CN105567696B - A method of cultivating anti-soybean mosaic virus genetically modified plants - Google Patents

A method of cultivating anti-soybean mosaic virus genetically modified plants Download PDF

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CN105567696B
CN105567696B CN201610142263.5A CN201610142263A CN105567696B CN 105567696 B CN105567696 B CN 105567696B CN 201610142263 A CN201610142263 A CN 201610142263A CN 105567696 B CN105567696 B CN 105567696B
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mosaic virus
soybean
dna fragmentation
soybean mosaic
genetically modified
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CN105567696A (en
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杨向东
郭东全
董英山
李启云
牛陆
张伟
杨静
杜茜
邢国杰
李海云
钱雪燕
姚瑶
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Jilin Academy of Agricultural Sciences
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Abstract

A method of cultivating anti-soybean mosaic virus genetically modified plants, platymiscium field of biotechnology.Method provided by the invention includes RNA segment shown in SEQ-1 or the sequence for being complementary to the segment.The RNA segment can interfere the duplication and movement of soybean mosaic virus, to inhibit the development of soybean mosaic virus symptom.The present invention also provides a kind of method for cultivating anti-soybean mosaic virus genetically modified plants, include the following steps: to express the RNA molecule in target plant, obtains the genetically modified plants significantly improved to soybean mosaic virus resistance.The present invention has important application value for cultivating the anti-soybean mosaic poisonous plant of wide spectrum.

Description

A method of cultivating anti-soybean mosaic virus genetically modified plants
Technical field
The invention belongs to plant biotechnology fields, and in particular, to a kind of to cultivate anti-soybean mosaic virus genetically modified plants Method.
Background technique
Soybean Mosaic (soybean mosaic virus, SMV) and it is one kind generation as caused by soybean mosaic virus One of most important disease of each major soybean production areas of criticality soybean diseases and China.SMV can generally cause soybean to be averaged the underproduction 10-30%, serious time and area even cause large area to have no harvest.SMV also often results in Seed mottling, seriously affects soybean seed The exterior quality and commodity value of grain.On taxology, SMV category marmor upsilon section (Potyviridae) potato Y disease Poison category (Potyvirus), mainly propagated by the seed infected, aphid and mechanical inoculation mode.With big in global range The exchange of beans germ plasm resource, the variation of the appearance of new virus strain and pathogenicity power, China's Soybean Resistance SMV breeding work face Situation it is also more serious.The problems such as due to chemical prevention difficulty and easily causing Environmental security produces to soybean mosaic virus Prevention and treatment depend on the cultivations of SMV resistance soybean varieties.Studies have shown that because there is complicated strain differentiations, soybean in SMV There is strain specialization again to the resistance of SMV, therefore, the resistance of host is easily lost with the variation of virus strain, cultivates wide The lasting disease-resistant variety of spectrum, resistance also becomes the fundamental way of prevention and treatment soybean Mosaic.
RNA silencing (RNA interference, RNAi) be plant resistant external nucleic acid (virus, transposons etc.) invasion with Keep a kind of defense mechanism of autogene group integrality.After poisoning intrusion host cell, double-stranded RNA (double-strand RNA, dsRNA) vsiRNA (virus short interfering is processed as by plant DCL (Dicer-like) nuclease RNA), subsequent vsiRNA and AGO protein binding, the long-chain viral RNA of degradation and vsiRNAs complementary pairing, to prevent disease Poison further infects.Many studies have shown that expressing fractionated viral genomic sequence fragment in plant, the dsRNA of generation can Effectively to inhibit infecting for virus.In terms of Soybean Resistance SMV research, Wang etc. will carry the coat protein gene of 3 '-UTR of SMVCPSoybean is imported, wherein 2 transgenic lines significantly improve SMV resistance level compared with receptor kind.Furutani etc. willSMV- CPGene Into Soybean, inoculated identification the result shows that, Transgenic soybean plants infect resistance level with higher to SMV.Point Analysis shows the entire stage of development in genetically engineered soybean,SMV-CPSequence specific tiny RNA is only present in true leaf stage, subsequent Growth phase is not expressed then.Zhang etc. and Kim etc. willSMV-CPGene order inverted repeat (inverted Repeat, IR) import soybean, inoculated identification the result shows that, genetically engineered soybean can effectively resist infecting for SMV, SMV resistance water It is flat to be significantly higher than receptor kind.Recently, Gao etc. will participate in posttranscriptional gene silencing (post-transcriptional gene Silencing, PTGS) negative regulatory factor SMV HC-Pro gene IR segment import soybean, inoculated identification shows that transgenosis is big Beans significantly improve SMV resistance.As it can be seen that mediating SMV encoding gene RNAi silencing using host is the effective of raising Soybean Resistance SMV Means, it is even more important that using RNAi perturbation technique to resist a variety of viruses and the different biological strain of virus to provide one simultaneously The more efficiently technological approaches of item.But existing research is mainly mediated by RNAi at presentSMV-CPOrSMV-HC-ProGene It is horizontal that silencing improves Soybean Resistance SMV, and participates in SMV to other and process keys gene RNAi silencing-mediated such as infect, replicate, move SMV Resistence research then there is not yet relevant report.
