中含有大量的病毒粒体和病毒基质。RDV的基因组总长度为25,617bp,由12条双链RNA组成，根据其在聚丙烯酰胺凝胶电泳上的迁移率由慢到快，依次命名为S1-S12。
目前对RDV病毒本身已有了较深入的认识，但对RDV与寄主间的互作了解甚少。这可能与RDV病毒粒子的本身的特性有关，不能进行有效的侵染性克隆，病毒的寄主范围仅限于禾本科植物，不能侵染烟草和拟南芥等容易操作的茄科和十字花科的植物。随着现代分子生物技术的发展和成熟，研究病毒与寄主的互作已经成为当前的热点。2005年Zhu首次揭示RDV致病的矮化机制，RDV编码的P2蛋白与水稻贝克杉烯氧化酶蛋白的互作使水稻赤霉素的合成能力大大下降，使得被RDV感染的水稻植株呈现矮缩症状。另外，贝克杉烯氧化酶可能参与了水稻植保素的合成，P2蛋白与其互作是为了阻止水稻植保素的合成从而使得寄主植物更加适合病毒粒子的复制和侵染(Zhu et al.,2005)。还有值得我们思考的是：外源GA可以减轻水稻矮缩症状并不能绝对的说明赤霉素的降低是水稻矮缩的直接原因，同样外源IAA不能减轻矮缩症状也不能绝对的排除生长素与矮缩症状无关，因为在RDV侵染的水稻中生长素等的信号传导可能也遭到了破坏。近期Liu等发现水稻的一个E3泛素连接酶OsRFPH2-10能够与RDV的P2蛋白发生互作，从而将P2蛋白通过泛素化降解途径进行降解，在病毒侵染早期发挥抗病毒功能(Liu et al.,2014)。Zhou等研究发现RDV P8蛋白与水稻体能的一个乙醇酸氧化酶互作(Zhou et al.,2007)，乙醇酸氧化酶是过氧化物酶体中的一个关键蛋白，P8与其互作可能使得P8也进入过氧化物酶体中，而过氧化物酶体可能是RDV的复制位点。利用水稻全基因组芯片技术对RDV侵染水稻进行了转录谱的分析显示，RDV的侵染水稻后引起了一系列的防御基因的表达，包括PR蛋白，WRKY转录因子等，这表明水稻对RDV的侵染启动了防御反应。有意思的是，在RDV侵染的水稻中，大量的细胞壁合成相关基因和叶绿体功能相关基因的表达显著下降了。细胞壁的合成是植物细胞伸长的关键因素，而细胞的伸长程度又决定了植株的高度，所以这些细胞壁合成相关基因的抑制很可能导致了细胞 壁合成的障碍，从而引起了矮缩的症状(Shimizu et al.,2007；Satoh et al.,2010)。先前研究显示在RDV侵染水稻后，病毒衍生的vsiRNA明显富集，且能够诱导某些AGO蛋白的高表达，表明RNA沉默信号通路在水稻抗RDV中发挥着重要的作用(Du et al.,2011；Wu et al.,2015)。
RNA沉默是真核生物基因表达调控的一种非常重要的策略，也是植物抵抗病毒侵染的一个方法(Hauptmann J and Gunter M,2013；Li et al.,2013a；Maillard,et al.,2013；Meister G 2013；Aliyari and Ding 2009；Ding 2010；Cao et al.,2014；Wu et al.,2015)。研究病毒和寄主之间在RNA沉默和RNA沉默的抑制对了解真核生物的基因表达调控将有重要的推动作用。该技术目前已被国际研究领域公认并应用于抗病基因工程育种以及功能基因研究。经典遗传学实验材料进行的RNA沉默实验显示了RNA沉默的几个特点：特异、高效、可扩散。与先前的正义或反义RNA技术相比，RNA沉默技术或人工构建miRNA等对同源基因表达的抑制效应至少高10倍以上。如何快速、高效地构建目标基因的反向重复序列，就成了当前利用RNA沉默机制获取对相关病毒免疫的工程植株的关键步骤。
在RNA沉默过程中包含了3个主要元件：分别为DCLs、AGO和RDR，其在植物抗病毒防御中都发挥重要作用。Dicer(DCLs)是一种Ⅲ型内切核酸酶，可以特异性识别生物体内的双链RNA，或单链RNA折叠形成的双链区域，产生长度为20～25nt的小RNA。所有的Dicer都具有一个同源的核酶结构域：RNaseⅢ结构域。这个结构域可以切割双链RNA产生duplex片段，切割产物一般具有5’磷酸集团及具有2个碱基突起的3’末端。大多数病毒在侵染复制过程中都会产生dsRNA形式，而这种dsRNA又会被生物体内的Dicer(DCL2、DCL3和DCL4等)识别并切割生成vsiRNA(virus-derived small interfering RNAs)，从而阻断病毒的复制和转录过程。Argonaute(AGO)蛋白，一个RNA结合蛋白，大小在90～130kDa，是RNA沉默过程中的核心组分可以与siRNA，miRNA或piRNA结合，形成有活性的RISC(RNA-induced silencing complex)。研究表明，AGO蛋白在参与植物抗病毒过程中发挥着重要的作用。模式生物拟南芥包含10个AGO蛋白，其中AtAGO1蛋白通过结合病毒衍生的vsiRNA而参与植物抗多种病毒的过程(Burgyan and Havelda,2011；Zhang et al.,2006)。另外，AGO2和AGO7也在抗病毒反应中发挥不同的作用(Carbonell et al.,2012；Ding,2010)。目前已知水稻具有19个AGO蛋白，但是关于水稻或其他重要作物AGO蛋白参与抗病毒防御反应的研究很少。RDR，RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase，RDR)，以RNA单链为模板合成dsRNA，在DCLs切割下产生small RNAs。最新的研究结果揭示，病毒和宿主RDRs合成的dsRNA作为主要的切割底物，以及源于R Vaistij and Jones DR6介导产生的dsRNA所产生的次级siRNAs具有更加有效的抗病毒反应。然而，这可能不是普遍现象，其结果可能取决于对特定的病毒。在病毒与寄主的长期进化过程中，RNA沉默(RNA silencing)成为植物抵抗病毒侵染的一种保守的防御机制。而作为相互竞争的另一方，病毒也不会坐以待毙，现在认为绝大多数的病毒都会编码一种抑制基因沉默的蛋白因子(VSR)，这种蛋白因子通过各种不同的方式作用于RNA沉默通路的不同位点从而抑制RNA沉默对病毒的切割。
突变体是研究功能基因组学的基础，是分离基因和鉴定基因功能的重要途径。自20世纪70年代以来，全球建立了一系列的突变体库。随着水稻全基因组测序工作的完成，水稻突变体已广泛应用于鉴定调控水稻形态和生理性状与基因的连锁分析及其相关基因的克隆和功能研究。与水稻表达谱数据相结合，通过对植物抗病相关基因的突变体进行筛选及转基因植物功能互补分析验证，是研究抗病相关基因及其作用的分子遗传机制的重要途径。
