CN102229948A - Method for increasing oil content in tobacco - Google Patents
Method for increasing oil content in tobacco Download PDFInfo
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- CN102229948A CN102229948A CN 201110117864 CN201110117864A CN102229948A CN 102229948 A CN102229948 A CN 102229948A CN 201110117864 CN201110117864 CN 201110117864 CN 201110117864 A CN201110117864 A CN 201110117864A CN 102229948 A CN102229948 A CN 102229948A
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Abstract
The invention relates to a method for increasing the oil content in tobacco, which is characterized by comprising the following steps: 1) cloning the rbcS promoter of tobacco and analyzing the sequence of the rbcS promoter; 2) cloning an SLC1-1 gene, performing the point mutation of the SLC1-1 gene and obtaining the SLC1-1 gene having the point mutation; 3) constructing a target expression vector rbcS:SLC1-1 to obtain plant expression vectors rbcS:pBI121 and rbcS:SLC1-1; and 4) constructing a transgenic line of tobacco and performing the polymerase chain reaction (PCR) detection of positive plants, namely directly placing a tobacco plate on a tobacco plate pre-culture and co-culture medium MS1, performing co-culture for 3 to 4 days, transferring the tobacco plate to a screening culture medium MS2 for screening culture, performing one time of subculture every other 2 to 3 weeks, cutting buds which are 1 centimeter long, placing the cut buds in a tobacco rooting culture medium MS3 for rooting culture, extracting DNA when the seedlings grow a little bit bigger, performing PCR detection, transplanting the buds, growing till matureness and obtaining T0 generation of tobacco transgenic plants. The method can improve the oil content in the tobacco.
Description
Technical field
The present invention relates to a kind of method that improves the tobacco leaf oleaginousness.
Background technology
Tobacco is that important leaf is used cash crop, only find at present that an example improves the method for tobacco leaf oleaginousness by gene engineering: the people such as Andrianov of U.S. Thomas Jefferson university use the DGAT (diacylglycerol acyltransferase) and LEC2 (the LEAFY COTYLEDON 2) gene of rbcS and Alc promoter expression Arabidopis thaliana respectively in tobacco, tobacco leaf per dry wt oleaginousness is brought up to 5.8-6.8%, oleaginousness than material before transforming has improved about 5 percentage points (Andrianov V et al., Plant Biotechnology Journal, 2010,8:277-287).
Summary of the invention
The object of the present invention is to provide a kind of method that improves the tobacco leaf oleaginousness, this method can improve the oleaginousness of tobacco leaf.
To achieve these goals, the technical solution used in the present invention is: a kind of method that improves the tobacco leaf oleaginousness is characterized in that it comprises the steps:
1), the clone of tobacco rbcS promotor and sequential analysis
With tobacco gene group DNA is that template is carried out pcr amplification, through the agarose gel electrophoresis demonstration of 1% (quality), goes out single band of 1000bp from tobacco gene group DNA cloning; This specific band is reclaimed, be connected to cloning vector pMD-18T vector, after importing host bacterium E.coli bacterial strain DH5 α, choose single bacterium colony and shake bacterium, the pcr amplification of recon pMD-18T-rbcS through screening and enzyme are cut the result and are shown, the purpose band all occurs, illustrate that amplified fragments successfully is connected among the pMD-18T;
Sequencing result shows that expanding fragment length is 979bp, and sequential analysis shows that this sequence is the rbcS promoter sequence;
2), the clone of SLC1-1 gene and point mutation thereof:
1. the clone of SLC1 gene coding region
Obtain barms from DSMZ of Wuhan University, the numbering of this barms: AY92007=AS2.159, source: Institute of Micro-biology of the Chinese Academy of Sciences, with the YM substratum 28 ℃ of overnight incubation, reclaim culture, its genomic dna of extracting uses high-fidelity Pyrobest archaeal dna polymerase and primer as the pcr amplification masterplate, and Primer 120 is 5 ' coding primer: 5 '-TTTCCCGGATCCGCCACCATGAGTGTGATAGGTAGGTTCTT-3 ' of SLC1; Primer 121 is 3 ' coding primer: 5 '-TTTCCCGAGCTCTTAATGCATCTTTTTTACAGATGAAC-3 ' of SLC1, carries out pcr amplification, and amplification condition is: 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 38cycles; 72 ℃ are extended 5min;
The YM medium component:
