CN106577771A - Method for preparing inhibitor for reducing tobacco nicotine synthesis by double-stranded RNA interference technology and application thereof - Google Patents
Method for preparing inhibitor for reducing tobacco nicotine synthesis by double-stranded RNA interference technology and application thereof Download PDFInfo
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- CN106577771A CN106577771A CN201611086981.1A CN201611086981A CN106577771A CN 106577771 A CN106577771 A CN 106577771A CN 201611086981 A CN201611086981 A CN 201611086981A CN 106577771 A CN106577771 A CN 106577771A
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- tobacco
- dsrna
- inhibitor
- putrescine
- stranded rna
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention discloses a method for preparing an inhibitor for reducing tobacco nicotine synthesis by a double-stranded RNA interference technology and an application thereof. The method comprises extracting RNA from tobacco, carrying out reverse transcription to obtain cDNA of a tobacco putrescine-N-methyltransferase (PMT) gene, amplifying a fragment of the gene cDNA through a PCR technology, connecting the fragment to a plasmid vector PET-21a for expressing RNA, transferring the plasmid into an RNAE3 enzyme-free Escherichia coli cell, expressing a hairpin dsRNA, crushing the cell to obtain a dsRNA crude preparation, and orderly adding glycerol, a protectant and a dispersant into the dsRNA crude preparation to obtain the inhibitor for reducing tobacco nicotine synthesis. The inhibitor is applied to tobacco according to a ratio of 5 to 15mL/plant so that tobacco nicotine synthesis is reduced. The method has the advantages of simple production processes, low cost, and no damage to human, domestic animals and higher animals, is suitable for large-scale application for tobacco, and has a wide industrial prospect.
Description
Technical field
The invention belongs to plant cultivation technology, more particularly to it is a kind of using double-stranded RNA perturbation technique preparation reduction tobacco smoke
The method of the inhibitor of alkali synthesis and its application.
Background technology
Nicotine, is called nicotine (nicotine), belongs to pyridine race alkaloid, is the main component of alkaloid in Nicotiana tabacum L.,
Its content height and quality of tobacco and its can be very close with sexual intercourse, be also one of index of quality main in Medicated cigarette.High-quality
Nicotine content in Nicotiana tabacum L. is typically 1.5～3.5%.Nicotine content of tobacco leaves is low, and flue gas strength is little, and fragrance is few；Nicotine content is high,
Flue gas strength is excessive, and zest strengthens, and produces acid.In China's flue-cured tobacco main producing region, nicotine content especially upper leaf nicotine
The higher problem of content is very prominent, and sale, exploitation and production thus to flue-cured tobacco produce serious adverse effect.
The structure of nicotine is quite similar with the chemical messenger material acetylcholine in human body, is entered after human body by flue gas,
90% in pulmonary absorption, can reach brain within 6 seconds into after blood, can play acetylcholine identical physiological action, energy
Excited spirit, allaying tiredness increases ability of thinking, improves work efficiency.But the nicotine of excess then plays suppression and paralysis effect.Jing
Often suction nicotine, rises the acceptor quantity that may participate in reaction, and reaction effect declines, and is referred to as drug resistance.It means that
In order to reach same perceived effect, smoker must suck more nicotine, so as to cause smoking addiction, constantly circulation.Cigarette
Addiction make smoker must not infrequently smoking, while many noxious substances of inspiration.In addition, nicotine has very strong toxicity.Medium dose
The nicotine of amount can make one accelerated breathing, vasodilation and vomiting substantially aggravation, and the nicotine of slightly larger dosage can cause and tremble and spasm.
Heavy smoker is sucked after more nicotine, shows as of short duration breathing enhancing and blood pressure rising, loss of appetite.Nicotine can also be damaged
Evil brain cell, makes smoker the series of symptoms such as central nervous system and respiratory system occur.Nicotine and its derivant can be right
Activation is played in " Akt " molecular channel, and " Akt " molecular channel can promote cell growth after being activated, and cell " suicide " is risen
To inhibitory action, cell amount reproduction may be induced and cause cancer.
The synthesis of nicotine is presented ascendant trend in the whole growthdevelopmental stage of Nicotiana tabacum L., from scratch, from less to more.It is smokeless in seed
Alkali, seed to be sprouted and initially form nicotine after 100h, and it is slow to be transplanted in squaring period Nicotiana tabacum L. nicotine accumulation, nicotine in Nicotiana tabacum L. after pinching
Accumulation increases sharply, until ripe harvesting reaches maximum.The height of nicotine synthetic quantity is relevant with many factors in Nicotiana tabacum L., such as cigarette
The inherited character of grass product kind, soil fertility and pH value, height above sea level etc..Still lack the effectively synthesis of reduction tobacco smoke alkaloid at present
Measure.
