CN103642827A - RNA interference vector suitable for herbicide selection marker Bar gene - Google Patents
RNA interference vector suitable for herbicide selection marker Bar gene Download PDFInfo
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Abstract
The invention relates to an RNA interference vector suitable for a herbicide selection marker Bar gene. The RNA interference vector includes a paddy rice GAS1 gene LOC-Os01g68860 intron, upstream multi-cloning sites and downstream multi-cloning sites, wherein the upstream multi-cloning sites and the downstream multi-cloning sites are respectively located at two sides of the paddy rice GAS1 gene intron; a nucleotide sequence of the paddy rice GAS1 gene LOC-Os01g68860 intron comprises 9621st-10099th nucleotides from the 5' terminal of a sequence 1 in a sequence table. Compared with an annular-structure RNA interference vector adopting a GUS gene as shRNA, the annular-structure RNA interference vector applying the gene intron as the shRNA has higher interference efficiency, at the same time, allows the experimental operation to become simple and easy to implement, so as to be beneficial for reducing errors and improving the work efficiency.
Description
Technical field
The rna interference vector that the present invention relates to a kind of applicable herbicide screening mark Bar gene, belongs to plant biotechnology field.
Background technology
Conventional plant RNA interference carrier is except the essential core parts of conventional binary vector, and as promotor, multiple clone site, outside resistant maker gene, also comprises section of DNA sequence as shRNA(short hairpin RNA, short hairpin RNA) ring texture.Plant RNA i interference carrier generally turns beta-Glucuronidase with the GUS(of bacterium) intron of gene fragment or plant gene is as ring texture.With with gus gene, as the rna interference vector of the ring texture of shRNA, compare, with thering is higher jamming effectiveness with the gene intron of splice site as the rna interference vector of the ring texture of shRNA.
In engineered development process, along with the science and technology development of each branch and people are to the deepening continuously of gene function understanding, selectable marker gene is also in the perfect process of development.The selectable marker gene of widely applying in genetically engineered operation till now can be divided into four classes substantially: be respectively antibiotics resistance gene, herbicide resistance gene, and compound detoxifying gene and plant carbohydrate metabolic enzyme gene.They are in the corresponding effect of selecting can play on substratum label, make to meet displaying that the cell of our condition can give prominence to.Wherein, Bar gene and careless fourth phosphine screening system are current most widely used classes of herbicides selective marker screening systems.
In the molecular biology operation that external source fragment is cloned into carrier, it is two kinds of conventional cloning process that homologous recombination directed cloning is connected with double digestion.Compare the latter, the former advantage is not need, with digestion with restriction enzyme object cloned sequence, to have overcome the restriction of restriction enzyme site, has reduced operation steps simultaneously, contributes to reduce workload, increases work efficiency.
Summary of the invention
The object of the present invention is to provide a kind of rna interference vector of applicable herbicide screening mark Bar gene, it comprises design, clone's target gene interference fragment, the recombinant vectors that screening contains target gene interference fragment, detects the standardized method that transgenosis is disturbed the sequence of operations such as plant; And in screening, contain target gene interference fragment recombinant vectors, detect the PCR that transgenosis used in disturbing the application processes such as plant and detect primer.When this rna interference vector of application, it is simple that experimental implementation becomes, and is conducive to reduce error, increases work efficiency.
Technical scheme of the present invention is achieved in that a kind of rna interference vector of applicable herbicide screening mark Bar gene, nucleotides sequence is classified the sequence 1 in sequence table as, comprise pTF101.1 carrier basic framework, 35s promotor and paddy rice GAS1 gene LOC_Os01g68860 intron, be positioned at upstream multiple clone site and the downstream multiple clone site of described paddy rice GAS1 gene intron both sides;
The nucleotides sequence of described paddy rice GAS1 gene intron is classified in sequence table sequence 1 as from 5 ' end 9621-10098 position Nucleotide;
Described upstream multiple clone site is BamHI, SmaI(XmaI) and MluI;
Described downstream multiple clone site is HpaI, SpeI and SacI;
The promotor of described interference carrier is 35s promotor.
Rna interference vector pLYZRNAi provided by the invention, is prepared as follows:
Described DNA fragmentation 1 comprises that the fragment a that contains upstream multiple clone site, paddy rice GAS1 gene (LOC_Os01g68860) intron, the fragment b that contains downstream multiple clone site form;
The nucleotides sequence of described paddy rice GAS1 gene (LOC_Os01g68860) intron is classified in sequence table sequence 1 as from 5 ' end 9621-10098 position;
The nucleotides sequence of described fragment a is classified in sequence table sequence 1 as from 5 ' end 9591-9620 position;
The nucleotides sequence of described fragment b is classified in sequence table sequence 1 as from 5 ' end 10099-10129 position,
The nucleotides sequence of described DNA fragmentation 1 is classified sequence 2 in sequence table as;
Described upstream multiple clone site is BamHI, SmaI(XmaI) and MluI;
Described downstream multiple clone site is HpaI, SpeI and SacI.
