CN108342409A - A kind of plant RNA i expression vectors and its construction method and application - Google Patents

Abstract

The invention belongs to gene engineering technology fields, and in particular to a kind of plant RNA i expression vectors and its construction method and application.The present invention is based on commercialized plant expression vector pCambia2301 and pHANNIBIAL, the plant RNA i expression vectors containing the opposite Gateway boxes of CaMV35S promoters, introne, OCS terminators, both direction are constructed, which can carry out the structure of plant target gene silent carrier using Gateway technologies；The present invention has also set up the construction method of the plant RNA i expression vectors, and carries out an application of the step Gateway reaction structures containing target gene RNAi silent carriers using fusion DNA vaccine.

Description

A kind of plant RNA i expression vectors and its construction method and application

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of plant RNA i expression vectors and its construction method and Using.

Background technology

Plant mutant is difficult to obtain, and the mutation of some genes may be lethal to plant, and gene silencing is relatively easy It obtains, it is a kind of posttranscriptional gene silencing technology that RNA, which interferes (RNA interference, RNAi), is a kind of effective research The reverse genetics tool of functional gene.RNAi is the posttranscriptional gene participated in by certain enzyme mediated by double-stranded RNA (dsRNA) Silencing phenomenon, the phenomenon are that Andrew Fire and Craig Mello etc. has found in nematode first, are that one kind is given birth in eukaryon Very conservative and generally existing mechanism in object, it is by by there are homologous complementary sequences with the transcription product mRNA of target gene RNA (dsRNA) with partially double stranded structure import cell after, be then degraded into mRNA homologous therewith is efficiently special The small fragment of 21~23nt promotes intracellular specific gene to lack, and finally reaches the purpose for preventing gene expression.RNAi genes are heavy In silent experiment, double-stranded RNA often exists in the form of hairpin RNA.

Conventional construction plant RNA i expression vectors, the method routinely used are digestion and connection, to target gene fragment into Row cuts and recycles, and is limited by the point of contact of restriction enzyme, has not only wasted time process but also cumbersome, and small fragment is difficult back It receives, multiple segments are not easy to connect, and the molecular weight of plasmid vector is all bigger, with the success of the method carrier construction of digestion and connection Rate is not high.

Gateway technologies are Invitrogen commercialized technologies, it is the locus specificity weight based on bacteriophage Group reaction adds recombination site in target fragment, and PCR product is reacted with the donor vehicle mixing progress BP containing recombination site and is obtained Entry vector (Entry clone) is obtained, entry vector can carry out LR reactions with the destination carrier of different purposes and obtain accordingly Expression vector, that is to say, that it obtains expression vector using the technology and usually requires to react two steps by BP reactions and LR, it is real Test that the period is longer, and cost is higher.

Invention content

The purpose of the present invention is to overcome the deficiency in the prior art, provides a kind of plant RNA i expression vectors and its construction method And application.

To achieve the above object, the present invention uses following technical scheme：

The present invention provides a kind of plant RNA i expression vectors, and the plant RNA i expression vectors contain target gene fragment expression member Part, it is opposite that the target gene fragment Expression element contains CaMV35S promoters, introne, OCS terminators, both direction Gateway boxes；The introne is located between the opposite Gateway boxes of both direction, for expressing in hairpin RNA structure Ring；The Gateway boxes contain the sites attR1, the sites attR2,CmRGene,ccdBGene and oneEcoRI digestions position Point, Gateway boxes constitute the arm in hairpin RNA structure for expressing the opposite target gene fragment in direction.

Further, the Gateway boxes areEcoV digestion pBS-Gateway-rfA carriers of R obtain, describedEcoRⅠ Restriction enzyme site distance attR1 site 453bp, distance attR2 site 12s 57bp.

Further, the target gene fragment Expression element is obtained by intermediate carrier of pHANNIBAL carriers；The plant Object RNAi expression vector is inserted into pCambia2301 using pCambia2301 carriers as skeleton, by target gene fragment Expression element and is carried It is obtained in the multiple cloning sites of body.

Further, the nucleotide sequence of the plant RNA i expression vectors is as shown in SEQ ID NO.1.

The present invention also provides the construction methods of above-mentioned plant RNA i expression vectors, include the following steps：

Step 1：It uses respectivelyEcoI Hes of RHinIII single endonuclease digestion pHANNIBAL carriers of d purify the product after digestion, recycle high protect True Taq enzyme filling-in point of contact, obtains E segments and H segments；

Step 2：WithEcoV digestion pBS-Gateway-rfA carriers of R, recycling obtain Gateway boxes, the Gateway boxes Containing the sites attR1, the sites attR2,CmRGene,ccdBGene and oneEcoRI restriction enzyme sites；

Step 3：E segments described in step 1 and H segments are connect with Gateway boxes described in step 2 respectively, then through screening Obtain pHANNIBAL-GW1.1 carriers and pHANNIBAL-GW1.2 carriers；The Gateway of the pHANNIBAL-GW1.1 carriers The sites attR1 in box are connect with CaMV35S promoters, and the sites attR2 are connect with introne；The pHANNIBAL- The sites attR1 in the Gateway boxes of GW1.2 carriers are connect with OCS terminators, and the sites attR2 are connect with introne；

Step 4：WithKpnI HeXhoI pair of pHANNIBAL-GW1.1 carrier and pHANNIBAL-GW1.2 carriers carry out double digestion, will Contain from what pHANNIBAL-GW1.1 carriers were cut outKpnI HeXhoThe Gateway boxes of I cohesive end, withKpnI HeXhoⅠ Segment connection after double digestion pHANNIBAL-GW1.2 carriers, obtains the recombination containing the opposite Gateway boxes of both direction Carrier pHANNIBAL-GW2；

Step 5：WithSacI HeSpeI double digestion pHANNIBAL-GW2 carriers, recycling obtain target gene fragment Expression element, so Afterwards withSacI HeXbaSegment connection after I double digestion pCambia2301 carriers, obtains pCambia2301-GW-RNAi loads Body, as plant RNA i expression vectors,SpeI HeXbaI is isocaudarner, can be attached between the cohesive end cut out.

Further, screening described in step 3 includes the following steps：It is selected first containing E segments and Gateway boxes Recombinant vector, the recombinant vector containing H segments and Gateway boxes, then to the recombination containing E segments and Gateway boxes Carrier is usedEcoI Hes of RKpnI digestion detects, and containing 1300bp or so size segments is pHANNIBAL-GW1.1 in electrophoresis result Carrier；Recombinant vector containing H segments and Gateway boxes I single endonuclease digestions of EcoR are detected, contain 2000bp in electrophoresis result Left and right size segment is pHANNIBAL-GW1.2 carriers.

