CN103468719A - RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and RNAi (RNA interfere) vector for coding same - Google Patents
RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and RNAi (RNA interfere) vector for coding same Download PDFInfo
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Abstract
The invention discloses an RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and an RNAi (RNA interfere) vector for coding the same. The RNA with high TuMV resistance has one of the following nucleotide sequences: 1) a nucleotide sequence as shown in SEQ ID No.2 in a sequence table; and 2) RNA sequences having more than 90% homology with the RNA sequence defined by the item 1), specifically more than 95% homology, more specifically more than 96% homology, further more specifically more than 97% homology, further more specifically more than 98% homology and the most specifically more than 99% homology, and having the same functions. The RNA and the RNAi vector for coding the RNA disclosed by the invention can be used for enhancing the resistance of plants to TuMV or glufosinate-ammonium herbicides, and simultaneously, also lay a foundation for breeding high-TuMV-resistance transgenic plants.
Description
Technical field
The invention belongs to the genetic engineering for plant virus resistance field, be specifically related to the RNA of a kind of high resistance TuMV and the RNAi carrier of this RNA of coding.
Background technology
Brassica 2 et 4 (Turnip Mosaic Virus, TuMV) belongs to marmor upsilon section (Potyviridae), Potyvirus (Potyviruse).The TuMV host range is very extensive, can infect 43 sections 156 and belong to 318 kind of plant.TuMV is the most serious virus of harm crop in cruciferae.Crop after infection virus also easily infects oidium and soft rot, causes Combined Infection, directly has influence on output and commodity value.
Rape is the main oil crops of China, and turnip mosaic virus is one of important disease of rape.In China middle and lower reach of Yangtze River, the rape due to illness viral disease underproduction generally reaches 20-30%, and the year of being very popular reaches 50% left and right.Susceptible rape is yield reducation not only, and oil yield rate and also obviously reduction of seed germination rate.Endanger because of TuMV the production loss that causes every year on average 5-10% according to another investigation China Chinese cabbage, the plot that disease is serious almost has no harvest.
TuMV propagates in the mode of perishability by aphid, has at least 89 kinds of aphids can propagate this virus.And adopt sterilant can not kill very soon whole aphids to stop viral propagation, and only depend on sterilant to kill the DeGrain that aphid prevents and treats virus disease, also easily cause environmental pollution.Therefore seeking new disease-resistant method becomes a urgent task.
Summary of the invention
An object of the present invention is to provide the RNA of a kind of high resistance TuMV and the RNAi carrier of this RNA of coding.
A kind of RNA provided by the invention has one of following nucleotide sequence:
1) nucleotide sequence shown in the SEQ ID № .2 in sequence table;
2) with 1) the RNA sequence that limits has 90% above homology, and has the RNA sequence of identical function; Concrete, described homology is more than 95%; Concrete is more than 96% again; Concrete is more than 97% again; Concrete is more than 98% again; Concrete is more than 99% again.
Described have identical function and specifically refer to have following arbitrary function:
1) strengthen the resistance of plant to TuMV;
2) strengthen the resistance of plant to weedicide.
Described plant is specially Arabidopis thaliana or crop in cruciferae; Described crop in cruciferae is specially rape or Chinese cabbage.
Described enhancing plant is copied realization to the resistance of TuMV by suppressing TuMV.
Described TuMV is specially TuMV-C4 and/or BJ-R01.
Described weedicide is specially careless ammonium phosphine weedicide.
The encoding gene of described RNA also belongs to protection scope of the present invention.
The recombinant vectors that contains described encoding gene, expression cassette, transgenic cell line or Host Strains also belong to protection scope of the present invention.
Described recombinant vectors is specially recombinant expression vector or recombinant cloning vector.
In the multiple clone site of described recombinant expression vector for carrier that the DNA fragmentation insertion is set out, obtain; Described DNA fragmentation is that X forward-joining region-X is reverse; The X forward is in SEQ ID No.1 shown in the Nucleotide of 15-380 position, and X is reversed the reverse complemental fragment of X forward.
