CN101838664A - Method for enhancing freezing resistance of tobacco by using genetic engineering method - Google Patents
Method for enhancing freezing resistance of tobacco by using genetic engineering method Download PDFInfo
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- CN101838664A CN101838664A CN200910047765A CN200910047765A CN101838664A CN 101838664 A CN101838664 A CN 101838664A CN 200910047765 A CN200910047765 A CN 200910047765A CN 200910047765 A CN200910047765 A CN 200910047765A CN 101838664 A CN101838664 A CN 101838664A
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- henbane
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Abstract
The invention belongs to the fields of molecular biology, enzymology, physiology, thremmatology, genetic engineering and the like, and in particular relates to a method for constructing an efficient plant expression vector and an engineered strain by using the cDNA of a henbane AOC gene to express the gene, screen a transformant and verify the freezing resistance in the tobacco. The method comprises the following steps of: cloning the AOC gene from the henbane; constructing the efficient plant expression vector; importing agrobacterium and genetically transforming the tobacco; and measuring the electric conductivity of transgenic tobacco leaves subjected to low-temperature treatment. The method has the advantages of improving the freezing resistance of the tobacco by excessively expressing the henbane AOC gene in the tobacco, and improving the germ plasma of the tobacco and enhancing the culture suitability by culturing the tobacco transformant having improved freezing resistance under a proper condition and obtaining transgenic progenies.
Description
Technical field
The invention belongs to fields such as molecular biology, zymetology, physiology, thremmatology and genetically engineered.Relate to a kind of method of utilizing genetic engineering technique to improve freezing resistance of tobacco, be specifically related to utilize the cDNA structure high-efficiency plant expression vector of henbane AOC gene and engineering strain in tobacco, to express the specific procedure of this gene, screening transformant, checking anti-freezing property.
Background technology
The jasmine acid is ubiquitous a kind of hormone in the vegitabilia, the signal conductive process of stress reaction under involved in plant development and the environment stress.Endogenous and jasmonic compounds external source can significantly improve plant to physical abuse, animal bait, the tolerance of unfavorable external environments such as disease and pest infects, low temperature, high salt.In plant, the route of synthesis of jasmine acid is the linolenic acid that discharges from cytolemma, (catalysis of a series of enzymes such as 12-oxo-(10,15Z)-phytodienoic acid) reductase enzyme, tryptophan side-chain alpha, jasmonic methylase generates jasmonic and methyl jasmonic at lipoxygenase, allene oxide synthase synthetic enzyme, allene oxide synthase cyclase, OPDA.Wherein because allene oxide synthase meeting spontaneous hydrolysis is particularly important in the route of synthesis that acts on the jasmine acid of allene oxide synthase cyclase thus.
As a kind of important model plant, and be easy to carry out genetic transformation, tobacco is the popular object during the signal pathway of plant is studied; As a kind of cash crop of extensive plantation, the anti-adversity of tobacco has very important significance on species quality and economic worth.The freeze proof research of tobacco relates to beta-glucuronidase enzyme (GUS) albumen at present, hemocuprein (SOD) albumen etc.
Comparatively speaking, the anti-freezing property that the genetically engineered of key enzyme allene oxide synthase cyclase that is used to come from the jasmine acid route of synthesis of henbane improves tobacco systemicly has very big advantage: (1) is in the synthetic and signal conduction studies field of jasmine acid, global researchist has carried out the further investigation of decades, its physiological action and biochemical mechanism are more clearly, for the gene that utilizes jasmine acid biosynthetic pathway in genetically engineered has been laid good theory and practice basis.(2) henbane is a kind of and the very close plant of Solanaceae of tobacco sibship, and the high reactivity effect can be expressed and reach to the allene oxide synthase cyclase that derives from henbane smoothly in tobacco.
At present, there is not using gene engineering technique mentioned in any and the present patent application that the enzyme gene of the jasmonic material biosynthetic pathway of henbane especially allene oxide synthase cyclase is forwarded in the tobacco as yet, improve the relevant report of freezing resistance of tobacco, comprise patent and document.
