CN100427602C - Plant bivalent anti-reverse gene bielement expression carrier - Google Patents
Plant bivalent anti-reverse gene bielement expression carrier Download PDFInfo
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Abstract
The present invention relates to an agro-bacillus bi-element expression carrier for the seed cultivation of the plant genetic engineering, particularly to a bivalent anti-reverse gene bi-element expression carrier comprising an arabidopis thaliana response gene transcription activation factor gene cbf 1 regulated by a corn ubiquitin promotor Pubi closely interlocked, and an iso-pentenyl transferase ipt gene regulated by an arabidopis thaliana specific aging gene promotor SAG12. The present invention comprises the following steps: firstly, the T-Nos in a sag 12-ipt fusion gene is inserted into a bi-element carrier; then, an ubi-cbf 1 fusion gene is inserted; finally, the sag 12-ipt fusion gene is inserted; thus, the complete divalent bi-element carrier is obtained. The carrier is favorable to the conversion of target plants from two function genes, obviously enhances the resistance of conversion plants to bad environment, and simultaneously delays the plant aging induced by the bad environment.
Description
Technical field
The present invention relates to a kind of Agrobacterium binary expression vector that is used for the plant genetic engineering breeding, specifically is the bivalent anti-reverse gene bielement expression carrier of prenyltransferase ipt gene that comprises the promotor SAG12 regulation and control of aging gene special in the activating transcription factor gene cbfl of Arabidopis thaliana cold-responsible genes of closely linked corn ubiquitin promoter Pubi regulation and control and the Arabidopis thaliana.The invention still further relates to the construction process of this binary vector, belong to plant genetic engineering field.
Technical background
Many freeze proof plants, as Arabidopis thaliana, rape, barley and rye etc., its common feature is the characteristic with cold domestication response, promptly through just having freeze proof ability more than 0 ℃ to the cold acclimation below 10 ℃.In this course, a series of physiology and biochemical the variation have taken place in plant.Gene expression in plants changes under low temperature stress, comprises the enhancing of original protein expression level, the expression of low temperature specificity inducible protein etc.Therefore, plant is actually expression and the metabolic environmental adaptation process of experiencing low-temperature signal, regulatory gene to cryogenic adaptation.((Cold responsive, COR) the COR expression of gene is induced in CRT/DRE element in the gene and combination with it to discern cold response for Cold responsive, COR) gene activating transcription factor CBF1 in cold response on the Arabidopis thaliana.The artificial constructed Agrobacterium binary expression vector that contains cbf1, and be that mediation is inserted cbf1 in the arabidopsis gene group and made its overexpression with the Agrobacterium, under non-cold condition, can activate the target gene expression that contains CRT/DRE, thereby (Jaglo-Ottosen etc. 1998 for frost resistance of raising plant and dehydration tolerance, Science,, 280:104-106; Liu etc. 1998, PlantCell, 10:1391-1046).