The biosynthesis pathway of tobacco smoke alkaloid is illustrated substantially.The synthesizing site of nicotine is mainly the root of Nicotiana tabacum L..Synthesis
The direct precursor thing of nicotine is nicotinic acid and N- crassitude salt.Participating in the enzyme of nicotine synthesis mainly has ODC Ornithine decarboxylase
(ODC), putrescine N-methyltransferase (PMT) and methylputrescine oxidase (MPO) etc..Saunders and Bush (1979) reports,
PMT and MPO amount increase after pinching in the Nicotiana tabacum L. of normal bio alkali gene type, but in the Nicotiana tabacum L. of low alkaloid genotypes, beat
MPO does not increase behind top.In the Nicotiana tabacum L. of several related and incoherent high and low alkaloid genotypes, the PMT in Tobacco Root
Activity be directly proportional to the nicotine amount in Nicotiana tabacum L. (Yoshida, 1973；Saunders and Bush, 1979).Pudliner (1980) sends out
Existing, the ODC and citrate synthetase in normal and low alkaloid genotypes Nicotiana tabacum L. increases, and citrate synthetase is metabolism
A kind of indicator of effect.These results indicate that PMT is the maximum enzyme of restriction effect in the route of synthesis.By PMT antisense genes
Nicotiana tabacum L. is imported, the controllable transgene tobacco kind of nicotine content has been cultivated.However, because transgene tobacco kind is in plantation
With it is edible present in greater risk, the controllable transgene tobacco kind of nicotine content is difficult popularization and application.
RNA interference (RNAinterference, RNAi) is to find first and prove within 1998 to belong in C. Elegans Automatic Screening to turn
After record level gene silencing mechanism (Fire et al., 1998).Its mechanism is using biological internal Dicer enzyme spcificitys
The double-stranded RNA (dsRNA) that ground degraded is introduced by external source importing or by various modes such as transgenic, virus infection becomes little interference
RNAs (small interfering RNAs, siRNAs), siRNA double-strand and induction silencing complex (RNA-induced
Silencing complex, RISC) combine after be cracked into the homology that siRNA is single-stranded, is combined with the single-stranded complete complementaries of siRNA
Said target mrna by RISC shear degradations, so as to specifically block the expression of its gene.This mechanism be widely present in from funguses to
In plant, the various biologies from invertebratess to mammal.Afterwards, the technology is in animals and plants and the functional gene of microorganism
Group is learned the aspects such as research, transgenic breeding and is applied rapidly.1998, Timmons etc. had found to there are in nematicide in vitro
RNA interference mechanism is realized using dsRNA.2003, Tenllado etc. applied the tobacco viruses of the great expression in antibacterial body
DsRNA sprays Nicotiana tabacum L. surface, can efficiently prevent and treat tobacco virus.We are according to the principle, the tobacco PMT gene that clone is obtained
Fragment and dsRNA expression plasmids build together the hair fastener dsRNA recombiant plasmid of an expression PMT genetic fragment, are transformed into
RNaseIII defective escherichia colis bacterial strain HT11 (DE3), by mass propgation engineered strain substantial amounts of PMT genetic fragments are obtained
Hair fastener dsRNA, hair fastener dsRNA is sprayed into Nicotiana tabacum L. before and after tobbaco, can effectively reduce the synthesis of tobacco smoke alkaloid, produce
Go out the Nicotiana tabacum L. of low content nicotine.The method is easy, efficiently and quick, low cost, it is not necessary to build transfer-gen plant.This method
Both make use of RNAi prevent and treat the high specificity of virosiss, it is efficient the characteristics of, be not required to again build transformed variety, be control tobacco smoke
The optimal path of alkali synthesis.
The content of the invention
It is an object of the invention to provide a kind of utilization double-stranded RNA perturbation technique prepares the suppression for reducing tobacco smoke alkaloid synthesis
The method of agent and its application, it can effectively reduce the nicotine amount in Nicotiana tabacum L..