Summary of the invention
First purpose of the present invention just provides a kind of method of utilizing genetic engineering technique to improve freezing resistance of tobacco, and the allene oxide synthase cyclase gene that this method will derive from henbane forwards in the tobacco.
Second purpose of the present invention provides a kind of efficient expression vector, and it comprises the allene oxide synthase cyclase gene segment of above-mentioned henbane.
The 3rd purpose of the present invention provides the above-mentioned efficient expression vector transformed host cells of a kind of usefulness.This host cell is a tobacco in example of the present invention.
The present invention also provides utilized raising that genetically engineered the obtains tobacco cell of anti-freezing property and filial generation, regeneration plant, plant tissue or the seed of plant and cultivation thereof.
The purpose of present method is achieved through the following technical solutions:
The isolated dna molecular of the present invention has the core segment of the nucleotide sequence of codifying for allene oxide cyclase, this core segment derives from the nucleotide sequence of the henbane allene oxide synthase cyclase gene of being logined by the applicant (AY708383) in GenBank, with primer P1 (5 '-GA
ACTAGTATG GCT ACT GCT TCC TCA G-3 ', the SpeI restriction enzyme site is represented with underscore) and P2 (5 '-GC
GGTGACCTCA ATT AGT GAA ATT TTT CAG-3 ', the BstEII restriction enzyme site is represented with underscore), adopt the PCR method can from henbane cDNA library, amplify this core segment, the cDNA sequence of henbane allene oxide synthase cyclase gene such as SEQ IDNO1:(henbane (Hyoscyamus niger) allene oxide synthase cyclase gene, total length 1045bp, Genbank accession number AY708383, the open reading frame position is the 71st bit base to the 817 bit bases).
The invention discloses a kind of method of utilizing transgenic technology to improve the tobacco freezing tolerance, it is characterized in that utilizing the pulsating expression vector of Nucleotide core, adopt any transgenic method in tobacco cell, tissue, organ, plant, to express with coding henbane allene oxide synthase cyclase:
Its step is as follows:
(1) adopt any possible means (as methods such as gene clone, synthetic genes) to obtain the core segment of henbane allene oxide synthase cyclase gene;
(2) henbane allene oxide synthase cyclase gene core segment operationally is connected in expression regulation sequence, forms expression vector;
(3) adopt any transgenic method to shift henbane allene oxide synthase cyclase gene core segment in tobacco cell, tissue, organ, plant;
(4) screen and identify transformant under given conditions;
(5) under the condition that is fit to, cultivate the tobacco transformant that anti-freezing property improves, obtain transgenic progeny.
The carrier that the present invention relates to comprises the pulsating DNA of core of the nucleotide sequence with coding henbane allene oxide synthase cyclase.
With the life entity that aforesaid method obtains, it is cell, tissue, organ, the plant of the anti-freezing property tobacco of having improved.
In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid etc.In the present invention, term " life entity " refers to cell, tissue, organ, the plant of tobacco.
In the present invention, term " any possible means " specifically comprises homology clone (as RT-PCR, RACE etc.), library screening then synthetic henbane allene oxide synthase cyclase gene and varient thereof for obtaining the method for henbane allene oxide synthase cyclase gene.
In the present invention, term " any transgenic method " comprises that the conversion of agrobacterium tumefaciens Ti-plasmids mediated gene, the plasmid-mediated gene transformation of Agrobacterium rhizogenes Ri, the conversion of plant viral vector mediated gene, the conversion of PEG mediated gene, liposome-mediated gene transformation, the conversion of electric shocking method mediated gene, ultrasonic-mediated gene transformation, microinjection mediated gene transform, the laser microbeam mediated gene transforms, the particle gun mediated gene transforms, the pollen tube channel mediated gene transforms, sexual cell infusion method mediated gene transforms.
In the present invention, term " screen under given conditions and identify transformant " is meant the transformant of selecting antibiotics resistance under the condition of isolated culture with microbiotic (kantlex, Totomycin, G418 etc.); Can use methods such as PCR, Southern hybridization, Northern hybridization and Western trace to identify transformant.
