CN101967491B - Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application - Google Patents
Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application Download PDFInfo
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Abstract
The invention relates to rice black-streaked dwarf virus (RBSDV) RNA interference vectors, a construction method and application, and belongs to the technical field of biology. RBSDV RNA interference vectors, namely pMCG161+/-B and pCMBIA1301+/-B, are constructed by using a sequence shown in SEQ ID NO:3 as positive-sense and antisense strands. The invention provides a virus gene interference expression vector and a construction method and application thereof for plant disease-resistant genetic engineering, and provides a novel way of thinking for application of transgenic technology and RNA interference in biological resistance breeding. By transferring the vector into rice, antiviral plants are successfully obtained, the expected goal for obtaining new anti-RBSDV materials of rice is achieved by using an RNA interference principle, successful examples for RNA interference-mediated plant resistance breeding and genetic engineering are provided and a gap of RNA interference-mediated RBSDV resistance in the research of the field is filled.
Description
Technical field
The invention belongs to gene engineering technology field, particularly relate to a kind of suitable monocotyledons genetic transformation, rice black-streaked dwarf virus RNA interference vector, construction process and use in paddy rice.
Background technology
Paddy rice is one of most important food crop in the world, accounts for 40% of grain-production and consumption in China paddy rice, and it occupies very important effect in grain security and national economy.Since two thousand, in Jiangsu, the rice district such as Zhejiang, Fujian, Jiangxi, by rice black-streaked dwarf virus (Rice black streaked dwarf virus, RBSDV) the rice black streak dwarf silence that causes for many years after eruption and prevalence once again, the rice field morbidity of Prevalent district about 60%, general field strain sickness rate 10～30%, serious field can be up to more than 40～80%.2004,800,000 mu of the middle and south, Zhejiang Province hybridisation rice plantation district onset areas, only the Taizhou plain onset area just reached 480,000 mu, accounts for 33% of rice growing area, more than 20,000 tons of lost units.2006～2007 years on the rice varieties such as regional No. 6, magnificent round-grained rice, the Huaihe River rice No. 5 such as Tongcheng, Jiangsu, Yancheng, Taizhou, II excellent 084, diseased plant rate is generally at 20-30%, serious about 80%, the grave illness field diseased plant rate that has even up to more than 90%, expand at present the ground such as Hunan, Shandong, therefore should disease become the another important virus disease that threatens China's Rice Production.This virus can also infect staple food crop corn and the wheat of China, and the maize rough dwarf virus that is caused by rice black-streaked dwarf virus (RBSDV) is the important virus disease that threatens Maize Production.
The epidemiology result of study shows that this disease is broken out, popular major cause is the forfeiture of varietal resistance and effectively passes the rising of virus mediator population quantity.Because passing instantaneity and persistence, the virus disease control medicament of poison, small brown rice planthopper lacks, so that pest control treatment is very difficult, also there are many disadvantages in the use of agricultural chemicals, and therefore the most cost-effective approach is to utilize the resistance of kind self to reach Initiative defence virus disease purpose.Yet traditional breeding method is owing to lack desirable anti-source (the domestic germ plasm resource of not yet finding anti-black-streaked dwarf virus), and the difficulty of dual nature kind that obtains disease-resistant, high yield and high quality is very large.
RNA interferes (RNA interference, RNAi) be the reticent phenomenon of a kind of important gene of discovered in recent years, it refers to the mRNA by the corresponding sequence of specific degraded of the mediation of double-stranded RNA, thereby suppressing specifically the expression of corresponding gene, is that plant is to external invader's a kind of important defense mechanism.The present invention take in recent years at the serious black-streaked dwarf virus (RBSDV) that occurs of China as object, utilize the transgenic plant genetic engineering technology with the part Nucleotide Introduced into Rice of RBSDV S10, disturb specifically, degrade or normal replication, accumulation and the propagation of reticent RBSDV virogene in rice plant by it, screening obtains the rice strain of anti-RBSDV, for further combined with conventional breeding work, improve existing good quality and high output but not disease-resistant rice varieties, so that realization utilizes the purpose of the rice new strain of transgenic technology adaptive immune or high resistance RBSDV.