To solve above-mentioned technical problem, the present invention is provided a kind of preparation using double-stranded RNA perturbation technique and reduces tobacco smoke alkaloid
The method of the inhibitor of synthesis, it is comprised the following steps：
1) RNA of Nicotiana tabacum L. is extracted, reverse transcription obtains the cDNA of tobacco putrescine-N- methyl transferase genes；
2) utilize round pcr, the cDNA with tobacco putrescine-N- methyl transferase genes as template, the tobacco putrescine for obtaining-
N- methyl transferase gene segments；
3) by step 2) in tobacco putrescine-N- methyl transferase gene segments be connected in carrier T；
4) by step 3) in carrier T tobacco putrescine-N- methyl transferase gene segments be connected to plasmid vector PET-21a
On, obtain recombiant plasmid PET-EH；
5) by step 4) in obtain recombiant plasmid PET-EH be transferred to disappearance RNAE3 enzymes Bacillus coli cells in,
6) by step 5) in Bacillus coli cells screened, when transformant；
7) by step 6) in transformant amplification culture in LB fluid mediums；
8) transformant that will be enlarged by cultivating adopts ultrasonic disruption, obtains the smudge cellses solution containing hair fastener dsRNA, i.e.,
For dsRNA crude preparation by using；
9) to step 8) in sequentially add and the wood that dsRNA crude preparation by using solution weight ratio is 2～5% in dsRNA crude preparation by using
Quality sodium sulfonate, 0.1～0.3% alkylbenzenesulfonate, 1～3% xanthan gum, 0.1～0.3% isoamyl alcohol, 2～5%
Glycerol, 0.01～0.1% tryptophan and 1～2% polyglycol ether, subpackage after then stirring, you can dropped
The inhibitor of low tobacco smoke alkaloid synthesis, dsRNA concentration is 25～35 μ g/mL in the inhibitor.
Further, the step 2) in, the length of tobacco putrescine-N- methyl transferase gene segments is 400～
700bp。
Yet further, the step 9) in, to the lignin sulfonic acid that dsRNA crude preparation by using solution weight ratio is 2～4%
Sodium, 0.2～0.3% alkylbenzenesulfonate, 1～2% xanthan gum, 0.1～0.2% isoamyl alcohol, 2～4% glycerol,
0.05～0.1% tryptophan and 1～2% polyglycol ether.
Present invention also offers a kind of application of the inhibitor for reducing tobacco smoke alkaloid synthesis.
Beneficial effect of the present invention：
1) production of the invention is easy, with low cost, harmless to people and animals and higher mammal, is suitable to be applied in a large number on Nicotiana tabacum L.
With with wide industrialization prospect.
2) instant invention overcomes the defect of wettable powder and emulsifiable concentrate, auxiliary agent is easily dispersed, and field applies more convenient, does not have
There are residual and environmental pollution.
3) sodium lignin sulfonate, alkylbenzenesulfonate, xanthan gum, isoamyl alcohol, glycerol, color ammonia are added in dsRNA crude preparation by using
Acid and polyglycol ether；They are used in mixed way the stability that can increase dsRNA crude preparation by using, so as to improve the activity of inhibitor.
Description of the drawings
Fig. 1 is PET-EH Vector maps of the present invention
Specific embodiment
In order to preferably explain the present invention, the main contents of the present invention are further elucidated below in conjunction with specific embodiment, but
Present disclosure is not limited solely to following examples.
Embodiment 1：
First, the extraction of tobacco root total serum IgE
With the main tobacco bred cloud and mist 87 of planting of China as material, after taking young root sterile water wash, using TRNzol Total
RNA Reagent extract the total serum IgE of Nicotiana tabacum L..
1) young tender cigarette root 0.1g sufficient grinding 55s in liquid nitrogen are taken, 1mLTRNzol is added；
2) homogenised sample is placed into 5min at 15～30 DEG C so that nucleic acid-protein compound is completely separated；
3) 4 DEG C, 12000rpm centrifugation 10m take supernatant；
4) add 0.2mL chloroforms, cover lid, fierceness vibration 15s, room temperature places 3min；
5) 4 DEG C, 12000rpm is centrifuged 10～15m, and sample can be divided into three layers：The organic faciess of yellow, intermediate layer and upper strata without
The water phase of color, RNA mainly in water phase, is mutually transferred to water in new centrifuge tube；
6) isopyknic isopropanol is added in the aqueous phase solution for obtaining, is mixed, room temperature places 20～30m.
7) 4 DEG C, 12000rpm centrifugation 10min remove supernatant, and RNA is often invisible before centrifugation, after centrifugation in tube side and
Ttom of pipe forms gelatinous precipitate；
8) 1mL75% ethanol, washing precipitation are added；
9) 4 DEG C, 10000rpm centrifugation 3min pour out liquid, are careful not to pour out precipitation, remaining a small amount of liquid it is of short duration from
The heart, is then suctioned out with pipette tips, is careful not to be drawn onto precipitation.
10) room temperature is placed and dried, and about dry in the air 2～3min, adds 100uLRNase-free ddH2O, repeats to blow and beat, and mixes
It is even, abundance dissolving RNA.
2nd, the synthesis of the chains of cDNA first
4 μ g total serum IgEs are taken, with the Reverse Transcriptase of SuperScriptTM III of Invitrogen cDNA is synthesized
First chain, concrete operations are as follows：
1) following components is added in the 0.2mL centrifuge tubes without RNase：
2) mix, 65 DEG C of heating 5min, cooled on ice at least 1min；
3) brief centrifugation, collects tube wall upper liquid and drops to ttom of pipe, and adds cDNA Synthesis Mix：
4) mix, 50 DEG C of insulation 50min；
5) 85 DEG C of placement 5min inactivate reaction；
6) 1 μ LRNaseH, 37 DEG C of placement 20min are added.