In the present invention, term " is cultivated the tobacco transformant that anti-freezing property improves, is obtained transgenic progeny " and is meant the transformant isolated culture through identifying under the condition that is fit to, and detects anti-freezing property, the good transformant that the screening anti-freezing property improves is cultivated, and obtains transgenic progeny.
In the present invention, clone's allene oxide synthase cyclase gene core segment from henbane, and made up high efficiency plant expression vector, import Agrobacterium and genetic transformation tobacco, to improve the anti-freezing property of tobacco, improved the germplasm of tobacco, strengthened the cultivation suitability.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these to implement only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, condition described in for example " molecular cloning " (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Embodiment 1
Henbane allene oxide synthase cyclase gene core is pulsating synthetic, and the structure of expression vector and engineering bacteria
1. the pulsating acquisition of henbane allene oxide synthase cyclase gene core
Full length sequence according to henbane allene oxide synthase cyclase gene; between the 71st base to the 817 bases of the inside of this gene, design primer; amplification 747bp sequence is as gene segment; and according to the polyclone restriction enzyme site of plant expression vector pCAMBIA1304; design two pcr amplification primers that contain SpeI and BstEII restriction endonuclease sites and protection base respectively, its primer sequence is:
P1:5 '-GA
ACTAGTATGGCTACTGCTTCCTCAG-3 ', the SpeI restriction enzyme site is represented with underscore
P2:5 '-GC
GGTGACCTCAATTAGTGAAATTTTTCAG-3 ', the BstEII restriction enzyme site is represented with underscore
Blade extracted total RNA (Shanghai China Shun biotechnology company limited plant RNA extracts test kit) from henbane; reverse transcription becomes cDNA (TaKaRa RNA PCR Kit); carry out pcr amplification then; the PCR reaction system is 50 μ L; comprise deionized water; 10 * PCR damping fluid, 5 μ L, dNTP 1 μ L, MgCl
23 μ L, each 1 μ L of primer, cDNA template 1 μ L, Taq enzyme 0.5 μ L.The PCR condition be 94 ℃ 3 minutes, carried out 28 circulations in 1 minute with 94 ℃ 50 seconds, 60 ℃ 45 seconds and 72 ℃ thereupon, extended 10 minutes at 72 ℃ at last.1% agarose electrophoresis detects pcr amplification product, and obtaining the amplification fragment length is 764bp.
After the electrophoretic separation; reclaim (Shanghai China Shun biotechnology PCR of company limited product reclaims test kit) purpose segment; change bacillus coli DH 5 alpha over to after getting an amount of recovery product and pMD 18-T carrier (TaKaRa PMD 18-T Vector test kit) being connected, at LB+ penbritin (100mgL
-1) resistance screening positive colony on the solid medium, PCR identifies back order-checking (Bo Ya bio-engineering corporation in Shanghai finishes).
2.CaMV35S the structure of constitutive promoter expression vector pCAMBIA-HnAOC
DH5 α (changed over to and had the pulsating pMD 18-T of henbane allene oxide synthase cyclase gene core carrier) after will be through sequence verification correct shakes bacterium, enlarged culturing, extract plasmid,, reclaim small pieces after 1% agarose electrophoresis with SpeI and BstEII double digestion.
PCAMBIA1304 with SpeI and BstEII double digestion, is reclaimed big segment after 1% agarose electrophoresis.
With the big segment of pCAMBIA1304 that reclaims with after henbane allene oxide synthase cyclase gene core segment is connected with the T4 ligase enzyme, conversion DH5 α competent cell, LB+ kantlex (100mgL
-1) the positive resistance clone of screening on the solid medium, PCR and double digestion are identified.Obtain to change henbane allene oxide synthase pulsating recombinant plasmid of cyclase gene core and called after pCAMBIA1304-HnAOC.
3.pCAMBIA1304-HnAOC transform Agrobacterium EHA105
PCAMBIA1304-HnAOC is transformed into agrobacterium strains EHA105, draws the positive single bacterium colony of plate picking and shake bacterium, the bacterium liquid extracting plasmid that takes a morsel carries out PCR and enzyme and cuts evaluation.Positive colony is defined as EHA105-HnAOC.