Summary of the invention
Based on above-mentioned purpose, the invention provides and can be used for the monocotyledonous 2 kinds of RNA that contain rice black-streaked dwarf virus (RBSDV) gene of Agrobacterium and particle gun genetic transformation and interfere (RNAi) carriers, these carriers comprise respectively the S10 Partial Fragment (RBSDV-HS10) of Causative virus RBSDV.
The present invention also provides these to interfere viral gene expression Vector construction method and application, for the Resistant breeding engineering provides a kind of new strategy and thinking.
Rice black-streaked dwarf virus RNA interference vector comprises skeleton carrier and contains the hairpin structure of positive and negative adopted chain, it is characterized in that: take the S10 Partial Fragment of RBSDV as positive and negative adopted chain, and the sequence of the S10 Partial Fragment of described RBSDV such as SEQ ID NO3.
Described skeleton carrier is the pMCG161 carrier, and described positive-sense strand is inserted between restriction enzyme site AscI and AvrII, and antisense strand is inserted between restriction enzyme site SpeI and the SacI, called after pMCG161+/-B, its structure is as shown in Figure 6A.
Described skeleton carrier is the pCMBIA1301 carrier that removes hygromycin gene, described hairpin structure be interference vector pMCG161+/-B cuts through BamHI, HindIII enzyme and obtains, called after pCMBIA1301+/-B, its structure is shown in Fig. 6 B.
PMCG161+/-construction process of B interference vector, step is as follows:
(1) obtains the S10 Partial Fragment of the RBSDV shown in sequence SEQ ID NO3; (2) with behind the Gene Partial sheet cracked ends AscI of step (1), the AvrII double digestion, be connected with the carrier pMCG161 that cuts through AscI, AvrII enzyme equally, obtain inserting the recombinant plasmid pMCG161+B of forward purpose fragment; With Gene Partial sheet cracked ends SpeI, the SacI double digestion of step (1), be connected with the carrier pMCG161+B that cuts through SpeI, SacI enzyme again, can obtain inserting the RNA interference vector pMCG161+ of positive antisense strand/-B.
Described step (1) is in the following way: to infect rice black-streaked dwarf virus RBSDV rice total RNA as template, take sequence shown in SEQ ID NO1 and the SEQ ID NO2 as primer pair, obtains through the RT-PCR amplification,
SEQ ID NO1：5’-AG
ACTAGT GGCGCGCCTAATGGCTGACATAAGACTC-3’，
SEQ ID NO2：5’-AG
GAGCTC CCTAGGGTTGCGTGATGTTGGGTAAAG-3’。
PCMBIA1301+/-construction process of B interference vector, step is as follows: (1) cuts the pCMBIA1301 carrier with restriction enzyme XhoI enzyme, obtains removing the pCMBIA1301 carrier of hpt marker gene, i.e. pCMBIA1301-hpt
-(2) with pMCG161+/-B cuts with BamHI, HindIII enzyme, the hairpin structure that obtains is connected into pCMBIA1301-hpt
-, obtain the RNA interference vector pCMBIA1301+ of marker-free/-B.
The application of described interference vector in transgenic plant.
Described being applied as transforms relevant recipient plant with the above-mentioned interference carrier, makes it rice black-streaked dwarf virus is produced resistance.
Described interference vector be pMCG161+/-B, the method for described conversion is particle bombardment.
Described interference vector be pCMBIA1301+/-B, the method for described conversion is agrobacterium-mediated transformation.
Described Agrobacterium is agrobacterium tumefaciens bacterial strain EHA105.
Described recipient plant is grass.
Described grass is paddy rice.
Described rice varieties is for liking to know the rising sun (Oryza sativa cv.Aichiasahi) or Anhui round-grained rice 97 (Wanjing 97).