Add 2U Taq enzyme and 1 μ l 2.5mM dATP in the 25 μ l pcr amplification products and add A 20min in 72 ℃, the clone inserts the pMD18-T carrier, transforms DH5 α and is applied to the Amp flat board; Screen with universal primer M13+ and M13-, program is 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 40cycles; 72 ℃ are extended 5min; In order to verify the direction of insertion sequence, once more with 120/121, M13+/120, M13-/121 verify, two clones of picking forward and oppositely insertion carry out unidirectional order-checking at random, found that 1 its sequence of clone and SLC1 gene order only have the difference of 1 Nucleotide, itself is exactly a genome mutation probably; 1 site so must suddenly change just can obtain and the identical sequence of mutant SLC1-1 gene function;
2. obtain the SLC1-1 gene after the SLC1 point mutation
Use Pyrobest archaeal dna polymerase and primer 120/126,121/127 and 1,20/,121 5 ' fragment, 3 ' fragment and the full length sequence of the above-mentioned SLC1 gene of pcr amplification in 25 μ l systems respectively earlier, the PCR program is: 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 35cycles; 72 ℃ are extended 5min; Primer 126 is the 5 ' mutant primer of SLC1: 5 '-CGCGCAGTAATCCACAGAGCCAAATGTTG-3 '; Primer 127 is the 3 ' mutant primer of SLC1: 5 '-TTGGCCTTGACGTCAAGGTCGTTGGCG-3 ';
5 ' fragment, 3 ' fragment and the full length sequence of SLC1 gene are mixed, carry out pcr amplification with the Pyrobest archaeal dna polymerase in 50 μ l systems, the PCR program is 94 ℃ of 1min; 94 ℃ of 30sec, 68 ℃ of 90sec, 20cycles; 72 ℃ are extended 5min; The PCR system is composed as follows:
Total length PCR product in the electrophoretic separation Pyrobest amplification system is cut glue recovery and order-checking; Order-checking shows that one of them clone (SLC-3-31) has completed successfully 1 target amino acid site mutation; Downcut the purpose fragment and insert the PBI121 carrier from SLC-3-31 with BamHI and SacI, promptly obtained the SLC1-1 gene after the point mutation;
3), the structure of purpose expression vector rbcS:SLC1-1
With HindIII and Xba I restriction enzymes double zyme cutting plasmid 35S:pBI121,35S:SLC1-1 and pMD-18T-rbcS, reclaim enzyme after finishing respectively and cut big fragment of carrier and promoter sequence fragment product, enzyme is cut the big fragment of carrier and promoter sequence fragment product to be mixed according to 4: 1 mass ratio, under T4DNA ligase effect more than 16 ℃ of incubation 12h, to connect product and import host bacterium E.coli DH5 α, cut evaluation by PCR evaluation and enzyme and show, obtain plant expression vector rbcS:pBI121 and rbcS:SLC1-1;
4), the PCR of the foundation of tobacco transformation system and positive seedling detects
Purpose expression vector rbcS:SLC1-1 is transformed Agrobacterium LBA4404, obtain containing the Agrobacterium of purpose expression vector rbcS:SLC1-1; Get the tender aseptic blade of tobacco children, produce the leaf dish with punch tool, and infect with containing purpose expression vector rbcS:SLC1-1 Agrobacterium;
With the leaf dish directly place tobacco leaf disc pre-, altogether on the substratum MS1, under the low light level pre-cultivate and cultivate 3-4 days altogether after; Cultivate altogether after 3-4 days the leaf dish is transferred to the enterprising row filter cultivation of screening culture medium MS2, every carrying out succeeding transfer culture 2-3 week one time; With carrying out root culture on the budlet cutting-out placement tobacco root media MS3, treat that the slightly bigger DNA of extraction of young plant growth carries out PCR and detects when waiting to occur the 1cm budlet;
The tobacco transgenosis adopts the During Agrobacterium method to transform, and agriculture bacillus mediated tobacco leaf disc conversion method is with reference to Agrobacterium protocols I; Minimum medium MS with additional AS
0Resuspended bacterium liquid, the 0D value reaches 0.5-1.0, infect through pre-cultivation or without pre-incubated tobacco leaf disc, infected 20-30 minute, cultivated altogether under the low light level 3 days, contain the 100mg/L card that, 400mg/L carboxylic benzyl substratum screening, per two weeks are changed a fresh culture, when growing to 1cm, resistant buds moves into tobacco root media MS3, additional that screening of 75mg/L card; The plant of growing way stalwartness is adopted NPTII to carry out PCR and detects;
Budlet is transplanted, grown to maturation, obtain T
0For the tobacco transfer-gen plant.