3rd, the amplification of tobacco putrescine-N- methyl transferase genes and detection
1st, design of primers
According to tobacco PMT gene primers in GeneBank：
Forward primer：Pmt-F：5'TGGGACATCCGAACAAC 3',
Downstream primer：Pmt-R：5'TGAATGCTGCTTTGTGAA 3'.
2nd, reaction system
Sequentially add in 0.2mL centrifuge tubes：
Mixing is flicked, the drop on brief centrifugation collection tube wall is to ttom of pipe
3rd, response procedures
Response procedures
Table1 The reaction programmes
4th, the recovery of purpose fragment
Reclaimed with the agarose gel DNA QIAquick Gel Extraction Kits of TIANGEN：
1) single target DNA band is cut (cut off redundance as far as possible) from agarose gel be put into it is clean from
In heart pipe, weight is weighed；
2) 3 times of volume sol solutionses PN are added in blob of viscose, and (if gel weight is 0.1g, its volume can be considered 100 μ L, then add
Enter 300 μ L sol solutionses), 10min is placed in 50 DEG C of water-baths, constantly centrifuge tube is leniently spun upside down therebetween, to guarantee that blob of viscose is abundant
Dissolving.If there is not molten blob of viscose, can again add some sol solutionses or continue to place a few minutes, until blob of viscose is completely dissolved
If (blob of viscose volume is excessive, first blob of viscose can be cut into into fragment)；
3) by previous step resulting solution addition one adsorb (adsorption column is put in collecting pipe) in CA2,13000rpm from
The heart 30 seconds, outwells the waste liquid in collecting pipe, and adsorption tube is reentered in collecting pipe；
4) 700 μ L rinsing liquid PW (please first check whether using before and added dehydrated alcohol) are added in adsorption column,
13000rmp is centrifuged 30 seconds, outwells waste liquid, and adsorption column is reentered in collecting pipe；
5) 500 μ L rinsing liquid PW, 13000rpm centrifugation 30s are added in adsorption column, waste liquid is outwelled；By centrifugal adsorbing column
CA2 is put back in collecting pipe, 13000rpm centrifugation 2min, and rinsing liquid is removed as far as possible；Adsorption column is placed in into room temperature or 50 DEG C of incubator numbers
Minute, thoroughly dry, to prevent the rinsing liquid for remaining from affecting the experiment of next step；
6) adsorption column is put in a clean centrifuge tube, to the appropriate 65-70 DEG C of water of the hanging Deca in adsorbed film centre position
The elution buffer EB of bath preheating, room temperature places 2min, and 13000rpm centrifugation 1min collect DNA solution；
7) in order to improve the yield of DNA, the solution that can be obtained centrifugation is again in add-back centrifugal adsorbing column, repeat step
6。
5th, recovery product and pMD18-T vector connect, with reference to TaKaRa description
Brief centrifugation after mixing, by the drop on tube wall ttom of pipe, 16 DEG C of connection 12h are collected.
6th, connection product Transformed E .coli DH5 α
1) take the μ L of connection product 5.0 to add in 100 μ L competence antibacterials, ice bath 30min.Set control simultaneously：Positive control, mark
Quasi- plasmid DNA and competence antibacterial；Negative control, is not added with the competent cell of plasmid；
2) 42 DEG C of heat shock 90sec, put back to immediately in ice bath.500mL LB fluid mediums are added after 5min (without antibiosis
Element), 37 DEG C, 200rpm, 40～60min of wave and culture；
3) take 200 μ L transformed bacterias to be spread evenly across on the LB solid mediums containing 50 μ g/mL ampicillin；
4) after liquid is absorbed on flat board, flat board is inverted, in 37 DEG C about 12h is cultivated, observe the growth feelings of antibacterial on flat board
Condition.
7th, a small amount of of recombinant plasmid dna is extracted
1) picking flat board single bacterium colony in containing 50 μ g/mL ampicillin 3mL LB liquid mediums in, 37 DEG C, 300rpm,
Wave and culture is overnight.
2) about 1.2mL bacterium solutions are shifted in the centrifuge tube of 1.5mL, 4 DEG C, 13000rpm centrifugation 1min, collects thalline is abandoned
Clearly, then 5s is centrifuged, sucks remaining supernatant；
3) solution I of 100 μ L ice pre-coolings is added in each centrifuge tube, thalline dispersion is mixed；
4) solution II that 200 μ L of each addition are newly prepared often is managed, it is slow to reverse 5 times, mix, ice bath 5min.