Solexa测序技术是深度测序技术中的一种，属于第二代测序技术，它们的兴起与发展为基因组与RNA研究提供了非常好的工具，通过该技术可以高通量测定核苷酸的序列，此方法不仅在小RNA测序中得到广泛应用，而且在研究基因组甲基化，转绿组和基因组重测序中也占据绝对优势。深度测序技术可以揭示RDV和水稻互作过程中的许多信息，比如水稻RNA沉默相关蛋白在抗病防御中的作用。在深度测序前需要构建小RNA库和mRNA库，首先提取分别提取感染RDV的水稻和健康水稻组织中的总RNA，分别构建mRNA和小RNA库，在其两端接上接头，进行逆转录和PCR。得到的cDNA进行深度高通量测序。
随着高通量分子生物学技术的广泛应用，它带给我们将是海量的数据信息，这些信息的分析必需依靠强大的生物信息学平台的支撑。水稻基因组和RDV基因组均已经测序完成，这为我们后续的生物信息学分析提供了很好的数据平台。
发明内容
本发明的目的是提供一种OsAGO18蛋白或其编码基因或含有所述编码基因的重组载体在调控植物对水稻矮缩病毒或其同科病毒的抗性中的应用。
本发明所提供的应用，具体如下：
OsAGO18基因或其编码蛋白或含有所述基因的重组载体在如下a1)或a2)中的应用：
a1)调控植物对水稻矮缩病毒或其同科病毒的抗性；
a2)选育对水稻矮缩毒或其同科病毒引发的植物病毒病害抗性增强的植物品种；
所述OsAGO18基因是以下1)～4)项中任何一项所述的DNA分子：
1)序列表中序列1所示的DNA分子；
2)序列表中序列3所示的DNA分子；
3)在严格条件下与1)或2)所限定的DNA分子杂交且编码所述蛋白的DNA分子；
4)与1)-3)任一限定DNA分子具有90％以上，优选95％以上，更优选99％以上同源性且编码所述蛋白的DNA分子。
上述严格条件可为用6×SSC，0.5％SDS的溶液，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
其中，序列1由3267个核苷酸组成，其中第1-3267位为所述OsAGO18基因的编码序列(ORF)，编码序列表中序列2所示的蛋白，序列2由1088个氨基酸残基组成。序列3由3300个核苷酸组成，其中第1-3300位为编码序列(ORF)，编码序列表中序列4所示的融合蛋白，序列4由1099个氨基酸残基组成。
在本发明中，以上所有a1)中的所述调控植物对水稻矮缩毒或其同科病毒引发的植物病 毒病害例如水稻矮缩病和黑条矮缩病的抗性均具体体现在：病原体侵染植物能够诱导该蛋白质及其编码基因的表达；促进所述蛋白质或其编码基因的表达，则所述植物对水稻矮缩病抗性增强。以上所有a2)中的所述选育对水稻矮缩病抗性增强的植物品种的方法，均具体可包括将所述蛋白质或其编码基因的表达量较高的植株作为亲本进行杂交的步骤。
本发明的另一个目的是提供一种培育对水稻矮缩毒或其同科病毒抗性增强的转基因植物的方法。
本发明所提供的培育对水稻矮缩毒或其同科病毒引发的植物病毒病害抗性增强的转基因植物的方法，具体可包括如下步骤：
a)向目的植物中导入由以下1)～4)中任何一项所述的OsAGO18蛋白的编码基因，得到表达所述编码基因的转基因植物：
1)序列表中序列1所示的DNA分子；
2)序列表中序列3所示的DNA分子；
3)在严格条件下与1)或2)所限定的DNA分子杂交且编码所述蛋白的DNA分子；
4)与1)-3)任一限定DNA分子具有90％以上，优选95％以上，更优选99％以上同源性且编码所述蛋白的DNA分子，
b)从步骤a)所得转基因植物中得到与所述目的植物相比，对水稻矮缩毒或其同科病毒抗性增强的转基因植物。
上述严格条件例如可为用6×SSC，0.5％SDS的溶液，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
其中，序列1由3267个核苷酸组成，其中第1-3267位为所述OsAGO18基因的编码序列(ORF)，编码序列表中序列2所示的蛋白，序列2由1088个氨基酸残基组成。序列3由3300个核苷酸组成，其中第1-3300位为编码序列(ORF)，编码序列表中序列4所示的融合蛋白，序列4由1099个氨基酸残基组成。
优选地，在上述的应用或方法中，所述蛋白由序列表中序列2所示的氨基酸序列组成或由序列表中序列4所示的氨基酸序列组成。其中，由序列表中序列2所示的氨基酸序列组成蛋白质命名为OsAGO18蛋白(编码基因为序列1，命名为OsAGO18基因)，由序列表中序列4所示的氨基酸序列组成蛋白质则为在OsAGO18蛋白的N端连接了MYC标签后得到的融合蛋白(编码基因为序列3)。
本领域的技术人员懂得，可以通过点突变、添加或缺失基因序列中的一个或多个碱基，影响该基因编码的蛋白功能。因此，本发明应该被理解为包括了对OsAGO18基因进行的上述变异。OsAGO18基因的序列不局限于序列表中序列1所示，还包括将其编码蛋白功能区中某氨基酸残基对应的任一密码子进行突变的DNA序列。主要是将有功能的氨基酸残基突变成一般被认为无特殊功能的丙氨酸。
在所述方法中，所述蛋白的编码基因是通过含有所述蛋白的编码基因的重组表达载体导入所述目的植物中的。
所述重组表达载体可用现有的植物表达载体构建。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等，如pCAMBIA3301、pCAMBIA2300、pCAMBIA2301、pCAMBIA1300、pCAMBIA 1301、pWM101、pGreen0029、pBI121、pBin19、pCAMBIA1301-UbiN等或其它衍生植物表达载体。所述植物表达载体还可包含外源基因的3’端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3’端。使用所述基因构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，例如花椰菜花叶病毒(CAMV)35S启动子、泛素基因Ubiquitin启动子(pUbi)、胁迫诱导型启动子Rd29A等，它们可单独使用或与其它的植物启动子结合使用；此外，使用本发明的基因构建重组表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用重组表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因、具有抗性的抗生素标记物或是抗化学试剂标记基因等。也可不加任何选择性标记基因，直接以逆境筛选转化植株。