WithPotyvirusIt is similar to belong to other virus panels, SMV genome is single stranded positive-sense RNA, and genome encodes 11 altogether The maturation protein of a different function.P3 albumen is a key protein by SMV genome encoding.Studies have shown that P3 albumen is not It only plays an important role in virus replication and motion process, while closely related with infection Symptoms on hosts phenotype, and The host range of virus infection is determined to a certain extent.Recent research indicate that P3 albumen is to carryRsv1Resistance locus soybean The nontoxic factor of determination of genotype.It carriesRsv4The nontoxic factor of determination of resistance locus soybean genotype also is located at P3 protein sequence Inside, and play an important role in SMV causes a disease.Studies have shown thatRsv1Coding albumen can identify P3 albumen, and induce Soybean generatesRsv1 The hypersensitization of gene mediated reacts (hypersensitive response, HR).Although not having still at present It is related with plant nonhost resistance mechanism that positive evidence shows P3 albumen, but also prompts P3 albumen in determining soybean host range It plays an important role, and pathogenic related with SMV.Therefore, inhibitSMV-P3The transcription and translation of gene can interfere or The pathogenic of SMV is influenced, and influences the host range of SMV to a certain extent.Based on above-mentioned understanding, this research is based on host RNAi is mediatedSMV-P3Gene silencing interferes the duplication and movement of SMV, and it is big to obtain the transgenosis significantly improved to SMV resistance Beans.On this Research foundation, the present invention is proposed.
Summary of the invention
The object of the present invention is to provide a kind of methods for cultivating anti-soybean mosaic virus genetically modified plants.
RNA segment provided by the invention can may be list for double stranded RNA fragment as shown in SEQ-1
Chain RNA segment.The RNA segment can interfere the duplication and movement of soybean mosaic virus, to inhibit big Tofu pudding The development of mosaic virus symptom.
Double chain DNA fragment provided by the invention is as shown in SEQ-2.The DNA fragmentation can be inverted by the RNA segment Record obtains cDNA segment, or passes through artificial synthesized acquisition.
The present invention also provides one kind to contain specific DNA fragment plasmid as shown in SEQ-3.The DNA fragment specific packet Include DNA fragmentation 1, DNA fragmentation 2, and the intervening sequence between the DNA fragmentation 1 and DNA fragmentation 2.The DNA fragmentation 1 With 2 reverse complemental of DNA fragmentation.The DNA1 segment 1 is as shown in SEQ-2.
The DNA fragment specific can successively include following element: promoter from upstream to downstream, the DNA fragmentation 1, The stuffer fragment, the DNA fragmentation 2 and terminator.The promoter can be composing type, and leaf expression Idiotype or virus lure Conductivity type promoter, the leaf expression specific promoter are preferably Phaseolus Leaves specific promoterRBSC2。
Recombinant bacterium or transgenic cell line containing above-mentioned plasmid also belong to the scope of protection of the invention.
The present invention also protects the RNA segment, and the double chain DNA fragment or any description above plasmid can be used for training Educate the application in anti-soybean mosaic virus genetically modified plants.
The present invention also provides a kind of methods for cultivating anti-soybean mosaic virus genetically modified plants, include the following steps: in mesh The RNA molecule is expressed in mark plant, obtains the genetically modified plants significantly improved to soybean mosaic virus resistance.It is described " in mesh Mark plant in express the RNA molecule " implementation it is specific as follows: by plasmid described above import target plant.The mesh It marks plant and genetically modified plants is dicotyledon, it more specifically can be the cultivated soybean that the dicotyledon, which is soybean,.The matter Grain can import target plant by agrobacterium-mediated transformation, particle bombardment, pollen tube mediated method etc..
