CN101962649A - Wild eggplant salt tolerant gene StP5CS and herbicide resistant plant expression vector thereof - Google Patents
Wild eggplant salt tolerant gene StP5CS and herbicide resistant plant expression vector thereof Download PDFInfo
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- CN101962649A CN101962649A CN2010105228783A CN201010522878A CN101962649A CN 101962649 A CN101962649 A CN 101962649A CN 2010105228783 A CN2010105228783 A CN 2010105228783A CN 201010522878 A CN201010522878 A CN 201010522878A CN 101962649 A CN101962649 A CN 101962649A
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Abstract
The invention belongs to the field of molecular biology and discloses wild eggplant salt tolerant gene StP5CS and an herbicide resistant plant expression vector thereof. The sequence of the wild eggplant salt tolerant gene StP5CS is SEQ ID NO.1. The herbicide resistant plant expression vector of the wild eggplant salt tolerant gene StP5CS is obtained by inserting the wild eggplant salt tolerant gene StP5CS into the locus BamH I and Sal I of an intermediate vector pCAMBIA3301-T800. The gene StP5CS applies the plant expression vector to the genetic transformation of crops; the gene StP5CS is expressed in quantity under the action of a promoter CaMV35S to synthesize a large number of praline biosynthesis key enzymes, then praline is accumulated in the plant so that the salt tolerance of the plant is improved. Meanwhile, the expression product Phosphinothricin N-transacetylase of the gene bar enables the plant to resist the herbicide using L-Phosphinothricin (PPT) as activate component.
Description
Technical field
The invention belongs to biology field, relate to wild eggplant resistant gene of salt StP5CS and herbicide resistant plants expression vector thereof.
Background technology
Along with the aggravation of water globe crisis of resource and soil salinization problem, salt stress has become influences plant growth, causes the important factor of crop failure.Nearly during ploughing, China have 1/10 to be salinization soil, mainly be distributed in major grain producing areas such as northwest, North China, salinification can cause the destruction of interior moisture of vegetable cell and ionic equilibrium, cause oxidative stress, destroy proteinic function, cause the serious variation of plant aspect form, physiology, biochemistry and molecule, influence growth and the output of crop.Transgenic technology is one of effective way that solves the salinification problem, with salt-resistant related gene clone and conversion crop, makes its overexpression in crop, will significantly improve the salt resistance ability of crop.
Δ '-pyrroline-5-carboxylic acid synthetase (Δ '-pyrroline-5-carboxylate synthetase, P5CS) be a key enzyme of proline(Pro) route of synthesis in the higher plant body, this enzyme has Glutamate kinase (γ-glutamylkinase, γ-GK) and gamma-glutamyl phosphoric acid reduction enzyme (γ-glutamyl phosphate reductase, the double activity of γ-GPR), the biosynthetic first two steps of catalysis proline(Pro): L-glutamic acid phosphorylation and glutamic acid-reduction (reference: Hu CAA, Delauney A J, Verma DPS.A bifunctional enzyme (Δ '-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in prolinebiosynthesis in plants.Proc Natl Acad Sci.1992,89:9354-9358).Resistant gene of salt P5CS transforms plant and improves P5CS that existing many reports: the Zhu etc. (1998) of salt tolerance of plant will derive from the aconite leaves cowpea and be connected rice transformation after the stress-inducing promotor, proline(Pro) is being coerced excess dynamic accumulation down, the biomass that its s-generation transfer-gen plant shows under osmotic stress increases apparently higher than non-transgenic group (reference: Zhu BC, Su J, Chang MC, Verma DPS, Fan YL, WU R.Overexpression of a Δ '-pyrroline-5-carboxylate synthetase gene and analysis of tolerance to water-andsalt-stress in transgenic rice.Plant Science.1998,139:41-48); Aida etc. (2005) with the P5CS of Arabidopis thaliana by the agrobacterium mediation converted potato, under 100mM NaCl culture condition, the proline(Pro) accumulation volume of transgenic Rhizoma Solani tuber osi is higher than control group, transgenic Rhizoma Solani tuber osi has shown higher tolerance (reference: Aida H S for high-salt stress, Radhia G B, Amira B, Lei L J, Arnould S, Samir J.Overexpression of Δ '-pyrroline-5-carboxylate synthetase increases proline productionand confers salt tolerance in transgenic potato plants.Plant Science.2005,169:746-752).