在本发明中，所述重组表达载体中启动所述编码基因转录的启动子具体为Actin启动子。
更为具体的，所述重组表达载体为将所述编码基因替换pCam23ACT:OCS载体的酶切位点Sma I和Sal I之间的小片段后得到的重组质粒。在该重组表达载体中，启动所述编码基因转录的启动子为所述Actin启动子。
在上述方法中，将携带有所述编码基因的所述重组表达载体导入所述目的植物，具体可为：通过农杆菌介导法、基因枪法、电击法、花粉管导入法、脂质体融合法以及其他任意可将质粒导入的方法转化植物细胞或组织，并将转化的植物组织培育成植株。
在上述应用或方法中，所述植物可为单子叶植物或双子叶植物。
所述单子叶植物为禾本科植物。
在本发明中，所述植物为水稻或玉米。进一步，水稻品种最好为对RDV敏感的品种，如中花11、秀水11、日本晴等。
更加具体的，在本发明的一个实施例中，所述植物为水稻品种日本晴。
在上述应用或方法中，所述水稻矮缩病的病原具体为水稻矮缩病毒(Rice Dwarf Virus)、水稻黑条矮缩病毒(Rice black-streaked dwarf virus)，玉米粗缩病毒(maiz rough dwarf virus)或水稻条纹病毒(Rice stripe virus)。以上所述的同科病毒优先选自水稻黑条矮缩病毒(Rice black-streaked dwarf virus),玉米粗缩病毒(maiz rough dwarf virus)或水稻条纹病毒(Rice stripe virus)。
实验证明，本发明发现OsAGO18是一个能够被病毒特异性诱导的抗病基因，在水稻矮缩病毒(RDV)、水稻条纹病毒(RSV)以及水稻黑条矮缩病毒(RBSDV)或玉米粗缩病毒 (maiz rough dwarf virus)侵染野生型日本晴水稻后，OsAGO18蛋白表达量明显积累。鉴于上述三类病毒均属植物呼肠孤病毒科，以水稻矮缩病毒为例，当水稻矮缩病毒侵染水稻ago18突变体后，病毒粒子积累量较水稻野生型明显增加，病症也更加严重，基本无产量；相反，RDV侵染过表达OsAGO18转基因水稻后，病毒粒子积累量明显低于野生型水稻，病症也较轻，发病率下降，表明OsAGO18基因具有提高水稻抗病毒的能力。鉴于水稻黑条矮缩病毒同时能够侵染玉米，引起玉米粗缩病，并且水稻黑条矮缩病毒侵染水稻后同样能够诱导OsAGO18mRNA水平和蛋白水平高表达，同时ago18突变体对该病毒抵抗能力较野生型减弱，表明AGO18蛋白或其编码基因能够提高玉米抗由RDV，RBSDV，MRDV等引起的玉米粗缩病的能力。
附图说明
图1为AGO18蛋白在水稻矮缩病毒、水稻黑条矮缩病毒以及水稻条纹病毒侵染野生型日本晴水稻后积累的实验结果。Western blot分析AGO18蛋白水平在多种病毒侵染后诱导。其中，Mock表示为被病毒侵染的野生型水稻品种日本晴；RDV表示被RDV侵染发病的野生型水稻品种日本晴；RBSDV表示被RBSDV侵染发病的野生型水稻品种日本晴；RSV表示RSV侵染发病的野生型水稻品种日本晴；Tubulin是上样control。
图2为OsAGO18mRNA在水稻不同病毒侵染后积累的实验结果。Realtime PCR分析OsAGO18mRNA在水稻不同病毒侵染后被诱导。其中，Mock表示未被相应病毒侵染的野生型水稻品种日本晴；RDV、RBSDV、RRSV、RGSV表示被相应病毒侵染发病的野生型水稻品种日本晴。以内参基因水稻EF的表达量为1。
图3为ago18突变体T-DNA插入位置信息与鉴定结果。其中，A为RT-PCR方法鉴定ago18突变体(NF6013)，LP+RP表示使用引物对6013_LP/6013_RP进行扩增的结果，LB+RP表示使用引物对tos17_tail6(LB)/6013_RP进行扩增的结果，AGO18表示使用引物对OsAGO18-F/OsAGO18-R进行扩增的结果，Actin表示使用引物对Actin-RT-F/Actin-RT-R进行扩增的结果；B为Tos17插入位点示意图。
图4为实施例2中各水稻品系接种RDV后的病症图。其中，WT-mock表示未被RDV侵染的野生型水稻品种日本晴；WT-RDV表示被RDV侵染发病的野生型水稻品种日本晴；ago18-mock表示未被RDV侵染的ago18突变体纯合子；ago18-RDV表示被RDV侵染的ago18突变体纯合子。
图5为实施例2中各水稻品系接种RBSDV后的病症图。其中，Mock表示未被病毒侵染的野生型水稻，WT-RBSDV表示被RBSDV侵染后的野生型水稻，ago18-RBSDV表示被RBSDV侵染后的ago18突变体水稻。
图6为感病ago18突变体水稻中RDV基因组RNA积累的结果。其中，WT-mock表示未被RDV侵染的野生型水稻品种日本晴；WT-RDV表示被RDV侵染发病的野生型水稻品种日本晴；ago18-mock表示未被RDV侵染的ago18突变体纯合子；ago18-RDV表示被RDV侵染的ago18突变体纯合子。
图7为OsAGO18过表达转基因水稻的鉴定结果。其中，A为RT-PCR鉴定结果；B为Western blot鉴定结果。
图8为实施例3中各水稻品系接种RDV病毒后的病症图。其中，Mock表示未被RDV侵染的野生型水稻品种日本晴；WT-RDV表示被RDV侵染发病的野生型水稻品种日本晴；Vector-RDV表示被RDV侵染的转空载体水稻；ago18-RDV示被RDV侵染的ago18突变体纯合子；AGO18OE-5-RDV表示被RDV侵染的转基因水稻株系5#；AGO18OE-2-RDV表示被RDV侵染的转基因水稻株系2#；AGO18OE-1-RDV表示被RDV侵染的转基因水稻株系1#。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
水稻(Oryza.sativa L.)品种日本晴：即日本晴水稻(Oryza sativa L.japonica.cv.Nipponbare)，参考文献：水稻品种“日本晴”.农业科技通讯，1973年02期。公众可从北京大学获得。
水稻矮缩病毒(Rice Dwarf Virus，RDV)：记载于“张仲凯，方琦，魏春红，丁铭，董佳红，俞立，李毅.水稻矮缩病毒云南、浙江分离株侵染水稻植株的细胞病理比较研究.电子显微学报，2006年第25卷第2期”一文，公众可从北京大学获得。