The present invention also protects the RNA segment, the double chain DNA fragment or any description above plasmid in preparation soybean Application in mosaic virus inhibitor.
The present invention also protects a kind of soybean mosaic virus inhibitor, its active constituent is the RNA segment, the double-strand DNA fragmentation or any description above plasmid.
Any description above soybean mosaic virus concretely soybean mosaic virus SC-3 strain.
Although the present invention has only carried out disease-resistant inoculated identification with SC-3 virus strain, the RNA segment is based on multiple The conserved sequence of Strains of Soybean Mosaic Virus has broad spectrum activity, so also having the same to other Strains of Soybean Mosaic Virus Inhibitory effect.
The present invention has important application for cultivating the anti-soybean mosaic virus genetically modified plants (especially soybean) of wide spectrum Value.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of recombinant plasmid pTF101.1-P3
Fig. 2 is T0For PAT test strips positive seedling PCR qualification result M, DNA standard molecular weight;Ctl+, plasmid pTF101.1-P3;Wt, non transformed plants;1~6 is the PAT test strips positive plant randomly selected
Fig. 3 is T1~T3Qualification result M, DNA standard molecular weight are tracked for genetically engineered soybean PCR;Ctl+, plasmid pTF101.1-P3;Wt, non transformed plants;1~6 is genetically engineered soybean B013 difference single plant;7~12 be genetically engineered soybean B115 difference single plant
Fig. 4 is T1Hybridize qualification result M, DNA standard molecular weight for genetically engineered soybean Southern;Ctl+, matter pTF101.1-P3;Wt, non transformed plants;1~6 is to turn T1For transgenic plant B013, B115, B127, B128, B163, B166. plant genome DNA (50 μ g) are usedHindAfter III digestion, marked with DIGbarGene probe carries out detection
Fig. 5 is T3Is showed for Transgenic soybean plants Field inoculation SMV SC-3 leaf symptom
Specific embodiment:
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from In the case where essence of the present invention, to modifications or substitutions made by the method for the present invention, all belong to the scope of the present invention.Following embodiments In experimental method, unless otherwise specified, the conventional method being well known to the skilled person.It is tried used in embodiment Material is tested, unless otherwise specified, can be obtained through commercial channels.
Plasmid pHANNIBAL-attB5-RBSC2-attB2: the public can be from Jilin Academy of Agricultural Science agro-ecology skill Art research institute obtains.
The cultivated soybean kind Williams82 and Shen Nong No. 9 numbers: the public can be from national crop germplasm resource database (http://www.cgris.net/query/croplist.php) is obtained.
No. 3 strains of soybean mosaic virus (SC-3 strain): for China soybean producing region Major Epidemic SMV strain, the public can be with It is obtained from Inst. of Plant Protection, Jilin Prov. Academy of Agricultural Sciences.Bibliography: the town Zheng Cuiming, Chang Ru, Qiu Lijuan, Wu Zongpu, Resistance Identification Soybean Science of the Gao Fenglan Soybean Germplasm to SMV3 strain, 2000,19 (4): 299-306..
Used various culture medium prescriptions are shown in Table 1 in embodiment.MS synthesizes salt and is purchased from Sigma company, and article No. is M5524.B5 synthesizes salt and is purchased from Sigma company, article No. G5768.
Embodiment 1. has the discovery for the segment for inhibiting soybean mosaic virus function
To the 26 SMV strains logged in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) Sequence alignment is carried out, finds soybean mosaic virusP3There are conserved regions for gene, have finally been determined 302bp's in the conserved region Segment is located atP3Gene is from 5 ' the 99-400 nucleotide in end, as shown in SEQ-2.The RNA for being contemplated by the fragment coding inhibits Soybean mosaic virus.The RNA sequence of the SEQ-2 segment transcription is single stranded RNA shown in SEQ-1.
The acquisition and identification of the anti-soybean mosaic virus genetically modified plants of embodiment 2.
1. the building of RNAi recombinant plasmid
(1) double chain DNA molecule shown in the SEQ-2 of composition sequence table.
(2) double chain DNA molecule synthesized using step 1 is template, the primer pair formed using SMV-P3-F and SMV-P3-R, PCR amplification is carried out with KOD FX high fidelity enzyme (Japanese TOYOBO company),
Obtain pcr amplification product.