It is template that the present invention adopts infection rice black-streaked dwarf virus (RBSDV) rice total RNA, according to the S10 gene design of very conservative RBSDV primer SEQ ID NO1 and SEQ ID NO2, it is RBSDV-HS10 that RT-PCR amplification obtains RBSDV-S10 Gene Partial fragment, it is inserted in the skeleton carrier as positive-sense strand and antisense strand obtain interference vector of the present invention.Interference vector of the present invention is changed in the recipient plant, will make RBSDV occur reticent and do not express, thereby make this receptor plant produce the RSV virus resistance.
RNA interference vector pMCG161+/-B, be suitable for the via Particle Bombardment Transformation monocotyledons, can obtain the transgenic progeny of anti-RBSDV, this carrier carries chloramphenicol resistance gene and herbicide screening marker gene.The skeleton carrier that adopts is two-way expression vector pMCG161, the positive-sense strand of RBSDV-HS10 is inserted between restriction enzyme site AscI and AvrII, antisense strand is inserted between restriction enzyme site SpeI and the SacI, form hairpin structure by the intro on the carrier (rice Waxy-a intron 1), can be used for like this via Particle Bombardment Transformation monocotyledons.
The present invention will cut the pCMBIA1301 carrier with restriction enzyme Xho I enzyme, obtain removing the pCMBIA1301 carrier of hpt marker gene, i.e. pCMBIA1301-hpt
-Then with pMCG161+/-the B interference vector cuts with BamHI, HindIII enzyme, the hairpin structure that obtains is connected into pCMBIA1301-hpt
-, obtain the RNA interference vector pCMBIA1301+ of marker-free/-B.Because this interference vector not only is suitable for super virulent strain, and does not contain antibiotic resistance gene, be conducive to the in the future safe release of transgenic plant.
The final RBSDV marker-free RNA interference vector pCMBIA1301+ that makes up/-B do not carry microbiotic or herbicide resistance gene, is fit to agriculture bacillus mediated monocotyledons genetic transformation, can obtain the transgenic progeny of anti-RBSDV; Carry out monocotyledonous genetic transformation contrast with the blank carrier of the pCMBIA1301 of the resistant gene that carries Totomycin and kalamycin resistance through Agrobacterium EHA105 mediation, can make things convenient for like this screening of resistant calli, can separate by the offspring again, conveniently obtain the transgenic regenerated plant of marker-free, reduce the biological safety hidden danger that transgenic plant bring.
The plant genetic transformation method of agrobacterium tumefaciens mediation is one of method for transformation that is most widely used at present, in the successful transgenic research that is applied to single, double cotyledon plant.The final interference vector pCMBIA1301+ that makes up of the present invention/-the agriculture bacillus mediated Genetic Transformation in Higher Plants such as the suitable super virulent strain EHA105 of B.Utilize agrobacterium tumefaciens EHA105 with its Introduced into Rice, resulting transfer-gen plant can make the Causative virus of invasion that the reticent phenomenon of RNA occurs, and makes the irreproducible or accumulation of virus, thereby makes plant possess disease resistance to virus.
The present invention utilizes the method for Agrobacterium tumefaciens mediated conversion to carry out the genetic transformation of paddy rice, Transcription by rice cell RdRp, produce the S10 gene of RBSDV, effect by DicerIII produces SiRNA again, when paddy rice is infected by RBSDV, just can produce SiRNA, utilize mediated rnai to lead the resistance to RBSDV thereby reach, finishing screen is selected the transgenic rice plant of immunity or high resistance RBSDV.