5) solution III of 150 μ L ice pre-coolings of each addition is often managed, reversing is mixed several times, ice bath 10min.
6) 4 DEG C, 12000rpm is centrifuged 10min.
7) supernatant is taken, plus the dehydrated alcohol of 2 times of volumes, -20 DEG C of placement 20min after mixing.
8) 4 DEG C, 12000rpm, centrifugation 10min obtains plasmid DNA precipitation.
9) 70% washing with alcohol is precipitated once, and 4 DEG C of 12000rpm are centrifuged 5min, abandons supernatant, precipitate vacuum drying.
10) precipitation is dissolved in into 20 μ L TE (pH8.0), adds 1 μ LRNase (10 μ g/ μ L) to eliminate microRNA, store in-
20 DEG C are standby.
8th, the identification of recombiant plasmid enzyme action and sequencing
Following reagent is added in 0.2mL centrifuge tubes：
After mixing, be centrifuged slightly, 37 DEG C temperature bath 3h, then by digestion products in 1% agarose gel electrophoresis, with PCR
Qualification result binding analysis determine whether corresponding fragment insertion.Sequencing is completed by Shanghai biological engineering company limited.
4th, tobacco putrescine-N- methyl transferase genes fragment amplification and detection
1st, design of primers
Two primers are designed according to tobacco putrescine-N- methyl transferase genes full length sequence:
Forward primer PF1：5'CGGAATTCCGCCAATTTTATAGGAAGA 3',
Downstream primer PR 1：5'GGGGTACCGGGTAAAGGCTGCTCAA 3',
2nd, reaction system
Sequentially add in 0.2mL centrifuge tubes：
Mixing is flicked, the drop on brief centrifugation collection tube wall is to ttom of pipe.
3rd, response procedures
Response procedures
Table1 The reaction programmes
4th, the recovery of purpose fragment
Reclaimed with the agarose gel DNA QIAquick Gel Extraction Kits of TIANGEN：
1) single target DNA band is cut from agarose gel and is put in clean centrifuge tube, weigh weight；
2) 3 times of volume sol solutionses PN are added in blob of viscose, if gel weight is 0.1g, its volume can be considered 100 μ L, then add
Enter 300 μ L sol solutionses, 50 DEG C of water-baths are placed 10min, constantly centrifuge tube leniently spun upside down therebetween, to guarantee that blob of viscose is fully molten
Solution.If there is not molten blob of viscose, can again add some sol solutionses or continue to place a few minutes, until blob of viscose is completely dissolved, if
Blob of viscose volume is excessive, first blob of viscose can be cut into into fragment；
3) previous step resulting solution is added into one to adsorb in CA2,13000rpm is centrifuged 30 seconds, in outwelling collecting pipe
Waste liquid, adsorption tube is reentered in collecting pipe；
4) add 700 μ L rinsing liquid PW, 13000rpm to be centrifuged in adsorption column 30 seconds, outwell waste liquid, by adsorption column again
In being put into collecting pipe；
5) 500 μ L rinsing liquid PW, 13000rpm centrifugation 30s are added in adsorption column, waste liquid is outwelled.By centrifugal adsorbing column
CA2 is put back in collecting pipe, 13000rpm centrifugation 2min, and rinsing liquid is removed as far as possible.Adsorption column is placed in into room temperature or 50 DEG C of incubator numbers
Minute, thoroughly dry, to prevent the rinsing liquid for remaining from affecting the experiment of next step；
6) adsorption column is put in a clean centrifuge tube, to appropriate 65～70 DEG C of water of the hanging Deca in adsorbed film centre position
The elution buffer EB of bath preheating, room temperature places 2min.13000rpm centrifugation 1min collect DNA solution；
7) in order to improve the yield of DNA, the solution that can be obtained centrifugation is again in add-back centrifugal adsorbing column, repeat step
6。
5th, recovery product and pMD18-T vector connect, with reference to TaKaRa description
Brief centrifugation after mixing, by the drop on tube wall ttom of pipe, 16 DEG C of connection 12h are collected.
6th, connection product Transformed E .coli DH5 α
1) take the μ L of connection product 5.0 to add in 100 μ L competence antibacterials, ice bath 30min.Set control simultaneously：Positive control, mark
Quasi- plasmid DNA and competence antibacterial；Negative control, is not added with the competent cell of plasmid；
2) 42 DEG C of heat shock 90sec, put back to immediately in ice bath.500mL LB fluid mediums are added after 5min (without antibiosis
Element), 37 DEG C, 200rpm, 40～60min of wave and culture；
3) take 200 μ L transformed bacterias to be spread evenly across on the LB solid mediums containing 50 μ g/mL ampicillin；
4) after liquid is absorbed on flat board, flat board is inverted, in 37 DEG C about 12h is cultivated, observe the growth feelings of antibacterial on flat board
Condition.
