CN102443602A - Construction method of RNA interference carriers for interfering in a plurality of genes of silencing rice black-streaked dwaf disease - Google Patents
Construction method of RNA interference carriers for interfering in a plurality of genes of silencing rice black-streaked dwaf disease Download PDFInfo
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Abstract
The invention relates to a construction method of RNA interference carriers for interfering in a plurality of genes of silencing rice black-streaked dwaf disease, which belongs to the molecular genetics field. The method is characterized in that rice black-streaked dwaf disease encoding gene can be fused to a segment by an overlapping PCR method, enzyme sites are added at the junction segment, and respectively connected to the upstream and the downstream of intron, excessive promoter expression is used for constructing the interference carriers for interfering in a plurality of genes of silencing rice black-streaked dwaf disease.
Description
Technical field
The invention provides rice black-streaked dwarf virus polygene interferential construction process, belong to the molecular genetics field, be exclusively used in the transgenic breeding of the anti-black streak dwarf of paddy rice.
Background technology
Black streaked dwarf virus of rice (Rice black-streaked dwaf disease) be a kind of be the virus disease that main amboceptor is propagated with small brown rice planthopper, mainly infect cereal crop and gramineous weeds.The diseased plant symptom is dark green stunting, and blade is stiff, do not ear or fringe little.The billet strumae is arranged on the vein of blade back and the cane, and this projection is cured white at the initial stage of a disease, after become chocolate.Morbidity field piece general underproduction 10%-40%, grave illness field even No kernels or seeds are gathered, as in a year of scarcity.Once the many areas of all provinces and cities extensively take place this disease in China East China in middle 1960s, and onset area will descend year by year in year after next in this, but in recent years, along with the great outburst of small brown rice planthopper, this disease is gone up popular again rapidly, and some areas, Jiangsu harm in recent years is serious.
Existing research confirm the black streaked dwarf virus of rice cause of disease be rice black-streaked dwarf virus (rice black-streaked dwaf virus, RBSDV).This virus belongs to Reoviridae (Reoviridae) Fijivirus and belongs to (Fijivirus), and genome sequencing work is accomplished, length overall 29141bp, and genome is made up of 10 double-stranded RNAs, presses molecular weight size called after S1-S10; Critical function genes such as the RNA polymerase that RNA relies on, coat protein, reticent inhibition are clear and definite.
This sick epidemiological study shows that rice black-streaked dwarf virus can not be delivered to the next generation by ovum, and RBSDV must could be with poison and transmitted virus by small brown rice planthopper behind the host plant food poison of band poison.Small brown rice planthopper worm amount radix was big in recent years owing to the Yangze river and Huai river basin, and weeds in field, gramineous crop is the host of RBSDV like corn, therefore passes through the food poison of small brown rice planthopper and passes poison, this disease maybe be in the big generation in paddy rice production area, Yangze river and Huai river basin.
Breeding for disease resistance is the effective means of preventing and treating black streaked dwarf virus of rice, and disease-resistant resource is the prerequisite of carrying out the paddy disease-resistant breeding.The inoculation authenticate technology system of black streaked dwarf virus of rice is not also set up fully at present, and disease-resistant resource evaluation work just starts to walk, and mainly relies on the field spontaneous induction to identify that breeding for disease resistance still belongs to blank.Therefore screen the disease-resistant resource of new black streaked dwarf virus of rice and create the new resources of anti-black streaked dwarf virus of rice by genetic engineering means significant for control RBSDV.
RNA disturbs a kind of fundamental mechanism of being not only the biological gene expression regulation, and RNA interference simultaneously also is a kind of defense mechanism of animal and plant disease resisting poison.The a certain sequences Design of virus is become can be transcribed into the double-stranded RNA structure import plant materials and make it to express, can bring out the RNA silence, make plant pass through RNA silencing virus gene and obtain anti-virus ability.But some study the phenomenon of finding that also the reticent disease-resistant efficient of RNA is not high even not reaching disease resisting effect at all.
Existing research all mainly is that the rna virus cdna group sequence to strand justice is that target site designs siRNA,, the gene of selection mainly is rdrp gene, coat protein gene.The reticent subbase that suppresses finds to disturb the reticent subbase that suppresses because of the better effects if than other gene because of after finding.Utilize the miRNA method in identical carrier, to design the approach of interfering virus of AIDS is had the good restraining effect to the target site of a plurality of genes.Also do not utilize at present the reticent mechanism of RNA to study the research report of the disease resistance of diplornavirus.