The present invention also provides the host cells containing above-mentioned plant RNA i expression vectors.

The present invention also provides application of the above-mentioned plant RNA i expression vectors in building target gene RNAi silent carriers.

Further, application of the plant RNA i expression vectors in building target gene RNAi silent carriers, including Following steps：

Step 1：Using the DNA sequence dna containing target gene CDS as template, PCR amplification is carried out using forward primer and reverse primer Joint sequence, nucleotide sequence difference are contained in target gene fragment, the ends 5' of the forward primer and the reverse primer As shown in SEQ ID NO.2 and SEQ ID NO.3；

Step 2：Using any entry vector as template, the DNA fragmentation in the sites attL1 is contained using L1-F and L1-R as primer amplification, Contain the DNA fragmentation in the sites attL2, the nucleotide sequence of L1-F, L1-R, L2-F and L2-R using L2-F and L2-R as primer amplification As shown in NO.4~7 SEQ ID；

Step 3：By the DNA fragmentation containing the sites attL1 of target gene fragment and step 2 gained obtained by step 1, contain The DNA fragmentation in the sites attL2 mixes, and carries out fusion DNA vaccine, obtains the target gene fragment containing attL recombination sites；

Step 4：By the target gene fragment containing attL recombination sites and the plant RNA i expression vectors obtained by step 3 into Row LR reactions, obtain target gene RNAi silent carriers.

Compared with prior art, the beneficial effects of the present invention are：

1, the present invention provides the improved plant RNA i expression vectors of a Gateway box, and a step is carried out using the empty carrier LR reacts the structure that target gene RNAi silent carriers can be completed：By the target gene fragment containing attL recombination sites and this The plant RNA i expression vectors of invention carry out a step LR reactions, you can obtain target gene RNAi silent carriers.

2, the present invention is using pCambia2301 carriers as framework construction plant RNA i expression vectors, pCambia2301 carrier sheets Body carries the Expression element of gus gene, using target gene RNAi silent carriers convert plant after can with GUS dyeing come Identify transfer-gen plant.

3, joint sequence is contained at the ends 5' of forward primer and reverse primer used in amplifying target genes segment in the present invention, For 22bp, the joint sequence than 29 bases of conventional Gateway reactions is slightly short.This method can greatly shorten experimental period, Save experimental cost half or so.

4, the present invention obtains the target gene fragment containing the sites attL using fusion DNA vaccine, and then directly carries out Gateway LR react without being reacted by BP, simplify the construction step of target gene RNAi silent carriers.

Description of the drawings

Fig. 1 is that fusion DNA vaccine obtains the target gene fragment schematic diagram containing attL recombination sites.

Fig. 2 is pHANNIBAL carrier schematic diagrames.

Fig. 3 is the electrophoresis detection that pHANNIBAL carriers use after EcoR I and the recycling of III single endonuclease digestions of Hind respectively in embodiment 1 Figure, M Marker, swimming lane 1 representEcoThe E segments obtained after I single endonuclease digestion pHANNIBAL carriers of R, swimming lane 2 representHinD III is single The H segments obtained after digestion pHANNIBAL carriers.

Fig. 4 is GW boxes both ends restriction enzyme site schematic diagram in pBS-Gateway-rfA carriers.

Fig. 5 is pHANNIBAL-GW1.1 carrier schematic diagrames in embodiment 1.

Fig. 6 is pHANNIBAL-GW1.2 carrier schematic diagrames in embodiment 1.

Fig. 7 is the electrophoresis detection figure of pHANNIBAL-GW1.1 and pHANNIBAL-GW1.2 carriers in embodiment 1, and M is Marker, swimming lane 1~4 are correct pHANNIBAL-GW1.1 carriers, and swimming lane 5~6 carries for correct pHANNIBAL-GW1.2 Body, 7~9 be the recombinant plasmid 1.2 being reversely inserted into.

Fig. 8 is pHANNIBAL-GW2 carrier schematic diagrames in embodiment 1.

Fig. 9 is the restriction enzyme digestion and electrophoresis figure of pHANNIBAL-GW2 carriers in embodiment 1, and M Marker, swimming lane 1~8 is PHANNIBAL-GW2 carriers.

Figure 10 is pCambia2301-GW-RNAi carrier schematic diagrames in embodiment 1.

Figure 11 is the restriction enzyme digestion and electrophoresis figure of pCambia2301-GW-RNAi carriers in embodiment 1, M Marker.

Figure 12 be in embodiment 2 fusion DNA vaccine obtain containing attL recombination sitesCEBiPGenetic fragment electrophoresis detection Figure, M Marker, swimming lane 1~2 is containing attL recombination sitesCEBiPGene fragment amplification product.

Protein expression accumulating level detection figure when Figure 13 is different bacterial concentrations in embodiment 2,0.7 represents OD₆₀₀=0.7 Contain 35ss-CEBiPThe Agrobacterium resuspended bacterium solution of -3HA plasmids, 0.35 represents OD₆₀₀=0.35 contains 35ss-CEBiP-3HA The Agrobacterium resuspended bacterium solution of plasmid.

Figure 14 is in embodiment 2CEBiPGene silencing vector pCambia2301-CEBiP- RNAi and over-express vector 35ss-CEBiPProtein accumulation level detection figure when -3HA converts this life Tobacco Leaves；α-HA indicate that the antibody of HA, Rubisco indicate Internal reference albumen ,-indicate unconverted ,+indicate conversion.

Specific implementation mode

Following embodiment is not used to limit protection scope of the present invention for illustrating the present invention.Unless otherwise specified, real Apply the conventional means that technological means used in example is well known to those skilled in the art.

The structure of 1 plant RNA i expression vectors pCambia2301-GW-RNAi of embodiment

PCambia2301 carriers, pHANNIBAL carriers, pBS-Gateway-rfA carriers are commercial carrier.