Described minimum medium MS
0: macroelement+trace element+organic composition+molysite composition+3% sucrose+0.7% agar;
Pre-, the common substratum MS1:MS of described tobacco leaf disc
0+ 2mg/L 6-BA+0.2mg/L IAA;
Described screening culture medium MS2:MS
0+ 2mg/L 6-BA+0.2mg/L IAA+500mg/L Carb+100mg/L Kan;
Described tobacco root media MS3:MS
0+ 0.2mg/L IAA+200mg/L Carb+75mg/L Kan;
The invention has the beneficial effects as follows: this method can improve the oleaginousness of tobacco leaf.Select transgenosis T
2Lower and RT-PCR result sample is preferably got the complete stool blade and is ground the mixing numbering for copy number in the transfer-gen plant, carries out the mensuration of crude fat, the crude fat content of sample slc5 than the crude fat content height of ck 5.9 percentage points.
Description of drawings
Fig. 1 rbcS promotor pcr amplification product electrophorogram, 1 DL2000marker; 2 negative controls; The 3PCR product.
Fig. 2 is that pMD-18T-rbcS is the PCR product electrophorogram of template, 1 DL2000Marker; 2 negative controls; 3-5 is the PCR product.
Fig. 3 is that the pMD-18T-rbcS enzyme is cut the product electrophorogram, and 1 Hind III and Xba I enzyme are cut product; 2 DGL2000 marker.
Fig. 4 is the rbcS promoter fragment sequence chart of being cloned into (is used for and reported sequence (GenBank:X02353) comparison result).
Fig. 5 is that plasmid 35S:SLC1-1 enzyme is cut evaluation figure, 1DL 2000marker; 2 BamH I and Sac I double digestion.
The SLC1-1 fragment sequence figure that Fig. 6 is cloned into [be used for reported sequence SLC1 (GenBank:Z74100.1) comparison result].
Fig. 7 rbcS:pBI121, rbcS:SLC1-1 positive colony PCR detect electrophorogram, 1DGL2000Marker; 2 negative controls; 3-21 PCR product.
Fig. 8 rbcS:pBI121, rbcS:SLC1-1 plasmid enzyme restriction electrophorogram, 1DL2000Marker; 2 plasmid rbcS:pBI121Hind III and Xba I enzyme are cut product; 3 plasmid rbcS:SLC1-1 Hind III and Xba I enzyme are cut product.
Fig. 9 is rbcS:pBI121, rbcS:SLC1-1 carrier collection of illustrative plates, LB, RB: be border, the left and right sides; NOSp, NOSt: be NOS promotor and terminator; NptII: for blocking that resistance screening gene; RbcS: be the rbcS of tissue-specific promoter of tobacco leaf portion; HindIII, XbaI, BamHI, SacI: be restriction enzyme digestion sites; GUS is a reporter gene.
Figure 10 is a tobacco genetic transformation procedure chart, and a cuts seedling, and b cultivates in advance, and c cultivates altogether, the d screening, and e is taken root, and f transplants.
The PCR of Figure 11 transgene tobacco detects electrophorogram, 1 DL2000Marker; 2-21 PCR product; 22 blanks; 23 negative controls; 24 positive plasmids.