Second object of the present invention is to provide a kind of recombinant vectors.
Recombinant vectors provided by the invention, according to following I or II method, prepare:
I comprises the steps:
1) DNA fragmentation 2 is inserted to the upstream multiple clone site of described interference carrier, obtain the intermediate carrier A that contains described downstream multiple clone site;
2) by DNA fragmentation 3 inserting steps 1) the described downstream multiple clone site of the intermediate carrier A that obtains, obtain recombinant vectors;
II comprises the steps:
A, DNA fragmentation 3 is inserted to the downstream multiple clone site of described interference carrier, obtain the intermediate carrier B that contains described upstream multiple clone site;
The described upstream multiple clone site of B, intermediate carrier B that DNA fragmentation 2 inserting step A are obtained, obtains recombinant vectors;
Described DNA fragmentation 2 comprises target protein encoding gene fragment and lays respectively at 2 kinds of DNA moleculars for insertion vector of target protein encoding gene both sides, described in lay respectively at target protein encoding gene fragment both sides for 2 kinds of DNA moleculars of insertion vector include described upstream multiple clone site at least one restriction enzyme site, with described interference carrier or with the Nucleotide of described intermediate carrier B homology;
Described DNA fragmentation 3 comprises the reverse complemental fragment of target protein encoding gene and lays respectively at 2 kinds of DNA moleculars for insertion vector of the reverse complemental fragment both sides of described target protein encoding gene, described in lay respectively at described target protein encoding gene reverse complemental fragment both sides for 2 kinds of DNA moleculars of insertion vector include described downstream multiple clone site at least one restriction enzyme site, with described interference carrier or with the Nucleotide of described intermediate carrier A homology;
The nucleotides sequence of described DNA fragmentation 2 is classified sequence 4 in sequence table as;
The nucleotides sequence of described DNA fragmentation 3 is classified sequence 5 in sequence table as.
Described DNA fragmentation 2 also can be inserted in interference carrier downstream multiple clone site, and described DNA fragmentation 3 also can be inserted in interference carrier upstream multiple clone site, as long as the opposite direction of just fragment and antisense fragment.
In the structure of above-mentioned recombinant vectors, in DNA fragmentation 2 and DNA fragmentation 3, existing homologous recombination sequence has again double enzyme site, can pass through the multiple clone site place of homologous recombination insertion vector, obtains recombinant vectors; Can cut with the same enzyme of process the carrier obtaining by double digestion again and be connected, obtain recombinant vectors.
The application in plant transgene breeding and/or identified gene function of described interference carrier and/or described recombinant vectors.
The expression that be applied as target gene in inhibition object plant of described recombinant vectors in plant transgene breeding and/or gene functional research, obtains transgenic plant; The plant height of described transgenic plant is lower than described object plant; The method of the expression of the target gene in described inhibition object plant realizes by proceed to recombinant expression vector in described object plant.
The nucleotides sequence of described target gene is classified sequence 3 in sequence table as;
Described object plant is monocotyledons or dicotyledons, and described dicotyledons is preferably soybean.
Experimental results show that, the present invention uses it to build key gene phosphoric acid enol pyruvic acid carboxylase (the Phosphoenolpyruvate carboxylase for regulation protein and fat content ratio in the metabolism of soybean oil route of synthesis, PEPc) rna interference vector, then in soybean transformation acceptor kind Williams82.Rna interference vector is expressed the shRNA forming and is cut into siRNA(Small interfering RNA by Dicer enzyme in acceptor, disturbs little RNA); SiRNA can specificity suppress the expression of target gene, thereby oil content in soybean kernel is increased, and then the object of GmPEPc gene function.
Compare with existing plant RNA interference carrier, positively effect of the present invention is:
1, use 35s strong promoter.Apply the expression amount that this rna interference vector can significantly improve shRNA, thereby improve the jamming effectiveness of target gene, strengthen interference effect, this carrier is specially adapted to the dicotyledonss such as Arabidopis thaliana, soybean.
2, use the intron of paddy rice GAS1 gene as the ring texture of shRNA.In intron montage process, in shRNA, the mutual pairing of target gene justice and antisense sequences is enhanced, and increased shRNA and by Dicer enzyme, cut into the probability of siRNA, thereby the jamming effectiveness of raising target gene strengthens interference effect.