The structure of plant RNA i expression vectors pCambia2301-GW-RNAi comprises the steps of：

Step 1：Single endonuclease digestion pHANNIBAL carriers

PHANNIBAL carriers are the intermediate carriers for being commonly used to structure RNAi expression vector, as shown in Figure 2.Traditional method be by The segment of 200~300bp long of target gene is inserted into more grams of introne both sides in pHANNIBAL carriers in a reverse direction Grand site, is then usedNotI scales off from CaMV35S promoters to the segment of OCS terminators, connects with plant expression vector It connects, is built into final RNAi expression vector.The present invention builds plant RNA i expression using pHANNIBAL carriers as intermediate carrier Carrier.It uses respectively firstEcoI Hes of RHinIII single endonuclease digestion pHANNIBAL carriers of d,EcoI Hes of RHinIII single endonuclease digestion pHANNIBAL of d Cohesive end outstanding is formed after carrier, for convenience connection reaction, high-fidelity Taq will be utilized after the product purification after single endonuclease digestion Enzyme respectively obtains E segments and H segments to point of contact filling-in, and recycling rear electrophoresis detection is as shown in Figure 3.

Step 2：Digestion pBS-Gateway-rfA carriers obtain Gateway boxes

PBS-Gateway-rfA carriers contain Gateway boxes, the Gateway boxes contain the sites attR1, attR2 Point,CmRGene,ccdBGene and oneEcoI restriction enzyme sites of R,EcoI restriction enzyme site distance attR1 site 453bp of R, distance AttR2 site 12 57bp, the position against the sites attR1 and attR2 haveEcoRV point of contact, as shown in figure 4, withEcoV enzymes of R PBS-Gateway-rfA carriers are cut, electrophoresis recycles the segment of about 1.7kb sizes.

Step 3：Product is connect with Gateway boxes after pHANNIBAL carrier single endonuclease digestion filling-in

PBS-Gateway-rfA carriers pass throughEcoThe segment that R V is recycled after cutting is flat end, can be with the E segments and H in step 1 Segment connects.Using T4DNA ligases, E segments and H segments are connect with Gateway boxes respectively.Contain in Gateway boxes HaveccdBVirus gene, for the gene outcome to Bacillus coli communis cell-lethal, the carrier containing the gene can be only present in DB3.1 In bacterial strain, in addition Gateway boxes contain the resistant gene of chloramphenicol, can assign host strain chlorampenicol resistant, simultaneously PHANNIBAL carriers contain ampicillin resistance gene, assign host strain amicillin resistance.Therefore by connection product Escherichia coli DB3.1 competent cells are converted respectively, and the bacterium solution after conversion is respectively coated containing ampicillin and chloramphenicol LB tablets on, by tablet be inverted 37 DEG C culture 12 hours with up to monoclonal is grown, picking monoclonal is containing ammonia benzyl mould The LB liquid medium overnight incubation of element extracts plasmid, is labeled as recombinant plasmid 1.1 and recombinant plasmid 1.2.

The monoclonal grown on the culture medium containing chlorampenicol resistant theoretically contains Gateway boxes, but flat It is indefinite that end connects direction, it is therefore desirable to filter out the carrier with correct connection direction.PHANNIBAL-GW1.1 carriers Gateway boxes correctly connect direction：The sites attR1 are connect with CaMV35S promoters, and the sites attR2 are connect with introne, As shown in Figure 5；Gateway boxes in pHANNIBAL-GW1.2 carriers correctly connect direction：The sites attR1 are terminated with OCS Son connection, the sites attR2 are connect with introne, as shown in Figure 6.There are one containing in Gateway boxesEcoThe digestion position of R I Point, the restriction enzyme site is away from the ends attR1 453bp, away from the ends attR2 1257bp, the other CaMV35S on pHANNIBAL carriers Have between promoter and introneKpnⅠ、EcoI Hes of RXhoI point of contact, so withEcoI Hes of RKpnI digestion recombinant plasmid 1.1, cuts It is pHANNIBAL-GW1.1 carriers to go out about 1300bp segments, and it is reversed insertion to cut out about 500bp, is used recombinant plasmid 1.2EcoI single endonuclease digestions of R, it is pHANNIBAL-GW1.2 carriers to cut out about 2000bp segments, and it is reversed insertion to cut out about 1300bp, As shown in Figure 7.

Step 4：It is connected after pHANNIBAL-GW1.1 carriers and pHANNIBAL-GW1.2 carrier double digestions

WithKpnI HeXhoI carries out double digestion to pHANNIBAL-GW1.1 carriers and pHANNIBAL-GW1.2 carriers respectively, from PHANNIBAL-GW1.1 carriers cut out containingKpnI HeXhoThe Gateway boxes of I cohesive end, withKpnI HeXhoI pair of enzyme It cuts the connection of the segment after pHANNIBAL-GW1.2 carriers and can be obtained the centre containing the opposite Gateway boxes of both direction Carrier.Obtained intermediate carrier is converted into Escherichia coli DB3.1 competent cells, the bacterium solution after conversion is coated on green containing ammonia benzyl On the LB tablets of mycin and chloramphenicol, the monoclonal grown was cultivated in the LB liquid medium containing ampicillin Night, extraction plasmid, as pHANNIBAL-GW2, as shown in Figure 8.

There are one above-mentioned pHANNIBAL-GW2 carriers containKpnI point of contact and twoEcoI point of contacts R, with Kpn I and EcoR I into Row double digestion obtains three segments, respectively may be about 6000bp, 2000bp and 1300bp, as shown in Figure 9.

Step 5：The structure of pCambia2301-GW-RNAi carriers

PHANNIBAL-GW2 carriers are usedSacI HeSpeThe segment at 6300bp is recycled in I digestion, which is target gene Fragment expression element contains the opposite Gateway boxes of CaMV35S promoters, both direction, an introne, an OCS Terminator, the introne are located between the opposite Gateway boxes of both direction.With I digestion of Sac I and Xba Then pCambia2301 carriers are connect with the target gene fragment Expression element of recycling,SpeI HeXbaI is isocaudarner, is obtained Cohesive end can be attached.Then connection product is converted into Escherichia coli DB3.1, be coated on mould containing kanamycins and chlorine On the LB tablets of the dual antibiotic of element, 37 DEG C of overnight incubations choose single bacterium colony and shake bacterium（37℃250rpm10h）, plasmid is extracted, i.e., For pCambia2301-GW-RNAi carriers.

There are three pCambia2301-GW-RNAi carriersEcoThe point of contact of R I is usedEcoI digestion carriers of R cut out three bands, About 12800bp, 3300bp and 1800bp, as shown in FIG. 10 and 11.