Even but the plant that under adverse environmental factors such as low temperature, can survive, and the decline of phytokinin in its blade (CTK) content (Yamada etc. 1985, J Japan Soc Hort Sci, and 53:419-426) plant is easy to aging.CTK can remove free radical directly or indirectly, and the minimizing lipid peroxidation (Leshem etc. 1981, PhysiolPlant, 53:9-12), the activity of film protective enzymes such as raising SOD (Wang Sangen and Liang Ying 1995, the rice in China science, 9:223-229).(isopentenyl transferase ipt) is the first crucial rate-limiting enzyme in the phytokinin biosynthesizing to prenyltransferase, and it impels 5`-AMP and the synthetic isopentenyl adenosine of isopentenyl gland purine tetra-sodium-5`-phosphoric acid.This material is converted to isopentenyl adenosine and isopentenyl gland purine very soon.Effect forms ribosylzeatin and zeatin respectively to these two kinds of materials through cytokinin oxidase.Relevant ipt transforms discovering of initial stage, the ipt that does not promptly change promotor with original ipt is in the transfer-gen plant of foreign gene acquisition, the phytokinin overexpression, leaf senile obviously delays, and causes plant to be downgraded simultaneously and grows thickly, lose apical dominance fully, blade is little and justify, and root system can not form, and plant can not normal growth grows that (Smigocki etc. 1988, PNAS, 85:5131-5135; Beinsberger etc. 1991, Plant CellPhysiology, 32:489-496).In order to eliminate the influence that the phytokinin overexpression produces in the plant tissue, the investigator has used many tissue-specific promoters and inducible promoters, and (Schmulling etc. 1989, FEBS Lett, 249:401-406 as heat-inducible promoter; Medford etc. 1987, PlantCell, 1:403-413; Smart etc. 1991, Plant Cell, 3:647-656; Van Loven 1993, J Exp Bot, 44:1671-1678), (Mckenzie etc. 1998 for the copper inducible promotor, Plant Physiology, 116:969-977), (Kunkel etc. 1999 for the tsiklomitsin evoked promoter, Nat Biotechnol, 116:969-977), (Smigocki etc. 1988 for photoinduction promoter, PNAS, 85:5131-5135), (Martineau etc. 1994 for fruit-specific promoter, Plant J, 5:11-19), (Gatz etc. 1998, Trends in Plant Sci. for the injury evoked promoter, 3:352-358), all obtained corresponding anti-ageing plant, but, limited the application of anti-ageing genetic transformation engineering in agriculture production greatly because they or inducible factor self are unfavorable to plant-growth.Nineteen ninety-five, the ipt of Ti-plasmids merges among the promotor of the aging gene SAG12 that Gan and Amasino is special with the Arabidopis thaliana camber and the Agrobacterium Agrobacteriumtumefaciens, has made up the self regulating and control system of a delaying sanility.P
SAG12The principle of work of-ipt is: when blade just senesced, the SAG12 promotor was activated, and ipt begins to express, and cytokinin concentration raises, and aging is suppressed; When cytokinin concentration reaches certain level, old and feeble transference cure, the SAG12 promotor no longer includes activity, and ipt no longer expresses, and the concentration of phytokinin is also controlled, thereby prevents the negative impact of excessive hormone to plant.Use P
SAG12(Can etc. 1995 for-ipt gene transformation tobacco, Science, 270:1986-1988), (Fu Yongcai etc. 1999 for paddy rice, Journal of Agricultural Biotechnology, 01:17-22), (Zhang Saiqun etc. 1999 for tomato, the gardening journal, 26:376-379), (Schroeder etc. 2001, Journal of the American Society for Horticultural, 126 for herbage Bai Sanye, 523-530)), (Chang etc. 2003 for Cauliflower, Plant physiology, 132:2174-2183), lettuce (McCabe etc., 2001), (Yuan Zheng etc. 2002 for green vegetables, Mol.Biol., 28:379-384) and wheat (Xi second places etc. 2004, Scientia Agricultura Sinica, 37:1236-1238), all obtain the transfer-gen plant that leaf senile delays, and do not influenced normal growth and the growth of plant substantially.
The resistance breeding is the important goal of plant breeding work always, because plant is regulated and control by minor-polygene the resistance of adverse circumstances such as low temperature, therefore, may make plant obtain better winter resistance and keep vigorous vitality if cbf1 and ipt gene change plant simultaneously over to.Only made up the binary vector that contains cbf1 or one of them functional gene of ipt in the research in the past.If must carry out cotransformation in the time of will changing two functional genes over to plant simultaneously, its result greatly raises the independent inheritance probability of two functional genes among the render transgenic plant offspring, is difficult to obtain the stable transgenic line of two functional gene linkage inheritances.
Summary of the invention
The objective of the invention is cbf1 and two functional genes of ipt are building up on the same Agrobacterium binary expression vector, so that cbf1 and ipt gene are mediation by Agrobacterium, change plant over to simultaneously and obtain the transgenic line of two functional gene close linkages, genetic stability, resistance of plant and anti-ageing ability are strengthened.