3, at the multiple clone site place, the left and right sides of carrier GAS1 gene intron, respectively add restriction enzyme digestion sites, facilitated the clone of target gene interference fragment.
4, when design target gene interference fragment PCR primer, in primer, introduce homologous recombination sequence and double enzyme site simultaneously.The visual particular case of subsequent experimental selects homologous recombination directed cloning or double digestion to connect cloning process, flexible operation not only, and by homologous recombination directed cloning method, overcome the restriction of restriction enzyme site.
5, the present invention comprises design, clone's target gene interference fragment, and the recombinant vectors that screening contains target gene interference fragment, detects the standardized method that transgenosis is disturbed the sequence of operations such as plant; And in screening, contain target gene interference fragment recombinant vectors, detect the PCR that transgenosis used in disturbing the application processes such as plant and detect primer.When this rna interference vector of application, it is simple that experimental implementation becomes, and is conducive to reduce error, increases work efficiency.
Accompanying drawing explanation
Fig. 1 is the structure schema of pLYZRNAi carrier.
Fig. 2 is the structural representation of pLYZRNAi carrier.
Fig. 3 is that the enzyme of pLYZRNAi recombinant vectors is cut evaluation electrophorogram.
Fig. 4 is the structure schema of pLYZRNAi-GmPEPc carrier.
Fig. 5 is pLYZRNAi-GmPEPc carrier structure schematic diagram.
Fig. 6 is that the enzyme of pLYZRNAi-GmPEPc recombinant vectors is cut evaluation electrophorogram.
PCR shown in Fig. 7 identifies pLYZRNAi-sense-GmPEPc recombinant vectors.
PCR shown in Fig. 8 identifies pLYZRNAi-GmPEPc recombinant vectors.
Fig. 9 is Genetic Transformation of Soybean schema.
Figure 10 is that PCR detects T1 for transgenic progeny material.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described: the experimental technique using in embodiment if no special instructions, is ordinary method; And in embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The structure of embodiment 1, rna interference vector pLYZRNAi
Rna interference vector pLYZRNAi is the method with homologous recombination directed cloning, the intron forward of paddy rice GAS1 gene is cloned into (Fig. 1 is experiment flow figure) in the plant expressing vector pTF101-35s with Bar mark, as shown in Figure 2, concrete grammar is as follows for the structural representation of rna interference vector pLYZRNAi:
1, the acquisition of paddy rice GAS1 gene LOC_Os01g68860 intron fragment
Utilizing the intron sequences of the synthetic paddy rice GAS1 gene of synthetic gene technology, and add multiple clone site a used and b two sections of sequences, is sequence 2; Then according to pTF101-35s carrier, select the primer sequence of BamI and SacI enzyme design amplification intron following (with the homologous recombination sequence of BamI and SacI restriction enzyme site both sides on the letter representation pTF101-35s carrier of underscore.）：
GAS1?intron-F：5'-?
TAGAAACAGAGGATCCGGATCCAACAGCCCCGGGAA-3'；
GAS1?intron-R：5'-
?GATCGGGGAAATTCGAGCTCGAGCTCTCTAGAACTAGTATCGATG-3'。
Pcr amplification program: 94 ℃ of 2 min; 98 ℃ of 10 sec again, 60 ℃ of 5 sec, 72 ℃ of 1.5 min, 30 circulations; Last 72 ℃ of 7 min.
The DNA of above-mentioned synthetic of take is template, adopts above-mentioned PCR amplification program, usings GAS1 intron-F and GAS1 intron-R to carry out pcr amplification as primer, and electrophoresis result, referring to the 5th swimming lane in Fig. 3, is the object band fragment of amplification gene.
Described upstream multiple clone site is (BamI, SmaI and MluI);
Described downstream multiple clone site is (SacI, SpeI and HpaI).
Reclaim PCR product standby.
2, the preparation that BamI and SacI enzyme are cut pTF101-35S linear carrier
Use BamI and the SacI restriction enzyme of NEBZhong Co., Ltd, enzyme is cut pTF101-35S carrier (electrophoresis detection is as 2 swimming lanes of Fig. 3) (Paz, M., Martinez, J. C., Kalvig, A., Fonger, T., Wang, K. (2010) the Agrobacterium-mediated transformation of soybean and recovery of transgenic soybean plants. public can obtain from Jilin Academy of Agricultural Science agro-ecology institute.), reclaim the pTF101-35S linear carrier of cutting through enzyme standby.Electrophoresis detection is as 3 swimming lanes of Fig. 3.