SMV-P3-F:5 '-CCGCTCGAGTCTAGATCTCTTGATGGGCTTGGTTTC-3’
SMV-P3-R:5 '-GGGGTACCAAGCTTGGTTGTTGGATGCTTTTCTTTC-3’
In SMV-P3-F and SMV-P3-R, underscore marks restriction endonuclease recognition sequence, wherein " CTCGAG " is restriction enzyme EnzymeXhoI restriction endonuclease recognition sequence, " TCTAGA " are restriction enzymeXbaI restriction endonuclease recognition sequence, " GGTACC " are limited Property restriction endonuclease processedKpnI restriction endonuclease recognition sequence, "AAGCTT" it is restriction enzymeHindIII restriction endonuclease recognition sequence.
PCR reaction condition are as follows: 95 DEG C of 2 min；94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 1 min, totally 35 recycle； 72℃ 5min。
With 1.0% agarose gel electrophoresis recovery purifying pcr amplification product, primer size 331bp.
(3) restriction enzyme is usedXbaI HeHindThe pcr amplification product of III double digestion step (2), and recycle digestion production Object.
(4) restriction enzyme is usedXbaI HeHindIII double digestion plasmid pHANNIBAL-attB5- RBSC2-attB2, It recycles vector backbone sequence (about 5.9kb).
(5) digestion products of step (3) are connected with the vector backbone sequence of step (4), obtains recombinant plasmid pHANNIBAL-RBSC2-revP3。
(6) restriction enzyme is usedXhoI HeKpnThe pcr amplification product of I double digestion step (2), and recycle digestion products.
(7) restriction enzyme is usedXhoI HeKpnI double digestion recombinant plasmid pHANNIBAL-RBSC2-revP3, recycling Vector backbone sequence (about 6.2kb).
(8) digestion products of step (6) are connected with the vector backbone sequence of step (7), obtains recombinant plasmid pHANNIBAL-RBSC2-P3 RNAi。
(9) restriction enzyme is usedSpeI HeEcoRI double digestion plasmid pHANNIBAL-RBSC2-P3 RNAi, recycling is about 2.88kb digestion small fragment (segment from upstream to downstream successively include Phaseolus Leaves specific promoterRBSC2, just RNA piece The coded sequence of section is spaced intron sequences, the coded sequence and OCS terminator of antisense RNA fragment).Digestion DNA fragmentation both ends It is viscous end.
(10) restriction enzyme is usedEcoRI HeXbaI double digestion plasmid pTF101.1, recycling vector backbone sequence is (about 9.5kb), DNA enzymatic slice section both ends are viscous end.
(11) the digestion small fragment of step (9) is connected into (restriction enzyme with the vector backbone sequence of step (10)Spe I withXbaI is isocaudarner, and the cohesive end sequence after digestion is identical), obtain RNAi recombinant plasmid pTF101.1-P3.
2. the acquisition of Transgenic soybean plants
(1) recombinant plasmid pTF101.1-P3 is imported by Agrobacterium EHA101 using freeze-thaw method, obtains recombinational agrobacterium.
(2) Agrobacterium is resuspended with the CCM fluid nutrient medium in subordinate list 1, the bacteria suspension for obtaining OD600nm=0.5-0.8 is standby With.
(3) the cultivated soybean Williams82 and Shen Nong ninth-seeded is chosen, sterilize 12- in the closed container containing chlorine 16 hours.
(4) seed in step (3) after sterilizing is taken, hilum is placed in downward on GM culture medium flat plate, and 23 DEG C of dark cultures 24 are small When.
(5) it is cut with sterile scalpel along hilum, and removes axillary bud, cotyledonary node position slightly scratches above axillary bud, so It is placed in the bacteria suspension that step (2) obtains and impregnates 30-40 minutes.
(6) after completing step (5), seed is moved to and is covered on the CCM culture medium flat plate of aseptic filter paper, 23 DEG C of dark culture 4- 5 days.
(7) after completing step (6), seed is transferred on SIM culture medium, 25 DEG C, 16h(light)/8h(is dark) under the conditions of train It supports, every 2-3 weeks subculture 1 time.