2 barley of embodimentCEBiPThe structure of the RNAi silent carriers of gene and detection

1. vegetable material and condition of culture：

Vegetable material：This life cigarette；Condition of culture：Hot-house culture；16h illumination (150 μm of ol/ (m²S)), 21 DEG C of temperature； 8h Dark, 19 DEG C of temperature；Relative humidity 55%.

2. experimental vehicle：

PCambia2301 carriers, pHANNIBAL carriers, pBS-Gateway-rfA carriers, pDONR207 carriers are commercialization Carrier.It, will using Gateway technologiesCEBiPGenetic fragment is integrated on donor vehicle pDONR207 and obtains entry vector pENTY-CEBiP；Then by entry vector pENTY-CEBiPLR is carried out with support C TAPi-35ss-GW-3HA, and load is obtained by the reaction Body 35ss-CEBiP- 3HA, carrier 35ss-CEBiP- 3HA expresses CEBiP and 3HA tag fusions, is convenient for late detection, wherein HA is antigenic tag, and for one section of amino acid sequence from influenza virus, the 3HA indicates 3 HA antigenic tag tandem sequence repeats, 35ss indicates two 35S series connection.

3. barleyCEBiPThe structure of RNAi gene RNAi silent carriers：

With over-express vector 35ss-CEBiP- 3HA is template amplification barleyCEBiPGenetic fragment, forward primer sequence are 5 '- ACTTTGTACAAAAAAGCAGGCTCCAAAGACCCTCAAGAAGGA-3 ', as shown in SEQ ID NO.8, reverse primer sequences It is 5 '-ACTTTGTACAAGAAAGCTGGGTAGCCGTTGGAATAACCACTG ' as shown in SEQ ID NO.9.

The DNA fragmentation for containing attL1 using L1-F and L1-R as primer amplification, is contained using L2-F and L2-R as primer amplification The DNA fragmentation of attL2, template are entry vector pENTY-CEBiP.PCR reactions use KAPA HiFi PCR Kits (Kapa Biosystems), PCR reaction systems（50 μL）It is prepared according to kit specification, often 0.5 μ L templates of pipe, it is forward primer, anti- To each 2.5 μ L of primer.PCR reaction conditions：95 DEG C of pre-degeneration 3min；98 DEG C of denaturation 20s, 60 DEG C of 20 s of annealing, 72 DEG C extend 30s, 30 cycles；72 DEG C of 5 min of extension, 4 DEG C of preservations.Amplified fragments are single band through electrophoresis detection, are then purified spare.

The barley expanded aboveCEBiPGenetic fragment, the DNA fragmentation containing attL1 and the DNA pieces containing attL2 Section respectively takes 1 μ L, as shown in Figure 1, containing the sites attL using fusion DNA vaccine acquisitionCEBiPGenetic fragment, electrophoresis detection result is such as Shown in Figure 12, amplified fragments are single band, and purifying is spare.Fusion DNA vaccine reaction uses KAPA HiFi PCR Kits (Kapa Biosystems), PCR reaction systems（50μL）It is prepared according to kit specification, barleyCEBiPGenetic fragment contains attL1 DNA fragmentation and DNA fragmentation containing attL2 respectively take 1 μ L, be first not added with primer.PCR reaction conditions：95 DEG C of 3 min of pre-degeneration； 98 DEG C of 20 s of denaturation, 60 DEG C of 20 s of annealing, 72 DEG C of extension 30s, 5 recycle；72 DEG C of 5 min of extension；PCR instrument is opened, is being reacted Each 2.5 μ L of primer L1-F and L2-R are added in system and carry out PCR programs, 95 DEG C of 3 min of pre-degeneration；98 DEG C denaturation 20 s, 60 DEG C Anneal 20 s, 72 DEG C of extension 30s, 25 cycles；72 DEG C of 5 min of extension, 4 DEG C of preservations.

With containing the sites attLCEBiPThe pCambia2301-GW-RNAi carriers that genetic fragment and embodiment 1 obtain exist LR reactions are carried out under the catalysis of LR enzymes, reaction condition is to contain the sites attLCEBiP3 μ L of genetic fragment, what the present invention obtained 1 μ L, LR enzyme of pCambia2301-GW-RNAi carriers 1 μ L, 25 DEG C of reaction 3h, reaction product convert Escherichia coli, be coated on containing On the LB solid mediums of kanamycins, it is inverted culture overnight for 37 DEG C and obtains barley to monoclonal, bacterium solution PCR identifications is grownCEBiPGene RNAi silent carrier pCambia2301-CEBiPIt is spare to protect bacterium upgrading grain by-RNAi.

4. Agrobacterium-mediated Transformation culture：

By 35ss-CEBiP- 3HA plasmids, pCambia2301-CEBiP- RNAi plasmids are converted respectively by freeze-thaw method in Agrobacterium GV3101 is coated with LB tablets（Contain rifampin 50mg/L, kanamycins 50mg/L, gentamicin 50mg/L）Culture, 28 DEG C are fallen Single bacterium colony is grown after setting culture 3 days, monoclonal is chosen and contains antibiotic to 1.5ml（Rifampin 50mg/L, kanamycins 50mg/L, Gentamicin 50mg/L）LB liquid medium, 28 DEG C of 200rpm are incubated overnight, and bacterium solution PCR identifications obtain Agrobacterium GV3101 It is spare to protect bacterium for positive strain.

5. raw Tobacco Leaves instantaneous conversion and detection：

35ss- will be containedCEBiPThe Agrobacterium of -3HA plasmids and contain pCambia2301-CEBiPThe Agrobacterium of-RNAi plasmids It preserves bacterium solution and is containing antibiotic respectively（Rifampin 50mg/L, kanamycins 50mg/L, gentamicin 50mg/L）LB tablets Upper scribing line, 28 DEG C are inverted culture 3 days to monoclonal is grown, respectively picking Agrobacterium monoclonal, in containing antibiotic（Rifampin 50mg/L, kanamycins 50mg/L, gentamicin 50mg/L）, acetosyringone（20 μM of final concentration）And MES（Final concentration 10mM） LB liquid medium in 28 DEG C of overnight incubations, then by bacterium solution respectively with LB liquid medium according to 1:200 volume ratios mix, Continue culture to OD₆₀₀=1.0~1.5.