Utilization of the present invention utilizes overlapping PCR that a plurality of virogene encode fragments are connected; Make up hairpin structure; The diplornavirus RBSDV complicated to genome carries out gene silencing; And be that target site carries out silence with a plurality of virogenes, in the hope of obtaining the new resources of the reticent anti-black streaked dwarf virus of rice of polygene, improve disease-resistant efficient.
Summary of the invention
Technical problem
The objective of the invention is: utilize overlapping PCR that a plurality of virogene encode fragments are connected; Make up hairpin structure; The diplornavirus RBSDV complicated to genome carries out gene silencing; And be that target site carries out silence with a plurality of virogenes, be mainly used in the efficient molecular breeding of transgenic of black streaked dwarf virus of rice.
Technical scheme
The reticent rna interference vector construction process of black streaked dwarf virus of rice polygene is characterized in that:
Be used to clone the primer of 4 gene fragments of rice black-streaked dwarf virus and overlapping PCR:
Fragment S1, the RNA polymerase (RdRp) that RNA relies on:
S1-For：5’GGTGGAACGAAAGTTCAGTAGATC?3’
S1-Rev：5’
GGTGCTTCAGGCAAAAAGTTGTCAGAATTTGGACTACACTTGGACGAA?3’
Fragment S2, core protein gene (CP1):
S2-For：5’
CTGACAACTTTTTGCCTGAAGCACCAGAGCGACAAGAAGAATCGAAA3’
S2-Rev：5’
TGGGATCAGACGAAAATATTGGACGCAAAAGTAGTTGTGTAAGCGGG?3’
Fragment S6, the reticent subbase that suppresses is because of (SS):
S6-For：5’
CGTCCAATATTTTCGTCTGATCCCACTCGAATCATCCGTCACTTCTGAGT?3’
S6-Rev：5’
TGCGTTTGTTGACCATTACCATGAAGGACAAAACCTTTCCAATTATCGAG?3’
Fragment S10, coat protein gene (CP2):
S10-For：5’
TTCATGGTAATGGTCAACAAACGCAGGAAACATTACTTTGAAGCCC?3’
S10-Rev：5’CCACCATAATGTGTAACATCCGTA?3’
Wherein, add identical segments at 5 of downstream primer S1-Rev and upstream primer S2-For ' end
GGTGCTTCAGGCAAAAAGTTGTCAG, be used to carry out overlapping PCR, fragment S1 is connected with S2; In like manner, add identical segments at 5 of downstream primer S2-Rev and upstream primer S6--For ' end
TGGGATCAGACGAAAATATTGGACGBe used to carry out overlapping PCR, fragment S2 is connected with S6; Add identical segments at 5 of downstream primer S6-Rev and upstream primer S10--For ' end
TGCGTTTGTTGACCATTACCATGAA, be used to carry out overlapping PCR, fragment S6 is connected with S10.
The cDNA of amplifying rice black-streaked dwarf virus, can obtain S1, S2, S6 and and S10 totally 4 fragments, size is respectively 235bp, 340bp, 317bp, 277bp; Utilizing primer S1-For and primer S2-Rev then, is that template is carried out PCR with fragment S1 and fragment S2, and fragment S1 is connected with fragment S2, obtains the 605bp fragment; Utilizing primer S6-For and primer S10-Rev, is that template is carried out PCR with fragment S6-For and S10-Rev, and S6 is connected with S10, obtains the 574bp fragment; With primer S1-For and primer S10-Rev, be that template is carried out PCR acquisition 1179bp fragment at last with 605bp fragment and 574bp.
Utilize this virus poly-gene to merge fragment and can be used for follow-up rna interference vector structure.
Beneficial effect
The black streaked dwarf virus of rice polygene interference constructing method that this institute provides has following advantage:
Obtained and to have carried out interferential rnai expression carrier construction method to a plurality of genes of rice black-streaked dwarf virus through the present invention.