(8) cotyledon on the explant for inducing Multiple Buds is cut, is placed on SEM culture medium flat plate, 25 DEG C, 16h (light)/8h(is dark) under the conditions of cultivate, every 2-3 weeks subculture 1 time, when subculture, cuts the callus of browning.
(10) when regeneration bud grows to 4-8cm, regeneration bud is cut, is then transferred on RM culture medium flat plate, 25 DEG C, 16h(light)/8h(is dark) under the conditions of cultivate.
(11) it when growing to resistant buds to 3-5 cm, and growing healthy and strong root, moves to seeding room and tames 3-5 days, then shift It is grown into greenhouse, obtains T0For plant.
(12) to the T of transplanting to greenhouse0For plant carry out PAT test strips (LibertyLink strips, Envirologix, USA) detection.The positive plant of test strips detection is initially identified as transgenic plant.
3. genetically engineered soybean Molecular Identification
(1) clip PAT test strips positive plant blade carries out PCR detection.The genomic DNA for mentioning plant leaf, using P3- The primer of F and P3-R composition carries out PCR amplification, and amplified fragments size is 727bp.
P3-F:5 '-ACCTCAACTCCACCAGCATC-3 '
P3-R:5 '-TACTCTCAACTTTTATCTTCTTCGTC-3 '
Part seedling PCR qualification result is shown in Fig. 2.
(2) PAT test strips and PCR detect positive T0For i.e. acquisition T after plant selfing1For strain.Using 500mg/L weeding Agent-glufosinate is sprayed, and yellowing leaf or withered phenomenon occurs in non-transgenic seedling after 7 days, and transgenic plant then shows just Often.
(3) clip T1Southern hybridization check is carried out for rotaring gene plant blade.It is extracted using CTAB method with high salt high-purity Spend Soybean genomic DNA.HindAfter III digestion total DNA (~50 μ g), endonuclease bamhi is separated with 0.8% Ago-Gel.Using height Endonuclease bamhi is transferred to positively charged Hybood TM-N by salt transfer method+On nylon membrane (Amersham, USA).With PTF101.1-P3 is template, is introduced using BAR-F and BAR-R to amplificationbarGenetic fragment, amplified fragments size are 441bp. Utilize DIG random primer labelling kit (Roche, USA) label probe.
BAR-F:5 '-GCACCATCGTCAACCACTACATCGAG-3 '
BAR-R:5 '-TGAAGTCCAGC TGCCAGAAACCCAC-3 '
Using the DIG High Prime DNA Labeling and Detection Starter Kit of Roche company II kit carries out molecule hybridization.Hybridization temperature is 42 DEG C, and washing film condition is 2 × SSC (containing 0.1% SDS), 37 DEG C of conditions Under wash film 2 times, 5 min every time；0.5 × SSC (contain 0.1% SDS), washes film 2 times under the conditions of 66 DEG C, every time 15 min.So BCIP/NBT chemical colour reaction on hybond membrane is utilized afterwards.Partial transgenic soybean Southern hybridization check result is shown in Fig. 3.
(4) the positive T of harvest Southern hybridization1For the seed obtained after plant selfing, T is after planting grown2For seedling, Continue to carry out tracing detection using 500mg/L herbicide-glufosinate and PCR, and remove nontransgenic plants, until obtaining pure The transgenic line of conjunction.Partial transgenic Soybean PC R tracing detection result is shown in Fig. 4.
4. genetically engineered soybean Disease Resistance Identification
Using artificial frictional inoculation method, to T2Generation and T3For genetically engineered soybean strain B115, B127, B128, B013, B163 Anti- soybean mosaic virus identification is carried out with B166.Wherein, strain B115, B127 and B128 derives from soybean varieties Williams82, strain B013, B163 and B166 derive from soybean varieties Shen Nong 9.Identify biological strain for the production of China soybean Area Major Epidemic SMV strain SC-3.Carry out inoculated identification when first trifoliolate leaf of soybean plant strain occurs, in SPRING WHEAT BEFORE AND AFTER FLOWERING into Row resistance investigation.Resistance Identification test is repeated 3 times, 30 plants of each repetitive identified plant.Soybean mosaic virus identifies the plant state of an illness Partition of the level standard is shown in Table 2.
Disease index calculation method is as follows:
Soybean mosaic virus assessment resistance standard are as follows: highly resistance (IM): without visible system symptom, disease index 0；It is disease-resistant (R): disease index is between 1%-20%；In resist (MR): disease index is between 21%-35%；Middle sense (MS): disease index exists Between 36%-50%；Susceptible (S): disease index is between 51%-70%；Height sense (HS): disease index is greater than 70%.