3, the acquisition of pLYZRNAi recombinant vectors
1) the homologous recombination directed cloning of GAS1 gene intron
The linear carrier of the recovery that the PCR product of the recovery that in use GenScript, the CloneEZ homologous recombination directed cloning test kit of Co., Ltd obtains step 1 obtains with step 2 carries out homologous recombination and reacts, and reaction system is as shown in table 1:
Table 1 homologous recombination reaction system
The operation steps of homologous recombination directed cloning is as follows:
(1) in 1.5 ml centrifuge tubes, prepare above-mentioned reaction system in each reactive component, slightly mix, by sample recombining reaction 30 min in 22 ℃ of water-baths;
(2) after reaction finishes, sample is placed on and hatches 5 min on ice.
(3) product step (2) being obtained proceeds in TransT1-1 competent cell (purchased from Beijing Quanshijin Biotechnology Co., Ltd), obtains transformant.
2) identify pLYZRNAi recombinant vectors, electrophoresis result is shown in Fig. 3
Select above-mentioned transformant, be inoculated in the LB liquid nutrient medium containing spectinomycin incubated overnight; Get the bacterium liquid of incubated overnight as PCR reaction template, use GAS1 intron-F and GAS1intron-R primer, carry out pcr amplification reaction.
Result as shown in Figure 3, cut and detect pTF101-35S carrier and the detection of GAS1 gene intron pcr amplification, 1:Trans 15K DNA marker by PCR and enzyme; 2:pTF101-35s does not contain the pTF101-35S empty carrier plasmid of GAS1 gene intron; 3: containing pTF101-35S recombinant vectors (pLYZRNAi) plasmid of GAS1 gene intron; 4:BamI and SacI enzyme are cut pLYZRNAi result; The PCR product of 5:GAS1 gene; 6:DL2000 DNA marker.
This plasmid is sent to order-checking, and sequencing primer is that the fragment on Left MCS-F and Right MCS-R(primer sequence and carrier pTF101-35S matches), primer sequence is as follows:
Left?MCS-F：5'-CGCACAATCCCACTATCCTT-3'；
Right?MCS-R：5'-AAGACCGGCAACAGGATTC-3'。
Sequencing result shows that this plasmid is for containing GAS1 gene LOC_Os01g68860 intron fragment by the sequence 2(in sequence table) be cloned into the BamI of pTF101-35S carrier and the recombinant vectors that SacI restriction enzyme site place obtains, and GAS1 gene LOC_Os01g68860 intron fragment is cloned into after the 35s promotor of pTF101-35S carrier, by this plasmid called after pLYZRNAi, be rna interference vector, sequence 1.As shown in Figure 2, as seen from the figure, this carrier has upstream multiple clone site and downstream multiple clone site to the structural representation of this carrier.
The application of embodiment 2, rna interference vector pLYZRNAi
One, the structure of GmPEPc gene RNA interference carrier pLYZRNAi-GmPEPc, builds flow process referring to Fig. 4, and structural representation is shown in Fig. 5, and PCR and enzyme are cut qualification result and seen Fig. 6.
1, the acquisition of GmPEPc gene interference fragment
Phosphoric acid enol pyruvic acid carboxylase PEPc participates in protein, the regulation and control of fat content ratio in plant seed, and the expression that suppresses PEPc is the effective way that improves grain oil content.
1) design of primers
1) amplification GmPEPc gene justice interference fragment primer used
In upstream multiple clone site with the letter representation pLYZRNAi carrier of underscore, select BamI and SmaI restriction enzyme cleavage site (can by homology exchange or double digestion connect be cloned on carrier), left oblique line represents that BamI and SmaI restriction enzyme cut, and primer sequence is as follows:
GmPEPc-RNAi-sense-F:5'-
tAGAAACAGAGGATC/
cthis primer of GCTGGAGATTCCATTGTGGT-3'(contains BamHI restriction enzyme site);
GmPEPc-RNAi-sense-R:5'-
gCGTGCTGTTCCCGG/
gthis primer of CCTTCCATGTTTTTGAAGCAA-3'(contains SamI restriction enzyme site).
In above primer, existing homologous recombination sequence has again restriction enzyme site, and the PCR product in later stage both can be connected with carrier by homologous recombination, also can be connected with carrier and is connected by double digestion.