水稻黑条矮缩病毒(Rice Black-Streaked Dwarf Virus，RBSDV)：记载于“陈声祥，张巧艳.我国水稻黑条矮缩病与玉米粗缩病研究进展.植物保护学报，2005年第32卷第1期”一文，公众可从北京大学获得。
水稻条纹病毒(Rice Stripe Virus，RSV)：记载于“蔡小卫,赵俊玲,邵英,桂青清,刘芳.灰飞虱传播水稻矮缩病毒的研究综述.中国植保导刊，2011年09期”一文，公众可从北京大学获得。
农杆菌EHA105：记载于“Zhu et al.,2005.The Rice Dwarf Virus P2Protein interacts with ent-Kaurene Oxidases in vivo,leading to reduced biosynthesis of Gibberellins and rice dwarf symptoms.Plant Physiology.139:1935-1945.”一文，公众可从北京大学获得。
pEASY-simple-T1载体：北京全式金生物技术有限公司产品，其产品目录号为CT111-01。
pCam23ACT:OCS载体：记载于“Liang Wu et al.,2009.Rice MrcroRNA Effector Complexes and Targets.Plant Cell.21(11):3421-3435.”一文，公众可从北京大学获得。
实施例1、发现OsAGO18基因能够被水稻多种病毒诱导表达
本实施例中所涉及的OsAGO18基因来源于水稻(Oryza.sativa L.)，OsAGO18基因的cDNA序列如序列表中序列1所示，序列1由3267个核苷酸组成，其中第1-3267位为编码序列(ORF)；序列1编码序列表中序列2所示的蛋白质，序列2由1088个氨基酸残基组成。
一、OsAGO18蛋白在水稻矮缩病毒、水稻黑条矮缩病毒以及水稻条纹病毒侵染后积累
取被水稻矮缩病毒(RDV)、水稻黑条矮缩病毒(RBSDV)以及水稻条纹病毒(RSV)侵染发病的野生型水稻(Oryza.sativa L.)品种日本晴和对照组水稻(未被水稻矮缩病毒侵染 的野生型水稻品种日本晴)叶片为材料，利用2×SDS上样缓冲液提取总蛋白，采用Western blot方法检测OsAGO18蛋白在两水稻中的积累情况。具体如下：
(1)SDS-PAGE电泳操作流程及注意事项参见《蛋白质的SDS－PAGE凝胶电泳》：
(2)SDS-PAGE凝胶转膜：
1)将蛋白胶放于玻璃板上，测量大小，剪取相同大小的PVDF膜，做好标记，将膜放于100％甲醇内浸润超过10秒；
2)剪取相同大小的六块滤纸，三张一份，与胶和PVDF膜放于转膜缓冲液内平衡超过10分钟；
3)在转膜缓冲液中将三张一份的滤纸放于最底层，依次放上蛋白胶、PVDF膜、另外三张滤纸，对齐后一手捏紧一角，另一手挤压滤纸，除去所有气泡，调整角度，重新挤压气泡；
4)将转膜槽放于冰盒内，灌注转膜缓冲液，将中间固定好蛋白胶和PVDF膜的双层滤纸放于海绵夹层中，置于转膜模具内，之后将PVDF膜朝向正极放入转膜槽内中，根据目的蛋白大小选择合适电流和时间恒流转膜；
(3)蛋白质的免疫检测：
1)取出PVDF膜放于平皿中，加入约25ml PBS-T，脱色摇床上摇动洗膜5分钟；
2)在平皿内加入50ml封闭液(含5％脱脂奶粉的50ml PBS-T)，脱色摇床摇动将膜封闭过夜；
3)大体积(约30ml)PBS-T洗膜5分钟，洗2次；
4)将膜放于三面剪开的杂交袋中，热封两边后按每平方厘米0.1ml加入稀释好的一抗(利用特异性较高的多肽AGO18N，即序列5所示多肽，作为免疫原，免疫兔子，制备获得的抗水稻OsAGO18蛋白的多克隆抗体)，封闭最后一边，室温反应2小时；其中一抗稀释液为含0.25％BSA的PBS-T，一抗的稀释比例取决于所用抗体的效价和转到膜上目的蛋白的量，期间要至少每隔5分钟混匀一次杂交袋；
5)剪开杂交袋，将膜放于平皿内加入20ml PBS-T洗10分钟，洗3次；后用20ml TBS-T洗膜10分钟，洗2次；
7)依照一抗反应的方法，将膜放于新的杂交袋中，加入稀释好的二抗(兔抗，Promega公司，货号：0000089056)在杂交袋中反应1小时，二抗稀释液为含0.25％BSA的TBS-T；
8)20ml TBS-T漂洗10分钟，漂洗4次；
9)利用Western blot显影kit(ImmobilonTM Western：MILLIPORE上海贸易有限公司，货号：1305701)进行显影，利用柯达X-OMAT BT医用X射线胶片检测结果；
(4)实验所需试剂：
1)2×SDS上样缓冲液10mL：甘油2Ml；溴酚蓝0.0202g；1MTris-HCl(pH6.8)1mL；β-巯基乙醇0.14mL；10％SDS 4mL；加ddH2O至10mL，保存于-20℃。
2)转膜缓冲液1L：39mM甘氨酸：2.9g；Tris 5.8g；SDS 0.37g；甲醇：200ml。
3)碱性磷酸酶缓冲液100ml：100mM NaCl；5mM MgCl2；100mM Tris-Cl pH9.5。
4)PBS-T 1L pH 7.5：1M磷酸二氢钠31.6ml；1M磷酸氢二钠68.4mlTween 20 0.1％1ml；水900ml；调pH后定容1L。
5)TBS-T 1L pH 7.6：Tris 2.42g；NaCl 8g；Tween 201ml；水900ml；调pH后定容1L。
6)封闭液100ml：100ml TBS-T内加入5克脱脂奶粉。
7)抗稀释液100ml：100ml PBS-T内加入0.25克BSA。
Western blot实验以tubulin蛋白为内参，其一抗为Tubulin单克隆抗体(鼠源，Sigma公司，货号：T6793)；二抗为鼠抗anti-mouse(Promega公司，货号：0000089661)。
如图1结果所示，OsAGO18蛋白在侵染的水稻材料中明显积累，表明不同的病毒侵染均可以诱导OsAGO18蛋白的积累，OsAGO18蛋白可能参与了水稻抗多种病毒的防御反应。
二、OsAGO18基因mRNA在水稻矮缩病毒、水稻黑条矮缩病毒、水稻条纹病毒以及水稻锯齿叶矮缩病毒侵染后积累