Description of drawings
Fig. 1 rice black-streaked dwarf virus S1, S2, S6, S10 fragment PCR amplified production are in 1% agarose gel electrophoresis result; (M is DNA ladder, and a is 100bp, and b is 300bp, and c is 500bp, and d is 700bp, and e is 1kb, and f is 1.5kb, and g is 2kb; 1,2,3,4 is the RT-PCR product of RBSDV S1, S2, S3, S4)
Fig. 2 S1 and S2, (M is DNAladder to the PCR product that S6 and S10 merge, and a is 100bp, and b is 300bp, and c is 500bp, and d is 700bp, and e is 1.0kb, and f is 1.5kb, and g is 2.0kb in 1% agarose gel electrophoresis result; The 1st, the overlapping PCR product of S6 and S10, the 2nd, the overlapping PCR product of S1 and S2)
The overlapping PCR product of Fig. 3 S1, S2, S6 and S10 is in 1% agarose gel electrophoresis result.(M is DNA ladder, and a is 100bp, and b is 300bp, and c is 500bp, and d is 700bp, and e is 1.0kb, and f is 1.5kb, and g is 2.0kb; 1 is 4 overlapping PCR products of fragment)
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
(1) the black streaked dwarf virus of rice polygene merges segmental construction process
1, total RNA extraction of RBSDV and cDNA are synthetic
Used glass, porcelain, ironware all adopt xeothermic (180 ℃) more than the sterilization 4h; Join reagent etc. and all use the 0.1%DEPC treating water.Experimental procedure is following:
The rice leaf tissue adding liquid nitrogen that 50-100mg infects black streaked dwarf virus of rice fully grinds; Treat to add 1ml Trizol before the sample freeze thawing, sample volume can not surpass 10% of Trizol, continues to grind until dissolving fully; Suck centrifuge tube; The centrifugal 10min of 1200g (4 ℃) gets supernatant, 15-30 ℃ of 5min; Add the 0.2ml chloroform (19: 1 chloroform: solution primary isoamyl alcohol), firmly shake 15s, 15-30 ℃ of 2-3min, the centrifugal 15min of 2-8 ℃＜1200g, upper phase is about 60% of TV; Get supernatant, add the 0.5ml Virahol, mixing, 15-30 ℃ of 10min, the centrifugal 10min of 2-8 ℃＜1200g; Remove supernatant, add 75% ethanol 1ml (preparation of 0.1%DEPC water), vibration, 2-8 ℃＜7500g 5min; Remove ethanol, dry air is with the water dissolution of 0.1%DEPC processing, in-70 ℃ of freezing preservations.
Carry out reverse transcription reaction by following reaction system and condition:
Reverse transcription reaction system (25 μ l) is: earlier with total RNA 1 μ l (2 μ g), 0ligo dT (15) primer 2 .5 μ l and pure water mix, behind 70 ℃ of sex change 5min, and rapid cooled on ice; And then add other composition successively.Comprise 5 * buffer, 2.5 μ l, dNTP (10mmol/L) 2.5 μ l, RNasin 0.5 μ l, reversed transcriptive enzyme (AMV) 1 μ l, pure water 12.5 μ l.42 ℃, 1h; 95 ℃, heating 5min termination reaction.
2, the clone of many Gene Partial coding regions of RBSDV, order-checking and overlapping PCR connect
Genome sequence design primer according to the RBSDV that has checked order.Respectively the RNA polymerase that relies on of the RNA of the synthetic RBSDV of design (RNA dependent RNA polymerase, RdRP), 4 pairs of primers of core protein (Core protein), reticent suppressor gene (Putative silencing suppressor), coat protein (Coat protein) encoding sox.4 fragments genome positions, place and the clip size that is used for the black streak dwarf virus of this research RT-PCR sees the following form 1, and (details please be landed: http://www.iah.bbsrc.ac.uk/dsRNA virus proteins/Fijivirus.htm).Add 20 identical bases at the downstream primer that designs with another upstream primer to primer, can be through overlapping PCR respectively with S1 and S2, S2 and S6, S6 is connected with S10, and at last with S1, S2, S6 and S10 connect into a fragment, send company's order-checking to confirm.