The bar gene obtains by separating in the streptomyces hygroscopicus (Streptomyce hygroscopicus) the earliest; its coded product phosphinothricin N-acetyl-transferase can make weedicide Basta; effective constituent PPT among the liberty (phosphinothricin) is degraded into acetyl derivatives and inactivation (reference: Thompson CJ; MovvaNR; Tizard R; Crameri R; Davies JE; Lauwereys M; Botterman J.Characterization ofthe herbicide-resistance gene bar from Streptomyces hygroscopicus.EMBO J.1987,6:2519-2523).At present, the bar gene is widely used in the production of antiweed genetically modified crops, as soybean (reference: Zhang Z, Xing A, Staswick P, Clemente T.The use of glufosinate as aselective agent in Agrobacterium mediated transformation of soybean.Plant CellTissue Organ Culture, 1999,56:37-46), paddy rice (Toki S, Susumu, Chyuhei M.Expression of a maize ubiquitin gene promoter-bar chimeric gene in transgenic riceplants.Plant Physiol.2002,100 (3): 1503-1507), wheat (Liang Hui, Tang Shunxue, tension and relaxation. improve the research of wheat cdna marksmanship transformation frequency. Acta Genetica Sinica .1999,26 (6): 643-648) etc.
Summary of the invention
The objective of the invention is provides a new wild eggplant resistant gene of salt StP5CS for solving the problem of raising farm crop salt tolerance.
Another object of the present invention provides this resistant gene of salt StP5CS herbicide resistant plants expression vector and construction process thereof.Constructed plant expression vector can be directly used in agriculture bacillus mediated crop genetic and transform, and initiative salt tolerant and antiweed new germ plasm can be used for the variety of crops improvement.
Technical problem of the present invention can solve by following technical solution:
Wild eggplant resistant gene of salt StP5CS (Δ '-pyrroline-5-carboxylic acid synthetase gene StP5CS), the sequence of this gene is SEQ ID NO.1.
Wild eggplant resistant gene of salt StP5CS herbicide resistant plants expression vector is made of wild eggplant resistant gene of salt StP5CS of the present invention and plant expression vector.
Wherein, described plant expression vector is preferably by using EcoR I/HindIII double digestion plant expression vector pCAMBIA3301 and complete sequence synthetic T800DNA fragment respectively, reclaim the big fragment of pCAMBIA3301 of 10986bp and the T800DNA small segment of 849bp, connect the intermediate carrier pCAMBIA3301-T800 that described two fragments obtain.
Wild eggplant resistant gene of salt and anti-herbicide gene plant expression vector, its construction process is as follows:
1) clone of wild eggplant resistant gene of salt StP5CS
Wild eggplant young leaflet tablet with extraction is a material, extracts total RNA, and reverse transcription is cDNA, design primer amplification StP5CS gene is a template with the cDNA of reverse transcription, carries out polymerase chain reaction PCR, product is connected to the pMD19-T carrier, transforms the TOP10 competent cell, carries out sequencing;
2) transformation of intermediate carrier pCAMBIA3301-T800
The synthetic T800 fragment SEQ ID NO.2 that contains CaMV35S promotor, multiple clone site, Nos terminator of complete sequence, wherein, the upstream of promotor and the downstream of terminator have been introduced EcoRI and HindIII restriction enzyme site respectively; EcoRI and HindIII double digestion pCAMBIA3301 (containing the bar gene) and described T800 fragment, reclaim the big fragment of pCAMBIA3301 (containing the bar gene) of 10986bp and the T800DNA small segment of 849bp, connect described two fragments and obtain intermediate carrier pCAMBIA3301-T800 (containing the bar gene), product transforms the TOP10 competent cell, extract plasmid, carry out enzyme and cut checking;
3) structure of plant expression vector pCAMBIA3301-T800-StP5CS
The design primer is a template with the wild eggplant cDNA of step 1) gained, carry out the PCR reaction with the high-fidelity enzyme, introduce BamH I and Sal I restriction enzyme site respectively at the upstream and downstream of StP5CS gene, with BamH I and Sal I double digestion PCR product and pCAMBIA3301-T800 plasmid, reclaim big fragment of plasmid pCAMBIA3301-T800 (11835bp) and StP5CS small segment (2250bp), connect described two fragments, connect product and transform the TOP10 competent cell, extract plasmid, carry out enzyme and cut checking, obtain described wild eggplant resistant gene of salt StP5CS herbicide resistant plants expression vector pCAMBIA3301-T800-StP5CS.
Beneficial effect:
1. wild eggplant resistant gene of salt StP5CS provided by the invention is a new resistant gene of salt, and this gene can improve the farm crop salt tolerance.