Qualification result shows after being inoculated with Strains of Soybean Mosaic Virus SC-3 that it is serious that blade occurs in control Non-transgenic soybean The SMV such as shrinkage, floral leaf and plant dwarfing infect classical symptom, and Transgenic soybean plants symptom then shows slightly or without obvious Symptom, as shown in Figure 5.In 6 strains of identification, B127 and B163 are most strong to SMV resistance, and disease index is reduced to 4.37~ 11.11%, in addition 4 strains also show higher resistance level to SMV.Continuous 2 generation (T2Generation and T3Generation) the equal table of qualification result Bright, the disease index of 6 strains is substantially less than control Non-transgenic soybean Williams82(disease index 36.81-45.24) With SN9(disease index 39.80-46.97%), specific Resistance Identification the results are shown in Table 3.
This experiment shows recombinant RNA i expression vector importing plant can significantly improve plant to soybean mosaic virus Resistance.
Sequence table
SEQ-1 sequence:
1 UCUCUUGAUG GGCUUGGUUU CACCUUCUAU UCUAAUUCAC AUGUAUCGUA UGAAGCAUUU
61 UGAGAAAGGG GUAGAGUUGU GGAUAAGUAA AGAACAUAGU GUGGCAAAGA UUUUCAUCAU
121 AUUGGAACAA CUCACCAAGA GGGUCGCUGC AAAUGACGUG UUACUUGAGC AACUUGAAAU
181 GAUUUCAGAA ACUUCUGAGA GAUUCAUGAG CAUUCUAGAG GACUGUCCUC AAGCGCCACA
241 GUCAUACAAG ACGGCAAAAG AUUUGUUGAC AAUAUACAUA GAAAGAAAAG CAUCCAACAA
301 CC
SEQ-2 sequence:
1 TCTCTTGATG GGCTTGGTTT CACCTTCTAT TCTAATTCAC ATGTATCGTA TGAAGCATTT
61 TGAGAAAGGG GTAGAGTTGT GGATAAGTAA AGAACATAGT GTGGCAAAGA TTTTCATCAT
121 ATTGGAACAA CTCACCAAGA GGGTCGCTGC AAATGACGTG TTACTTGAGC AACTTGAAAT
181 GATTTCAGAA ACTTCTGAGA GATTCATGAG CATTCTAGAG GACTGTCCTC AAGCGCCACA
241 GTCATACAAG ACGGCAAAAG ATTTGTTGAC AATATACATA GAAAGAAAAG CATCCAACAA
301 CC
SEQ-3 sequence:
1 TCTCTTGATG GGCTTGGTTT CACCTTCTAT TCTAATTCAC ATGTATCGTA TGAAGCATTT
61 TGAGAAAGGG GTAGAGTTGT GGATAAGTAA AGAACATAGT GTGGCAAAGA TTTTCATCAT
121 ATTGGAACAA CTCACCAAGA GGGTCGCTGC AAATGACGTG TTACTTGAGC AACTTGAAAT
181 GATTTCAGAA ACTTCTGAGA GATTCATGAG CATTCTAGAG GACTGTCCTC AAGCGCCACA
241 GTCATACAAG ACGGCAAAAG ATTTGTTGAC AATATACATA GAAAGAAAAG CATCCAACAA
301 CCCAATTGGT AAGGAAATAA TTATTTTCTT TTTTCCTTTT AGTATAAAAT AGTTAAGTGA
361 TGTTAATTAG TATGATTATA ATAATATAGT TGTTATAATT GTGAAAAAAT AATTTATAAA
421 TATATTGTTT ACATAAACAA CATAGTAATG TAAAAAAATA TGACAAGTGA TGTGTAAGAC
481 GAAGAAGATA AAAGTTGAGA GTAAGTATAT TATTTTTAAT GAATTTGATC GAACATGTAA
541 GATGATATAC TAGCATTAAT ATTTGTTTTA ATCATAATAG TAATTCTAGC TGGTTTGATG
601 AATTAAATAT CAATGATAAA ATACTATAGT AAAAATAAGA ATAAATAAAT TAAAATAATA
661 TTTTTTTATG ATTAATAGTT TATTATATAA TTAAATATCT ATACCATTAC TAAATATTTT
721 AGTTTAAAAG TTAATAAATA TTTTGTTAGA AATTCCAATC TGCTTGTAAT TTATCAATAA
