CN107841511B - One kind turning extremely halophilic archaea NaSOD genetic tobacco - Google Patents
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Abstract
The invention discloses a kind of extremely halophilic archaea (Natrinema altunense sp.) NaSOD genes to reduce the application in tobacco nicotine, and the base sequence of the extremely halophilic archaea NaSOD gene is as shown in SEQ ID NO.3.Extremely halophilic archaea NaSOD gene is transferred in tobacco by the present invention, can significantly reduce Nicotine in Tobacco content.
Description
Technical field
The present invention relates to one kind to turn extremely halophilic archaea NaSOD genetic tobacco, and in particular to extremely halophilic archaea NaSOD base
Because in the application for reducing Nicotine in Tobacco content.
Background technique
Improper with the deterioration of ecological environment, the development of industrialized agriculture and fertilizer application, the soil salinization has become one
A global resource problem and ecological problem, the area in salt-soda soil increase year by year (Mahajan S, Tuteja N.Cold,
salinity and drought stresses:an overview[J].Arch.).According to incompletely statistics, whole world salt-soda soil
About 9.54 hundred million hm of area2(FAO,2008).China's salinized soil area is big, widely distributed, type multiplicity, and all kinds of saline alkali land areas are total
Count 9913.3 ten thousand hm2, wherein modern saline-alkali soil area is 36,930,000 hm2, about 44,870,000 hm of remaining saline-alkali soil2, and still had
About 17,330,000 hm2Potential saline-alkali soil (such as Li Bin China's saline alkali land resource and the arid area Study on Sustainable Use [J] agricultural
Study .2005,23:154-158.).The master that salt stress, which has become, to be influenced plant growth, lead to grain and the industrial crops underproduction
Want limiting factor.It solves the soil salinization and generally takes following two measure, first is that with draining and irrigating the physical methods such as the desalinization of soil by flooding or leaching
Or the chemical methodes such as gypsum and sulphur improve soil；Second is that cultivating salt tolerant crop product by conventional breeding or biotechnological method
Kind.The former input cost is high.It is most positive that biological modification measure at present (such as plantation salt-tolerant plant) has become improvement salt-soda soil
And permanently effective approach, the salt tolerance for improving plant especially crop has been one of the key subjects of the future of agriculture development.
The excavation and utilization of functional gene are the commanding elevations of world today's living resources competition.Once who possesses more function
Energy genetic resources, who will have the initiative in hands in biological economy competition.Submarine hydrothermal solution mouth and high mountain salt lake are two on the earth
Extreme environment the most typical.High mountain salt lake is the residual product of fossil sea floor, same as salt pond to have solar radiation strong, windy few
The exceedingly odious environmental conditions such as rain, evaporation capacity be big so that biosphere abundant disappears originally, and have evolved high mountain salt lake
Distinctive biological species-halophilic microorganism.These biologies with height salt resistance ability are constantly complete during long-term evolution
Their metabolic pathway has been apt to it to adapt to by caused huge osmotic pressure with high salt.From the last century 50's, people just carry out
The research of its coping mechanism, but up to the present there has been no breakthrough for the development and utilization of the resistance to genetic resources with high salt in high mountain salt lake
Progress.
Biologist and breeder improve the salt resistance ability of plant by traditional breeding method cultivation salt-enduring cultivars, from
And mitigate the saline and alkaline influence to agriculture and forestry production, certain progress is obtained, but develop slowly.Transgenic technology has broken object
Reproduction sovereignty nuisance between kind, widens the genetic background of plant resources, directly the hereditary object of plant modification at the genetic level
Matter, the inhereditary feature or characteristic of directional transformation plant make up conventional breeding methods to effectively improve the salt tolerance of plant
It is insufficient.So far the research of plant anti-salt transgenic engineering achieves considerable progress, shows tempting prospect.Such as Ohta
(2002) by vacuole type Na+/H+Antiporter protein (SOS1) gene is transferred in rice, is remarkably improved transgenic plant offspring
Salt tolerance.Intercellular vesicular transporter is overexpressed in arabidopsis, genetically modified plants reduce the accumulation of ROS, improve
Salt resistance ability (Alexander M, Yehoram L, Budhi ST, Alex L.Induction of salt and osmotic
stress tolerance by overexpression of an intracellular vesicle trafficking
protein AtRab7(AtRabG3e)[J].Plant Physiol,2004,134:118–128.).P5CS (2- hydrogen pyrroles 5-
Carboxylic acid synthetase) gene is intermediary enzyme necessary to proline biosynthesis, P5CS gene is transferred to tobacco, wheat, potato and rice,
The salt tolerance of transgenic plant is obviously higher than control.