Embodiment 2
Obtain the transgene tobacco that freezing tolerance improves
1. Agrobacterium EHA105-HnAOC.Take out from refrigerator before using, be inoculated in 50ml YEB liquid (Rif
+, Str
+, Kan
+), 28 degree, twice of 200rpm shaking culture.
2. activate for the second time OD
600Reach at 0.3 o'clock and add 100 μ mol L
-1Syringylethanone continues 28 degree, 200rmp shaking culture, OD
600Reached under 0.6 o'clock room temperature 4000rpm centrifugal 10 minutes.
3. abandon supernatant, thalline (adds 100 μ mol L with MS
-1Syringylethanone) liquid nutrient medium suspends, and is diluted to 5-20 times of original volume, makes the OD of bacterium liquid
600About=0.3, claim conversion fluid.
4. get young tender, the healthy and strong blade of tobacco aseptic seedling, remove master pulse, blade is cut into leaf dish about 0.8cm * 0.8cm, be placed on MS and (add 1mg L
-16-benzyl aminopurine, 1mg L
-1Naphthylacetic acid) on the substratum, anti-uppermost leaf dish dehydration; The leaf dish is put into the Agrobacterium nutrient solution, and 190rpm contaminates 10min on 28 ℃ of shaking tables; The medication spoon is pulled the leaf dish out, is placed on the thieving paper of sterilization, blots the unnecessary bacterium liquid of Ye Panshang; The leaf dish that blots is lain in the MS that adds lid layer filter paper (add 1mg L
-16-benzyl aminopurine, 1mg L
-1Naphthylacetic acid) on the substratum, about 25 ℃, cultivated altogether about 2 days in the dark place.
5. after cultivating altogether, according to the following steps the Agrobacterium of leaf panel surface is washed off, is shaken frequently therebetween, make the leaf dish fully contact solution:
A) sterilized water is 15 minutes
B) sterilized water+Pyocianil (500mg L
-1) 15 minutes
C) MS+ Pyocianil (500mg L
-1) 20 minutes
Then the medication spoon is pulled the leaf dish out, is placed on the thieving paper, blots excessive moisture; The leaf dish is placed on MS (contains 30mg L
-1Totomycin and 250mg L
-1Pyocianil) on the substratum, the leaf plate edge gently is pressed in the substratum; About 25 ℃, the 16h/24h illumination cultivation; Visible differentiation bud grows after about 20 days, treat that bud is grown up after, downcut, place MS (to contain 0.5mg L
-1Naphthylacetic acid, 30mg/L
-1Totomycin and 250mg/L
-1Pyocianil) carries out root culture on the substratum; Seedling replanting is grown under 25 ℃, the condition of 14h light/24h in the small-sized plastic flowerpot of the peat soil, vermiculite and the perlite mixture that are equipped with 1: 1: 1 after about 14 days.
6. detect hygromycin gene and the integration of henbane allene oxide synthase cyclase gene in the genome of transgene tobacco respectively with PCR, confirm transgenic event.
Embodiment 3
The freezing tolerance of transgene tobacco and the contrast of non-transgenic tobacco detects
1. compare transgenosis and non-transgenic tobacco freezing tolerance, characterize it to cryogenic tolerance performance with the ion leakage rate after the tobacco leaf subzero treatment.(reference: Wallis, J.G., Wang, H.Y., and Guerra, D.J. (1997) Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures.Plant Mol.Biol.35,323-330; Nunes, M.E.S.and Smith, G.R. (2003) .Electrolyte leakage assay capable of quantifying freezing resistance in rose clover.Crop Sci.43,1349-1357)
2. take by weighing contrast and the transgene tobacco vegetative period blade more consistent respectively and restrain for every part 0.5,, put into-20 ℃ of processing 30 minutes with deionized distilled water flushing three times with growth conditions.
3. thawed 1 hour in room temperature, add the 30ml ultrapure water in every duplicate samples, again in 25 ℃, 100rpm vibration 2 hours, with the specific conductivity Rc of digital conductivitimeter mensuration solution.