The present invention obtained take paddy rice pattern kind like to know the rising sun as the paddy rice T0 of acceptor for regeneration plant, transform RBSDV carrier pCMBIA1301+/-plant of B is 122 strains, method by artificial inoculation has been carried out Resistance screening in the greenhouse, 7 strains reveal any symptoms (0 grade) not wherein, 23 strains performance light symptoms (I level), 54 strains show medium symptom (II level), 38 strain serious symptoms (III).Detect through PCR, all amplification is not to goal gene in 7 strains of reveal any symptoms, and having in 23 strains of performance light symptoms only has 8 strains that positive band is arranged, acceptor is liked to know the rising sun 28 strains simultaneously, and except 1 strain was not fallen ill, all the other morbidities were serious, all be II or III level, diseased plant rate is 96%., this explanation transgenic rice plant has obvious disease resistance than acceptor.
The present invention provides a kind of interference virogene expression vector and construction process and application for genetic engineering of plant for disease resistance, and the application in the biotic resistance breeding provides new thinking for transgenic technology and RNA interference.This carrier is changed in the paddy rice, successfully obtained disease-resistant plant, realized utilizing the RNA interference principle to obtain the re-set target of anti-RSV Rice New Material, plant resistance to environment stress breeding and the genetically engineered led for mediated rnai provide successful example, have remedied the vacancy of RBSDV resistance in the research of this area that mediated rnai is led.
The resistance organism that utilizes interference carrier of the present invention to transform gained can be any microorganism, plant or its tissue, cell, and the microorganism, the plant that obtain to have thus any anti-disease activity, and such plant offspring's seed, hybridization and transformation offspring.
The construction process of the RNA interference vector of transforming monocots of the present invention comprises the steps:
1) acquisition of RBSDV specificity nucleotide sequence (RBSDV-HS10)
To infect rice black-streaked dwarf virus (RBSDV) rice total RNA as template, take SEQ ID NO.1 (upstream primer) and SEQ ID NO.2 (downstream primer) as primer pair, through RT-PCR amplification RBSDV-HS10, obtain the PCR product, the purpose fragment is 290bp.Reclaim the PCR product, be connected with the pMD18-T carrier, transform intestinal bacteria DH 5 α (available from the full formula in Beijing King Company) and obtain positive plasmid pMD18-T-HS10, carry out the sequence checking, the correct purpose fragment that checks order namely can be used for the structure of RNA interference vector.The sequence of described SEQ ID NO1 and SEQ ID NO2 is as follows:
SpeI AscI
SEQ ID NO 1：5’-AG
ACTAGT GGCGCGCCTAATGGCTGACATAAGACTC-3’
SacI AvrII
SEQ ID NO2：5’-AG
GAGCTC CCTAGGGTTGCGTGATGTTGGGTAAAG-3’
2) structure of viral RNA interference vector (intermediate carrier)
The PCR product of above-mentioned recovery behind AscI, AvrII double digestion, is connected with the carrier pMCG161 that cuts through AscI, AvrII enzyme equally, obtains inserting the recombinant plasmid-pMCG161+B of forward purpose fragment.Equally with above-mentioned PCR product through SpeI, SacI double digestion, be connected with the carrier pMCG161+B that cuts through SpeI, SacI enzyme, can obtain inserting the RNA interference vector pMCG161+ of positive and negative adopted chain/-B, through pcr amplification detection, nucleotide sequence analysis, the correct interference vector of purpose fragment sequence can be used for the via Particle Bombardment Transformation monocotyledons, obtains the transfer-gen plant of monoclonal antibody RBSDV.These interference vectors carry chloramphenicol resistance gene and herbicide screening marker gene.
Because the pMCG161 carrier is with chloramphenicol resistance gene, and Agrobacterium EHA105 chloramphenicol resistance, therefore the interference vector of the suitable via Particle Bombardment Transformation paddy rice of above-mentioned structure can not be directly used in agriculture bacillus mediated rice transformation.And the pCAMBIA1301 carrier is the common carrier of agrobacterium co-cultivation rice transformation, has hygromycin resistance and kalamycin resistance.