Concrete, the described carrier that sets out is pBBBast.
In described recombinant expression vector, described X forward-joining region-reverse fragment of X has the SEQ ID described 1365-2919 of № .3 position nucleotide sequence in sequence table.
Another object of the present invention is to provide following at least one application of the encoding gene of described RNA, described RNA, the described recombinant vectors that contains described encoding gene, expression cassette, transgenic cell line or Host Strains:
1) prepare the product of anti-TuMV;
2) strengthen the resistance of plant to TuMV;
3) strengthen the resistance of plant to careless ammonium phosphine weedicide.
In described application, described plant is specially Arabidopis thaliana or crop in cruciferae; Described crop in cruciferae is specially rape or Chinese cabbage.
Described enhancing plant is copied realization to the resistance of TuMV by suppressing TuMV.
In described application, described TuMV is specially TuMV-C4 and/or BJ-R01.
In described application, described weedicide is specially careless ammonium phosphine weedicide.
A further object of the present invention is to provide a kind of method of cultivating transgenic plant, is that described recombinant expression vector is proceeded to the purpose plant, obtains transgenic plant; Described transgenic plant are compared with described purpose plant, have following at least one proterties: 1) resistance of TuMV strengthened; 2) resistance of weedicide strengthened.
The described enhancing of the resistance to TuMV copies realization by suppressing TuMV.
In described method, described TuMV is specially TuMV-C4 and/or BJ-R01.
In described method, described weedicide is specially careless ammonium phosphine weedicide.
In described method, described purpose plant is specially Arabidopis thaliana or crop in cruciferae; Described crop in cruciferae is specially rape or Chinese cabbage.
The accompanying drawing explanation
The enzyme that Fig. 1 is intermediate carrier is cut the result, and wherein M is molecular weight marker; Swimming lane 1 is cut result for the enzyme of pHannibal-CI carrier; Swimming lane 2 is cut result for the enzyme that is connected into reverse fragment pHannibal-CI (-) carrier; Swimming lane 3 is cut result for the enzyme of pHannibal empty carrier.
The structural representation that Fig. 2 is carrier pBBBTu-CI.
The MluI enzyme that Fig. 3 is plant expression vector pBBBTu-CI is cut detected result figure, and wherein M is molecular weight marker; Swimming lane 1 is cut result for the enzyme of pBBBTu-CI carrier.
Fig. 4 is T
1spray result after careless ammonium phosphine weedicide for seedling, survival be the Herbicid resistant strain.
Fig. 5 is part transgenosis T
1for seedling PCR qualification result figure, wherein swimming lane 2 is molecular weight marker; Swimming lane 1 is the result of contrast col-0; Swimming lane 3-9 is the transgenosis T that herbicide screening goes out
1the result of Dai Miao.
Fig. 6 is the picture after different transgenosis high resistance strain inoculation TuMV-C4, and wherein col represents the result of wild-type col-0.
Fig. 7 is picture when after different transgenosis high resistance strain inoculation TuMV-C4, seed is gathered in the crops, and wherein col represents the result of wild-type col-0.
Fig. 8 is the picture after transgenosis high resistance strain # 07 and #48 inoculation BJ-R01, and wherein col represents the result of wild-type col-0.
Fig. 9 is the disease index statistics after different transgenosis high resistance strain inoculation TuMV-C4, and wherein col represents the result of wild-type col-0, #07, and #12, #19, #22, #30, #34, #48 represents respectively the result of 7 transgenosis high resistance strains.
After Figure 10 is TuMV-C4 inoculation Arabidopis thaliana, RT-PCR detects the expression of TuMV coat protein gene in transgenic arabidopsis, and wherein swimming lane 1 is the result of contrast Col-0; Swimming lane 2-6 is followed successively by transgenosis high resistance strain # 07, #12, #22, #34, the result of #48.