Then bacterium solution at 3000rpm/min is centrifuged into 15min respectively, collects thalline, has been hanged to OD with re-suspension liquid₆₀₀= 0.7, then by OD₆₀₀=0.7 contains 35ss-CEBiPThe Agrobacterium resuspended bacterium solution and re-suspension liquid of -3HA plasmids mix in equal volume Make OD₆₀₀=0.35.The re-suspension liquid ingredient is：The sucrose of 2% weight ratio, 5g/L MS salt, 200 μM of acetosyringones, 10mM's MES.Bacterium solution after resuspension is placed 1~3 hour in the dark, is used for this life Tobacco Leaves instantaneous conversion.

By above-mentioned OD₆₀₀=0.7, contain 35ss-CEBiPThe Agrobacterium resuspended bacterium solution and OD of -3HA plasmids₆₀₀=0.7, contain pCambia2301-CEBiPThe Agrobacterium resuspended bacterium solution 1 of-RNAi plasmids:1 mixing, obtains mixed bacteria liquid.1ml syringes are taken, are removed Syringe needle draws OD respectively₆₀₀=0.7、OD₆₀₀=0.35 contains 35ss-CEBiPThe Agrobacterium resuspended bacterium solution of -3HA plasmids and above-mentioned Mixed bacteria liquid is injected from the back side of this life Tobacco Leaves, is marked, low light culture three days.The egg of extraction injection blade after three days In vain, Western Blot detections are carried out with HA antibody.As a result as shown in figure 13, various concentration（OD₆₀₀=0.7 and OD₆₀₀=0.35） Contain 35ss-CEBiPThe Agrobacterium resuspended bacterium solution of -3HA plasmids injects this life Tobacco Leaves, and the accumulation of CEBiP does not have in blade There is notable difference.Illustrate the accumulation of CEBiP in bacterium solution concentration range intra vane used in the present invention not to contain 35ss-CEBiP- The Agrobacterium of 3HA plasmids is resuspended the concentration variation of bacterium and changes.As shown in figure 14, CEBiP in the blade of mixed bacteria liquid injection Accumulation significantly reduces, explanationCEBiPGene is by success silence.

The embodiment of the above, only presently preferred embodiments of the present invention, is only used to explain the present invention, not limit The scope of the present invention processed to those of ordinary skill in the art certainly can be according to skill disclosed in this specification Art content, makes other embodiments easily by way of replacing or changing, therefore all in the principle of the present invention and technique item The changes and improvements etc. that part is done, should all be included in scope of the present invention patent.

SEQUENCE LISTING

<110>Zhoukou Normal University

<120>A kind of plant RNA i expression vectors and its construction method and application

<130>Nothing

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 17965

<212> DNA

<213>Artificial sequence

<400> 1

catggtagat ctgagggtaa atttctagtt tttctccttc attttcttgg ttaggaccct 60

tttctctttt tatttttttg agctttgatc tttctttaaa ctgatctatt ttttaattga 120

ttggttatgg tgtaaatatt acatagcttt aactgataat ctgattactt tatttcgtgt 180

gtctatgatg atgatgatag ttacagaacc gacgactcgt ccgtcctgta gaacgtgaaa 240

tcaaaaaact cgacggcctg tgggcattca gtctggatcg cgaaaactgt ggaattgatc 300

agcgttggtg ggaaagcgcg ttacaagaaa gccgggcaat tgctgtgcca ggcagtttta 360

acgatcagtt cgccgatgca gatattcgta attatgcggg caacgtctgg tatcagcgcg 420

aagtctttat accgaaaggt tgggcaggcc agcgtatcgt gctgcgtttc gatgcggtca 480

ctcattacgg caaagtgtgg gtcaataatc aggaagtgat ggagcatcag ggcggctata 540

cgccatttga agccgatgtc acgccgtatg ttattgccgg gaaaagtgta cgtatcaccg 600

tttgtgtgaa caacgaactg aactggcaga ctatcccgcc gggaatggtg attaccgacg 660

aaaacggcaa gaaaaagcag tcttacttcc atgatttctt taactatgcc ggaatccatc 720

gcagcgtaat gctctacacc acgccgaaca cctgggtgga cgatatcacc gtggtgacgc 780

atgtcgcgca agactgtaac cacgcgtctg ttgactggca ggtggtggcc aatggtgatg 840

tcagcgttga actgcgtgat gcggatcaac aggtggttgc aactggacaa ggcactagcg 900

ggactttgca agtggtgaat ccgcacctct ggcaaccggg tgaaggttat ctctatgaac 960

tgtgcgtcac agccaaaagc cagacagagt gtgatatcta cccgcttcgc gtcggcatcc 1020

ggtcagtggc agtgaagggc gaacagttcc tgattaacca caaaccgttc tactttactg 1080

gctttggtcg tcatgaagat gcggacttac gtggcaaagg attcgataac gtgctgatgg 1140

tgcacgacca cgcattaatg gactggattg gggccaactc ctaccgtacc tcgcattacc 1200

cttacgctga agagatgctc gactgggcag atgaacatgg catcgtggtg attgatgaaa 1260

ctgctgctgt cggctttaac ctctctttag gcattggttt cgaagcgggc aacaagccga 1320

aagaactgta cagcgaagag gcagtcaacg gggaaactca gcaagcgcac ttacaggcga 1380

ttaaagagct gatagcgcgt gacaaaaacc acccaagcgt ggtgatgtgg agtattgcca 1440

acgaaccgga tacccgtccg caagtgcacg ggaatatttc gccactggcg gaagcaacgc 1500

gtaaactcga cccgacgcgt ccgatcacct gcgtcaatgt aatgttctgc gacgctcaca 1560

ccgataccat cagcgatctc tttgatgtgc tgtgcctgaa ccgttattac ggatggtatg 1620

tccaaagcgg cgatttggaa acggcagaga aggtactgga aaaagaactt ctggcctggc 1680

aggagaaact gcatcagccg attatcatca ccgaatacgg cgtggatacg ttagccgggc 1740

tgcactcaat gtacaccgac atgtggagtg aagagtatca gtgtgcatgg ctggatatgt 1800

atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga acaggtatgg aatttcgccg 1860

attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa caagaaaggg atcttcactc 1920

gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa acgctggact ggcatgaact 1980