It is one of important indicator of plant salt endurance that ROS, which removes system activity or the height of content, in plant.SOD
(superoxide dismutase, superoxide dismutase) is the first line of defence in antioxidant system, in enzyme protection system
In be in core status.Many is the study found that under Salt Strees Condition, and the enzymes such as SOD is active anti-oxidant with plant in plant
Stress ability is positively correlated.Sod gene is transferred in Different Crop, such as rice, tobacco, Chinese cabbage and capsicum, transgenic line
Salt tolerance significantly increase.
Chinese Patent Application No. 200810163748.8 discloses Natrinema altunense sp (Natrinema altunense sp.)
The sequence of high-salt tolerance relevant protein and its encoding gene and application, the gene is shown in sequence table SEQ .ID.NO.3, and sequence is complete
A length of 603bp, the albumen of coding are the MnSOD of halophilic archaea.It is resistance to that in expression in escherichia coli Escherichia coli can be improved in the gene
By the ability of hypersaline environment.
Nicotine is commonly called as nicotine, is a kind of distinctive alkaloid of tobacco, it is a kind of most important alkaloid of commercial Tobacco,
Account for 2% one the 5% of dry cell weight.Nicotine is synthesized by pyrrole ring and pyridine ring, is a kind of pyrroles's alkali, can be acted on humans and animals
Nervous system, stimulate neurons secrete dopamine, make one to feel pleasant sensation.Dopamine is the main chemical for causing to be addicted to smoking
Matter.In view of the business and medical value of nicotine, the regulation of the nicotine content in tobacco is all the time by the pass of scientist
Note.Nicotine content removes outside the influence for being extractd the general agricultural operation such as axillary bud by water and fertilizer management, topping, other more effective drops
The method of low Nicotine in Tobacco content becomes new project, always by expectation.
Summary of the invention
In order to solve the above problems, the present inventor is by extremely halophilic archaea (Natrinema altunense sp.)
It after NaSOD channel genes tobacco, is surprised to find and turns in NaSOD genetic tobacco, the content of nicotine is lower than wild strain, thus complete
At the present invention.
Therefore the present invention provides a kind of method for reducing Nicotine in Tobacco content, the method is by extreme halotolerant Gu
Bacterium (Natrinema altunense sp.) NaSOD channel genes tobacco, cultivation turn NaSOD genetic tobacco kind.
The base sequence of the extremely halophilic archaea NaSOD gene is as shown in SEQ ID NO.3.
It specifically includes:
Building includes the expression vector of the extremely halophilic archaea NaSOD gene, by mediated by agriculture bacillus by extreme halotolerant
Archaeal (Natrinema altunense sp.) NaSOD gene is transferred to tobacco cell, and culture generates Tobacco Salt plant.
The expression vector includes the promoter and terminator of initial carrier and insertion initial carrier, the original load
Body is pCAMBIA1301.
The promoter is CaMV 35S promoter.
The terminator is NOS terminator.
It is overexpressed NaSOD transgene tobacco ROS Scavenging activity with higher, can reduce film rouge mistake caused by salt treatment
Oxidation, to ensure that higher photosynthetic rate, the present invention is by extremely halophilic archaea (Natrinema altunense sp.)
NaSOD gene is transferred in tobacco, not only significantly improves Tobacco Salt performance.