The PCR electrophorogram of Figure 12 transgene tobacco, 1 DL2000Marker; 2 negative controls; 3 positive plasmids; The 4-13PCR product.
Figure 13 contrast, change rbcs:pBI121, change the active histochemical stain figure of different sites GUS of 35S:pBI121 plant, A1, A2, A3 are respectively not that root, stem, the leaf of transfer-gen plant (negative control) carry out GUS dyeing back body formula anatomical lens observation figure among the figure; B1, B2, B3 are respectively the tobacco root, stem, the leaf that change rbcS:pBI121 and carry out GUS dyeing after body formula dissecting microscope observation figure; C1, C2, C3 are respectively the tobacco root, stem, the leaf that change 35S:pBI121 and carry out GUS dyeing back body formula dissecting microscope observation figure.
Figure 14 utilizes RT-PCR technical Analysis tobacco leaf SLC1-1 expression of gene spirogram.
Embodiment
In order to understand the present invention better, further illustrate content of the present invention below in conjunction with embodiment, but content of the present invention not only is confined to the following examples.
One, English initialism
CaMV35S: cauliflower mosaic virus promotor
GUS: beta-Glucuronidase
NPTII: aminoglycoside-3 '-phosphotransferase (kalamycin resistance)
RbcS: ribulose-1,5-bisphosphate, 5-bisphosphate oxygenase/carboxylase small subunit
SLC gene: the gene of coding yeast sn-2 Acyltransferase
IPCR: inverse PCR
TaiL-PCR: hot asymmetric interlaced PCR
CTAB: cetyl trimethylammonium bromide
The 6-BA:6-benzyladenine
NAA: α-Nai Yisuan
Car: Pyocianil
Rif: Rifampin
Amp: penbritin
DEPC: baycovin
X-gluc:5-bromo-4 chloro-3-indoles glucosides.
Two, experiment material
1.1 carrier and bacterial strain
E.coli bacterial strain DH5 α, Agrobacterium LBA4404, plant expression vector 35S:pBI121,35S:SLC1-1 are provided by oil crops institute of the Chinese Academy of Agricultural Sciences.
1.2DNA it is the Fermentas product that purifying reclaims test kit, plasmid extraction kit is available from vast Tyke, Beijing biological gene technology limited liability company, and pMD-18T vector, T4DNA ligase enzyme, Hind III, Xba I, BamH I, Sac I, Taq I and Mst I restriction enzyme are purchased in Takara, Promega company.
1.3 substratum
The MS culture medium prescription:
Above solution all is made into 100 * mother liquor.
(1) minimum medium MS
0: macroelement+trace element+organism (organic composition)+molysite+3% sucrose+0.7% agar;
(2) pre-, the common substratum MS1:MS of tobacco leaf disc
0+ 2mg/L 6-BA+0.2mg/L IAA;
(3) screening culture medium MS2:MS
0+ 2mg/L 6-BA+0.2mg/L IAA+500mg/L Carb+100mg/L Kan;
(4) tobacco root media MS3:MS
0+ 0.2mg/L IAA+200mg/L Carb+75mg/L Kan;
(5) YEB substratum: extractum carnis (5g/L), yeast extract (1g/L), peptone (5g/L), sucrose (5g/L), MgSO
4(2mmol/L) pH7.2, solid medium contain agar 15g/L;
(6) LB substratum: with following components dissolved in 0.9L water: peptone 10g, yeast extract 5g, sodium-chlor 10g adjusts PH to 7.0, supplies water again to 1L, solid medium contains agar 15g/L;
(7) tobacco pollen germination medium: boric acid 0.01%, sucrose 10%, PH 6.0.
1.4 hormone and microbiotic
6-benzyl aminopurine (6-BA): U.S. SIGMA is original-pack, is made into the mother liquor of 1mg/mL with sterile distilled water, and 4 ℃ of preservations are standby.
α-Nai Yisuan (NAA): U.S. SIGMA is original-pack, is made into the mother liquor of 1mg/mL with sterile distilled water, and 4 ℃ of preservations are standby.