tcggtgaaaa accgcagcag ggaggcaaac aagctagcca ccaccaccac caccacgtgt 2040

gaattacagg tgaccagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc 2100

ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac 2160

gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg 2220

attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac 2280

taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt aaactatcag 2340

tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat tagaataacg 2400

gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc 2460

acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct atagtgcagt 2520

cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt 2580

tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg 2640

cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca agagcgccgc 2700

cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga ccaaccaacg 2760

ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca ccggcaccag 2820

gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg acgttgtgac 2880

agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca ttgccgagcg 2940

catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg acaccaccac 3000

gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg agcgttccct 3060

aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg 3120

cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga tcgaccagga 3180

aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga ccctgtaccg 3240

cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg 3300

tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac gccaagagga 3360

acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc 3420

gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg 3480

cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc 3540

ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac 3600

agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag 3660

gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca 3720

tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt 3780

ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg 3840

ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact 3900

tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag 3960

ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc 4020

tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg 4080

tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg 4140

ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg 4200

ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc 4260

aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat 4320

gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc 4380

aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc 4440

ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct 4500

gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat 4560

gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg 4620

tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgtct 4680

gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga cggtcgcaaa 4740

ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga gaagttgaag 4800

gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg 4860

caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg 4920

tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc gatgctctat 4980

gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag 5040

cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca cgtagaggtt 5100

tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact gatggcggtt 5160

tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa gcccggccgc 5220

gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga tggcggaaag 5280

cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt tgccatgcag 5340

cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga agccttgatt 5400

agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga gatcgagcta 5460

gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct gacggttcac 5520

cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct ggcacgccgc 5580

gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg cagtggcagc 5640

gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg 5700

ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt catgcgctac 5760

cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca gatgctaggg 5820

caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac 5880

attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa cccaaagccg 5940

tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa aggcgatttt 6000

tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc ctgtgcataa 6060

ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc 6120

tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc aaaaatggct 6180

ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc actcgaccgc 6240

cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg 6300

acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 6360

agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc 6420

acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg 6480

agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 6540

aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 6600

gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 6660

ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 6720

ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 6780

cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 6840

ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 6900

tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 6960

gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 7020

tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 7080

gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 7140

tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 7200

ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 7260

agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 7320

gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 7380

attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata atattttatt 7440

ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata ctgttcttcc 7500

ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt gtccgccctg 7560

ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa gatgttgctg 7620

tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt ctttaaaaaa 7680

tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc gcaatccaca 7740

tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc taagctattc 7800

gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc cgcatacagc 7860

tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag gacgccatcg 7920

gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag gacctttgga 7980

acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac atcataggtg 8040

gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc tcccaccagc 8100

ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta tttttcgatc 8160

agttttttca attccggtga tattctcatt ttagccattt attatttcct tcctcttttc 8220

tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc aattcactgt 8280

tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg ttttcaaagt 8340

tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca caggcagcaa 8400

cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt gtttcaaacc 8460

cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag tctgccgcct 8520

tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat cgagtggtga 8580

ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg 8640

tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg 8700

aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg gttttaggaa 8760

ttagaaattt tattgataga agtattttac aaatacaaat acatactaag ggtttcttat 8820

atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg ggaactactc 8880

acacattatt atggagaaac tcgagcttgt cgatcgactc tagctagagg atcgatccga 8940

accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga 9000

atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc 9060

ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg 9120

gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc 9180

atcgccatgt gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa 9240

cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc 9300

ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca 9360

ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc 9420

ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca 9480

gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc 9540

cagccacgat agccgcgctg cctcgtcctg gagttcattc agggcaccgg acaggtcggt 9600

cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca 9660

gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga 9720

acctgcgtgc aatccatctt gttcaatccc catggtcgat cgacagatct gcgaaagctc 9780

gagagagata gatttgtaga gagagactgg tgatttcagc gtgtcctctc caaatgaaat 9840

gaacttcctt atatagagga aggtcttgcg aaggatagtg ggattgtgcg tcatccctta 9900

cgtcagtgga gatatcacat caatccactt gctttgaaga cgtggttgga acgtcttctt 9960

tttccacgat gctcctcgtg ggtgggggtc catctttggg accactgtcg gcagaggcat 10020

cttgaacgat agcctttcct ttatcgcaat gatggcattt gtaggtgcca ccttcctttt 10080

ctactgtcct tttgatgaag tgacagatag ctgggcaatg gaatccgagg aggtttcccg 10140

atattaccct ttgttgaaaa gtctcaatag ccctttggtc ttctgagact gtatctttga 10200

tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt atcacatcaa tccacttgct 10260

ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat 10320

ctttgggacc actgtcggca gaggcatctt gaacgatagc ctttccttta tcgcaatgat 10380

ggcatttgta ggtgccacct tccttttcta ctgtcctttt gatgaagtga cagatagctg 10440

ggcaatggaa tccgaggagg tttcccgata ttaccctttg ttgaaaagtc tcaatagccc 10500

tttggtcttc tgagactgta tctttgatat tcttggagta gacgagagtg tcgtgctcca 10560

ccatgttggc aagctgctct agccaatacg caaaccgcct ctccccgcgc gttggccgat 10620

tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 10680

aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 10740

tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 10800

tgattacgaa ttcgagctcg tcgagcggcc gctcgacgaa ttaattccaa tcccacaaaa 10860

atctgagctt aacagcacag ttgctcctct cagagcagaa tcgggtattc aacaccctca 10920

tatcaactac tacgttgtgt ataacggtcc acatgccggt atatacgatg actggggttg 10980

tacaaaggcg gcaacaaacg gcgttcccgg agttgcacac aagaaatttg ccactattac 11040

agaggcaaga gcagcagctg acgcgtacac aacaagtcag caaacagaca ggttgaactt 11100

catccccaaa ggagaagctc aactcaagcc caagagcttt gctaaggccc taacaagccc 11160

accaaagcaa aaagcccact ggctcacgct aggaaccaaa aggcccagca gtgatccagc 11220

cccaaaagag atctcctttg ccccggagat tacaatggac gatttcctct atctttacga 11280

tctaggaagg aagttcgaag gtgaaggtga cgacactatg ttcaccactg ataatgagaa 11340

ggttagcctc ttcaatttca gaaagaatgc tgacccacag atggttagag aggcctacgc 11400

agcaggtctc atcaagacga tctacccgag taacaatctc caggagatca aataccttcc 11460

caagaaggtt aaagatgcag tcaaaagatt caggactaat tgcatcaaga acacagagaa 11520

agacatattt ctcaagatca gaagtactat tccagtatgg acgattcaag gcttgcttca 11580

taaaccaagg caagtaatag agattggagt ctctaaaaag gtagttccta ctgaatctaa 11640

ggccatgcat ggagtctaag attcaaatcg aggatctaac agaactcgcc gtgaagactg 11700

gcgaacagtt catacagagt cttttacgac tcaatgacaa gaagaaaatc ttcgtcaaca 11760

tggtggagca cgacactctg gtctactcca aaaatgtcaa agatacagtc tcagaagacc 11820

aaagggctat tgagactttt caacaaagga taatttcggg aaacctcctc ggattccatt 11880

gcccagctat ctgtcacttc atcgaaagga cagtagaaaa ggaaggtggc tcctacaaat 11940

gccatcattg cgataaagga aaggctatca ttcaagatct ctctgccgac agtggtccca 12000

aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt 12060

caaagcaagt ggattgatgt gacatctcca ctgacgtaag ggatgacgca caatcccact 12120

atccttcgca agacccttcc tctatataag gaagttcatt tcatttggag aggacacgct 12180

cgaggaatta tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat 12240

atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa 12300

catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg 12360

gctcgtataa tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag 12420

ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 12480

aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 12540

tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 12600

ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag 12660

acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa 12720

ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca 12780

tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 12840

ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 12900

acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc 12960

aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct 13020

tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg 13080

cgtaaacgcg tggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg 13140

atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta 13200

tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc 13260

atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt 13320

ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt 13380

attgaaatga acggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt 13440

ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat 13500

tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa 13560

agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac 13620

caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca 13680

ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg 13740

ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac 13800

agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat 13860

tttacgtttc tcgttcagct ttcttgtaca aagtggtgat aattcggtac cccaattggt 13920

aaggaaataa ttattttctt ttttcctttt agtataaaat agttaagtga tgttaattag 13980

tatgattata ataatatagt tgttataatt gtgaaaaaat aatttataaa tatattgttt 14040

acataaacaa catagtaatg taaaaaaata tgacaagtga tgtgtaagac gaagaagata 14100

aaagttgaga gtaagtatat tatttttaat gaatttgatc gaacatgtaa gatgatatac 14160

tagcattaat atttgtttta atcataatag taattctagc tggtttgatg aattaaatat 14220

caatgataaa atactatagt aaaaataaga ataaataaat taaaataata tttttttatg 14280

attaatagtt tattatataa ttaaatatct ataccattac taaatatttt agtttaaaag 14340

ttaataaata ttttgttaga aattccaatc tgcttgtaat ttatcaataa acaaaatatt 14400

aaataacaag ctaaagtaac aaataatatc aaactaatag aaacagtaat ctaatgtaac 14460

aaaacataat ctaatgctaa tataacaaag cgcaagatct atcattttat atagtattat 14520

tttcaatcaa cattcttatt aatttctaaa taatacttgt agttttatta acttctaaat 14580

ggattgacta ttaattaaat gaattagtcg aacatgaata aacaaggtaa catgatagat 14640

catgtcattg tgttatcatt gatcttacat ttggattgat tacagttggg aaattgggtt 14700

cgaaatcgat aagctatcac cactttgtac aagaaagctg aacgagaaac gtaaaatgat 14760

ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa 14820

acacaacata tccagtcact atggtcgacc tgcagactgg ctgtgtataa gggagcctga 14880

catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct 14940

gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat 15000

catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc 15060

tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat 15120

atcactctgt acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt 15180

aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc 15240

gggcgacctc agccatccct tcctgatttt ccgctttcca gcgttcggca cgcagacgac 15300

gggcttcatt ctgcatggtt gtgcttacca gaccggagat attgacatca tatatgcctt 15360

gagcaactga tagctgtcgc tgtcaactgt cactgtaata cgctgcttca tagcatacct 15420

ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg 15480

cgcaaatacg catactgtta tctggctttt agtaagccgg atccacgcgt ttacgccccg 15540

ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca 15600

tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta 15660

taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa 15720

tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac 15780

cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt 15840

agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc 15900

tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc 15960

attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc 16020

ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga 16080

acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga 16140

tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc 16200

ttagctcctg aaaatctcga cggatcctaa ctcaaaatcc acacattata cgagccggaa 16260

gcataaagtg taaagcctgg ggtgcctaat gcggccgcca tagtgactgg atatgttgtg 16320

ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta 16380

tatcatttta cgtttctcgt tcagcttttt tgtacaaact tgtgatagct tggatcctct 16440

agagtcctgc tttaatgaga tatgcgagac gcctatgatc gcatgatatt tgctttcaat 16500

tctgttgtgc acgttgtaaa aaacctgagc atgtgtagct cagatcctta ccgccggttt 16560

cggttcattc taatgaatat atcacccgtt actatcgtat ttttatgaat aatattctcc 16620

gttcaattta ctgattgtac cctactactt atatgtacaa tattaaaatg aaaacaatat 16680

attgtgctga ataggtttat agcgacatct atgatagagc gccacaataa caaacaattg 16740

cgttttatta ttacaaatcc aattttaaaa aaagcggcag aaccggtcaa acctaaaaga 16800

ctgattacat aaatcttatt caaatttcaa aaggccccag gggctagtat ctacgacaca 16860

ccgagcggcg aactaataac gttcactgaa gggaactccg gttccccgcc ggcgcgcatg 16920