取被水稻矮缩病毒(RDV)、水稻黑条矮缩病毒(RBSDV)、水稻条纹病毒(RSV)以及水稻锯齿叶矮缩病毒(RRSV)侵染发病的野生型水稻(Oryza.sativa L.)品种日本晴和对照组水稻(未被水稻矮缩病毒侵染的野生型水稻品种日本晴)各0.5g，在液氮中研磨后，依照Invitrogen公司的TRIzol Reagent说明书(Invitrogen Trizol Reagent，cat No.15596-018)提取总RNA。测定总RNA浓度后，取10μg总RNA，按照RQ1Dnase(Promega，货号：M610A)的说明书对总RNA中的水稻基因组DNA进行消化。消化反应体系：总RNA 10μg，10×Dnase缓冲液10μl，DNase 10μl，DEPC水补足100μl。将整个消化反应体系于37℃孵育35min。孵育后向体系中加入4μl RQ1DNase终止反应液，65℃孵育10min，使DNase灭活。
消化基因组DNA后，再利用氯仿抽提法浓缩总RNA，测定RNA浓度，取2μg RNA参照Invitrogen公司的SuperScript II逆转录酶进行反转录，所用引物为16个核苷酸的Oligod(T)引物，具体方法参见invitrogen M-MLV Reverse Transcriptase(货号：28025-021)。以反转录获得水稻cDNA为模板，利用realtime PCR的方法对OsAGO18基因的转录水平进行检测，实验方法参照TOYOBO
Figure PCTCN2015080183-appb-000001
Realtime PCR Master Mix(货号QPK-201)说明书，引物为：
OsAGO18-F：5’-TGTTCGTCCAGGCACAGTAG-3’(序列1的第2901-2920位)；
OsAGO18-R：5’-GCGGTGAAGTTGTTGTCGTC-3’(序列1的第3022-3041位的反向互补序列)。
内参基因为水稻EF，引物为：
OsEF-1a-F：5’-GCACGCTCTTCTTGCTTTCACTCT-3’；
OsEF-1a-R：5’-AAAGGTCACCACCATACCAGGCTT-3’。
数据处理方法，参照bio-rad公司CFX96型号实时定量荧光PCR仪自带软件CFX managerTM Software(Version2.1)。
结果如图2所示，可见在mRNA水平上，OsAGO18基因在上述呼肠孤病毒科不同病毒侵 染水稻后明显富集，表明多种呼肠孤病毒科病毒侵染水稻后能够诱导OsAGO18基因的表达，使该基因的转录水平提高。
实施例2、OsAGO18基因参与水稻抗RDV防御反应
一、ago18水稻突变体纯合子的获得
ago18突变体种子(NF6013)在Tos17数据库中购买
(https://tos.nias.affrc.go.jp/～miyao/pub/tos17/)。
T-DNA插入序列为：
tos17_tail6(LB)：5’-AGGTTGCAAGTTAGTTAAGA-3’。
插入位点如图3中B所示。
利用数据库中提供的方法鉴定ago18突变体的纯合子
(https://pc7080.abr.affrc.go.jp/cgibin/tos17/ricegenome.cgi？action＝getTarget&chr＝7&pos＝16895280&primer＝y&version＝7)。
以上述鉴定得到的水稻ago18突变体(NF6013)纯合子的cDNA为模板，以
6013_LP/6013_RP引物对、tos17_tail6(LB)/6013_RP引物对、OsAGO18-F/OsAGO18-R引物对，分别进行半定量RT-PCR反应。同时设置野生型水稻(Oryza.sativa L.)品种日本晴作为对照。
引物序列如下：
6013_LP：5’-GATCGAGGGAACTCGACAAG-3’；
6013_RP：5’-CAAGATCAACTCCACGCAAA-3’。
tos17_tail6(LB)、OsAGO18-F和OsAGO18-R序列见上文。
以Actin为内参，引物如下：
Actin-RT-F：5’-CTTCGTCTCGACCTTGCTGGG-3’；
Actin-RT-R：5’-GAGAAACAAGCAGGAGGACGG-3’。
结果如图3中A所示，由图可见水稻ago18突变体(NF6013)纯合子仅采用引物对tos17_tail6(LB)/6013_RP可以扩增相应的目的条带，而采用6013_LP/6013_RP引物对和OsAGO18-F/OsAGO18-R引物对均未扩增出相应的目的条带。而作为对照的野生型水稻(Oryza.sativa L.)品种日本晴采用6013_LP/6013_RP引物对和OsAGO18-F/OsAGO18-R引物对均扩增出相应的目的条带，但采用tos17_tail6(LB)/6013_RP引物对未扩增出相应的目的条带。与预期结果一致。
二、OsAGO18基因参与水稻抗RDV防御反应
1、RDV侵染ago18突变体水稻后，水稻发病率提高，症状加重
以带RDV病毒的叶蝉侵染同时期(分蘖期)的野生型水稻(Oryza.sativa L.)品种日本晴和步骤一获得的ago18突变体纯合子(实验组)。以无毒叶蝉侵染相应的水稻作为对照组。
2周后检测水稻发病情况(水稻发病情况鉴定参照文献《稻麦主要病毒病识别与控制》一书中1.2节)，分别统计实验组与对照组的发病率。统计发病率的同时，观察水稻发病后的表型在不同的实验组之间的区别。
实验组中，各水稻品系的发病率统计结果如表1所示。可见，RDV病毒侵染ago18突变体后，水稻发病率较高；而RDV病毒侵染野生型水稻后，水稻发病率较低。与预期结果一致。进一步，各水稻品系接种RDV病毒后的病症图如图4所示，RDV侵染ago18突变体后，水稻植株更加矮小，在叶片或叶鞘上出现的白色斑点增多。
表1 RDV侵染各水稻品系后发病率统计
水稻品系 Na Db Pc
日本晴 30 22 73.3％
ago18突变体 30 26 86.7％
注：a:被观测的水稻总株数；b:侵染两周后具发病表型的水稻株数；c:发病水稻占全部被侵染水稻的比例。
2、Northern blot实验检测RDV基因组RNA链在发病水稻中的积累情况
以步骤1中获得的发病的野生型水稻品种日本晴、发病的ago18突变体水稻、未发病的野生型水稻品种日本晴、未发病的ago18突变体水稻为实验材料。分别取各水稻材料叶片2g，在液氮中研磨成粉末，依照Invitrogen公司的TRIzol Reagent说明书(Invitrogen Trizol Reagent，cat No.15596-018)提取总RNA，测定浓度后，备用。