The present invention with wild eggplant Δ '-pyrroline-5-carboxylic acid synthetase resistant gene of salt StP5CS is cloned among the intermediate carrier pCAMBIA3301-T800 that contains anti-herbicide gene (bar gene), the StP5CS gene is overexpression under the startup of CaMV35S promotor, a large amount of proline biosynthesis biosynthesizing key enzymes, the plant interior accumulation proline(Pro) improves plant salt endurance.Simultaneously, bar expression of gene product phosphinothricin N-acetyl-transferase, (phosphinothricin PPT) is the weedicide of activeconstituents with L-Phosphinothricin to make the plant opposing.
3. the wild eggplant resistant gene of salt StP5CS herbicide resistant plants expression vector of the present invention's structure is a reported first, but the render transgenic farm crop have antiweed and improve the salt tolerance double effects, can be directly used in agriculture bacillus mediated farm crop genetic transformation, initiative salt tolerant and antiweed new germ plasm, for the cultivation of good crop new variety and widespread use aborning, significant.
Description of drawings
Fig. 1 StP5CS agarose gel electrophoresis is analyzed
1：DNAMarker(2Kb/1Kb/0.75Kb/0.5Kb/0.25Kb/0.1Kb)
2：StP5CS；
Fig. 2 intermediate carrier pCAMBIA3301-T800 plasmid double digestion detects the agarose gel electrophoresis analysis
1：DNAMarker(2Kb/1Kb/0.75Kb/0.5Kb/0.25Kb/0.1Kb)
2：pCAMBIA3301-T800/Hind?III/EcoR?I；
3：pCAMBIA3301-T800
Fig. 3 plant expression vector pCAMBIA3301-T800-StP5CS plasmid double digestion detects the agarose gel electrophoresis analysis
1：DNAMarker(15Kb/10Kb/7.5Kb/5Kb/2.5Kb/1Kb/0.25Kb)
2，3：pCAMBIA3301-T800-StP5CS/BamH?I/Sal?I；
Fig. 4 plant expression vector pCAMBIA3301-T800-StP5CS collection of illustrative plates
Embodiment
The clone of embodiment 1.StP5CS:
' as material, the seedling phase (4-5 sheet true leaf) is used 100mmolL to Torvum Vigor ' (for Japanese stock variety, available from Japanese Takii seedling Co., Ltd.) to select wild eggplant (Solanum torvum Swartz) for use
-1NaCl coerces continuously and handled 5 days, gets young leaflet tablet, with reference to TaKaRa company total RNA extraction reagent box specification sheets method, extracts the total RNA of blade, spins (Shanghai) cDNA of Bioisystech Co., Ltd synthetic agent box ReverTraAce-a-according to Japan
TMIllustrate that getting the total RNA reverse transcription of 2 μ g becomes cDNA, with RNase digested cdna product, with reference to tomato salt-tolerant gene P5CS sequence information (NCBI accession number: U60267), design primer amplification StP5CS through the Oligo6.0 software analysis;
Upstream primer ZHPs:SEQ ID NO.3, downstream primer ZHPa:SEQ ID NO.4 is a template with the leaf cDNA of extracting, and carries out the PCR reaction: 50 μ L reaction systems: 10 * RCR Buffer, 5.0 μ L, ZHPs, each 1.0 μ L (20 μ molL of ZHPa primer
-1), dNTP mix 4.0 μ L (2.5mmolL
-1), Taq DNAPolymerase 0.2 μ L, cDNA template 1 μ L, ddH
2O 37.8 μ L; Response procedures: 95 ℃ of pre-sex change 4min, 94 ℃ of 30sec that unwind then, 52.7 ℃ of annealing 30sec, 72 ℃ are extended 2min 30sec, reacts 30 circulations, 72 ℃ of extension 10min; The PCR product reclaims test kit (AXYGEN) with gel and reclaims agarose gel electrophoresis analysis (collection of illustrative plates is seen Fig. 1) behind the purifying, uses T
4Dna ligase (TaKaRa) is connected to pMD19-T carrier (TaKaRa), transforms TOP10 competent cell (sky, Nanjing is a Bioisystech Co., Ltd), carries out sequencing, and sequence is SEQ ID NO.1.