Binary expression vector of the present invention is to comprise the activating transcription factor gene cbf1 of closely linked Arabidopis thaliana cold-responsible genes COR and the bivalent anti-reverse gene bielement expression carrier of isopentenyl transferase genes ipt.
The present invention has successfully made up the activating transcription factor gene cbf1 that comprises closely linked Arabidopis thaliana cold-responsible genes COR and the bivalent anti-reverse gene bielement expression carrier of isopentenyl transferase genes ipt.Utilize this carrier to transform target plant, the transfer-gen plant that is obtained has obviously strengthened the resistance to adverse circumstance, and has delayed the aging that adverse circumstance is brought out.
The binary expression vector that the present invention is constructed, its functional gene cbf1 are that functional gene ipt is the promoter Ps ag by aging gene special in the Arabidopis thaliana by corn ubiquitin promoter Pubi regulation and control
12Regulate and control, its structure is referring to Fig. 1.
The binary expression vector that the present invention is constructed, the about 17.3kb of base pair, wherein the about 11kb in T-DNA district contains inducible promoter Psag
12The ipt gene of regulation and control, the cbf1 gene of constitutive promoter Pubi regulation and control, and the anti-herbicide gene bar and the reporter gene gus of constitutive promoter CaMV35s regulation and control.Respectively it is carried out enzyme with Sac I and KpnI and cut, product is on 1.5% the glue at agarose concentration, and with the voltage electricity of 3v/cm 1h forever, after EB dyeing, should obtain its cracking collection of illustrative plates on the ultraviolet light imaging instrument is Fig. 2.
The construction process of the bivalent anti-reverse gene bielement expression carrier that the present invention makes up is with the middle cloning vector with fusion gene Pubi-cbf1, has fusion gene Psag
12The middle cloning vector of-ipt and binary expression vector p3301 are that initial carrier makes up.
The middle cloning vector that wherein has fusion gene Pubi-cbf1 is that to contain the plasmid transformation escherichia coli of fusion gene Pubi-cbf1 resulting.Classification called after: intestinal bacteria Escherichia (Escherichiacoli), depositary institution's name is called: China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City. Institute of Microorganism, Academia Sinica, preservation date is on August 18th, 2005, deposit number is CGMCCNo.1440, and the preservation name is called: pUBC/DH5a.
The structure iron of plasmid is Fig. 3 among the pUBC/DH5a, and the about 5.2kb of base pair can obtain three fragments of 2.3+2.0+0.9kb with the HindIII single endonuclease digestion.
Has fusion gene Psag
12The middle cloning vector of-ipt is to contain fusion gene Psag
12The plasmid transformation escherichia coli of-ipt is resulting.Classification called after: intestinal bacteria Escherichia (Escherichiacoli), depositary institution's name is called: China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City. Institute of Microorganism, Academia Sinica, preservation date is on August 18th, 2005, deposit number is CGMCCNo.1441, and the preservation name is called: pSAGI/DH5a.
The structure iron of pSAGI/DH5a is Fig. 4, and the about 5.62kb of base pair can obtain two fragments of 2.9+2.72kb with the SacI single endonuclease digestion.
Binary expression vector p3301 buys from PCAMBIA company.
The construction process of bivalent anti-reverse gene bielement expression carrier of the present invention is earlier with Psag
12T-nos in the-ipt fusion gene is inserted on the binary vector, inserts the Pubi-cbf1 fusion gene then, inserts the Psag that does not contain T-nos at last
12-ipt fusion gene, thus complete divalence binary vector obtained.
Specifically be with an EcoR I and SacI enzyme carrier pSAGI that hits, reclaim the T-nos fragment of 0.3kb, cut binary vector p3301 with EcoR I and SacI enzyme simultaneously, reclaim the 11.3kb fragment as carrier, connect on the two segmental corresponding sites with the T4-DNA ligase enzyme, obtain carrier p3301-nos, the new T-nos fragment of inserting 5 ' end is Sac I site and Hind III site successively, be the single endonuclease digestion site, referring to Fig. 5.