ggtgagattc cttgaagttg agtattggcc gtccgctcta ccgaaagtta cgggcaccat 16980

tcaacccggt ccagcacggc ggccgggtaa ccgacttgct gccccgagaa ttatgcagca 17040

tttttttggt gtatgtgggc cccaaatgaa gtgcaggtca aaccttgaca gtgacgacaa 17100

atcgttgggc gggtccaggg cgaattttgc gacaacatgt cgaggctcag caggacctgc 17160

aggcatgcaa gctagcttac tagagtcgac ctgcaggcat gcaagcttgg cactggccgt 17220

cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 17280

acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 17340

acagttgcgc agcctgaatg gcgaatgcta gagcagcttg agcttggatc agattgtcgt 17400

ttcccgcctt cagtttagct tcatggagtc aaagattcaa atagaggacc taacagaact 17460

cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa 17520

aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac 17580

agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct 17640

cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg 17700

tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc 17760

cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 17820

tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 17880

cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 17940

ggagagaaca cgggggactc ttgac 17965

<210> 2

<211> 22

<212> DNA

<213>Artificial sequence

<400> 2

actttgtaca aaaaagcagg ct 22

<210> 3

<211> 22

<212> DNA

<213>Artificial sequence

<400> 3

actttgtaca agaaagctgg gt 22

<210> 4

<211> 25

<212> DNA

<213>Artificial sequence

<400> 4

tcgcgttaac gctagcatgg atctc 25

<210> 5

<211> 21

<212> DNA

<213>Artificial sequence

<400> 5

agcctgcttt tttgtacaaa c 21

<210> 6

<211> 21

<212> DNA

<213>Artificial sequence

<400> 6

acccagcttt cttgtacaaa c 21

<210> 7

<211> 24

<212> DNA

<213>It is artificial synthesized

<400> 7

gtaacatcag agattttgag acac 24

<210> 8

<211> 42

<212> DNA

<213>Artificial sequence

<400> 8

actttgtaca aaaaagcagg ctccaaagac cctcaagaag ga 42

<210> 9

<211> 42

<212> DNA

<213>Artificial sequence

<400> 9

actttgtaca agaaagctgg gtagccgttg gaataaccac tg 42

Claims (9)

1. a kind of plant RNA i expression vectors, which is characterized in that the plant RNA i expression vectors contain target gene fragment table Up to element, the target gene fragment Expression element contains CaMV35S promoters, introne, OCS terminators, both direction phase Anti- Gateway boxes；The introne is located between the opposite Gateway boxes of both direction, for expressing hairpin RNA knot Ring in structure；The Gateway boxes contain the sites attR1, the sites attR2,CmRGene,ccdBGene and oneEcoRI enzymes Enzyme site, Gateway boxes constitute the arm in hairpin RNA structure for expressing the opposite target gene fragment in direction.

2. a kind of plant RNA i expression vectors according to claim 1, which is characterized in that the Gateway boxes areEcoV digestion pBS-Gateway-rfA carriers of R obtain, describedEcoI restriction enzyme site distance attR1 site 453bp of R, distance AttR2 site 12s 57bp.

3. a kind of plant RNA i expression vectors according to claim 1, which is characterized in that the target gene fragment expression Element is obtained by intermediate carrier of pHANNIBAL carriers；The plant RNA i expression vectors are using pCambia2301 carriers as bone Target gene fragment Expression element is inserted into the multiple cloning sites of pCambia2301 carriers and obtains by frame.

4. a kind of plant RNA i expression vectors according to claim 1, which is characterized in that the plant RNA i expression vectors Nucleotide sequence as shown in SEQ ID NO.1.

5. the construction method of any one of Claims 1 to 4 plant RNA i expression vectors, which is characterized in that including following step Suddenly：

Step 1：It uses respectivelyEcoI Hes of RHinIII single endonuclease digestion pHANNIBAL carriers of d purify the product after digestion, recycle high protect True Taq enzyme filling-in point of contact, obtains E segments and H segments；

Step 2：WithEcoV digestion pBS-Gateway-rfA carriers of R, recycling obtain Gateway boxes, the Gateway boxes Containing the sites attR1, the sites attR2,CmRGene,ccdBGene and oneEcoRI restriction enzyme sites；

Step 3：E segments described in step 1 and H segments are connect with Gateway boxes described in step 2 respectively, then through screening Obtain pHANNIBAL-GW1.1 carriers and pHANNIBAL-GW1.2 carriers；The Gateway of the pHANNIBAL-GW1.1 carriers The sites attR1 in box are connect with CaMV35S promoters, and the sites attR2 are connect with introne；The pHANNIBAL- The sites attR1 in the Gateway boxes of GW1.2 carriers are connect with OCS terminators, and the sites attR2 are connect with introne；

Step 4：WithKpnI HeXhoI pair of pHANNIBAL-GW1.1 carrier and pHANNIBAL-GW1.2 carriers carry out double digestion, will Contain from what pHANNIBAL-GW1.1 carriers were cut outKpnI HeXhoThe Gateway boxes of I cohesive end, withKpnI HeXhoⅠ Segment connection after double digestion pHANNIBAL-GW1.2 carriers, obtains the recombination containing the opposite Gateway boxes of both direction Carrier pHANNIBAL-GW2；

Step 5：WithSacI HeSpeI double digestion pHANNIBAL-GW2 carriers, recycling obtain target gene fragment Expression element, so Afterwards withSacI HeXbaSegment connection after I double digestion pCambia2301 carriers, obtains pCambia2301-GW-RNAi loads Body, as plant RNA i expression vectors,SpeI HeXbaI is isocaudarner, can be attached between the cohesive end cut out.

6. construction method according to claim 5, which is characterized in that screening described in step 3 includes the following steps：First The recombinant vector containing E segments and Gateway boxes, the recombinant vector containing H segments and Gateway boxes are selected, it is then right It is used containing the recombinant vector of E segments and Gateway boxesEcoI Hes of RKpnI digestion detects, left containing 1300bp in electrophoresis result Right size segment is pHANNIBAL-GW1.1 carriers；It is single with EcoR I to the recombinant vector containing H segments and Gateway boxes Digestion detects, and containing 2000bp or so size segments is pHANNIBAL-GW1.2 carriers in electrophoresis result.

7. the host cell containing any one of Claims 1 to 4 plant RNA i expression vectors.

8. any one of Claims 1 to 4 plant RNA i expression vectors answering in building target gene RNAi silent carriers With.

9. application of the plant RNA i expression vectors in building target gene RNAi silent carriers according to claim 8, It is characterized in that, includes the following steps：

Step 1：Using the DNA sequence dna containing target gene CDS as template, PCR amplification is carried out using forward primer and reverse primer Joint sequence, nucleotide sequence difference are contained in target gene fragment, the ends 5' of the forward primer and the reverse primer As shown in SEQ ID NO.2 and SEQ ID NO.3；

Step 2：Using any entry vector as template, the DNA fragmentation in the sites attL1 is contained using L1-F and L1-R as primer amplification, Contain the DNA fragmentation in the sites attL2, the nucleotide sequence of L1-F, L1-R, L2-F and L2-R using L2-F and L2-R as primer amplification As shown in NO.4~7 SEQ ID；

Step 3：By the DNA fragmentation containing the sites attL1 of target gene fragment and step 2 gained obtained by step 1, contain The DNA fragmentation in the sites attL2 mixes, and carries out fusion DNA vaccine, obtains the target gene fragment containing attL recombination sites；

Step 4：By the target gene fragment containing attL recombination sites and the plant RNA i expression vectors obtained by step 3 into Row LR reactions, obtain target gene RNAi silent carriers.