3) marker-free carrier pCMBIA1301-hpt
-Acquisition
Cut the pCMBIA1301 carrier with restriction enzyme XhoI enzyme, after electrophoresis detection, recovery, purifying large fragment, spend the night through 16 ℃ of connections of T4DNA ligase enzyme again, can obtain removing the pCMBIA1301 carrier of hpt marker gene, i.e. pCMBIA1301-hpt
-
4) structure of unmarked vector gene viral RNA interference vector
With carrier pMCG161+/-B and pCMBIA1301-hpt
-Cut with BamHI, HindIII enzyme, reclaim large fragment, (16 ℃ of connections are spent the night) is connected into pCMBIA1301-hpt with the hairpin structure on the intermediate carrier under the effect of T4DNA ligase enzyme
-, obtain the RNA interference vector pCMBIA1301+ of marker-free/-B.Namely can be used for agriculture bacillus mediated monocotyledons genetic transformation through the interference vector that PCR detects, sequential analysis is correct, acquisition does not contain the transgenic regenerated plant of the monoclonal antibody RBSDV of marker gene.
Description of drawings
The RT-PCR amplified production of the S10 Partial Fragment (HS10) of Fig. 1 .RBSDV;
Swimming lane 1.DL2000,2. healthy water rice plants, 3-5.RBSDV isolate
Fig. 2. be connected into the PCR qualification result of viral positive-sense strand recombinant plasmid pMCG161+B
Fig. 3. be connected into positive and negative adopted chain recombinant plasmid pMCG161+/-qualification result of B
Fig. 4. marker-free carrier pCMBIA1301-hpt
-The PCR detected result
Swimming lane 1.DL2000,2. blank, 3-15.pCMBIA1301-hpt
-
Fig. 5. recombinant plasmid pCMBIA1301+/-the PCR qualification result of B
Swimming lane 1.DL2000,2. blank, 3. blank plasmid pCMBIA1301,4-11. recombinant plasmid
Fig. 6 A.pMCG161+/-B interference vector collection of illustrative plates
Fig. 6 B pCMBIA1301+/-B interference vector collection of illustrative plates
The PCR qualification result of Fig. 7 .RBSDV-HS10 restructuring Agrobacterium
Swimming lane 1.DL2000,2. blank, 3.DH5 α, 4-10. recombinant plasmid
Fig. 8. the inducing culture of Mature Embryos of Rice callus
Fig. 9. the succeeding transfer culture of Mature Embryos of Rice callus
Figure 10. the common cultivation of callus and Agrobacterium
Figure 11. the screening of resistant calli
Figure 12. the differentiation of resistant calli
Figure 13 breaks up the strong seedling culture of seedling
Figure 14 .T0 transgenic paddy rice seedling
Figure 15 .T0 identifies for the PCR of transgenic regenerated plant
Swimming lane 1.DL2000,2. healthy water rice plants, T0 is for plant for the 3-12. transgenosis
Figure 16 T0 likes to know rising sun plant for transgenosis and the disease resistance that impinges upon the land for growing field crops is showed
A. transfer-gen plant, B. non-transgenic plant
Embodiment
PMD18-T used in the experiment is available from the precious biotech firm (Takara company) in Dalian, and competent cell DH 5 α are available from the full formula in Beijing King Company.
The carrier pMCG161 that uses in the experiment is kept at Plant Protection institute, Chinese Academy of Agricultral Sciences plant virus laboratory (can to public's Free distribution); to be specially adapted for the rna interference vector that grass transforms; it contains the Rice waxy-a intron 1 that derives from paddy rice; be positioned at 5442bp-6576bp; contain two restriction enzyme sites of AscI and AvrII in its upstream; be used for connecting the forward fragment of goal gene; contain SpeI and SacI site in its downstream; can be used to connect the reverse fragment of goal gene, finally obtain double-stranded hairpin structure.
Carrier pCMBIA1301 is the common carrier (this carrier is kept in the Plant Protection institute, Chinese Academy of Agricultral Sciences virus group laboratory, can to public's Free distribution) of agrobacterium-mediated transformation rice transformation, has hygromycin resistance and kalamycin resistance.