After Figure 11 is TuMV-C4 inoculation Arabidopis thaliana, the quantitative fluorescent PCR analytical results, wherein col represents the result of wild-type col-0, #07, #12, #22, #34, #48 represents respectively the result of 5 transgenosis high resistance strains.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
1, material
This tests wild-type Arabidopis thaliana Col-0 used purchased from Arabidopsis Biological Resource Center.
The Brassica 2 et 4 TuMV main popular strong strain TuMV-C4 that causes a disease in Beijing area is available from vegetable or flower institute of the Chinese Academy of Agricultural Sciences; Reference: Feng Lanxiang, Xu Ling, Liu Jia, button heart lattice, Li Xiusheng, the evaluation of Beijing area Turnip Mosaic Virus of Chinese Cabbage strain, China's Vegetable, 1988,4:11-13; The public can obtain this strain from Beijing Agricultural Biological Technology Rsearch Centre.
TuMV radish pathogenic strain BJ-R01 by force is that the contriver gathers and preserves from field.Reference " Ye Yanying, Zeng Gang, Yan Xiaohong, Ma Rongcai, Wu Caijun, Yao Lei, Agricultural University Of Jiangxi's journal, 2012,34 (2): 264-269 ".The public can obtain from Beijing Agricultural Biological Technology Rsearch Centre.
Intestinal bacteria (Escherichia coli) DH5 α bacterial strain is purchased from Tiangen company, and catalog number is CB101;
Agrobacterium (Agrobacterium tumefacieus) GV3101(pMP90), reference: Koncz, C.and Schell, J. (1986) The promoter of TL-DNA gene5controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector.Mol.Gen.Genet.204,383 – 396.The public can obtain this bacterial strain from Beijing Agricultural Biological Technology Rsearch Centre.
The RNAi carrier pHannibal public can be from CSIRO(
http:// www.csiro.au/pi) obtain; Reference: Wesley S V, Helliwell C A, Smith N A.Construct design for efficient, effective and high-throughput gene silencing in plants[J] .The Plant Journal, 2001,27 (6): 581-590.
High-fidelity enzyme Fast pfu DNA Polymerase, Easy Taq enzyme and molecular weight marker are purchased from full formula King Company.The little extraction reagent kit of plasmid, glue reclaim test kit, the PCR purification kit is the century bio tech ltd purchased from health.M-MLV ThermoScript II, quantitative PCR reagent, restriction enzyme and Klenow Fragment fill enzyme purchased from TaKaRa company.DNTP, dGTP and dCTP are purchased from Promega company.
The preparation of embodiment 1, RNAi carrier
(1) preparation of CI gene conservative fragments
According to the conservative property of listed 118 the TuMV strains of GenBank, the conservative fragments of choosing the common 366bp of 882-1247 position nucleotide sequence of CI gene is that RNA disturbs target.
Extract total RNA of TuMV-C4 bacterial strain, and reverse transcription is cDNA, take this cDNA as template, take following CI366F and CI366R(underscore is partly restriction enzyme site Xho I, BamH I and EcoR I, Hind III) be primer, carry out pcr amplification with high-fidelity enzyme Fastpfu DNA Polymerase:
CI366 F 5’-cc
ctcgagggatccTGGAACACCTAGCAAGAAGCACT-3’；
CI366 R 5’-cg
gaattcaagcttCGTGCCTGCTTTGCCGTTAC-3'。
Pcr amplification program: 94 ℃ of 5min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 7min.
The PCR product reclaims after the agarose electrophoresis purifying.Gained PCR product is correct through order-checking proof nucleotide sequence, and its nucleotide sequence has the nucleotide sequence shown in SEQ ID № .1 in sequence table, and the RNA of its coding has a nucleotide sequence shown in SEQ ID № .2 in sequence table.