Detailed description of the invention
Fig. 1 is that plant expression vector p1301-NaSOD constructs schematic diagram；
Fig. 2 is p1301-NaSOD transgene tobacco T0It is detected for PCR, M:DL 2000DNA ladder；NC: water；WT: wild
Raw type；1-14: transgenic plant；
Fig. 3 is nicotine example chromatogram, and upper figure is standard diagram, and following figure A is reference substance, and B is sample of the invention；
Fig. 4 is nicotine content in transgene tobacco strain, and NC is negative control, and WT is wild strain.
Specific embodiment
1 expression vector p1301-NaSOD of embodiment building
The Escherichia coli for cultivating pGEX-SOD containing plasmid (patent application 200810163748.8), with halophilic archaea NaSOD
The specific primer (adding Pst I and Kpn I restriction enzyme site respectively) of gene carries out bacterium solution PCR, by the PCR product of recovery purifying
It is connected on intermediate vector pMD19-T, converts bacillus coli DH 5 alpha.Picking positive colony, PCR and sequencing identification.It shakes and takes correctly
Bacterium solution extract plasmid pMD19-NaSOD.Pst I and Kpn I double digestion plasmid pMD19-NaSOD, is inserted into
In the multiple cloning sites of pCAMBIA1301, and it is inserted into CaMV 35S promoter and NOS terminator respectively before and after target gene,
It obtains plant expression vector pCAMBIA1301-NaSOD (as shown in Figure 1).For recon after digestion is identified, freeze-thaw method imports agriculture bar
In bacterium EHA105.
NaSOD specific primer:
NaSOD-F:5 '-CGGGGTACCATGACTGATCACGAACTTCCAC-3’(Kpn I)
NaSOD-R:5 '-AAACTGCAGTTACTCGAAGTGGTCGAG GCAG-3’(Pst I)
The tobacco genetic transformation of 2 mediated by agriculture bacillus of embodiment
2.1 tobacco Aseptic seedling cultures
1) in aseptic superclean bench, tobacco seed is put into the Eppendorf pipe of 1.5mL, is washed with 75% ethyl alcohol
1min is washed, with aseptic water washing 3~4 times；
2) 10%NaClO immersion 10min is added and removes the NaClO of trace with aseptic water washing 4~5 times；
3) seed is placed on sterile blotting paper and sucks traces of moisture, be seeded on MS0 culture medium, 25 DEG C of (16h/8h=
It is light/dark), when plant length to 6~8 leaves, it is used to prepare the explant of Transformation of tobacco.
2.2 leaf disk method transformation of tobacco
1) the Agrobacterium bacterium solution containing pCAMBIA1301-NaSOD is inoculated in containing 50mg/L Kam (kanamycins) and
In 40mg/L Rif (rifampin) LB liquid medium, 28 DEG C are shaken bacterium (200rpm) overnight, to bacterium it is long to (OD600=0.5~
0.8) in 28 DEG C of centrifugation (5000rpm) 8min when, the isometric 1/2MS0 fluid nutrient medium of thallus will be collected and be resuspended, 28 DEG C are shaken
It is converted after bed (180rpm) culture 30min for tobacco leaf disc.
2) master pulse is gone to be cut into 0.5 × 0.5cm the sterile tobacco tender leaf being grown in MS0 culture medium2Small pieces；
3) tobacco leaf disc 6min is sheared with the dip dyeing of ready engineering bacteria (step 1), shakes frequently infect bottle therebetween, then
Remaining bacterium solution is gone to the greatest extent, and explant taking-up is placed in MS1 and co-cultures co-cultivation 36h (25 DEG C of dark conditions) on base；
4) explant is transferred to MS2 culture medium after cotransformation to be screened and broken up, and with without expression vector
For EHA105 transformation of tobacco leaflet tablet as negative control, every 10~12d subculture is primary；
5) after 30~35d, regeneration bud it is long to 3~4cm when cut and move into MS3 culture medium and take root；
6) it is transferred to 7~10d of maintenance in the small alms bowl equipped with perlite after seedling rooting to be regenerated, then plants into greenhouse for subsequent
Research.