The structure of embodiment 2 plant bivalent expression vector pCAMBIA3301-T800-StP5CS
1) transformation of intermediate carrier pCAMBIA3301-T800
The synthetic T800 fragment (seeing SEQ ID NO.2) that contains CaMV35S promotor, multiple clone site, Nos terminator of complete sequence is introduced EcoRI (Promega) and HindIII (Promega) restriction enzyme site (the prosperous bio tech ltd of Beijing ancient cooking vessel state) respectively at two ends; EcoRI (Promega) and HindIII (Promega) double digestion pCAMBIA3301 (the international agriculture molecular biology of Australia is used the center) and T800 fragment, enzyme are cut product in 1: 4 ratio T
4Dna ligase (TaKaRa) connects, and product transforms TOP10 competent cell (sky, Nanjing is a Bioisystech Co., Ltd), extracts plasmid, and enzyme is cut checking (collection of illustrative plates is seen Fig. 2):
1. the synthetic T800 fragment (seeing SEQ ID NO.2) that contains CaMV35S promotor, multiple clone site, Nos terminator of complete sequence is introduced EcoRI (Promega) and HindIII (Promega) restriction enzyme site (the prosperous bio tech ltd of Beijing ancient cooking vessel state) respectively at two ends; Get each 15 μ L of plasmid vector pCAMBIA3301 and T800 fragment, use EcoRI (Promega) and HindIII (Promega) double digestion respectively, 50 μ L enzymes are cut system: 10 * Buffer E, 5 μ L, BSA0.5 μ L, plasmid pCAMBIA3301 or T800 15 μ L, EcoR I 1.25 μ L, Hind III 1.25 μ L, ddH
2O 27 μ L; 37 ℃ of endonuclease reaction 3h, enzyme cut product and carry out the agarose gel electrophoresis analysis, reclaim test kit (AXYGEN) with gel and reclaim big fragment of plasmid pCAMBIA3301 and T800 small segment.
2. reclaim big fragment of plasmid pCAMBIA3301 (10986bp) and T800 small segment (849bp), according to 1: 4 ratio T
4DNA enzyme (TaKaRa) connects, ligation system (25 μ L): 10 * T
4LigaseBuffer 2.5 μ L, the big fragment 2 μ L of pCAMBIA3301, T800 small segment 8 μ L, T
4Dna ligase 1 μ L, ddH
2O 11.5 μ L; 16 ℃ of reaction overnight are got 10 μ L and are connected product conversion TOP10 competent cell, 37 ℃ of incubated overnight of coated plate; After the picking mono-clonal enlarged culturing, extract plasmid, carry out enzyme and cut checking; The transformation of pCAMBIA3301-T800 intermediate carrier is finished;
2.) the structure of plant expression vector pCAMBIA3301-T800-StP5CS
The design primer carries out the PCR reaction, upstream and downstream at goal gene StP5CS is introduced restriction enzyme site BamH I and SmlI respectively, BamH I (Promega) and Sal I (Promega) double digestion pCAMBIA3301-T800 intermediate carrier and PCR product, reclaim big fragment of pCAMBIA3301-T800 and StP5CS small segment, in 1: 4 ratio T
4Dna ligase (TaKaRa) connects, and product transforms TOP10 competent cell (sky, Nanjing is a Bioisystech Co., Ltd), extracts plasmid, carries out double digestion checking (collection of illustrative plates is seen Fig. 3):
1. with wild eggplant leaf cDNA as template, with high-fidelity enzyme (PrimeSTAR
TMHS DNAPolymerase TaKaRa) carries out the PCR reaction, 50 μ L reaction systems: 10 * RCR Buffer, 5.0 μ L, ZHPMBs2 (SEQ ID NO.5), each 1.0 μ L (20 μ molL of ZHPMSa2 (SEQ ID NO.6) primer
-1), dNTP mix 4.0 μ L (2.5mmolL
-1), PrimeSTAR
TMHS DNAPolymerase 0.4 μ L, cDNA template 1 μ L, ddH
2O 37.6 μ L; Response procedures: 95 ℃ of pre-sex change 4min, 98 ℃ of 10sec that unwind then, 68 ℃ are extended 2min 30sec, reacts 30 circulations, 72 ℃ of extension 10min.The PCR product reclaims test kit (AXYGEN) with gel and reclaims, and carries out sequencing;
2. 1. the plasmid vector pCAMBIA3301-T80015 μ L and that gets reincarnate goes on foot resulting StP5CS with BamH I (Promega) and Sal I (Promega) double digestion, double digestion system (50 μ L): 10 * Buffer E, 5 μ L, BSA 0.5 μ L, plasmid pCAMBIA3301-T800 or StP5CS 15 μ L, BamHI 1.25 μ L, SalI 1.25 μ L, ddH
2O 28.25 μ L; 37 ℃ of reaction 3h; The double digestion product carries out the agarose gel electrophoresis analysis, reclaims test kit (AXYGEN) with gel and reclaims big fragment of plasmid pCAMBIA3301-T800 and StP5CS small segment;
3. the big fragment of plasmid pCAMBIA3301-T800 and the StP5CS small segment of Hui Shouing according to 1: 4 ratio, used T
4Dna ligase (TaKaRa) connects, ligation system (25 μ L): 10 * T
4LigaseBuffer 2.5 μ L, the big fragment 2 μ L of pCAMBIA3301-T800, StP5CS small segment 8 μ L, T
4Dna ligase 1 μ L, ddH
2O 11.5 μ L; 16 ℃ of ligations of spending the night are got 10 μ L and are connected product conversion TOP10 competent cell.37 ℃ of incubated overnight, picking mono-clonal enlarged culturing is extracted plasmid, carries out enzyme and cuts checking.Successfully constructing of plant expression vector pCAMBIA3301-T800-StP5CS (collection of illustrative plates being seen Fig. 4).