With restriction endonuclease Hind III enzyme cutting clone carrier pUBC, reclaim the fusion gene Pubi-cbf1 fragment of about 3kb, cut carrier p3301-nos with restriction endonuclease Hind III enzyme simultaneously, reclaim the 11.6kb fragment as carrier, fusion gene ubi-cbf1 is received on the Hind III site of p3301-nos, obtain carrier p3301C-nos, wherein fusion gene ubi-cbf1 is a clockwise direction, and 5 ' of the new T-nos that inserts end has SacI single endonuclease digestion site on p3301C-nos.Referring to Fig. 7.
With a restriction endonuclease Sac I enzyme cloning vector pSAGI that hits, reclaim the fusion gene Psag that does not have T-nos of about 2.7kb
12-ipt fragment is cut carrier p3301C-nos with restriction endonuclease Sac I enzyme simultaneously, reclaims enzyme and cuts that big fragment will not have the fusion gene Psag of T-nos as carrier in the product
12-ipt fragment, forward are inserted into the SacI site of p3301C-nos, obtain divalence binary vector p3301IC.Referring to Fig. 9.
The invention has the advantages that:
1. the present invention is incorporated into the adversity gene cbf1 of corn ubiquitin promoter regulation and control and the anti-ageing gene ipt of senescence-specific promoter regulation on the same binary vector, be beneficial to two functional genes and transform target plant simultaneously, with one of them binary vector institute plant transformed of two functional genes only arranged compare the resistance of significant raising plant adverse circumstance, and delayed the The Plant Senescence that adverse circumstance is brought out, make plant in adverse circumstance, vegetative period and green phase all obviously prolong.
2. after this binary vector transformed plant, transfer-gen plant was obviously short strong, was contrast so that one of them binary vector institute plant transformed of two functional genes only to be arranged, the obvious overstriking of cane; Leaf thickness increases 10.8-18.5%; The leaf look dark green, and chlorophyll a+b contrasts in the blade increases 22.3-30.6%, and vegetative period, contrast prolonged 30-45 days.Under low temperature or drought stress, proline(Pro) and soluble sugar level are all apparently higher than contrast in the rotaring gene plant blade, and plant also shows stronger winter resistance and drought resistance.
3. vector construction step of the present invention is simple.If according to the conventional method, successively two complete fusion gene Pubi-cbf1, Psag
12-ipt is inserted into relatively difficulty of p3301, because promotor Pubi, Psag
12And the cleavage site that a large amount of restriction restriction endonucleases of using always are arranged on the functional gene ipt dna sequence dna, be structured in them on the same binary vector and just must avoid these restriction enzyme sites.Method commonly used in vector construction is to connect new restriction enzyme site at the two ends of target fragment, and finds suitable intermediate carrier equally very difficult.By analysis, we have adopted three steps successfully two fusion genes to be incorporated on the binary vector.Promptly at first Psag
12T-nos in the-ipt fusion gene is inserted on the binary vector, inserts the Pubi-cbf1 fusion gene then, inserts Psag at last
12-ipt fusion gene, thus complete divalence binary vector obtained.
Description of drawings
The design of graphics of Fig. 1 divalence binary vector p3301IC
Fig. 2 p3301IC enzyme is cut evaluation figure.1 cuts for the KpnI enzyme among the figure: 10.0kb+3.1kb+2.7kb+0.86kb+0.74kb; 2 cut for the SacI enzyme: 14.4kb+2.9kb; M is that Marker is followed successively by from top to bottom: 4.5,3.0,2.0,1.2,0.8,0.5, and 0.2kb.
The design of graphics of Fig. 3 pUBC/DH5a
The design of graphics of Fig. 4 pSAGI/DH5a
The design of graphics of Fig. 5 carrier p3301-nos
Fig. 6 p3301-nos enzyme is cut evaluation figure.1 cuts for the KpnI enzyme among the figure: 0.8+1.8kb; M is that Marker is followed successively by from top to bottom: 4.5,3.0,2.0,1.2,0.8,0.5, and 0.2kb.