Used in the process of tobacco genetic transformation to culture medium prescription be this field conventional reagent.
The identification of 3 transgenic plant of embodiment
Hygromycin selection: after regrowth is transplanted to greenhouse, choosing with position young leaves, take 2~3cm, is soaked in containing 50mg/L tide
In the sterile water of mycin and 1.0mg/L 6-BA.Non-transgenic tobacco blade flavescence browning after 5d, false positive plant leaf also become
Huang, transformed plant blade keep green.
The PCR of transgene tobacco is detected: SDS method extracts tobacco leaf total DNA, with NaSOD primer amplified, 14 plants
Transgene sample has band (as shown in Figure 2) at 621bp.
The nicotine content of 4 transgene tobacco of embodiment is analyzed
Above-mentioned 14 plants of transgene tobaccos, are cultivated according to conventional method to seed collecting.Harvest time take respectively middle part tobacco leaf, under
Portion's tobacco leaf 6-8 piece, with leaf green decline blade face jaundice, fine hair falls off, and when blade tip leaf margin is sagging, petiole and stem are in close to straight
Angle is best collecting time.
The tobacco leaf of harvesting is put into the baking oven to heat up in advance, 130 DEG C are toasted 2 minutes；Baking oven is opened, is cooled to 80 DEG C, is dried
It is 16 hours or so roasting.
The measuring method of nicotine content
Japanese Shimadzu Corporation GCMS-QP2010 type gas chromatograph-mass spectrometer (GC-MS)；Agilent DB-5MS(30m×
0.32mm × 0.25 μm) capillary gas chromatographic column；AS3120 Ultrasound Instrument (120W, 40kHz).
4.1 experimental condition
Chromatographic condition uses DB-5MS (30m × 0.32mm × 0.25 μm) capillary gas chromatographic column；It is with high-purity helium
Carrier gas, flow velocity 3mLmin-1；Injector temperature: 280 DEG C；Input mode: split sampling, split ratio 10: 1；Temperature programming:
60 DEG C of initial temperature, 2min is kept, with 10 DEG C of min-1Rate is warming up to 280 DEG C, keeps 6min；Sample volume: 1 μ L.
Mass Spectrometry Conditions interface temperature: 280 DEG C；EI source temperature: 200 DEG C；Electron bombardment energy: 70eV；Nicotine detect from
Sub- m/z84；Acquisition delay: 3min.
4.2 solution are prepared
Reference substance stock solution precision weighs nicotine about 100mg in 10mL measuring bottle, and ethyl acetate is added sufficiently to dissolve and dilute
It releases to scale, shakes up, up to being 1.0mgmL containing nicotine-1Stock solution.
4.3 chromatographic behavior
Under above-mentioned chromatographic condition, full ion scan chromatogram, mass spectrogram and the molecular formula of nicotine are shown on Fig. 4, Ni Gu
The selection ion detection figure of fourth and tobacco is shown under Fig. 4.The retention time of nicotine is 10.6min.
4.4 linear relationships and detection limit
It is appropriate that precision draws reference substance stock solution, be respectively 0.01 with ethyl acetate compound concentration, 0.05,0.1,0.5,
5.0、10mg·mL-1Control series product solution, 1 μ L of sample introduction, record chromatogram, using peak area Y as ordinate, concentration X (mg
mL-1) it is abscissa, carry out linear regression, regression equation are as follows:
Y=6.144 × 105X+5.558×104R=0.9998
The result shows that nicotine concentration linear relationship within the scope of 0.01~10mgmL-1 is good.
The measurement of 4.5 samples
Precision weighs tobacco 1.0g, sets in 10mL measuring bottle, add ethyl acetate ultrasound (120W, 40kHz) 30min dissolve and it is dilute
It releases to scale, shakes up, up to sample.