(2) structure of RNAi carrier
1, the preparation of intermediate carrier pHannibal-CI
1) preparation process
PCR product with Hind III and BamH I difference double digestion pHannibal carrier and above-mentioned preparation.Enzyme is cut by purifying and is reclaimed the carrier framework fragment of about 5818bp and the PCR product fragment of about 372bp, and 4 ℃ of connections are spent the night.Connect product and transform bacillus coli DH 5 alpha through thermal shock.The picking clone, extract plasmid and cut checking with PstI and XhoI enzyme, obtains the intermediate carrier pHannibal-CI (-) containing reverse fragment.
With XhoI and EcoRI respectively the above-mentioned preparation of double digestion the PCR product and be connected into pHannibal-CI (-) intermediate carrier of reverse fragment.Enzyme is cut the PCR product fragment of the purified rear recovery about 6184bp carrier framework fragment of product and about 384bp, and 4 ℃ of connections are spent the night.Connect product and transform bacillus coli DH 5 alpha, the picking clone, the extraction plasmid also, with PstI and the checking of XhoI double digestion, obtains the intermediate carrier pHannibal-CI that simultaneously contains reverse and forward fragment.
2) enzyme of constructed carrier is cut proof procedure
With XhoI and PstI double digestion pHannibal empty carrier, the pHannibal-CI (-) that is connected into reverse fragment and pHannibal-CI intermediate carrier respectively, enzyme is cut rear electrophoresis and is detected, and carries out confirmatory experiment.Experimental result is shown in Fig. 1.Fig. 1 result shows: the pHannibal empty carrier produces 4276bp and 1548bp two bar segment after double digestion, pHannibal-CI (-) produces 4276bp and 1914bp two bar segment after double digestion, and pHannibal-CI produces 4276bp and 2292bp two bar segment after double digestion.Enzyme is cut and electrophoresis result shows, intermediate carrier pHannibal-CI builds correct.
2, the preparation of plant expression vector pBBBTu-CI
1) preparation process
1 NotI restriction enzyme site is respectively contained at the RNAi hairpin structure two ends of pHannibal.Cut the intermediate carrier pHannibal-CI containing hairpin structure with the NotI enzyme, same enzyme is cut in system and is respectively mended two bases G with Klenow enzyme and dGTP at two sticky ends of endonuclease bamhi again, product is in 1% agarose gel electrophoresis purifying, and glue reclaims containing the fragment of the 3708bp of hairpin structure standby.Through order-checking, the fragment of the described 3708bp containing hairpin structure has the described nucleotide sequence of SEQ ID № .3 in sequence table, and described hairpin structure fragment has the SEQ ID described 1365-2919 of № .3 position nucleotide sequence in sequence table.
Plant binary vector pBBBast can be and will have the SspI restriction enzyme site of the double chain DNA molecule insertion vector pBBR1MCS-2 of nucleotide sequence shown in SEQ ID № .4 in sequence table, the recombinant plasmid that the small segment between the SspI restriction enzyme site of replacement plasmid pBBR1MCS-2 obtains.
The plasmid pBBR1MCS-2 public can obtain from Beijing Agricultural Biological Technology Rsearch Centre; Reference: Kovach ME; Elzer PH; Hill DS; Robertson GT; Farris MA; Roop RM2nd, Peterson KM.Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.Gene.1995Dec1; 166 (1): 175-6.
With XmaI single endonuclease digestion plant binary vector pBBBast, respectively mend two base C with Klenow enzyme and dCTP at two sticky ends again, product is in 1% agarose gel electrophoresis, glue reclaims carrier segments 6559bp, again with containing 4 ℃ of the 3708bp fragments of hairpin structure, being connected and spending the night of above-mentioned preparation, obtain expression of plants RNAi plasmid pBBBTu-CI, its structural representation is shown in Fig. 2.