A.在通风厨内制备1.2％的琼脂糖甲醛变性胶120ml：87.6ml DEPC水中加入1.44g琼脂糖，微波炉加热使琼脂糖融化，后冷却至温度为60℃左右，后加入12ml 10×mops母液和20.4ml甲醛。摇晃混合均匀后，迅速倒入胶槽内并插入梳子。
B.在10-20μg的RNA样品中加入RNA loading buffer，100℃加热10分钟，后放置在冰上变性2-3min，上样前离心1-2min。
C.移液器将变性后的样品加入到冷却好的琼脂糖甲醛变性胶中，电泳液为1×mops溶液，电压100V，电泳3-4个小时。切胶并放于20×SSC溶液平衡10-20min。
D.电泳完毕用两种方法转膜：真空转移法和毛细管转移。毛细管转移的基本方法：在培养皿倒入20×SSC溶液，在玻璃板上搭上2-3层的滤纸形成纸桥。再把胶放在纸桥上，PDVF膜放在胶的上面，再放上3层滤纸以及10-25cm后的吸水纸。再上压重物，转膜24-36小时。真空转移的方法原理和毛细管转移一样，使用抽真空仪器，较快溶液转移的速度。
E.紫外交联：能量为1800对膜进行交联。之后可放80℃烤膜30min。处理好的膜可用亚甲基蓝染色，及检测之前步骤中RNA的有无降解及上样量是否一致。染色得到的rRNA的条带可用作control。
F.将膜放于含有预杂液(Sigma公司，货号为SLBG7228V)的杂交瓶，65℃条件下预杂1-2小时。
G.将标记好的探针(用于扩增RDV的三条RNA片段探针的引物序列见下文)放置于100℃变性10min，后放置于冰上3min冷却。将其添加到预杂液中，65℃条件下杂交过夜(24小时以上)。标记探针随机引物法反应体系参照TAKARA公司探针标记试剂盒提供的方法(货号D6045)：
ddH2O补加体积到50μl 29μl
Labeling 5×buffer 10μl
未标记的dNTPs混合物 2μl
变性的RNA模板(30-50ng) 1μl
BSA 2μl
α-32P dCTP(50μCi，3000Ci/mmol) 5μl
DNA聚合酶I Klenow大片段(5U) 1μl
扩增四条RNA链探针所用的引物如下所示(5’-3’)：
RDV-S2-F：5’-CCGGGACGTGCCAAACCGTGAAC-3’；
RDV-S2-R：5’-ACGCCATCAACAGAGCAGAATCCATTA-3’。
RDV-S8-F：5’-TCAATAGCGATACCAAGCCTACCGTTTC-3’；
RDV-S8-R：5’-ATCGATGTCGGGGTCTGAGTTATCGAGTTCAAT-3’。
RDV-S11-F：5’-CATGCCATGGCAATGAGTGGAACATTACCCTTG-3’；
RDV-S11-R：5’-AGAATGCGGCCGCTTACTTACGCTTTGATTTGCGAG-3’。
H.杂交完毕，用2×洗膜液(2×SSC，加入SDS至终浓度为1g/L)在65℃洗膜两次，每次20分钟。然后用0.1×洗膜液(0.1×SSC，加入SDS至终浓度为1g/L)，65℃洗膜1次，约20分钟。
I.膜晾干，并用保鲜膜包好，检测放射强度。压片(X光片或磷屏)，时间根据放射强度决定压片时间长短。
实验同时以rRNA作为对照。
实验结果如图6中B所示，RDV基因组三个RNA片段在ago18突变体水稻中富集量均高于野生型水稻日本晴，这进一步表明RDV在ago18突变体中复制量增加。与步骤1中测得的ago18突变体较野生型水稻品种日本晴更感病，抗病性减弱相一致。
以上结果均证明，OsAGO18基因参与水稻抗病毒防御反应，水稻失去该基因后更加容易感病，且病症更加严重，病毒粒子复制量增加。
三、OsAGO18基因参与水稻抗RBSDV防御反应
RBSDV侵染ago18突变体水稻后，水稻发病率提高，症状加重
以带RBSDV病毒的灰飞虱侵染同时期(分蘖期)的野生型水稻(Oryza.sativa L.)品种日本晴和步骤一获得的ago18突变体纯合子(实验组)。以无毒叶蝉侵染相应的水稻作为对照组。
2周后检测水稻发病情况(水稻发病情况鉴定参照文献《稻麦主要病毒病识别与控制》一书中1.3节)，观察水稻发病后的表型在不同的实验组之间的区别。
实验组中，各水稻品系的发病病症如图5所示。可见，RBSDV病毒侵染ago18突变体较侵染野生型水稻后的发病病症更加严重，水稻植株更加矮小，叶片短宽僵直浓绿，表明AGO18蛋白在水稻抗RBSDV中发挥着重要的作用。
实施例3、过表达OsAGO18的转基因水稻抗病性增强
一、OsAGO18基因植物表达载体的构建
1、水稻cDNA模板的获得
依照说明书，用Invitrogen公司的TRIzol Reagent提取日本晴水稻(Oryza sativa L.japonica.cv.Nipponbare)总RNA，用该公司的SuperScript II逆转录酶进行逆转录。逆转录所用引物为16个核苷酸的Oligod(T)引物，最终获得逆转录所得的水稻cDNA模板。
2、水稻OsAGO18基因的获得
以步骤1获得的水稻cDNA为模板，用如下引物对进行PCR扩增。
AGO18cds-F：5’-ATAATGGCGAGCCGAGGAGGAGGC-3’(该序列的第4-24位为序列1的第1-21位，前三位的ATA为保护碱基)；
AGO18cds-R：5’-GACCTAGCAAAAGAACATGGACTTTTTC-3’(该序列的第4-28位为序列1的第3243-3267位的反向互补序列，前三位的ATA为保护碱基)。
PCR得到片段后，将全长的序列连接pEASY-simple-T1载体上，转化大肠杆菌，提质粒进行测序。将经测序表明在pEASY-simple-T1载体中正向连入“ATA+序列1+GTC”所示DNA片段后的重组质粒命名为pEASY-OsAGO18。
3、OsAGO18基因植物表达载体的构建
以步骤2获得的重组质粒pEASY-OsAGO18为模板，以如下引物进行PCR扩增。
AGO18cds-Sma 1-MYC-F：5’-ATA cccggg ATG GAG CAG AAG CTG ATC TCA GAG  GAG GAC CTG-ATG GCG AGC CGA GGA GGA GGC-3’(下划线小写字母部分为Sma I的识别序列，下划线大写字母部分为MYC标签序列，-后的序列为序列1的第1-21位)；
AGO18cds-Sal 1-R：5’-GAC gtcgac CTAGCAAAAGAACATGGACTTTTTC-3’(下划线小写字母部分为Sal I的识别序列，-后的序列为序列1的第3243-3267位的反向互补序列)。