The design of graphics of Fig. 7 carrier p3301C-nos
Fig. 8 p3301C-nos enzyme is cut evaluation figure.1 cuts for the KpnI enzyme among the figure: 11.0+2.8+1.8kb; 3 cut for the HindIII enzyme: 11.6+2.0+1.0kb; M is that Marker is followed successively by from top to bottom: 4.5,3.0,2.0,1.2,0.8,0.5, and 0.2kb.
The design of graphics of Fig. 9 divalence binary vector p3301IC
Embodiment
Further illustrate the present invention in the following embodiments, this does not limit the scope of the invention.
One, experiment material and test kit
Used restriction enzyme and ligase enzyme test kit are NEB company and produce, and dna fragmentation reclaims and adopts Tsing-Hua University sky is the centrifugal test kit of post that Time Inc. produces.Competent escherichia coli cell is a Time Inc. available from Tsing-Hua University sky.It is the test kit that Time Inc. produces that plasmid extracts with Tsing-Hua University sky, and all operations program is all undertaken by the test kit specification sheets.
Two, experiment content
With an EcoR I and SacI enzyme carrier pSAGI that hits, reclaim the T-Nos fragment of 0.3kb, cut binary vector p3301 with EcoR I and SacI enzyme simultaneously, reclaim the 11.3kb fragment as carrier, connect on the two segmental corresponding sites with the T4-DNA ligase enzyme, obtain carrier p3301-nos, the new T-nos fragment of inserting 5 ' end is Sac I site and Hind III site successively, is the single endonuclease digestion site.Referring to Fig. 3,4.
Three, experimental procedure
1, plasmid extracts
(1) required equipment: 0.5 μ l-1000 μ l different size pipettor, whizzer.
(2) extract plasmid: program is referring to the test kit specification sheets.
2, plasmid enzyme restriction reaction:
(1) required equipment: 0.5-100 μ l different size pipettor, whizzer, 37 ℃ of incubators:
(2) reaction system is EcoR I and SacI double digestion system:
Plasmid 5 μ l (about 1 μ g)
10×buffer?1 0.5μl
EcoR I 0.5 μ l (about 5 units)
SacI 0.5 μ l (about 5 units)
10×BSA 0.5μl
ddH
2O 43μl
Total 50μl
After above sample was mixed, the complete degestion sample was put 37 ℃ and is incubated overnight.Not exclusively enzyme is cut and is taken out after sample is put 7 ℃ of incubation 4min, adds Loadimg buffer termination reaction immediately.
3, fragment reclaims
(1) required equipment: 0.5 μ l-1000 μ l different size pipettor, DNA electrophoresis apparatus, ultraviolet light imaging system, whizzer.
(2) the target segment reclaims: in concentration is that electrophoresis enzyme is cut product on the 0.8-1.5% agar gel, reclaims the fragment that needs.
4, DNA ligation
(1) required equipment: 0.5-100 μ l different size pipettor, whizzer, PCR instrument (being used to provide 16 ℃ of isoperibols).
(2) reaction system:
Target fragment 10 μ l
10×buffer 1.5μl
T4 ligase enzyme 1.0 μ l (about 10 units)
ddH
2O 0.5μl
Total 15μl
The molar ratio of target fragment and carrier segments is 4: 1 in the reaction system.The sample mixing is placed on 16 ℃ of following ligations 2 hours. and the ligation product is directly used in transformed into escherichia coli or is stored in 4 ℃ of refrigerators standby.
5, connect the product transformed into escherichia coli
(1) required equipment: water-bath, 0.5 μ l-1000 μ l different size pipettor, whizzer.
(2) transformed into escherichia coli: program is referring to the test kit specification sheets.