2) enzyme of constructed carrier is cut proof procedure
The plant expression vector pBBBTu-CI of above-mentioned preparation is carried out to the MluI enzyme and cut detection.The pBBBast carrier itself has a MluI restriction enzyme site, connects on the CaMV promotor of the above-mentioned fragment containing hairpin structure a MluI restriction enzyme site is separately arranged, and enzyme is cut the positive clone of rear demonstration 2 bands.According to the large I judgement of enzyme slitting band insertion, be that clockwise forward connects or counterclockwise Opposite direction connection.Forward ligase enzyme slitting band is 9715bp and 542bp, and the Opposite direction connection band is 6494bp and 3773bp band.The MluI enzyme is cut detected result and is seen Fig. 3.Fig. 3 result shows to be connected in the XmaI restriction enzyme site of plant binary vector pBBBast for the RNAi structure of CI gene conservative fragments, and is Opposite direction connection.
The functional verification of the RNAi carrier of the RNA of embodiment 2, high resistance TuMV and this RNA that encodes
(1) screening of the genetic transformation of Arabidopis thaliana and positive seedling
Utilize electric shocking method that the pBBBTu-CI carrier is proceeded in Agrobacterium GV3101 (pMP90), adopt agriculture bacillus mediated inflorescence pickling process to infect Arabidopis thaliana.Arabidopis thaliana T by results
0in the culture tray that fills Nutrition Soil, be placed in 22 ℃ of (daytime)/18 ℃ (night) for planting seed, periodicity of illumination is 16h(light)/8h(is dark) phytotron.Spray 500 times of careless ammonium phosphine weedicides of by volume number dilution of water (described careless ammonium phosphine weedicide is the commercial weedicide that market is bought, and its medium-height grass ammonium phosphine concentration is 200mg/L) and screen positive seedling after seedling grows two cotyledons.
After spraying 2-3 careless ammonium phosphine herbicide screening, the non-transgenic seedling can not continued growth, withered and yellow death gradually; And the transgenic seedling growing way is healthy and strong, it is green that blade keeps, the part seedling the results are shown in Figure 4.Obtain altogether 126 strain T
1for the Herbicid resistant plant.Randomly draw the blade of 68 strain resistance seedlings, with a small amount of rapid method, extract plant genome DNA.Carry out the pcr amplification detection with primer CI366F and CI366R, reaction conditions is: 94 ℃ of 5min; 94 ℃ of 45s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 7min.The PCR product carries out electrophoresis detection on 1% sepharose, and what amplify goal gene fragment 394bp is the transgenic positive seedling.Detected result shows whole positive seedlings.The PCR qualification result of part seedling is shown in Fig. 5.
Choose the transgenosis T1 that is accredited as positive seedling and reserve seed for planting for the seedling selfing, T
2in generation, randomly drawed 32 strain sowings, after 500 times of careless ammonium phosphine weedicides of by volume number dilution of 2-3 water of seedling sprinkling (described careless ammonium phosphine weedicide is the commercial weedicide that market is bought, and its medium-height grass ammonium phosphine concentration is 200mg/L), adds up survival/Mortality Ratio.There are 18 strains to separate than being about 3:1, tentatively confirm as single copy strain, and individual plant selfing sowing.The offspring continues screening and does not separate strain and be homozygous lines.
(2) the disease-resistant evaluation of transfer-gen plant
Randomly draw 12 homozygous lines and carry out disease-resistant evaluation.By homozygous lines, wild-type Col-0 with turn the empty carrier Arabidopis thaliana and sowed simultaneously, treat that seedling grows to TuMV-C4 or the BJ-R01 strain of 8-10 sheet lotus throne leaf frictional inoculation in period TuMV.Inoculation liquid is in the about 1g/10mL ratio preparation of sick leaf weight/phosphoric acid buffer volume (pH=7.0).Each homozygous lines inoculation 8-12 strain, two larger blades of every strain inoculation.With non-transgenic wild-type Arabidopis thaliana Col-0 with turn the empty carrier Arabidopis thaliana and inoculate in contrast.Rinse blade with clear water immediately after the inoculation several minutes.Middle net cover is observed in the controlled environment chamber, carries out disease-resistant evaluation and disease index statistics after 20 days.