The pCMBIA1301-hpt that transforms through this laboratory
-Also can provide to the public, by single endonuclease digestion site BamHI and HindIII can with intermediate carrier pMCG161+/-hairpin structure of goal gene on the B inserts wherein, obtain marker-free, and be fit to the RNA interference vector of agriculture bacillus mediated monocotyledons heredity.
The structure of the rice black-streaked dwarf virus rna interference vector of embodiment 1, suitable agrobacterium mediation converted
The RNA of step 1, design RBSDV S10 fragment interferes primer
According to the sequence (NCBI No.AJ297433) of the RBSDV S10 gene of having announced, and there is the multiple clone site analysis on the pMCG161 carrier, utilizes AscI and AvrII the forward fragment of goal gene can be connected to carrier; And utilize SpeI and SacI the reverse fragment of S10 part nucleotide fragments can be connected to carrier.Therefore designed following interference primer:
SpeI AscI
SEQ ID NO1：5’-AG
ACTAGT GGCGCGCCTAATGGCTGACATAAGACTC-3’
SacI AvrII
SEQ ID NO2：5’AG
GAGCTC CCTAGGGTTGCGTGATGTTGGGTAAAG-3’
The acquisition of step 2:RBSDV S10 Partial Fragment specificity nucleotide sequence
To gather infection rice black-streaked dwarf virus (RBSDV) rice total RNA from Jining of Shandong Province as template, take SEQ ID NO1 (upstream primer) and SEQ ID NO2 (downstream primer) as primer pair, obtain the band (Fig. 1) of about 287bpd through the RT-PCR amplification, reclaim the PCR product, be connected with the pMD18-T carrier, transform intestinal bacteria DH 5 α, obtain positive plasmid pMD18-T-HS10, carry out the sequence checking, the sequencing results shows, the purpose fragment is the part nucleotide fragments of RBSDV S10, and the part nucleotide fragments of the RB SDV S10 that the present invention obtains is described.
The PCR reaction conditions: 94 ℃ of 4min, (94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 1min) 30 circulations, 72 ℃ are extended 10min.The PCR product is electrophoresis detection on 1.0% sepharose.
With above-mentioned PCR product and carrier pMCG161 respectively behind AscI, AvrII double digestion, process 20min for 65 ℃, make the restriction endonuclease inactivation, under the T4DNALigase effect, connect, with forward purpose fragment insertion vector pMCG161, transform intestinal bacteria DH 5 α, identify that through PCR (Fig. 2), the recombinant plasmid that sequential analysis is correct are exactly the recombinant plasmid that contains the forward fragment of RBSDV-HS10, called after pMCG161+B.
Equally with above-mentioned PCR product through SpeI, SacI double digestion, be connected under T4DNA Ligase effect with the carrier pMCG161+B that cuts through SpeI, SacI enzyme, can obtain inserting the RNA interference vector pMCG161+ of the forward and reverse fragment of RBSDV-HS10/-B, the PCR detected result is seen Fig. 3, and the carrier collection of illustrative plates is seen Fig. 6-A.This interference vector can be used for the via Particle Bombardment Transformation monocotyledons, obtains the transfer-gen plant of anti-RBSDV, also can be used as the intermediate carrier that makes up unmarked RNA interference vector.This interference vector carries chloramphenicol resistance gene and herbicide screening marker gene.
Cut the pCMBIA1301 carrier with restriction enzyme Xho I enzyme, behind the recovery of large fragment post, purifying, spend the night through 16 ℃ of connections of T4 dna ligase again, can obtain removing the pCMBIA1301 carrier of hpt marker gene, i.e. pCMBIA1301-hpt
-The purpose fragment that detects the carrier amplification of hpt marker gene by PCR is 189bp (Fig. 4), and the purpose fragment that contains to fall the carrier amplification of hpt marker gene is about 1200bp.