Precision draws reference substance solution (0.3mgmL-1) and each 1 μ L sample introduction measurement of test solution, records chromatogram,
The content of nicotine is calculated with external standard method.Measurement result is as shown in Figure 4.In Fig. 4, NC is negative control, and WT is wild strain, Buddhist nun
Ancient fourth content is in 2.7mg/g or so, and nicotine content is reduced to 2mg/g or so in transgenic line T1-1 to T1-14.Its
Reach in middle T1-1, T1-2, T1-4, T1-5 and T1-8 the level of signifiance (p < 0.05), has more reached extremely aobvious in other strains
Write horizontal (p < 0.01).Inventor will carry out segregating generation genetic analysis to positive strain tobacco, obtain stable transgenosis
Tobacco line.
SEQUENCE LISTING
<110>China's metering university
<120>one kind turns extremely halophilic archaea NaSOD genetic tobacco
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 31
<212> DNA
<213>artificial sequence
<400> 1
cggggtacca tgactgatca cgaacttcca c 31
<210> 2
<211> 31
<212> DNA
<213>artificial sequence
<400> 2
aaactgcagt tactcgaagt ggtcgaggca g 31
<210> 3
<211> 603
<212> DNA
<213>Natrinema altunense sp (Natrinema altunense sp.)
<400> 3
atgactgatc acgaacttcc accactcccg tacgattacg acgcgctcga accggcactg 60
tccgaacagg tactgacctg gcatcacgat acgcaccacc agggctacgt caacggcctc 120
aacgccgccg aggagaccct cgcggagaac cgcgaggagg gcgacttcgg ctcgacgccc 180
ggtgccctca aaaacgttac tcacaacggc tgtggtcact atctccacac gctgttctgg 240
gagaacatgt cccccaacgg cggcggcgag ccggacggcg acctcgccga ccgcatcgag 300
gaggacttcg gatcctacga gggctggaaa ggcgagttcg aggccgctgc cggtgccgcc 360
ggtggctggg cactgctggt gtacgatccg gttgcgaagc aacttcgcaa cgtcgcggtc 420
gacaagcacg accagggcgc gctctggggc gcacatccag tgctcgcgct ggacgtctgg 480
gagcactcct actactacga ctacggtccg gaccgcggag acttcatcga cgccttcttc 540
gacgtcgtca actgggagaa ggccgaagag gagtaccaga cctgcctcga ccacttcgag 600
taa 603
Claims (6)
1. a kind of method for reducing Nicotine in Tobacco content, which is characterized in that by extremely halophilic archaea (Natrinema
Altunense sp.) in NaSOD channel genes tobacco, and express that the NaSOD gene in tobacco, the NaSOD gene
Base sequence as shown in SEQ ID NO.3.
2. the method as described in claim 1 characterized by comprising
Building includes the expression vector of the extremely halophilic archaea NaSOD gene, by mediated by agriculture bacillus by extremely halophilic archaea
(Natrinema altunense sp.) NaSOD gene is transferred to tobacco cell, and culture generates Tobacco Salt plant.
3. method according to claim 2, which is characterized in that the expression vector includes initial carrier and the original load of insertion
The promoter and terminator of body, the initial carrier are pCAMBIA1301.
4. method as claimed in claim 3, which is characterized in that the promoter is CaMV35S promoter.
5. method as claimed in claim 4, which is characterized in that the terminator is NOS terminator.
6. extremely halophilic archaea (Natrinema altunense sp.) NaSOD gene is in reducing tobacco nicotine content
Using the base sequence of the NaSOD gene is as shown in SEQ ID NO.3.
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| CN101768212B (en) * | 2008-12-30 | 2012-07-25 | 浙江大学 | Natrinema altunense sp. high-salt tolerance relevant protein, coding gene and application thereof |
| CN102399816A (en) * | 2011-11-15 | 2012-04-04 | 中国计量学院 | Application of extremely halophilic archaea NaSOD gene in improving rice salt tolerance |
| CN103012572B (en) * | 2012-12-05 | 2013-11-27 | 北京师范大学 | Gene capable of reducing nicotine content of tobacco and application thereof |
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