Pyocianil (Car) 100mg/ml:1g is dissolved in sterile distilled water, and constant volume is in 10ml, 0.22 μ m membrane filtration, and-20 ℃ of preservations are standby.
Kantlex (Kan) 100mg/ml:1g is dissolved in sterile distilled water, and constant volume is in 10ml, 0.22 μ m membrane filtration, and-20 ℃ of preservations are standby.
Penbritin (Amp) 100mg/ml:1g is dissolved in sterile distilled water, and constant volume is in 10ml, 0.22 μ m membrane filtration, and-20 ℃ of preservations are standby.
Streptomycin sulphate 50mg/ml:0.5 gram streptomycin sulfate is dissolved in the dehydrated alcohol, is settled to 10ml at last, and-20 ℃ of preservations are standby.
Rifampin (Rif) 20mg/ml:0.2 gram is dissolved in methyl alcohol or NaOH, and the dissolving back is settled to 10ml with sterile distilled water, and-20 ℃ of preservations are standby.
1.5 main solution
(1)1mol/L?Tris-HCl
Be dissolved between Tris 121g with 0.9L water in, add a certain amount of concentrated hydrochloric acid according to desired pH value again, water is adjusted final volume to 1L.
(2)5mol/L?NaCl
Dissolve 29.2g sodium-chlor in the capacity distilled water, constant volume 100mL.
(3)3mol/L?NaAC，pH?5.2
The sodium acetate trihydrate of dissolving 40.8g with glacial acetic acid adjust pH to 5.2, adds the water constant volume to 100mL again in about 90mL water.
(4) 0.2mol/L phosphate buffered saline buffer (pH 7.0)
A liquid: 0.2mol/L NaH2PO42H2O (31.2g/L)
B liquid: 0.2mol/L Na2HPO4 (28.39g/L)
(5) 10mg/mL Rnase (no Dnase)
The RNA enzyme (pH5.0) in the aqueous sodium acetate solution of the 10mmol/L of 1mL of dissolving 10mg, 15min is boiled in the dissolving back in water-bath, make the DNA enzyme deactivation.Tris-HCl with 1mol/L transfers pH to 7.5, stores in-20 ℃ of refrigerators (will be with gloves in the process for preparation).
(6) DEPC (baycovin) treating water
Add 100 μ LDEPC in the 100mL distilled water, the volume fraction that makes DEPC is 0.1%.Bathe 12h at least 37 ℃ of temperature, autoclaving 20min under the 104.7kPa condition then is so that remaining DEPC inactivation.DEPC can react with amine, and unavailable DEPC handles the Tris damping fluid.
(7)50×TAE
Take by weighing 242gTris and be dissolved in an amount of distilled water, add the EDTA of 57.1mL glacial acetic acid and 100mLpH8.0, the distilled water constant volume is in 1L.
(8) the saturated phenol of Tris
(9)1mol/L?HCl
The concentrated hydrochloric acid that adds 8.6mL is to the distilled water of 91.4mL.
(10) phenol/chloroform: saturated phenol: chloroform (1: 1) mixed solution.
(11) phenol/chloroform/primary isoamyl alcohol (25: 24: 1): with phenol, chloroform, primary isoamyl alcohol mixed, 4 ℃ of preservations by 25: 24: 1.
(12) restriction enzyme and damping fluid: available from Promega, Takara company.Endonuclease reaction carries out according to specification sheets.
(13)1mol/L?MgCl
2
Dissolving 20.3MgCl
26H
2O is in the distilled water of capacity, and constant volume is to 100mL, filtration sterilization.
(14) 10mol/L sodium hydroxide (NaOH)
Dissolving 400g sodium hydrate particle (magnetic stirrer) in the beaker that about 0.9L water is housed, sodium hydroxide dissolve the back fully and are settled to 1L with distilled water.
(15) X-gluc (1mg/ μ l) solution: claim 10mg X-gluc to be dissolved in the 100 μ l N-N-dimethyl formamides ,-20 ℃ of preservations are standby;
(16) phenol/chloroform (1: 1): phenol and chloroform equal-volume are mixed 4 ℃ of preservations.