Reagent equipment and experimental procedure are identical with embodiment 1, and experiment content is as follows:
With restriction endonuclease Hind III enzyme cutting clone carrier pUBC, reclaim the fusion gene Pubi-cbf1 fragment of about 3kb, cut carrier p3301-nos with restriction endonuclease Hind III enzyme simultaneously, reclaim the 11.6kb fragment as carrier, fusion gene Pubi-cbf1 is received on the Hind III site of p3301-nos, obtain carrier p3301C-nos, wherein fusion gene Pubi-cbf1 is a clockwise direction, and 5 ' of the new T-nos that inserts end has SacI single endonuclease digestion site on p3301C-nos.Referring to Fig. 5,6.
Reaction system is the single enzyme system of Hind III:
Plasmid 5 μ l (about 1 μ g)
10×buffer?2 0.5μl
Hind III 0.5 μ l (about 5 units)
ddH
2O 44μl
Total 50μl
Embodiment 3 makes up divalence binary vector p3301IC
Reagent equipment and experimental procedure are identical with embodiment 1, and experiment content is as follows:
With a restriction endonuclease Sac I enzyme cloning vector pSAGI that hits, reclaim the fusion gene Psag that does not have T-nos of about 2.7kb
12-ipt fragment is cut carrier p3301C-nos with restriction endonuclease Sac I enzyme simultaneously, reclaims enzyme and cuts that big fragment will not have the fusion gene Psag of T-nos as carrier in the product
12-ipt fragment, forward are inserted into the SacI site of p3301C-nos, obtain divalence binary vector p3301IC.Referring to Fig. 7,2.
Reaction system is a Sac I single endonuclease digestion system:
Plasmid 5 μ l (about 1 μ g)
10×buffer 10.5μl
SacI 0.5 μ l (about 5 units)
10×BSA 0.5μl
ddH
2O 43.5μl
Total 50μl
Embodiment 4 divalence binary vector p3301IC transformation of tobacco
According to aseptic technique, one of picking contains the single colony lift of Agrobacterium LBA4404 of p3301IC in 3mL liquid LB substratum (table one), this substratum also contains 100mg/L kantlex and 25mg/L Rifampin, 200rmp, cultivated under 28 ℃ of conditions 24 hours, and therefrom got 1mL bacterium liquid again and transferred in the 250mL triangular flask that contains 50 LB nutrient solution expanding propagation 6 hours.When OD/600nm is 0.35, collect bacterium liquid with the 50ml centrifuge tube, with the centrifugal 10min of 4000rmp, precipitate with the resuspended nutrient solution of isopyknic Agrobacterium, greatly different again thalline makes to transform reserve liquid.Medium component also contains the Syringylethanone of 100 μ g/L in addition except that identical with the above-mentioned liquid LB medium component that contains kantlex and Rifampin.
Get the aseptic tobacco leaf that newly sends, draw 3-5 time in vertical master pulse direction with scalper, to draw disconnected vein but the blade branch is fragmentated is degree, be put into then in the reserve liquid and soaked 4 minutes, take out blade, bacterium liquid is blotted with aseptic filter paper in the back, is inoculated on the tobacco division culture medium (it is 0.5mg/L6-benzylamino VITAMIN B4 (6-BA), 0.05mg/L naphthylacetic acid (NAA) that Ms substratum (table two) adds hormone in addition).Cultivate 4 days altogether in 25 ℃ of following dark after, clarify with sterile water wash blade to scavenging solution, aseptic filter paper blots excess liquid, forwards new tobacco division culture medium (containing the 250mg/L cephamycin in addition) to and goes up cultivation 8 days, changes over to then and selects to cultivate on the substratum.Selecting substratum is to add 2.5mg/L grass fourth phosphine (production of Sigma company) and 250mg/L cephamycin in the tobacco division culture medium respectively.Select regeneration induction transformed plant on the substratum, then the regenerated plantlet is being forwarded on the tobacco root media (the additional hormone of Ms substratum (table two) is 0.02mg/L indolylacetic acid (IAA), 0.02mg/L naphthylacetic acid (NAA)), turning out whole plant.