State of an illness grade classification: 0 grade complete asymptomatic; 1 grade of 1-2 sheet is not inoculated leaf floral leaf or jaundice, the energy bolting; 3 grades of 3-4 sheets are not inoculated leaf floral leaf or jaundice energy bolting; 5 grades of 5-6 sheets are not inoculated leaf floral leaf or jaundice, affect bolting; 7 grades of most blade floral leaves or jaundice, can not bolting; 9 grades of most of blades are withered, and plant is at death's door.
Disease index=100 * ∑ (diseased plant numbers at different levels * typical values at different levels)/(investigating total strain number * highest typical value)
The partial results of disease-resistant inoculation is shown in Fig. 6,7,8, and the disease index statistics is shown in Fig. 9.Fig. 6 result shows, after inoculation TuMV-C4 about 20 days, contrast wild-type Col-0 is withered death substantially, turn empty carrier results of comparison and Col-0 basically identical, and transgenic line shows different resistances, 7 strain (#07, #12, #19, #22, #30, #34, #48) after the inoculation, can grow preferably, and the later stage can be normally solid, shows the highly resistant to TuMV-C4.Fig. 7 result shows, inoculates the high resistance transgenic line of TuMV-C4 to the seed results period and can normally set seeds; And the plant of contrast Col-0 is all dead, without seed, can gather in the crops.Fig. 9 result shows, after inoculation TuMV-C4, the disease resistance of disease index statistical result showed high resistance strain improves approximately 80% than contrast plant Col-0 resistance, turns empty carrier results of comparison and Col-0 basically identical.
After high resistance transgenic line # 07 and #48 inoculation BJ-R01, Fig. 8 result shows, the aobvious disease of contrast wild-type Col-0 morbidity, and high resistance transgenic line # 07 and #48 show growing way preferably, it has the resistance of height equally to BJ-R01.Illustrate that the present invention has certain broad spectrum to the resistance of the different strains of TuMV.
The high resistance transgenic line is reserved seed for planting, and the offspring proceeds disease-resistant evaluation.Result shows that these transgenic lines can genetic stability to the high resistance of TuMV.
(3) sxemiquantitative and relative quantification detect viral accumulation
5 high resistance strain # 07, #12, #22, #34, #48, contrast Col-0 and turn empty carrier contrast inoculation TuMV-C4 after approximately 20 days, choose each strain and do not inoculate blade and extract respectively total RNA by the TRIzol method, be inverted to cDNA with M-MLV.The 196bp conservative fragments of TuMV coat protein gene of take is detected object, and the 298bp fragment of Arabidopis thaliana SAND gene (AT2G28390) of take is carried out sxemiquantitative and quantitative fluorescence analysis as internal reference.
The primer sequence of the conservative section of 196bp of amplification TuMV-CP gene is as follows:
CP F 5’-AAAGCGTAACCAAGACCGACCAT-3'；
CPR 5’-TCCATCCAAGCCGAACAAAT-3'。
The primer sequence of amplification reference gene SAND gene 298bp fragment is as follows:
SAND F 5'-AATGTGGATGGTGACACTGCCTCG-3；
SAND R 5'-ACCTGGTGATTTCCTGCCTTGAC-3'。
RT-PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 57 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 7min.The PCR product carries out electrophoresis detection on 1.2% sepharose.
It is template that the cDNA that dilutes 40 times is take in the reaction of quantitative fluorescent PCR, and SYBR-green I makees fluorescent indicator, and response procedures adopts the two-step approach amplification.Amplification condition is: 95 ℃ of 30s; 95 ℃ of 5s, 57 ℃ of 30s, 72 ℃ of 30s, 40 circulations.Each strain sample carries out repeated experiments 3 times, averages.