In sum, the present invention has made up the plant expression vector pCAMBIA3301-T800-StP5CS that contains resistant gene of salt and anti-herbicide gene, wherein StP5CS reported first.Constructed carrier can import in soybean and other crops, improves the salt tolerance of crop.
Claims (4)
1. wild eggplant resistant gene of salt StP5CS, the sequence that it is characterized in that this gene is SEQ ID NO.1.
2. wild eggplant resistant gene of salt StP5CS herbicide resistant plants expression vector is characterized in that being made of described wild eggplant resistant gene of salt StP5CS of claim 1 and plant expression vector.
3. wild eggplant resistant gene of salt StP5CS herbicide resistant plants expression vector according to claim 2, it is characterized in that described plant expression vector is by using EcoR I/Hind III double digestion plant expression vector pCAMBIA3301 and complete sequence synthetic T800 dna fragmentation respectively, reclaim the big fragment of pCAMBIA3301 of 10986bp and the T800 DNA small segment of 849bp, connect the intermediate carrier pCAMBIA3301-T800 that described two fragments obtain.
4. the construction process of the described wild eggplant resistant gene of salt StP5CS herbicide resistant plants expression vector of claim 3 is characterized in that comprising the steps:
1) clone of wild eggplant resistant gene of salt StP5CS
Wild eggplant young leaflet tablet with extraction is a material, extracts total RNA, and reverse transcription is cDNA, design primer amplification StP5CS gene is a template with the cDNA of reverse transcription, carries out polymerase chain reaction PCR, product is connected to the pMD19-T carrier, transforms the TOP10 competent cell, carries out sequencing;
2) transformation of intermediate carrier pCAMBIA3301-T800
The synthetic T800 fragment SEQ ID NO.2 that contains CaMV35S promotor, multiple clone site, Nos terminator of complete sequence, wherein, the upstream of promotor and the downstream of terminator have been introduced EcoRI and HindIII restriction enzyme site respectively; EcoRI and HindIII double digestion pCAMBIA3301 and described T800 fragment, reclaim the big fragment of pCAMBIA3301 of 10986bp and the T800DNA small segment of 849bp, connect described two fragments and obtain intermediate carrier pCAMBIA3301-T800, product transforms the TOP10 competent cell, extract plasmid, carry out enzyme and cut checking;
3) structure of plant expression vector pCAMBIA3301-T800-StP5CS
The design primer is a template with the wild eggplant cDNA of step 1) gained, carry out the PCR reaction with the high-fidelity enzyme, introduce BamH I and Sal I restriction enzyme site respectively at the upstream and downstream of StP5CS gene, with BamHI and Sal I double digestion PCR product and pCAMBIA3301-T800 plasmid, reclaim the big fragment of plasmid pCAMBIA3301-T800 of 11835bp and the StP5CS small segment of 2250bp, connect described two fragments, connect product and transform the TOP10 competent cell, extract plasmid, carry out enzyme and cut checking, obtain described wild eggplant resistant gene of salt StP5CS herbicide resistant plants expression vector pCAMBIA3301-T800-StP5CS.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017824A (en) * | 2014-06-19 | 2014-09-03 | 南京农业大学 | Culture method of transgenic soybean with OsPT6 phosphorus being efficiently utilized |
| CN109640631A (en) * | 2016-06-17 | 2019-04-16 | 积水化学工业株式会社 | The method for improving plant salt tolerance |
-
2010
- 2010-10-28 CN CN2010105228783A patent/CN101962649A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017824A (en) * | 2014-06-19 | 2014-09-03 | 南京农业大学 | Culture method of transgenic soybean with OsPT6 phosphorus being efficiently utilized |
| CN109640631A (en) * | 2016-06-17 | 2019-04-16 | 积水化学工业株式会社 | The method for improving plant salt tolerance |
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