The short shape of transformation of tobacco plant, dark green leaf color.Under environment stresses such as arid, low temperature, the cured matter layer of the very fast formation one deck of transfer-gen plant leaf surface, the blade face is glossy, and the plant dehydration is slow, shows tangible drought resisting, cold-resistant proterties, delays plant senesecence.
Embodiment 5 divalence binary vector p3301IC transform Dianthus caryophyllus L.
Acceptor material is the new leave of Dianthus caryophyllus L..During Agrobacterium, transformed plant regeneration and screening process are with embodiment 4.
It is short according to plant apart from comparison to transform Dianthus caryophyllus L. plant internode, and branch increases, and blade is thicker relatively.Under low temperature stress, apparently higher than unconverted adjoining tree, mda content significantly is lower than adjoining tree to dried meat by acid, soluble sugar content in the rotaring gene plant blade, illustrates that the plant cold tolerance strengthens, and has kept vigorous vitality.
Table one:
LB substratum: Nacl 10g/L, yeast extract 5g/L, peptone 10g/L, pH 7.0.
Table two:
MS minimum medium (Murashige﹠amp; Skoog, 1962) composition
Claims (9)
1. a binary expression vector is characterized in that described binary expression vector is loaded with closely linked divalence adversity gene Arabidopis thaliana cold-responsible genes activating transcription factor gene cbf1 and isopentenyl transferase genes ipt.
2. according to the described binary expression vector of claim 1, it is characterized in that gene cbfl is that gene ipt is by the special aging gene promoter Ps of Arabidopis thaliana ag by corn ubiquitin promoter Pubi regulation and control
12Regulation and control, described binary expression vector structure is:
3. according to the construction process of the described binary expression vector of claim 1, it is characterized in that described binary expression vector be with have corn ubiquitin promoter Pubi and Arabidopis thaliana cold-responsible genes activating transcription factor gene cbf1 mutually fusion gene middle cloning vector, have the special aging gene promoter Ps of Arabidopis thaliana ag
12The middle cloning vector and the binary expression vector p3301 of fusion gene are that initial carrier makes up mutually with isopentenyl transferase genes ipt.
4. according to the described construction process of claim 3, it is characterized in that described corn ubiquitin promoter Pubi and the Arabidopis thaliana cold-responsible genes activating transcription factor gene cbf1 middle cloning vector of fusion gene mutually that has, be to contain corn ubiquitin promoter Pubi and Arabidopis thaliana cold-responsible genes activating transcription factor gene cbf1 the plasmid transformation escherichia coli of fusion gene is resulting mutually, described intestinal bacteria deposit number is CGMCCNo.1440.
5. according to the described construction process of claim 3, it is characterized in that the described special aging gene promoter Ps of the Arabidopis thaliana ag that has
12With the isopentenyl transferase genes ipt middle cloning vector of fusion gene mutually, be to contain the special aging gene promoter Ps of Arabidopis thaliana ag
12The plasmid transformation escherichia coli of fusion gene is resulting mutually with isopentenyl transferase genes ipt, and described intestinal bacteria deposit number is CGMCCNo.1441.
6. according to the described construction process of claim 3, the construction process that it is characterized in that this bivalent anti-reverse gene bielement expression carrier is earlier with the special aging gene promoter Ps of Arabidopis thaliana ag
12With isopentenyl transferase genes ipt mutually the T-Nos in the fusion gene be inserted on the binary vector, insert corn ubiquitin promoter Pubi and Arabidopis thaliana cold-responsible genes activating transcription factor gene cbf1 fusion gene mutually then, insert the special aging gene promoter Ps of the Arabidopis thaliana ag that does not contain T-Nos at last
12With isopentenyl transferase genes ipt fusion gene mutually, thereby obtain complete divalence binary vector.