(2) amplification GmPEPc gene antisense interference fragment primer used
In downstream multiple clone site with the letter representation pLYZRNAi carrier of underscore, the homologous recombination sequence of SpeI and SacI restriction enzyme site both sides (can by homology on the same group directed cloning to carrier), left oblique line represents SpeI and SacI restriction enzyme cleavage site (can be connected and be cloned on carrier by double digestion), and primer sequence is as follows:
GmPEPc-RNAi-antisense-F：
5'-
tAACATCGATACTAGTthis primer of/GCTGGAGATTCCATTGTGGT-3'(contains SpeI restriction enzyme site);
GmPEPc-RNAi-antisense-R：
5'-
gATCGGGGAAATTCG/
aGCTCthis primer of CCTTCCATGTTTTTGAAGCAA-3'(contains SacI restriction enzyme site).
In above primer, existing homologous recombination sequence has again restriction enzyme site, and the PCR product in later stage both can be connected with carrier by homologous recombination, also can be connected with carrier and is connected by double digestion.
2) soybean varieties Williams 82 extractions of seedling RNA and the preparation of cDNA
Use RNA prep pure plant total RNA extraction reagent box (purchased from TIANGEN Biotech (Beijing) Co., Ltd.), extract 30 days seedling RNA of soybean Williams 82, reverse transcription obtains cDNA.
3) pcr amplification GmPEPc gene interference fragment
Take cDNA as template, with GmPEPc-RNAi-sense-F and GmPEPc-RNAi-sense-R primer, carry out pcr amplification, obtain PCR product 1.Take cDNA as template, with GmPEPc-RNAi-antisense-F and GmPEPc-RNAi-antisense-R primer, carry out pcr amplification, obtain PCR product 2.
Reclaim respectively PCR product 1 and 2, and send to order-checking, result shows that PCR product 1 has Nucleotide shown in sequence 4 in sequence table (this sequence is the part in sequence 3 namely, adds that the BamHI of carrier pLYZRNAi upstream multiple clone site that cloned sequence is required is, the homologous recombination sequence of SmaI restriction enzyme site both sides); Sequence 4 from 5 ' end 17-316 position Nucleotide, be the sequence from 5 ' end 701-1414 position Nucleotide of sequence 3.), PCR product 1 is just GmPEPc gene interference fragment; PCR product 2 has in sequence table Nucleotide shown in sequence 5, and (this sequence is the part in sequence 3, and the required SpeI of carrier pLYZRNAi downstream multiple clone site and the homologous recombination sequence of SacI double enzyme site both sides of the added cloned sequence in sequence two ends), be the reverse complemental fragment of sequence 4.Sequence 5 from 5 ' end 19-732 position Nucleotide, be the reverse complemental fragment from 5 ' end 701-1414 position Nucleotide of sequence 3, so PCR product 2 is antisense GmPEPc gene interference fragment.
2, with the acquisition of the pLYZRNAi-sense-GmPEPc recombinant vectors of GmPEPc gene justice interference fragment
1) the homologous recombination directed cloning of GmPEPc gene justice interference fragment
With BamHI and SmaI enzyme, cut the pLYZRNAi carrier that example 1 obtains, obtain linear pLYZRNAi, reclaim stand-by.
Adopt two methods of cutting doubly-linked or homologous recombination directed cloning (concrete steps and reaction system see embodiment 1 3), first the PCR product 1 of recovery and linear pLYZRNAi are carried out to homologous recombination reaction, again homologous recombination product is proceeded to TransT1-1 competent cell, obtain transformant.
2) identify pLYZRNAi-sense-GmPEPc recombinant vectors
Select 1) in the transformant of gained, be inoculated in the LB liquid nutrient medium containing spectinomycin incubated overnight; Get the bacterium liquid of incubated overnight as PCR reaction template, use Left MCS-F and Left MCS-R primer, carry out pcr amplification reaction.As shown in Figure 7, PCR identifies pLYZRNAi-sense-GmPEPc recombinant vectors, M:Trans 2K plus marker to result; 1-12: template is cloned from pLYZRNAi-sense GmPEPc recombinant vectors.Can find out, obtain the positive colony that is of 520bp DNA band, primer sequence is as follows:
Left?MCS-F：5'-CGCACAATCCCACTATCCTT-3'；
Left?MCS-R：5'-AGGCGGTACAATGATCAACC-3'。
Extract the plasmid of positive colony, electrophoresis detection is as cut the pLYZRNAi-sense GmPEPc carrier of detection for passing through BamHI and SamI enzyme in 4 swimming lanes in Fig. 6.6 swimming lanes are the PCR product of sense GmPEPc.