4. then sample is boiled 30min in original solution, measure the specific conductivity Rc ' of solution again.
5. define relative conductivity=Rc/Rc ' * 100%.
6. six detected transgenic lines all demonstrate the tolerance higher to subzero treatment.After-20 ℃ of processing, the leakage rate of the leaves ions of six transgenic lines plant is respectively 25.04% (5.81%), and 15.57% (1.96%), 11.82% (0.98%), 17.30% (3.82%), 12.07% (0.86%), 13.45% (0.96%); Ion leakage rate after the wild-type tobacco-20 ℃ processing is 77.95% (3.82%); Ion leakage rate after ℃ processing of commentaries on classics empty carrier contrast tobacco-20 is 73.26% (3.82%).It in its bracket standard deviation.The ion leakage rate level of transgene tobacco is about 1/4th of blank and empty carrier contrast.
SEQUENCE?LISTING
＜110〉Fudan University
＜120〉a kind of method of utilizing henbane AOC gene transformation to improve freezing resistance of tobacco
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Claims (6)
1. method of utilizing gene engineering method to strengthen freezing resistance of tobacco, it is characterized in that utilizing the plant expression vector of nucleotide sequence (Genbank No.:AY708383), adopt the dna molecular of transgenic method overexpression henbane allene oxide synthase cyclase gene in tobacco cell, tissue, organ, plant with coding henbane allene oxide synthase cyclase; Comprise the steps:
(1) adopt gene clone or synthetic gene method to obtain the segment of henbane allene oxide synthase cyclase gene;
(2) henbane allene oxide synthase cyclase gene segment is connected in expression regulation sequence, forms high efficiency plant expression vector;
(3) adopt transgenic method to shift henbane allene oxide synthase cyclase gene segment in cell, tissue, organ or the plant of tobacco;
(4) screening and evaluation transformant;
(5) cultivate transformant, obtain transgenic progeny;
(6) anti-freezing property of mensuration transgenic progeny.
2. an expression vector is characterized in that it comprises the allene oxide synthase cyclase gene segment of the henbane of claim 1.
3. a host cell is characterized in that it transforms above-mentioned expression vector.
4. by the described host cell of claim 3, it is characterized in that it being tobacco.
5. a life entity is characterized in that it being cell, tissue, organ, the plant of improving with the anti-freezing property that the described method of claim 1 obtains.
6. the offspring of a life entity is characterized in that it being the offspring who obtains cell, tissue, organ or the plant transgenosis life entity of anti-freezing property raising with the described method of claim 1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181458A (en) * | 2011-03-31 | 2011-09-14 | 复旦大学 | Method for increasing nicotine content in tobacco by utilizing camptotheca acuminate AOC (Allene Oxide Cyclase) gene transformation |
| CN108374020A (en) * | 2017-12-28 | 2018-08-07 | 南京农业大学 | A kind of agriculture bacillus mediated soybean transformation in planta method of improvement |
| CN111748579A (en) * | 2020-08-07 | 2020-10-09 | 浙江华缔药业集团医药开发有限公司 | Method for in vitro culture expression of protein by plant leaf |
| CN113287506A (en) * | 2021-06-15 | 2021-08-24 | 河南农业大学 | Method for improving freezing resistance of tobacco seedlings |
-
2009
- 2009-03-18 CN CN200910047765A patent/CN101838664A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181458A (en) * | 2011-03-31 | 2011-09-14 | 复旦大学 | Method for increasing nicotine content in tobacco by utilizing camptotheca acuminate AOC (Allene Oxide Cyclase) gene transformation |
| CN108374020A (en) * | 2017-12-28 | 2018-08-07 | 南京农业大学 | A kind of agriculture bacillus mediated soybean transformation in planta method of improvement |
| CN111748579A (en) * | 2020-08-07 | 2020-10-09 | 浙江华缔药业集团医药开发有限公司 | Method for in vitro culture expression of protein by plant leaf |
| CN113287506A (en) * | 2021-06-15 | 2021-08-24 | 河南农业大学 | Method for improving freezing resistance of tobacco seedlings |
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Application publication date: 20100922 |