Be used to clone the primer of 4 gene fragments and overlapping PCR:
The RNA polymerase (RdRp) that RNA relies on:
S1-For：5’GGTGGAACGAAAGTTCAGTAGATC?3’
S1-Rev：5’
GGTGCTTCAGGCAAAAAGTTGTCAGAATTTGGACTACACTTGGACGAA?3’
Core protein gene (CP1):
S2-For：5’
CTGACAACTTTTTGCCTGAAGCACCAGAGCGACAAGAAGAATCGAAA3’
S2-Rev：5’
TGGGATCAGACGAAAATATTGGACGCAAAAGTAGTTGTGTAAGCGGG?3’
The reticent son (SS) that suppresses:
S6-For：5’
CGTCCAATATTTTCGTCTGATCCCACTCGAATCATCCGTCACTTCTGAGT?3’
S6-Rev：5’
TGCGTTTGTTGACCATTACCATGAAGGACAAAACCTTTCCAATTATCGAG?3’
Coat protein (CP2):
S10-For：5’
TTCATGGTAATGGTCAACAAACGCAGGAAACATTACTTTGAAGCCC?3’
S10-Rev：5’CCACCATAATGTGTAACATCCGTA?3’
Rna polymerase gene (S1), core protein gene (S2), silence that table 1 is used to make up the RNA dependence of disturbing RBSDV suppress subbase because of (S6) and coat protein gene (S10) place genomic information
Primer is to S1-For and S1-Rev (S1), S2-For and S2-Rev (S2), and S6-For and S6-Rev (S6), S10-For and S10-Rev (S10) amplify 4 target fragment respectively, utilize test kit to reclaim then, see Fig. 1.Respectively getting 1 μ L S1 and S2 is masterplate, is primer PCR with S1-For and S2-Rev, and respectively getting 1 μ L S6 and S10 is masterplate, is that primer carries out PCR with S6-For and S10-Rev, obtains target fragment such as Fig. 2.The PCR product is reclaimed, carry out PCR with these two PCR products for touching version then, S1-For and S10-Rev are primer, obtain the fragment (Fig. 3) of 1179bp.The fragment that connects is connected with the T carrier.Utilize universal primer M13 order-checking.
(2) make up the reverse RNA of repetition of expression polygene and disturbed the double base transgene expression vector
The hairpin structure of RBSDV Gene Partial coding region makes up and binary expression vector makes up
Intermediate carrier pHurricane carrier has Arabidopis thaliana FAD2 gene 1.1kb intron, and there are restriction enzyme site BamHI and NotI in the intron upper reaches, and there are restriction enzyme site XhoI, EcoRI and KpnI in downstream.At the enzyme-added site BamHI that cuts of fusion fragment S1-For that step 1 makes up, the enzyme-added site NotI that cuts of downstream primer S10-Rev is connected to the upper reaches of intron; The enzyme-added site XhoI that cuts of S1-For, the enzyme-added site EcoRI that cuts of downstream primer S10-Rev is connected to the downstream of intron, and so reverse repeating structure makes up successfully.The reverse repetition plasmid that builds is utilized BamHI and KpnI double digestion and recovery.
Be used to make up the primer of reverse repeating structure:
iS1-For：5’GATGGATCCGGTGGAACGAAAGTTCAGTAGATC3’(BamHI)
iS10-Rev：5’GTAGCGGCCGCCCACCATAATGTGTAACATCCGTA?3’(NotI)
iS10-For：5’GAT?CTCGAGCCACCATAATGTGTAACATCCGTA?3’(XhoI)
iS1-Rev：5’GATGAATTCGGTGGAACGAAAGTTCAGTAGATC3’(EcoRI)
Have multienzyme at the back at binary expression vector pVec8Ubi promotor Ubi and cut site BamHI and KpnI and Nos terminator; This carrier is utilized BamHI and KpnI double digestion; The big fragment that reclaims is connected with above-mentioned reverse repeated fragment, promptly is built into the binary vector of a plurality of virogenes of interference of overexpression.Utilize BamHI and KpnI double digestion binary expression vector, can obtain size and be the target fragment of 3.2kb.
The nucleotide sequence of goal gene and deduced amino acid thereof;
RNA disturbs hairpin structure total length 3375bp, no protein expressioning product.