将PCR产物用SmaI和SalI酶切(NEB公司产品，货号分别为：R0141和R0138)，胶回收后与经过同样双酶切的pCam23ACT:OCS载体的骨架大片段相连，得到重组质粒。
将经SmaI和SalI双酶切初步鉴定正确的重组质粒(得到大小约为10300bp和3300bp的条带)送样测序。将经测序表明将pCam23ACT:OCS载体的酶切位点Sma I和Sal I之间的小片段替换为序列表中序列3所示DNA片段的重组质粒命名为pCambia2300-Actin-MYC-OsAGO18。序列3与序列1相比，在序列1所示的OsAGO18基因的5’端多出了MYC标签序列。序列3编码序列表中序列4所示的蛋白质(MYC标签与OsAGO18的融合蛋白)。
在重组表达载体pCambia2300-Actin-MYC-OsAGO18中，启动序列3所示DNA片段转录的启动子为Actin启动子。
二、过量表达转OsAGO18基因水稻的获得
1、愈伤组织的诱导培养
将日本晴水稻(Oryza sativa L.japonica.cv.Nipponbare，以下简称为野生型水稻)种子去壳，先用70％(体积分数)乙醇浸泡10min，再用0.1％(体积分数)升汞浸泡30min；进行 表面除菌。用大量无菌水洗去种子表面的溶液，用无菌滤纸吸去种子表面的水分。将种子置于成熟胚愈伤诱导培养基(培养基配方见下文)平板上，用Parafilm膜封闭平皿边缘，于26℃温箱内避光培养。大约15天后，小心取下长出的愈伤组织，转移到成熟胚继代培养基(培养基配方见下文)上，同样条件继续进行培养。每两周需进行一次继代培养。用于转化时，需挑选继代培养5天左右、呈淡黄色的颗粒状愈伤组织。
其中，所需要的培养基配方如下：
a)NB基本培养基：
Figure PCTCN2015080183-appb-000002
b)成熟胚愈伤诱导培养基和继代培养基：
Figure PCTCN2015080183-appb-000003
Figure PCTCN2015080183-appb-000004
2、农杆菌的培养
将步骤一获得的重组表达载体pCambia2300-Actin-MYC-OsAGO18电转入农杆菌EHA105中，筛选能够在加了抗生素利福平和卡那霉素的LB平板上生长，而且以引物AGO18-PCR-F和AGO18-PCR-R进行PCR扩增，得到793bp的PCR产物的为阳性菌，将其命名为EHA105/pCambia2300-Actin-MYC-OsAGO18。
AGO18-PCR-F：5’-CCCAACTATTATATTTGGTGCTGAT-3’(序列1的第2475-2499位)；
AGO18-PCR-R：5’-CTAGCAAAAGAACATGGACTTTTTC-3’(序列1的第3243-3267位的反向互补序列)。
将重组农杆菌EHA105/pCambia2300-Actin-MYC-OsAGO18在含有抗生素(50mg/L Kan，50mg/L Rif)的LB平板上划线，28℃培养2天。挑取单菌落接入液体LB培养基中，28℃振荡培养至OD600约为0.5，加入乙酰丁香酮至终浓度100mM，得到用于转化水稻愈伤组织的农杆菌悬液。
3、水稻愈伤组织与农杆菌的共培养
将步骤1准备好的继代愈伤组织放入灭过菌的锥形瓶中，倒入步骤2获得的农杆菌悬液使之浸没愈伤组织。室温放置20min，并不时轻轻晃动使愈伤组织与菌液充分接触。用无菌的镊子轻轻取出愈伤组织，放于无菌滤纸上吸去多余的菌液，转移到铺有一层无菌滤纸的共培养培养基(培养基配方见下文)平板上。28℃暗培养2-3天，得到经过共培养的愈伤组织。
4、抗性愈伤组织的筛选与分化
将经过共培养的愈伤组织用适量无菌水清洗，除去表面残余的农杆菌，放在筛选培养基(培养基配方见下文)上，26℃避光培养进行筛选，两周后转移到新的筛选培养基上继续筛选两周。挑选经过两轮筛选后状态较好的愈伤组织，将其转移到分化培养基(培养基配方见下文)平板上，先避光培养3天，然后再转至光照培养箱中(15h/day)进行光照培养。一个月后可见分化出的小苗。当分化的小苗长至约2cm时，将其转移到锥形瓶中的生根培养基(培养基配方见下文)上，继续培养两周左右。选择长势较好、根系发达的小苗，用自来水洗去根部的培养基后移栽入土壤中，收取种子，得到T0代转OsAGO18基因水稻种子。
其中所需要的培养基配方如下：
a)共培养培养基：
Figure PCTCN2015080183-appb-000005
Figure PCTCN2015080183-appb-000006
(注意：液体共培养培养基中不添加2,4-D)
b)筛选培养基：
Figure PCTCN2015080183-appb-000007
c)分化培养基：
Figure PCTCN2015080183-appb-000008
d)生根培养基：
Figure PCTCN2015080183-appb-000009
采用同样的方法，将pCam23ACT:OCS空载体转入野生型水稻中，得到转空载体水稻，作为对照。
三、OsAGO18基因过表达的水稻抗RDV能力增强。
1、OsAGO18转基因水稻的鉴定
将步骤二获得的T0代转OsAGO18基因水稻种子播种，获得T1代水稻幼苗，从中随机选取6株，编号为1#、2#、3#、4#、5#和6#。取部分叶片，在液氮中研磨，一部分做半定量RT-PCR，一部分做Western blot鉴定。
(1)半定量RT-PCR
从T1代转OsAGO18基因水稻幼苗中提取RNA，并反转录得到cDNA。以cDNA为模板，以OsAGO18-F/OsAGO18-R引物对进行半定量RT-PCR反应。同时设置未转基因的野生型水稻(Oryza.sativa L.)品种日本晴以及步骤二获得的转空载体水稻作为对照。
引物序列如下：
OsAGO18-F：5’-TGTTCGTCCAGGCACAGTAG-3’(序列1的第2901-2920位)；
OsAGO18-R：5’-GCGGTGAAGTTGTTGTCGTC-3’(序列1的第3022-3041位的反向互补序列)。
以Actin为内参，引物如下：
Actin-RT-F：5’-CTTCGTCTCGACCTTGCTGGG-3’；
Actin-RT-R：5’-GAGAAACAAGCAGGAGGACGG-3’。
结果如图7中A所示，由图可见，与未转基因的野生型水稻(Oryza.sativa L.)品种日本晴相比，6个T1代转OsAGO18基因水稻植株中OsAGO18基因的表达量更高。而转空载体水稻植株中OsAGO18基因的表达量与未转基因的野生型水稻(Oryza.sativa L.)品种日本晴相比基本一致，无统计学意义。