This plasmid is sent to order-checking, sequencing primer is Left MCS-F and Left MCS-R, sequencing result shows, this plasmid is by the sequence 3(GmPEPc gene justice interference fragment in sequence table) be cloned into the upstream multiple clone site BamHI of pLYZRNAi carrier and the recombinant vectors that SamI place obtains, by plasmid called after pLYZRNAi-sense-GmPEPc carrier
3, with the acquisition of the pLYZRNAi-GmPEPc recombinant vectors of GmPEPc gene justice and antisense interference fragment
1) the homologous recombination directed cloning of GmPEPc gene antisense interference fragment
With SpeI and SacI enzyme, cut step 2,2) in the pLYZRNAi-sense-GmPEPc that obtains, reclaim linear pLYZRNAi-sense-GmPEPc.Electrophoresis is shown in shown in 5 swimming lanes in Fig. 6, cuts the enzyme of pLYZRNAi-sense GmPEPc carrier cut detected result for SpeI and SacI enzyme.
Adopt the method (concrete steps and reaction system see embodiment 1 3) of homologous recombination directed cloning, first the PCR product 2 of recovery and linear pLYZRNAi-sense-GmPEPc are carried out to homologous recombination reaction, again homologous recombination product is proceeded to TransT1-1 competent cell, obtain transformant.
2) identify pLYZRNAi-GmPEPc recombinant vectors
Select 1) in the transformant of gained, be inoculated in the LB liquid nutrient medium containing spectinomycin incubated overnight; Get the bacterium liquid of incubated overnight as PCR reaction template, use Right MCS-F and Right MCS-R primer, carry out pcr amplification reaction.As shown in Figure 8, PCR identifies pLYZRNAi-GmPEPc recombinant vectors, M:DL 2000 DNA marker to result; 1-6: template is from pLYZRNAi-GmPEPc recombinant vectors.Can find out, obtain the positive colony that is of 425bp DNA band.Primer sequence is as follows:
Right?MCS-F：5'-TATGACACGGCTGTTTCGAG-3'；
Right?MCS-R：5'-GAATCCTGTTGCCGGTCTT-3'。
This plasmid is sent to order-checking, with Left MCS-F, Left MCS-R, Right MCS-F and Right MCS-R are as sequencing primer.Sequencing result shows, this plasmid is for by the sequence 4(GmPEPc gene antisense interference fragment in sequence table) be cloned into the recombinant vectors that pLYZRNAi-sense-GmPEPc carrier S peI and SacI restriction enzyme site place obtain; Also can say that this plasmid is for by the sequence 3(GmPEPc gene justice interference fragment in sequence table) be cloned into BamHI and SamI place in the upstream multiple clone site of pLYZRNAi carrier, and by the sequence 4(GmPEPc gene antisense interference fragment in sequence table) be cloned into the recombinant vectors that SpeI in the downstream multiple clone site of pLYZRNAi carrier and SacI restriction enzyme site place obtain.By this plasmid called after pLYZRNAi-GmPEPc.The structural representation of pLYZRNAi-GmPEPc as shown in Figure 4, builds flow process as shown in Figure 3.
Also can adopt the basic skills of above-mentioned homologous recombination directed cloning, first GmPEPc gene antisense interference fragment is carried out to homologous recombination directed cloning and obtain intermediate carrier, again GmPEPc gene justice interference fragment is inserted to above-mentioned intermediate carrier, obtain pLYZRNAi-GmPEPc recombinant vectors.Through order-checking, identify, vector construction is correct.Also the method that can use enzyme to cut connection builds pLYZRNAi-GmPEPc recombinant vectors:
Specific as follows: first PCR product 1 use SpeI and SacI enzyme are cut, the enzyme obtaining is cut product and is connected with the pLYZRNAi carrier of cutting through same enzyme, obtains intermediate carrier; PCR product 2 use BamH I and SmaI enzyme are cut, the enzyme obtaining is cut product and is connected with the intermediate carrier of cutting through same enzyme, obtains pLYZRNAi-GmPEPc recombinant vectors again.Through order-checking, identify, vector construction is correct.
Two, the genetic transformation of rna interference vector pLYZRNAi-GmPEPc
PLYZRNAi-GmPEPc carrier is proceeded in EHA101 Agrobacterium, obtain transformant.Transformant is extracted to plasmid and send to order-checking, result shows that plasmid is pLYZRNAi-GmPEPc, therefore by the transformant called after EHA101/pLYZRNAi-GmPEPc that contains pLYZRNAi-GmPEPc.
Utilize soybean cotyledon node genetic transformation method to proceed to (Fig. 9, the conversion process of soybean cotyledon node genetic transformation method in soybean acceptor kind Williams82 EHA101/pLYZRNAi-GmPEPc.) in, concrete steps are as follows:
(1) from flat board, get single bacterium colony, access 10mlYEB liquid nutrient medium adds in 50mg/L Kan, and 26 ℃, 160r/min shaking culture is to logarithmic phase, and OD600=0.6, by 2 times of MSO liquid nutrient medium dilutions for bacterium liquid, as the engineering bacteria liquid of Agrobacterium.