Forward sequence and reverse sequence are 1123bp
ggtggaacgaaagttcagtagatctgcagaaatttgcttaaagaaatttgaatcaatccaacaatttagacaaaagagacaacaacaagaagcaagcacagataaatcacattctcaaaacgaaaaaactaaatcacccggcccccgtacttccgacagtaaaaaagattcttcaacacgacaaaatgaaaattcgtccaagtgtagtccaaattctgacaactttttgcctgaagcaccagagcgacaagaagaatcgaaagatgaaaagaagaaaaatcaatccgaaacagaacaaacgtcatcacccgaccccaaacttataaacgaccaagaaggtataactaatgactcaagacccgaagaaattacttatcagagtggtaacacaattcaagctaaaacagcattaactgttgaagcgataatgaatattcaaggacatctcaaacaatcagaccagaaattagttgatgaacaaaccagtcaatatcttacttatctaacttcaaatgttcccgcttacacaactacttttgcgtccaatattttcgtctgatcccactcgaatcatccgtcacttctgagtcttctaattcgaaaaaacgctacgcacgtccatctaacaaagcttctcctcaacctaactcttccgaatcatctaatgagactaaactgcttcacaatttgatttcttctgcttcaaattcctccgtggtgatgaaacctaattcagacgtactcaacctgtctaaaactcaaactatccttccttctttagttgtgaatgccacttgccagttacgaatgaaaaatctcgataattggaaaggttttgtccttcatggtaatggtcaacaaacgcaggaaacattactttgaagcccttttccgctgatggtttagaagtcattctcgatgatagttatattggtatagttggtaaaattccaggtttagaagttcataaattgttagataaatgttgtcgtgaagttcctgctcaaatgggaatacttactgatgaagttaaacttttgatgcgtactggtaaattaagaattgatggtggttacgattttaattgtcctgcaagcactacggatgttacac?attatggtgg
Claims (1)
1.RNA disturb the carrier construction method of a plurality of genes of reticent rice black-streaked dwarf virus; It is characterized in that: utilize overlapping PCR method the encode fragment of a plurality of genes of a black-streaked dwarf virus fragment that permeates; Be built into direction multiple rna interference vector; The a plurality of genes of virus are disturbed, to reach disease-resistant purpose.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103276006A (en) * | 2013-05-28 | 2013-09-04 | 扬州大学 | Anti-BSDV (Black-Streaked Dwarf Virus) carrier and anti-BSDV transgenic rice cultivation |
| CN105229021A (en) * | 2013-03-15 | 2016-01-06 | 加利福尼亚大学董事会 | The RNA interference of antiviral immunity effect is played in Mammals |
| WO2019015223A1 (en) * | 2017-07-20 | 2019-01-24 | 中国检验检疫科学研究院 | Agent against plant virus |
| CN111172308A (en) * | 2019-12-06 | 2020-05-19 | 扬州大学 | Detection method of S6RNAi gene-transferred black-streaked dwarf resistant rice strain WLJ1-US6-11-5 |
| CN111690766A (en) * | 2020-07-14 | 2020-09-22 | 扬州大学 | Detection method of black-streaked dwarf resistant rice line WLJ1-US6-10-5 |
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| CN101967491A (en) * | 2010-11-01 | 2011-02-09 | 中国农业科学院植物保护研究所 | Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application |
| CN101974550A (en) * | 2010-11-01 | 2011-02-16 | 中国农业科学院植物保护研究所 | RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as construction method and application thereof |
| CN102220374A (en) * | 2011-05-15 | 2011-10-19 | 扬州大学 | Method for cultivating black streaked dwarf-resistant rice by using RNAi (ribonucleic acid interference) technology |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101967491A (en) * | 2010-11-01 | 2011-02-09 | 中国农业科学院植物保护研究所 | Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application |
| CN101974550A (en) * | 2010-11-01 | 2011-02-16 | 中国农业科学院植物保护研究所 | RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as construction method and application thereof |
| CN102220374A (en) * | 2011-05-15 | 2011-10-19 | 扬州大学 | Method for cultivating black streaked dwarf-resistant rice by using RNAi (ribonucleic acid interference) technology |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105229021A (en) * | 2013-03-15 | 2016-01-06 | 加利福尼亚大学董事会 | The RNA interference of antiviral immunity effect is played in Mammals |
| CN103276006A (en) * | 2013-05-28 | 2013-09-04 | 扬州大学 | Anti-BSDV (Black-Streaked Dwarf Virus) carrier and anti-BSDV transgenic rice cultivation |
| CN103276006B (en) * | 2013-05-28 | 2014-12-10 | 扬州大学 | Anti-BSDV (Black-Streaked Dwarf Virus) carrier and anti-BSDV transgenic rice cultivation |
| WO2019015223A1 (en) * | 2017-07-20 | 2019-01-24 | 中国检验检疫科学研究院 | Agent against plant virus |
| CN109275662A (en) * | 2017-07-20 | 2019-01-29 | 中国检验检疫科学研究院 | A kind of medicament of Antiphytoviral |
| CN111172308A (en) * | 2019-12-06 | 2020-05-19 | 扬州大学 | Detection method of S6RNAi gene-transferred black-streaked dwarf resistant rice strain WLJ1-US6-11-5 |
| CN111690766A (en) * | 2020-07-14 | 2020-09-22 | 扬州大学 | Detection method of black-streaked dwarf resistant rice line WLJ1-US6-10-5 |
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Application publication date: 20120509 |