7th, a small amount of of recombinant plasmid dna is extracted
1) picking flat board single bacterium colony in containing 50 μ g/mL ampicillin 3mL LB liquid mediums in, 37 DEG C, 300rpm,
Wave and culture is overnight；
2) about 1.2mL bacterium solutions are shifted in the centrifuge tube of 1.5mL, 4 DEG C, 13000rpm centrifugation 1min, collects thalline is abandoned
Clearly, then 5s is centrifuged, sucks remaining supernatant；
3) solution I of 100 μ L ice pre-coolings is added in each centrifuge tube, thalline dispersion is mixed；
4) solution II that 200 μ L of each addition are newly prepared often is managed, it is slow to reverse 5 times, mix, ice bath 5min；
5) solution III of 150 μ L ice pre-coolings of each addition is often managed, reversing is mixed several times, ice bath 10min；
6) 4 DEG C, 12000rpm is centrifuged 10min；
7) supernatant is taken, plus the dehydrated alcohol of 2 times of volumes, -20 DEG C of placement 20min after mixing；
8) 4 DEG C, 12000rpm, centrifugation 10min obtains plasmid DNA precipitation；
9) 70% washing with alcohol is precipitated once, and 4 DEG C of 12000rpm are centrifuged 5min, abandons supernatant, precipitate vacuum drying；
10) precipitation is dissolved in into 20 μ LTE (pH8.0), adds 1 μ LRNase to eliminate microRNA, store in -20 DEG C it is standby.
8th, the identification of recombiant plasmid enzyme action and sequencing
Following reagent is added in 0.2mL centrifuge tubes：
After mixing, be centrifuged slightly, 37 DEG C temperature bath 3h, then by digestion products in 1% agarose gel electrophoresis, with PCR
Qualification result binding analysis determine whether corresponding fragment insertion.Sequencing is completed by Shanghai biological engineering company limited.
4th, construction recombination plasmid PET-EH
Based on PET-21a carriers, the tobacco putrescine-N- methyl transferase gene fragments of sequencing are inserted into into promoter
Downstream
1st, the amplification of purpose fragment
The plasmid of T-vector is properly inserted into as mould with the tobacco putrescine-N- methyl transferase gene fragment sequences being sequenced
Plate expands purpose fragment, and reaction system is identical with above-mentioned amplification tobacco putrescine-N- methyl transferase gene fragments with response procedures.
2nd, the enzyme action of purpose fragment
Following reagent is added in 0.2mL centrifuge tubes：
After mixing, it is centrifuged slightly, 37 DEG C of temperature baths are overnight.
3rd, the recovery again of purpose fragment
With Tiangeng glue reclaim test kit recycling step 2 again) in purpose fragment after enzyme action, its method is with above-mentioned purpose piece
Duan Huishou.
4th, the acquisition of PET-21a carriers
Plasmid vector PET-21a, purchased from German Merck companies, enzyme action, enzyme action is carried out with reference to above-mentioned enzymatic cleavage methods to plasmid
Plasmid afterwards is reclaimed with agarose gel QIAquick Gel Extraction Kit, and method is ibid.
5th, the connection of purpose fragment and PET-21a carriers
The 5th, recombiant plasmid PET-EH is transferred to the Bacillus coli cells of disappearance RNAE3 enzymes
The 1st, recombiant plasmid PET-EH is transferred to the Bacillus coli cells of disappearance RNAE3 enzymes
1) the μ L of colibacillary competent cell 100 are placed in ice, after ice melts, 10 μ L enzyme connect products things are added and is experienced
In state cell, gently mix, 30min is placed in ice.
2) after 42 DEG C of thermal shock 90s, 1min is placed in ice.
3rd, 890 μ L LB liquid mediums, 37 DEG C, 220rpm shaken cultivation recovery 60min are added.
4th, 200～300 μ L liquid spreadings are taken in the LB solids containing 100 μ g/mL ampicillin and 20 μ g/mL chloromycetin
On plating medium, 37 DEG C of culture 16h.