So far the present invention has obtained 2 kinds of RNA interference vectors that suitable unifacial leaf transforms, and wherein a kind of intermediate carrier of genetic transformation of suitable particle gun is another kind of for being fit to the final carrier of agrobacterium-mediated transformation conversion.In 2 kinds of constructed interference vectors, the forward fragment is had an appointment as the paddy rice intron fragment of 1100bp with oppositely sheet is intersegmental, is conducive to keep the formation of hairpin structure.The final a kind of RNA interference vector that makes up do not contain anti-herbicide gene, reduced the hidden danger that there is biological safety in transgenic plant in the future.
Because recombinant plasmid pCMBIA1301+/-B is very low to the efficient that Agrobacterium EHA105 carries out the thermal shock conversion, and the present invention has taked the method for electric shock conversion, has obtained to contain the restructuring Agrobacterium of goal gene.Concrete steps are as follows:
The PCR of step 5, restructuring Agrobacterium identifies
Bacterium colony PCR qualification result (seeing Fig. 7) shows, positive transformant and vector plasmid are increase respectively band about about 290bp of the positive control of template; Negative control take the EHA105 bacterium liquid of unconverted sky as template does not obtain the purpose fragment.
The application of the rna interference vector of embodiment 3RBSDV-HS10.
Relevant recipient plant-the Ai of restructuring Agrobacterium-mediated Transformation that uses the above-mentioned RBSDV of containing purpose fragment hairpin structure knows rising sun rice varieties, makes it to produce the resistance (Fig. 8-14) to the rice black-streaked dwarf virus virus disease.
The present invention obtained take paddy rice pattern kind like to know the rising sun as the paddy rice T0 of acceptor for regeneration plant, transform RBSDV carrier pCMBIA1301+/-plant of B is 122 strains (Figure 16).
The PCR of embodiment 4, transfer-gen plant (T0 generation) identifies
The present invention is take SEQ ID NO1/SEQ ID NO2 as primer pair, to knowing that take love the rising sun carries out PCR as reuse water rice plants T0 generation 122 strains that turn the RBSDV-HS10 interference vector of acceptor and detects, the result shows: 7 strains of reveal any symptoms are not all increased and are arrived goal gene, the purpose band (Figure 15) that only has 8 strains amplification to be about 290bp is arranged in 23 strains of performance light symptoms, positive rate is 12.3%, and the illustration purpose gene integration has advanced the genome of paddy rice.
The present invention is by the method for artificial inoculation, in the greenhouse to obtaining 122 strain transgenosis T0 for the regeneration plant Resistance screening, inoculate after 40 days, 7 strains are reveal any symptoms (0 grade) not, 23 strains performance light symptoms (I level), 54 strains show medium symptom (II level), 38 strain serious symptoms (III).Inoculate simultaneously acceptor 28 strains, except 1 strain is 0 grade, 2 strains are outside the I level, and all the other are all fallen ill seriously, all are II or III level, and diseased plant rate is 96%, and this explanation transgenic rice plant has obvious disease resistance (Figure 16) than acceptor.
Although the present invention only adopted pCMBIA1301+/-B transformed paddy rice, those of ordinary skill in the art can learn according to conventional knowledge, adopt particle bombardment equally can with pMCG161+/-B transforms other acceptor plant, obtains identical effect.
Claims (10)
1. rice black-streaked dwarf virus RNA interference vector, comprise skeleton carrier and the hairpin structure that contains positive and negative adopted chain, described skeleton carrier is the pCMBIA1301 carrier that removes hygromycin gene, described hairpin structure be RNA interference vector pMCG161+/-B cuts and obtains through BamHI, HindIII enzyme; Described RNA interference vector pMCG161+/-structure of B is: skeleton carrier is the pMCG161 carrier, take the S10 Partial Fragment of RBSDV as positive and negative adopted chain, the sequence of the S10 Partial Fragment of described RBSDV such as SEQ ID NO3, described positive-sense strand is inserted between restriction enzyme site AscI and AvrII, and antisense strand is inserted between restriction enzyme site SpeI and the SacI.