781 ACAAAATATT AAATAACAAG CTAAAGTAAC AAATAATATC AAACTAATAG AAACAGTAAT
841 CTAATGTAAC AAAACATAAT CTAATGCTAA TATAACAAAG CGCAAGATCT ATCATTTTAT
901 ATAGTATTAT TTTCAATCAA CATTCTTATT AATTTCTAAA TAATACTTGT AGTTTTATTA
961 ACTTCTAAAT GGATTGACTA TTAATTAAAT GAATTAGTCG AACATGAATA AACAAGGTAA
1021 CATGATAGAT CATGTCATTG TGTTATCATT GATCTTACAT TTGGATTGAT TACAGTTGGG
1081 AAATTGGGTT CGAAATCATC AAATTGGGTT GTTGGATGCT TTTCTTTCTA TGTATATTGT
1141 CAACAAATCT TTTGCCGTCT TGTATGACTG TGGCGCTTGA GGACAGTCCT CTAGAATGCT
1201 CATGAATCTC TCAGAAGTTT CTGAAATCAT TTCAAGTTGC TCAAGTAACA CGTCATTTGC
1261 AGCGACCCTC TTGGTGAGTT GTTCCAATAT GATGAAAATC TTTGCCACAC TATGTTCTTT
1321 ACTTATCCAC AACTCTACCC CTTTCTCAAA ATGCTTCATA CGATACATGT GAATTAGAAT
1381 AGAAGGTGAA ACCAAGCCCA TCAAGAGA
Sequence table
SEQ-1 sequence:
1 UCUCUUGAUG GGCUUGGUUU CACCUUCUAU UCUAAUUCAC AUGUAUCGUA UGAAGCAUUU
61 UGAGAAAGGG GUAGAGUUGU GGAUAAGUAA AGAACAUAGU GUGGCAAAGA UUUUCAUCAU
121 AUUGGAACAA CUCACCAAGA GGGUCGCUGC AAAUGACGUG UUACUUGAGC AACUUGAAAU
181 GAUUUCAGAA ACUUCUGAGA GAUUCAUGAG CAUUCUAGAG GACUGUCCUC AAGCGCCACA
241 GUCAUACAAG ACGGCAAAAG AUUUGUUGAC AAUAUACAUA GAAAGAAAAG CAUCCAACAA
301 CC
SEQ-2 sequence:
1 TCTCTTGATG GGCTTGGTTT CACCTTCTAT TCTAATTCAC ATGTATCGTA TGAAGCATTT
61 TGAGAAAGGG GTAGAGTTGT GGATAAGTAA AGAACATAGT GTGGCAAAGA TTTTCATCAT
121 ATTGGAACAA CTCACCAAGA GGGTCGCTGC AAATGACGTG TTACTTGAGC AACTTGAAAT
181 GATTTCAGAA ACTTCTGAGA GATTCATGAG CATTCTAGAG GACTGTCCTC AAGCGCCACA
241 GTCATACAAG ACGGCAAAAG ATTTGTTGAC AATATACATA GAAAGAAAAG CATCCAACAA
301 CC
SEQ-3 sequence:
1 TCTCTTGATG GGCTTGGTTT CACCTTCTAT TCTAATTCAC ATGTATCGTA TGAAGCATTT
61 TGAGAAAGGG GTAGAGTTGT GGATAAGTAA AGAACATAGT GTGGCAAAGA TTTTCATCAT
121 ATTGGAACAA CTCACCAAGA GGGTCGCTGC AAATGACGTG TTACTTGAGC AACTTGAAAT
181 GATTTCAGAA ACTTCTGAGA GATTCATGAG CATTCTAGAG GACTGTCCTC AAGCGCCACA
241 GTCATACAAG ACGGCAAAAG ATTTGTTGAC AATATACATA GAAAGAAAAG CATCCAACAA
301 CCCAATTGGT AAGGAAATAA TTATTTTCTT TTTTCCTTTT AGTATAAAAT AGTTAAGTGA
361 TGTTAATTAG TATGATTATA ATAATATAGT TGTTATAATT GTGAAAAAAT AATTTATAAA
421 TATATTGTTT ACATAAACAA CATAGTAATG TAAAAAAATA TGACAAGTGA TGTGTAAGAC
481 GAAGAAGATA AAAGTTGAGA GTAAGTATAT TATTTTTAAT GAATTTGATC GAACATGTAA
541 GATGATATAC TAGCATTAAT ATTTGTTTTA ATCATAATAG TAATTCTAGC TGGTTTGATG
601 AATTAAATAT CAATGATAAA ATACTATAGT AAAAATAAGA ATAAATAAAT TAAAATAATA
661 TTTTTTTATG ATTAATAGTT TATTATATAA TTAAATATCT ATACCATTAC TAAATATTTT
721 AGTTTAAAAG TTAATAAATA TTTTGTTAGA AATTCCAATC TGCTTGTAAT TTATCAATAA
781 ACAAAATATT AAATAACAAG CTAAAGTAAC AAATAATATC AAACTAATAG AAACAGTAAT
841 CTAATGTAAC AAAACATAAT CTAATGCTAA TATAACAAAG CGCAAGATCT ATCATTTTAT
901 ATAGTATTAT TTTCAATCAA CATTCTTATT AATTTCTAAA TAATACTTGT AGTTTTATTA
961 ACTTCTAAAT GGATTGACTA TTAATTAAAT GAATTAGTCG AACATGAATA AACAAGGTAA