(2) transgenic method adopts agriculture bacillus mediated soybean cotyledon node conversion method.After soybean seeds sterilization, in adding MSB5 substratum, sprout 1d, kind of a skin is removed, along plumular axis, seed is cut into 2 half, often half is all with a slice cotyledon and 3～5 mm hypocotyls.Explant is immersed in ready engineering bacteria liquid to 30 minutes, taking out the explant of invading after bacterium inserts on filter paper again, sop up unnecessary bacterium liquid, go to common substratum and cultivate, after 5 days, proceed to again bud inducing culture, after 28 days, proceed to bud elongation medium, every two weeks subcultures once, treat that resistant buds is stretched to 3～4cm, and resistant buds is cut and puts into root media root induction, after inducing root, transplanted, put into greenhouse it is blossomed and beared fruit.
(3) adopt to use the same method empty carrier pLYZRNAi is proceeded in soybean acceptor kind Williams82, as the contrast in experiment.
Three, GmPEPc gene T1 is for disturbing soybean genotype and phenotypic evaluation
1, genotype identification
Extract T1 and for GmPEPc gene, disturb the DNA of plant, it is primer that forward fragment be take Left MCS-F and Left MCS-R, carries out PCR detection, obtains the positive colony that is of 520bp DNA band.Oppositely to take Right MCS-F and Right MCS-R be primer to fragment, and the T1 that is that obtains 425bp DNA band disturbs paddy rice (Figure 10) for GmPEPc gene.Obtain altogether 9 strain PCR and be accredited as positive T1 for GmPEPc gene interference soybean.
2, phenotypic evaluation
We for transgenic positive material, carry out the mensuration of oil content to the positive T1 of this 9 strain, adopt the soybean acceptor kind Williams82 that proceeds to empty carrier pLYZRNAi to contrast.
Test-results shows, turns in GmPEPc gene T2 9 transgenosis familys in generation and has 8 oleaginousness in family to increase than contrast, wherein has oleaginousness in 3 transgenosis family seeds to improve more than 10 (table 1)
Table 1 T2 is for oil content content in transgenic progeny seed
| Kind (plant line) | Oil content content % oil | Compared with CK ± % |
| CK （Williams 82） | 20.6 ± 0.5 | 0 |
| 1 | 21.1 ± 1.8 | +4.6 |
| 2 | 22.7 ± 1.3 | +10.12 |
| 3 | 24.3 ± 0.9 | +17.96 |
| 4 | 22.6 ± 0.9 | +9.7 |
| 5 | 23.4 ± 1.5 | +13.6 |
| 6 | 21.7 ± 0.8 | +5.3 |
| 7 | 21.3 ± 0.9 | +3.4 |
| 8 | 21.8 ± 2.2 | +5.82 |
| 9 | 18.8 ± 1.7 | -8.7 |
These results suggest that, plant RNA interference carrier provided by the invention is expressed the shRNA forming and is cut into siRNA by Dicer enzyme in acceptor; SiRNA can suppress the expression of target gene GmPEPc specifically, thereby reaches the content of controlling soybean seeds kind oil content, the object of research GmPEPc gene function.
The invention discloses a kind of rna interference vector and application thereof.Aspect plant transgene breeding and gene functional research, have important using value, the present invention provides new technique means for plant transgene breeding and gene functional research.
Claims (10)
1. a rna interference vector for applicable herbicide screening mark Bar gene, comprises paddy rice GAS1 gene LOC_Os01g68860 intron, is positioned at upstream multiple clone site and the downstream multiple clone site of described paddy rice GAS1 gene intron both sides;
The nucleotides sequence of described paddy rice GAS1 gene LOC_Os01g68860 intron is classified in sequence table sequence 1 as from 5 ' end 9621-10099 position Nucleotide.
2. the rna interference vector of a kind of applicable herbicide screening mark Bar gene according to claim 1, is characterized in that described upstream multiple clone site is BamHI, SmaI, XmaI, MluI.
3. the rna interference vector of a kind of applicable herbicide screening mark Bar gene according to claim 1, is characterized in that described downstream multiple clone site is HpaI, SpeI and SacI.
4. the rna interference vector of a kind of applicable herbicide screening mark Bar gene according to claim 1, is characterized in that the promotor of described interference carrier is 35s promotor.