Half-quantitative detection the results are shown in Figure 10.Figure 10 result shows, in the situation that the reference gene expression amount is similar consistent, a large amount of virus accumulations can be detected in contrast Col-0 plant body, turn empty carrier results of comparison and Col-0 basically identical, and micro-virus can only be detected in high resistance transgenic line body.
The detected result of quantitative fluorescent PCR is shown in Figure 11.The demonstration of Figure 11 result, consistent with semiquantitative result, a large amount of virus detected in contrast Col-0 body, turn empty carrier results of comparison and Col-0 basically identical, and almost can't detect copying of virus in 5 high resistance strain plant materialss.Illustrate and utilize the RNAi carrier of CI gene fragment structure to bring into play vital role in transgenic plant are antiviral, the disease resistance of transfer-gen plant significantly improves.
Claims (8)
1. a RNA has one of following nucleotide sequence:
1) nucleotide sequence shown in the SEQ ID № .2 in sequence table;
2) with 1) the RNA sequence that limits has 90% above homology, and has the RNA sequence of identical function; Concrete, described homology is more than 95%; Concrete is more than 96% again; Concrete is more than 97% again; Concrete is more than 98% again; Concrete is more than 99% again.
2. the encoding gene of RNA claimed in claim 1.
3. the recombinant vectors, expression cassette, transgenic cell line or the Host Strains that contain encoding gene claimed in claim 2; Described recombinant vectors is specially recombinant expression vector or recombinant cloning vector.
4. recombinant expression vector according to claim 3, is characterized in that: in the multiple clone site of described recombinant expression vector for carrier that the DNA fragmentation insertion is set out, obtain; Described DNA fragmentation is that X forward-joining region-X is reverse; The X forward is in SEQ ID No.1 shown in the Nucleotide of 15-380 position, and X is reversed the reverse complemental fragment of X forward.
5. recombinant expression vector according to claim 4, it is characterized in that: described X forward-joining region-reverse fragment of X has the SEQ ID described 1365-2919 of № .3 position nucleotide sequence in sequence table.
6. following at least one application of RNA claimed in claim 1, encoding gene claimed in claim 2, the arbitrary described recombinant vectors of claim 3-5, expression cassette, transgenic cell line or Host Strains:
1) prepare the product of anti-TuMV;
2) strengthen the resistance of plant to TuMV;
3) strengthen the resistance of plant to careless ammonium phosphine weedicide.
7. a method of cultivating transgenic plant, be that the described recombinant expression vector of claim 4 or 5 is proceeded to the purpose plant, obtains transgenic plant; Described transgenic plant are compared with described purpose plant, have following at least one proterties: 1) resistance of TuMV strengthened; 2) resistance of careless ammonium phosphine weedicide strengthened.
8. method according to claim 7, it is characterized in that: described purpose plant is specially Arabidopis thaliana or crop in cruciferae; Described crop in cruciferae is specially rape or Chinese cabbage.
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| CN102559666A (en) * | 2010-12-18 | 2012-07-11 | 中国科学院上海生命科学研究院 | Plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof |
| CN102796739A (en) * | 2012-08-16 | 2012-11-28 | 北京农业生物技术研究中心 | Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant |
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| CN102559666A (en) * | 2010-12-18 | 2012-07-11 | 中国科学院上海生命科学研究院 | Plant virus inhibitory artificial miRNA (microRNA) and construction and application thereof |
| CN102796739A (en) * | 2012-08-16 | 2012-11-28 | 北京农业生物技术研究中心 | Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant |
Non-Patent Citations (3)
| Title |
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| HQ446217.2: "Turnip mosaic virus strain BJ-C4, complete genome", 《GENBANK》 * |
| 于占东 等: "TuMV-Nib反义基因对大白菜的遗传转化研究", 《园艺学报》 * |
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