(17) chloroform/primary isoamyl alcohol (24: 1): with chloroform and primary isoamyl alcohol with 24; 1 volume mixture, 4 ℃ of preservations.
(18) phenol/chloroform/primary isoamyl alcohol (25: 24: 1): with phenol, chloroform, primary isoamyl alcohol mixed, 4 ℃ of preservations by 25: 24: 1.
(19) plasmid extracts solution I, II, III
Solution I: 50mM glucose, 25mM TrisHCl pH8.0,10mM EDTA.
The configuration of solution I: system 100mL.Take by weighing 0.99g glucose, add 2.5mL M Tris-Cl, 2mL0.5M EDTA, use the aqua sterilisa constant volume to 100mL.
Solution II: 0.2mol/L NaOH, 1%SDS.Matching while using.
The configuration of solution II: 0.2mL 1M NaOH, 50uL 20%SDS, 750uL aqua sterilisa.
Solution III: 3mM Glacial acetic acid potassium.
The configuration of solution III: 60mL 5M Potassium ethanoate, 11.5mL Glacial acetic acid, 28.5mL aqua sterilisa.
(20) GUS dye liquor: 50mmol/L phosphoric acid buffer, pH7.0; 10mmol/L Na
2-EDTA; 0.001%Triton X-100; 0.5mmol/L yellow prussiate of potash; 0.5mmol/L the Tripotassium iron hexacyanide; 20% methyl alcohol; 0.5mg/mLX-gluc.X-gluc (1mg/ μ l) solution: claim 10mg X-gluc to be dissolved in the 100 μ lN-N-dimethyl formamides ,-20 ℃ of preservations are standby; Na
2HPO
4(1mol/l) solution: take by weighing Na
2HPO
4.2H
2O 35.814 grams are dissolved in the 100ml water, and 4 ℃ of preservations are standby; NaH
2PO
4(1mol/l) solution: take by weighing NaH
2PO
4.2H
2O 15.6 grams are dissolved in the 100ml water, and 4 ℃ of preservations are standby; Tripotassium iron hexacyanide K
3[Fe (CN)
6] (50mmol/l): take by weighing K
3[Fe (CN)
6] 1.646 gram constant volumes are in 100ml water, 4 ℃ of preservations are standby; Yellow prussiate of potash K
4[Fe (CN)
6] (50mmol/l): take by weighing K
4[Fe (CN)
6] .2H
2O 2.1 gram constant volumes are in 100ml water, and 4 ℃ of preservations are standby;
EDTA (0.5mol/l PH8.0): take by weighing EDTA 14.6 grams and be dissolved in the 80ml water, transfer PH to 8.0, be settled to 100ml, 4 ℃ of preservations are standby.
Three, a kind of method that improves the tobacco leaf oleaginousness, it comprises the steps:
1, the clone of tobacco rbcS promotor and sequential analysis
With tobacco gene group DNA is that template is carried out pcr amplification, and the agarose gel electrophoresis through 1% shows, is with (Fig. 1) from the list that tobacco gene group DNA cloning goes out about 1000bp; This specific band is reclaimed, be connected to cloning vector pMD-18T vector, after importing host bacterium E.coli DH5 α (E.coli bacterial strain DH5 α), choose single bacterium colony and shake bacterium, pcr amplification and the enzyme of recon pMD-18T-rbcS through screening are cut result (Fig. 2, Fig. 3) show, the purpose band all occurs, illustrate that amplified fragments successfully is connected among the pMD-18T;
Sequencing result shows that expanding fragment length is 979bp, and sequential analysis shows that this sequence is the rbcS promoter sequence; RbcS promoter sequence result such as Fig. 4;
PMD-18T-rbcS sequencing result (Fig. 4) shows with the comparison of NCBI reported sequence (GenBank:X02353), do not find the change of base in critical function zones such as TATA-box, CAAT-box, G-box from tobacco NC89 clone's rbcs promoter sequence, but there is the sudden change of 7 place's bases in other zones, comprise that 4 place's bases are replaced and 3 place's bases are inserted, the corresponding zone homology is 99.08%;
2, the clone of SLC1-1 gene and point mutation thereof:
2.1SLC1 the clone of gene coding region