2nd, E. coli transformant in step 1 is screened
1) single bacterium colony grown in picking step 1 middle plateform, is inoculated in the LB liquid cultures containing 100mg/L ampicillin
In base, shaken cultivation 16h in 37 DEG C of constant-temperature shaking incubators；
2) plasmid extraction is using raw work UNIQ-10 pillar small amount plasmid extraction agent box.1.5～5mL bacterium solutions are taken,
12000rpm is centrifuged 2minm collects thallines, uses up or blot culture fluid supernatant；
3) 250 μ L SolutionI, suction are added to beat or vibrate to thorough suspension thalline in bacterial sediment；
4) 250 μ L SolutionII are added, 5～10 mixings of centrifuge tube is gently overturned immediately, be stored at room temperature 2-4m；
5) 350 μ L SolutionIII are added, centrifuge tube is gently overturned immediately and is fully mixed for 5～10 times；
6) 12000rpm centrifugations 10min；
7) supernatant is all carefully moved into adsorption column, 8000rpm centrifugation 30s.Outwell the liquid in collecting pipe；
8) 500 μ L Wash Solution, 10000rpm centrifugation 1min are added in adsorption column.Outwell the liquid in collecting pipe
Body, adsorption column is put in same collecting pipe；
9) repeat step 8) once；
10) suction attached column and collecting pipe are put into into centrifuge, 12000rpm centrifugation 2min；
11) adsorption column is put in clean 1.5mL centrifuge tubes, in adsorbed film central authorities 50～100 μ LElution is added
Buffer, is stored at room temperature 2min, 10000rpm centrifugation 1min；Resulting plasmid DNA solution is placed in into -20 DEG C to preserve or be used for
Follow-up test.
12) plasmid PET-EH is contained with the detection of 1% sepharose electrophoresis and in sequence verification conversion daughter cell.
3rd, transformant amplification culture in LB fluid mediums；
1) will verify that the transformant for obtaining is inoculated in step 2 mould containing 100 μ g/mL ampicillin and 20 μ g/mL chlorine
The LB fluid mediums of element, 37 DEG C, 220rpm cultivates 10h；
2) bacterium solution is transferred in same fresh LB fluid mediums with 1: 100 dilution ratio, is cultivated to OD600
=0.4, isopropyl-beta D-thio galactopyranoside (IPTG) is added to final concentration 0.4mM, 37 DEG C of 5～6h of culture.
6th, the detection of dsRNA crude preparation by using and ultrasonic disruption cell
1st, dsRNA is detected
(1) qualitative method
1) E. coli broth of 5mL expression dsRNA is taken in the centrifuge tube of 10mL, the cell for adding appropriate 3mL splits
Solution liquid (1M ammonium acetates/10mMEDTA), 65 DEG C of water-bath 1h, period rocks bacterium solution frequently；
2) add isopyknic phenol/chloroform (1: 1), 12000rpm that 15min is centrifuged；
3) supernatant is taken, the dehydrated alcohol of 2 times of volumes is added, -20 DEG C, is stood overnight；
4) 4 DEG C, 12000rpm, 20min abandon supernatant；
5) often pipe adds the dsRNA buffer (10mM Tris/1mM EDTA) of 150 μ L, -20 DEG C of preservations；
6) 8uL steps 5 are taken respectively) the dsRNA solution for preparing, it is each to add DNAase I, RNaseA (low salt buffer),
RNaseA (high-salt buffer), 37 DEG C of water-bath 3h carry out enzyme action；
7) 10uL steps 5 are respectively taken) and step 6) solution 10uL, detected with 1.2% agarose gel electrophoresiies.DNAase and
RNaseA (low salt buffer) can digest and band that RNaseA (high-salt buffer) can not be digested be expression dsRNA.
(2) quantitative method
Take after 1mL bacterium solution centrifugation thalline with 200 μ L lysates smudge cellses to extract dsRNA, finally with 100 μ L
TE dissolving gained dsRNA.DsRNA solution to obtaining is located successively with DNAase I, RNaseA in the buffer of high salt concentration
Reason, to remove DNA, single stranded RNA impurity, it is to avoid impact to dsRNA ultraviolet detection.Then, existed with ultraviolet spectrophotometer
The light absorption value of detection sample under 260nm wavelength, calculates the concentration of dsRNA, in 40 μ g/mL.
2nd, detect after dsRNA, the transformant that will be enlarged by cultivating adopts ultrasonic disruption, obtain broken containing hair fastener dsRNA
Broken cell solution, as dsRNA crude preparation by using；
7th, inhibitor is prepared
Account for cell solution weight 5% sulfomethylated lignin is sequentially added in the smudge cellses solution containing hair fastener dsRNA
Sour sodium, 0.2% alkylbenzenesulfonate, 2% xanthan gum, 0.2 isoamyl alcohol, 2% glycerol, 0.1% tryptophan and 1% poly- second two
Alcohol ether, subpackage after stirring；Can be inhibited agent.The solubility of dsRNA is 30 μ g/mL wherein in inhibitor.Preparation will be kept away
Light is stored in brown reagent bottle, if long storage time need to be stored in less than 4 degrees Celsius.
Embodiment 2
Using inhibitor nornicotine,
2 days before tobbaco, in the morning before 9 points or after at 5 points in afternoon by the inhibitor containing 30ug/mLdsRNA spray in
The root face of Nicotiana tabacum L. in experimental group, amount of application is 10mL/ strains.Experimental group and matched group respectively have 4 groups, each group of all 100 plants Nicotiana tabacum L.s, just
Often picking and roasting after maturation, is measured by sampling nicotine content in tobacco leaf.Experimental result, compared with the control, middle leaf nicotine content drop
Low by 43.36%, upper leaf nicotine content reduces by 55.42%, is shown in Table 1.