2. the construction process of rice black-streaked dwarf virus RNA interference vector claimed in claim 1, step is as follows: (1) cuts the pCMBIA1301 carrier with restriction enzyme Xho I enzyme, obtain removing the pCMBIA1301 carrier of hpt marker gene, i.e. pCMBIA1301-hpt
-(2) with RNA interference vector pMCG161+/-B cuts with BamHI, HindIII enzyme, the hairpin structure that obtains is connected into pCMBIA1301-hpt
-, obtain the RNA interference vector pCMBIA1301+ of marker-free/-B;
Described RNA interference vector pMCG161+/-construction process of B is:
1) obtains the S10 Partial Fragment of the RBSDV shown in sequence SEQ ID NO3;
2) with behind the Gene Partial sheet cracked ends AscI of step 1), the AvrII double digestion, be connected with the carrier pMCG161 that cuts through AscI, AvrII enzyme equally, obtain inserting the recombinant plasmid pMCG161+B of forward purpose fragment; With Gene Partial sheet cracked ends SpeI, the SacI double digestion of step 1), be connected with the carrier pMCG161+B that cuts through SpeI, SacI enzyme again, can obtain inserting the RNA interference vector pMCG161+ of positive antisense strand/-B.
3. method according to claim 2, described step 1) be in the following way: to infect rice black-streaked dwarf virus RBSDV rice total RNA as template, take sequence shown in SEQ ID NO1 and the SEQ ID NO2 as primer pair, obtain through the RT-PCR amplification,
SEQ ID NO1：5’-AGACTAGT GGCGCGCCTAATGGCTGACATAAGACTC-3’，
SEQ ID NO2：5’-AGGAGCTC CCTAGGGTTGCGTGATGTTGGGTAAAG-3’。
4. the application of rice black-streaked dwarf virus RNA interference vector claimed in claim 1 in the preparation transgenic plant.
5. application according to claim 4 for rice black-streaked dwarf virus RNA interference vector claimed in claim 1 is transformed relevant recipient plant, makes it rice black-streaked dwarf virus is produced resistance.
6. application according to claim 5, described interference vector are rice black-streaked dwarf virus RNA interference vector claimed in claim 1, and the method for described conversion is agrobacterium-mediated transformation.
7. application according to claim 6, described Agrobacterium are agrobacterium tumefaciens bacterial strain EHA105.
8. application according to claim 5, described recipient plant are grass.
9. application according to claim 8, described grass are paddy rice.
10. application according to claim 9, described rice varieties is for liking to know the rising sun or Anhui round-grained rice 97.
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| CN102443602A (en) * | 2011-10-27 | 2012-05-09 | 江苏省农业科学院 | Construction method of RNA interference carriers for interfering in a plurality of genes of silencing rice black-streaked dwaf disease |
| CN103255166B (en) * | 2013-05-20 | 2014-12-10 | 中国农业科学院植物保护研究所 | Ultra-binary vector as well as construction method and application thereof |
| CN109504704B (en) * | 2018-12-07 | 2022-07-12 | 华南农业大学 | Method for enhancing resistance of monocotyledon against RNA virus infection |
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| CN101713000A (en) * | 2009-12-09 | 2010-05-26 | 湖南农业大学 | RT-PCR detection method of ordinary rice black streaked dwarf virus (RBSDV) |
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Non-Patent Citations (2)
| Title |
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| 代玉华;王锡锋;.一种抗水稻条纹病毒RNAi载体的构建.《中国植物病理学会2006年学术年会论文集》.2006,182-183. * |
| 王迅;江蓓蓓;李晓颖;陈妮;公娇芬;竺晓平.RBSDV-S10基因和SCMV-CP基因双价植物表达载体的构建.《山东农业科学》.2010,2. * |
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