Obtain barms (numbering: AY92007=AS2.159 from DSMZ of Wuhan University, source: Institute of Micro-biology of the Chinese Academy of Sciences), with YM (Yeast Malt Agar) substratum 28 ℃ of overnight incubation, reclaim culture, its genomic dna of extracting is as the pcr amplification masterplate, (Primer 120, i.e. 5 ' of SLC1 coding primer: 5 '-TTTCCCGGATCCGCCACCATGAGTGTGATAGGTAGGTTCTT-3 ' to use high-fidelity Pyrobest archaeal dna polymerase and primer; Primer 121, i.e. 3 ' of SLC1 coding primer: 5 '-TTTCCCGAGCTCTTAATGCATCTTTTTTACAGATGAAC-3 '), carry out pcr amplification, amplification condition is: 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 38cycles; 72 ℃ are extended 5min;
YM (Yeast Malt Agar) medium component (1L):
Add 2U Taq enzyme and 1 μ l 2.5mM dATP in the 25 μ l pcr amplification products and add A 20min in 72 ℃, the clone inserts the pMD18-T carrier, transforms DH5 α and is applied to the Amp flat board; Screen with universal primer M13+ and M13-, program is 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 40cycles; 72 ℃ are extended 5min; In order to verify the direction of insertion sequence, once more with 120/121, M13+/120, M13-/121 verify, two clones of picking forward and oppositely insertion carry out unidirectional order-checking at random, found that 1 its sequence of clone and SLC1 gene order only have the difference of 1 Nucleotide, itself is exactly a genome mutation probably; 1 site (131A → T), just can obtain and the identical sequence of mutant SLC1-1 gene function so must suddenly change;
2.2SLC1 obtain the SLC1-1 gene after the point mutation
(Primer 126 to use Pyrobest archaeal dna polymerase and primer 120/126 earlier, 5 '-CGCGCAGTAATCCACAGAGCCAAATGTTG-3 '), 121/127 (Primer 127 be the 5 ' mutant primer of SLC1:, 5 '-TTGGCCTTGACGTCAAGGTCGTTGGCG-3 ') and 1,20/,121 5 ' fragment, 3 ' fragment and the full length sequence of the above-mentioned SLC1 gene of pcr amplification in 25 μ l systems respectively be the 3 ' mutant primer of SLC1:, the PCR program is: 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 35cycles; 72 ℃ are extended 5min;
5 ' fragment, 3 ' fragment and the full length sequence of SLC1 gene are mixed, carry out pcr amplification with the Pyrobest archaeal dna polymerase in 50 μ l systems, the PCR program is 94 ℃ of 1min; 94 ℃ of 30sec, 68 ℃ of 90sec, 20cycles; 72 ℃ are extended 5min; The PCR system is composed as follows:
Total length PCR product in the electrophoretic separation Pyrobest amplification system is cut glue recovery and order-checking; Order-checking shows that one of them clone (SLC-3-31) has completed successfully 1 target amino acid site mutation; Downcut the purpose fragment and insert the PBI121 carrier from SLC-3-31 with BamHI and SacI, promptly obtained the SLC1-1 gene after the point mutation;
Yeast bacillus SLC1 gene fragment is long to be 912bp, has report to point out the sn-2Acyltransferase enzymic activity height of the sn-2Acyltransferase enzymic activity of mutant SLC1-1 coded by said gene than the SLC1 coded by said gene; Contain the SLC1-1 gene in the 35S:SLC1-1 plasmid that preserve in this laboratory; The plant expression vector 35S:SLC1-1 that the laboratory has been built carries out BamHI and the evaluation of Sac I double digestion, and 1% gel electrophoresis shows that endonuclease bamhi meets purpose clip size (as shown in Figure 5), and the vector construction success tentatively is described; To the SLC1-1 of plasmid 35S:SLC1-1 order-checking (as Fig. 6) result result that compares;
The order-checking comparison result shows, the sheet segment length who links carrier is 912bp, yeast 1-acyl-sn-gylcerol-3-phosphate acyltransferase gene order (GenBank:Z74100.1) matching rate of including with Genebank is 99%, there is base mutation in 3 sites, wherein the sudden change at the 131st base place is artificial purpose sudden change, purpose is to improve the activity of this enzyme, is about to glutamine (Q) and changes leucine (L) into; The position of base replacement takes place respectively at the 555th, 612 base places in other two places, its codon is changed into atc and is changed into ttt by ttc by att, although there is base mutation, but codon att and atc are the coding Isoleucines, codon ttc and ttt coding phenylalanine be not so its amino acid sequence coded changes; The base sequence that illustrates in the carrier to be linked is correct, can utilize this plasmid to carry out the structure of rbcS:SLC1-1 carrier;