1, table applies PMT Gene Partials sequence hair fastener dsRNA preparation nicotine content in tobacco leaf (%)
Note：* represent that same column data reaches significant level (p ＜ 0.05) with the difference for compareing
Embodiment 3
In the present embodiment, the inhibitor 10mL/ strains containing 30ug/mLdsRNA are imposed on the blade face of Nicotiana tabacum L..Other and reality
Apply example 2 identical.Experimental result, compared with the control, middle leaf nicotine content reduces by 18.92%, and upper leaf nicotine content is reduced
23.31%.
Foliar spray PMT Gene Partials sequence hair fastener dsRNA preparation nicotine content in tobacco leaf (%) of table 2
Embodiment 4
In the present embodiment, the inhibitor 5mL/ strains containing 30ug/mLdsRNA are imposed on the blade face of Nicotiana tabacum L., 5mL/ strains are applied
In root face.Other are same as Example 2.Experimental result, compared with the control, middle leaf nicotine content reduces by 23.53%, upper leaf
Nicotine content reduces by 34.41%.
The foliar spray of table 3 and Gen Shi PMT Gene Partials sequence hair fastener dsRNA preparation nicotine content in tobacco leaf (%)
Claims (4)
1. a kind of method that utilization double-stranded RNA perturbation technique prepares the inhibitor for reducing tobacco smoke alkaloid synthesis, it includes following step
Suddenly：
1) RNA of Nicotiana tabacum L. is extracted, reverse transcription obtains the cDNA of tobacco putrescine-N- methyl transferase genes；
2) utilize round pcr, the cDNA with tobacco putrescine-N- methyl transferase genes as template, the tobacco putrescine-N- first for obtaining
Based transferase gene segment；
3) by step 2) in tobacco putrescine-N- methyl transferase gene segments be connected in carrier T；
4) by step 3) in carrier T tobacco putrescine-N- methyl transferase gene segments be connected on plasmid vector PET-21a,
Obtain recombiant plasmid PET-EH；
5) by step 4) in obtain recombiant plasmid PET-EH be transferred to disappearance RNAE3 enzymes Bacillus coli cells in,
6) by step 5) in Bacillus coli cells screened, when transformant；
7) by step 6) in transformant amplification culture in LB fluid mediums；
8) transformant that will be enlarged by cultivating adopts ultrasonic disruption, obtains the smudge cellses solution containing hair fastener dsRNA, as
DsRNA crude preparation by using；
9) to step 8) in sequentially add and the lignin that dsRNA crude preparation by using solution weight ratio is 2～5% in dsRNA crude preparation by using
Sodium sulfonate, 0.1～0.3% alkylbenzenesulfonate, 1～3% xanthan gum, 0.1～0.3% isoamyl alcohol, 2～5% it is sweet
Oil, 0.01～0.1% tryptophan and 1～2% polyglycol ether, subpackage after then stirring, you can be reduced cigarette
The inhibitor of tobacco smoke alkaloid synthesis, dsRNA concentration is 25～35 μ g/mL in the inhibitor.
2. utilization double-stranded RNA perturbation technique according to claim 1 prepares the side of the inhibitor for reducing tobacco smoke alkaloid synthesis
Method, the step 2) in, the length of tobacco putrescine-N- methyl transferase gene segments is 400～700bp.
3. utilization double-stranded RNA perturbation technique according to claim 1 prepares the side of the inhibitor for reducing tobacco smoke alkaloid synthesis
Method, the step 9) in, to sodium lignin sulfonate, 0.2～0.3% alkane that dsRNA crude preparation by using solution weight ratio is 2～4%
Base benzene sulfonate, 1～2% xanthan gum, 0.1～0.2% isoamyl alcohol, 2～4% glycerol, 0.05～0.1% tryptophan
Polyglycol ether with 1～2%.
4. a kind of application of the inhibitor for reducing tobacco smoke alkaloid synthesis according to claim 1, it is characterised in that：The suppression
Agent is imposed on Nicotiana tabacum L. with 5～15mL/ strains.
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| CN112639105A (en) * | 2018-07-26 | 2021-04-09 | 奥驰亚客户服务有限公司 | PMT-based engineered compositions and methods for producing tobacco plants and products with altered levels of alkaloids |
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| CN112639105A (en) * | 2018-07-26 | 2021-04-09 | 奥驰亚客户服务有限公司 | PMT-based engineered compositions and methods for producing tobacco plants and products with altered levels of alkaloids |
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