5. the rna interference vector of a kind of applicable herbicide screening mark Bar gene according to claim 1, is characterized in that described rna interference vector is prepared as follows:
DNA fragmentation 1 and pTF101-35s carrier framework are cultivated altogether to the carrier obtaining;
Or DNA fragmentation 1 and pTF101.1 carrier framework are cultivated altogether to the carrier obtaining;
Wherein DNA fragmentation 1 comprises that the fragment a that contains upstream multiple clone site, paddy rice GAS1 gene LOC_Os01g68860 intron, the fragment b that contains downstream multiple clone site form;
The nucleotides sequence of described fragment a is classified in sequence table sequence 1 as from 5 ' end 9591-9620 position;
The nucleotides sequence of described fragment b is classified in sequence table sequence 1 as from 5 ' end 10099-10129 position,
The nucleotides sequence of described DNA fragmentation 1 is classified sequence 2 in sequence table as.
6. the rna interference vector of a kind of applicable herbicide screening mark Bar gene according to claim 1, is characterized in that the recombinant vectors of described rna interference vector, according to following I or the preparation of II method:
I comprises the following steps:
1) DNA fragmentation 2 is inserted to the upstream multiple clone site of described interference carrier, obtain the intermediate carrier A that contains described downstream multiple clone site;
2) by DNA fragmentation 3 inserting steps 1) the described downstream multiple clone site of the intermediate carrier A that obtains, obtain recombinant vectors;
II comprises the following steps:
A, DNA fragmentation 3 is inserted to the downstream multiple clone site of described interference carrier, obtain the intermediate carrier B that contains described upstream multiple clone site;
The described upstream multiple clone site of B, intermediate carrier B that DNA fragmentation 2 inserting step A are obtained, obtains recombinant vectors;
Described DNA fragmentation 2 comprises target protein encoding gene fragment and lays respectively at 2 kinds of DNA moleculars for insertion vector of target protein encoding gene both sides, described in lay respectively at target protein encoding gene fragment both sides for 2 kinds of DNA moleculars of insertion vector include described upstream multiple clone site at least one restriction enzyme site, with described interference carrier or with the Nucleotide of described intermediate carrier B homology;
Described DNA fragmentation 3 comprises the reverse complemental fragment of target protein encoding gene and lays respectively at 2 kinds of DNA moleculars for insertion vector of the reverse complemental fragment both sides of described target protein encoding gene, described in lay respectively at described target protein encoding gene reverse complemental fragment both sides for 2 kinds of DNA moleculars of insertion vector include described downstream multiple clone site at least one restriction enzyme site, with described interference carrier or with the Nucleotide of described intermediate carrier A homology;
The nucleotides sequence of described DNA fragmentation 2 is classified sequence 4 in sequence table as;
The nucleotides sequence of described DNA fragmentation 3 is classified sequence 5 in sequence table as;
Described DNA fragmentation 2 also can be inserted in interference carrier downstream multiple clone site, and described DNA fragmentation 3 also can be inserted in interference carrier upstream multiple clone site, as long as the opposite direction of just fragment and antisense fragment.
7. the recombinant vectors of a kind of rna interference vector according to claim 6, it is characterized in that in the structure of described recombinant vectors, in DNA fragmentation 2 and DNA fragmentation 3, existing homologous recombination sequence has again double enzyme site, can pass through the multiple clone site place of homologous recombination insertion vector, obtain recombinant vectors; Can cut with the same enzyme of process the carrier obtaining by double digestion again and be connected, obtain recombinant vectors.
8. a rna interference vector or its recombinant vectors application in plant transgene breeding and/or identified gene function.
9. the recombinant vectors of a kind of rna interference vector according to claim 8, is characterized in that the expression for the target gene in inhibition object plant in plant transgene breeding of described recombinant vectors, obtains transgenic plant; The plant height of transgenic plant is lower than described object plant; The method that suppresses the expression of the target gene in object plant realizes by proceed to recombinant expression vector in described object plant.
10. the recombinant vectors of a kind of rna interference vector according to claim 8, is characterized in that the nucleotides sequence of described target gene is classified sequence 2 in sequence table as.
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| CN102191262A (en) * | 2011-03-08 | 2011-09-21 | 中国农业科学院作物科学研究所 | RNA (Ribonucleic Acid) interference vector and application thereof |
| CN103146745A (en) * | 2013-03-13 | 2013-06-12 | 辽宁大学 | Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector |
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| CN102191262A (en) * | 2011-03-08 | 2011-09-21 | 中国农业科学院作物科学研究所 | RNA (Ribonucleic Acid) interference vector and application thereof |
| CN103146745A (en) * | 2013-03-13 | 2013-06-12 | 辽宁大学 | Plant RNA (Ribose Nucleic Acid) interference vector for inhibiting lignin from synthesizing, and construction method and application of plant RNA interference vector |
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