1021 CATGATAGAT CATGTCATTG TGTTATCATT GATCTTACAT TTGGATTGAT TACAGTTGGG
1081 AAATTGGGTT CGAAATCATC AAATTGGGTT GTTGGATGCT TTTCTTTCTA TGTATATTGT
1141 CAACAAATCT TTTGCCGTCT TGTATGACTG TGGCGCTTGA GGACAGTCCT CTAGAATGCT
1201 CATGAATCTC TCAGAAGTTT CTGAAATCAT TTCAAGTTGC TCAAGTAACA CGTCATTTGC
1261 AGCGACCCTC TTGGTGAGTT GTTCCAATAT GATGAAAATC TTTGCCACAC TATGTTCTTT
1321 ACTTATCCAC AACTCTACCC CTTTCTCAAA ATGCTTCATA CGATACATGT GAATTAGAAT
1381 AGAAGGTGAA ACCAAGCCCA TCAAGAGA

Claims (11)

  1. RNA segment shown in 1.SEQ-1 or the sequence for being complementary to the segment.
  2. Single-stranded DNA fragments shown in 2.SEQ-2.
  3. 3. the plasmid containing specific fragment, the DNA fragment specific includes DNA fragmentation 1, DNA fragmentation 2, and is located at institute State the intervening sequence between DNA fragmentation 1 and DNA fragmentation 2,2 reverse complemental of the DNA fragmentation 1 and DNA fragmentation, the DNA fragmentation 1 as shown in SEQ-2.
  4. 4. plasmid described in claim 3, it is characterised in that: the DNA fragment specific successively includes as follows from upstream to downstream Element: promoter, the DNA fragmentation 1, the stuffer fragment, the DNA fragmentation 2 and terminator.
  5. 5. recombinant bacterium or transgenic cell line containing plasmid described in claim 3 or claim 4.
  6. 6. RNA segment described in claim 1, DNA fragmentation described in claim 2, plasmid described in claim 3 or claim 4 Cultivating the application in anti-soybean mosaic virus genetically modified plants.
  7. 7. a kind of method for cultivating anti-soybean mosaic virus genetically modified plants includes the following steps: to express power in target plant Benefit require 1 described in RNA molecule, obtain the genetically modified plants that significantly improve to soybean mosaic virus resistance.
  8. 8. according to the method described in claim 7, it is characterized by: expressing RNA molecule described in claim 1 in target plant Implementation it is as follows: by plasmid described in claim 3 or claim 4 import target plant.
  9. 9. according to claim 7 or method according to any one of claims 8, it is characterised in that: the target plant and genetically modified plants For dicotyledon, the dicotyledon is soybean.
  10. 10. RNA segment described in claim 1, DNA fragmentation described in claim 2, plasmid described in claim 3 or claim 4 Preparing the application in soybean mosaic virus inhibitor.
  11. 11. a kind of soybean mosaic virus inhibitor, active constituent is RNA segment described in claim 1, described in claim 2 DNA fragmentation, plasmid described in claim 3 or claim 4.
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