RT-PCR检测中OsAGO18mRNA明显积累的株系作为RT-PCR阳性苗。
(2)Western blot鉴定
具体操作参见实施例1步骤一进行。
结果如图7中B所示，由图可见，与未转基因的野生型水稻(Oryza.sativa L.)品种日本晴相比，6个T1代转OsAGO18基因水稻植株中OsAGO18蛋白的表达量更高。而转空载体水稻植株中OsAGO18蛋白的表达量与未转基因的野生型水稻(Oryza.sativa L.)品种日本晴相比基本一致(含量较低，基本检测不到OsAGO18的表达)，无统计学意义。
Western blot检测结果中，蛋白条带较野生型明显积累(野生型水稻在非侵染条件下，基本检测不到OsAGO18的表达)的株系为western blot阳性苗。
2、RDV侵染OsAGO18过表达水稻后，水稻发病率降低，病症减轻
以带RDV病毒的叶蝉侵染同时期(分蘖期)的未转基因的野生型水稻品种日本晴以及经步骤1鉴定阳性的T1代转OsAGO18基因水稻株系1#、2#和5#，以及步骤二获得的转空载体水稻(实验组)。以无毒叶蝉侵染相应的水稻作为对照组。
2周后检测水稻发病情况(水稻发病情况鉴定参照文献《稻麦主要病毒病识别与控制》一书中第二章)，分别统计实验组与对照组的发病率。统计发病率的同时，观察水稻发病后的表型在不同的实验组之间的区别。
实验组中，各水稻品系的发病率统计结果如表2所示。由表可见，RDV病毒侵染T1代转OsAGO18基因水稻株系1#、2#和5#后水稻植株的发病率均显著低于RDV病毒侵染未转基因的野生型水稻品种日本晴后水稻植株的发病率。进一步，各水稻品系接种RDV病毒后的病症图如图8所示，RDV侵染OsAGO18转基因阳性株系后，病症较野生型水稻明显变轻，沿叶脉分布的白斑减少，矮缩减弱，发病的叶片减少。与之相对应，RDV侵染ago18突变体水稻后， 沿叶脉分布的白斑明显增多，叶片浓绿，植株矮缩明显。
表2 RDV侵染OsAGO18过表达水稻后发病率统计
水稻品系 Na Db Pc
WT 30 23 76.7％
AGO18OE-5# 30 4 13.3％
AGO18OE-2# 30 7 23.3％
AGO18OE-1# 30 5 16.7％
注：a:被观测的水稻总株数；b:侵染两周后具发病表型的水稻株数；c:发病水稻占全部被侵染水稻的比例；AGO18OE-5#、AGO18OE-2#和AGO18OE-1#分别表示被RDV侵染的转基因水稻株系5#、2#和1#。
以上实验结果表明，OsAGO18过表达后增强了植物的抗病性，使水稻不易被RDV侵染。

Claims (10)

  1. OsAGO18基因或其编码蛋白或含有该基因的重组载体在如下a1)或a2)中的应用：
    a1)调控植物对水稻矮缩病毒或其同科的病毒的抗性；
    a2)选育对水稻矮缩病毒或其同科病毒引发的病毒病害抗性增强的植物品种；
    所述OsAGO18蛋白的编码基因是如下1)至4)中任一所述的DNA分子：
    1)序列表中序列1所示的DNA分子；
    2)序列表中序列3所示的DNA分子；
    3)在严格条件下与1)或2)所限定的DNA分子杂交且编码所述蛋白质的DNA分子；
    4)与1)-3)任一限定的DNA分子具有90％以上同源性且编码所述蛋白质的DNA分子。
  2. 培育对水稻矮缩病毒或其同科病毒引发的病毒病害抗性增强的转基因植物的方法，包括如下步骤：
    a)向目的植物中导入由如下1)～4)项中任何一项所述的OsAGO18蛋白的编码基因，得到表达所述编码基因的转基因植物：
    1)序列表中序列1所示的DNA分子；
    2)序列表中序列3所示的DNA分子；
    3)在严格条件下与1)或2)所限定的DNA分子杂交且编码所述蛋白质的DNA分子；
    4)与1)-3)任一限定的DNA分子具有90％以上同源性且编码所述蛋白质的DNA分子，
    b)从步骤a)所得转基因植物中得到与所述目的植物相比，对水稻矮缩病抗性增强的转基因植物。
  3. 根据权利要求1所述的应用，或权利要求2所述的方法，其特征在于：所述OsAGO18蛋白由序列表中序列2所示的氨基酸序列组成或由序列表中序列4所示的氨基酸序列组成。
  4. 根据权利要求2或3所述的方法，其特征在于：所述编码基因是通过含有所述蛋白质的编码基因的重组表达载体导入所述目的植物中的。
  5. 根据权利要求2-4中任一项所述的方法，其特征在于：所述重组表达载体中启动所述编码基因转录的启动子为Actin启动子。
  6. 根据权利要求1所述的应用或权利要求2-5中任一项所述的方法，其特征在于：所述同科的病毒选自水稻黑条矮缩病毒、玉米粗缩病毒或水稻条纹病毒。
  7. 根据权利要求1-6中任一所述的应用或方法，其特征在于：所述植物为单子叶植物或双子叶植物。
  8. 根据权利要求7所述的应用或方法，其特征在于：所述单子叶植物为禾本科植物。
  9. 根据权利要求8所述的应用或方法，其特征在于：所述禾本科植物为水稻或玉米，优选水稻品种中花11、秀水11、日本晴。
  10. 根据权利要求2-9中任一所述的应用或方法，其特征在于：水稻矮缩病毒或其同科病毒引发的病毒病害选自水稻矮缩病、水稻黑条矮缩病、水稻条纹叶枯病或玉米粗缩病。
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WO2018027713A1 (zh) * 2016-08-11 2018-02-15 北京大学 OsAO基因在提高水稻对水稻条纹病毒及水稻黑条矮缩病毒或其同科病毒抗性中的应用
CN108315333B (zh) * 2017-10-19 2021-07-23 江苏省农业科学院 水稻黑条矮缩病抗病基因RBSDV-6c及其编码蛋白
CN112390866B (zh) * 2019-08-14 2022-07-15 北京大学 OsARF12基因在提高水稻对水稻矮缩病毒抗性中的应用
CN111253480B (zh) * 2020-03-04 2021-08-06 宁波大学 水稻转录因子OsARF17基因及其在抗黑条矮缩病毒植物育种中的应用
CN112812162B (zh) * 2021-02-08 2022-07-19 江苏省农业科学院 一种水稻抗性相关基因及其应用

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