7. according to the described construction process of claim 6, it is characterized in that cutting the special aging gene promoter Ps of Arabidopis thaliana ag with EcoR I and SacI enzyme
12The intermediate carrier that merges mutually with isopentenyl transferase genes ipt, reclaim the T-Nos fragment of 0.3kb, cut binary vector p3301 with EcoR I and SacI enzyme simultaneously, reclaim the 11.3kb fragment as carrier, connect on the two segmental corresponding sites with the T4-DNA ligase enzyme, obtain p3301 and the carrier that Nos merges mutually, the T-Nos fragment 5 ' end that newly inserts has the restriction enzyme site of restriction endonuclease Sac I and Hind III.
8. according to the described construction process of claim 6, it is characterized in that the cloning vector that merges mutually with restriction endonuclease Hind III enzyme jade cutting rice ubiquitin promoter Pubi and Arabidopis thaliana cold-responsible genes activating transcription factor gene cbf1, reclaim the gene fragment that Pubi and cbf1 merge mutually, cut the carrier that p3301 and Nos merge mutually with restriction endonuclease Hind III enzyme simultaneously, reclaim the 11.6kb fragment as carrier, with corn ubiquitin promoter Pubi and Arabidopis thaliana cold-responsible genes activating transcription factor gene cbf1 mutually fusion gene receive p3301 and NOS merges on the segmental Hind III site mutually, obtain the carrier that p3301C and NOS merge mutually, wherein corn ubiquitin promoter Pubi and Arabidopis thaliana cold-responsible genes activating transcription factor gene cbfl mutually fusion gene be clockwise direction, merge mutually at p3301C and NOS that the 5 ' end of the new T-Nos that inserts has SacI single endonuclease digestion site on the fragment.
9. according to the described construction process of claim 6, it is characterized in that cutting the special aging gene promoter Ps of Arabidopis thaliana ag with restriction endonuclease Sac I enzyme
12With the isopentenyl transferase genes ipt middle cloning vector of fusion gene mutually, reclaim the special aging gene promoter Ps of the Arabidopis thaliana ag that does not have T-Nos
12With isopentenyl transferase genes ipt fusion gene fragment mutually, cut the carrier that p3301C and Nos merge mutually with restriction endonuclease Sac I enzyme simultaneously, reclaim enzyme and cut in the product big fragment as carrier, with the special aging gene promoter Ps of the Arabidopis thaliana that does not have T-Nos ag
12With isopentenyl transferase genes ipt fusion gene fragment mutually, forward is inserted into p3301C and merges segmental SacI site mutually with NOS, obtains following divalence binary vector p3301IC,
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| CN101372692B (en) * | 2008-04-23 | 2010-08-18 | 吉林农业大学 | Ice flower low temperature stress transcription factor AaCBF gene sequence, and clone and use thereof |
| CN102206641B (en) * | 2010-04-23 | 2013-02-27 | 深圳华大基因科技有限公司 | Promoter SbUbi1, its preparation method and use |
| CN102206640B (en) * | 2010-04-23 | 2012-10-10 | 深圳华大基因科技有限公司 | Promoter SbUbi2, its preparation method and use |
| CN102234646B (en) * | 2010-04-23 | 2014-10-22 | 深圳华大基因科技有限公司 | Promoter SbUbi1, preparation method and application thereof |
| CN102191253B (en) * | 2011-04-03 | 2013-04-17 | 浙江大学 | Plant disease-resistant regulation and control gene UEP and application thereof |
| CN102329812B (en) * | 2011-09-21 | 2014-03-19 | 西南大学 | Multi-gene binary expression vector constructed by using homologous recombination and preparation method and application of multi-gene binary expression vector |
| CN102816790B (en) * | 2012-09-04 | 2014-02-19 | 河北农业大学 | Plant expression vector pCamE and application thereof in gene transformation |
| CN108486142A (en) * | 2018-04-11 | 2018-09-04 | 大连民族大学 | A kind of construction method of the genetic engineering bacterium of expression prenyltransferase ComQ and application |
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