3, the structure of purpose expression vector rbcS:SLC1-1
With HindIII and Xba I restriction enzymes double zyme cutting plasmid 35S:pBI121,35S:SLC1-1 and pMD-18T-rbcS, reclaim enzyme after finishing respectively and cut big fragment of carrier and promoter sequence fragment product, enzyme is cut the big fragment of carrier and promoter sequence fragment product to be mixed according to 4: 1 mass ratio, under T4DNA ligase effect more than 16 ℃ of incubation 12h, to connect product and import host bacterium E.coli DH5 α, cut evaluation (as Fig. 8) by PCR evaluation (Fig. 7) and enzyme and show, obtain plant expression vector rbcS:pBI121 and rbcS:SLC1-1; Carrier collection of illustrative plates such as Fig. 9;
4, the PCR of the foundation of tobacco transformation system and positive seedling detects
Purpose expression vector rbcS:SLC1-1 is transformed Agrobacterium LBA4404, obtain containing the Agrobacterium of purpose expression vector rbcS:SLC1-1; Get the tender aseptic blade of tobacco children, produce the leaf dish with punch tool, and infect with containing purpose expression vector rbcS:SLC1-1 Agrobacterium;
With the leaf dish directly place tobacco leaf disc pre-, altogether on the substratum MS1, under the low light level pre-cultivate and cultivate 3-4 days altogether after, the water stain putrefactive phenomenon of leaf dish often appears, so the leaf dish is placed on the filter paper, at covering one deck filter paper, water stain putrefactive phenomenon significantly reduces on it; Cultivate altogether after 3-4 days the leaf dish is transferred to the enterprising row filter cultivation of screening culture medium MS2, every carrying out succeeding transfer culture 2-3 week one time; With carrying out root culture on the budlet cutting-out placement tobacco root media MS3, treat that the slightly bigger DNA of extraction of young plant growth carries out PCR and detects when waiting to occur 1cm left and right sides budlet;
The tobacco transgenosis adopts the During Agrobacterium method to transform, and agriculture bacillus mediated tobacco leaf disc conversion method is with reference to Agrobacterium protocols I; Minimum medium MS with additional AS
0Resuspended bacterium liquid, the OD value reaches 0.5-1.0, infect through pre-cultivation or without pre-incubated tobacco leaf disc, infected 20-30 minute, cultivated altogether under the low light level 3 days, contain the 100mg/L card that, 400mg/L carboxylic benzyl substratum screening, per two weeks are changed a fresh culture, when growing to the 1cm left and right sides, resistant buds moves into tobacco root media MS3, additional that screening of 75mg/L card; The plant of growing way stalwartness is adopted NPTII to carry out PCR and detects (Figure 11,12);
Budlet is transplanted, grown to maturation, obtain T
0For the tobacco transfer-gen plant.
5, the activity identification of rbcS gene promoter
Be the expression pattern of gus reporter gene in transgene tobacco of analysis tobacco rbcS promotor control, the transgenic tobacco plant of choosing through the PCR tests positive carries out the GUS staining analysis.Get root, stem and leaf on each plant respectively and dye, respectively get five strains, not genetically modified as negative control (CK), the plant that changes 35S:pBI121 is as positive control.As can see from Figure 13, the root of negative control, stem, leaf (CK1, CK2, CK3) do not observe blueness, and observe tangible blueness at root, stem, the Ye Junneng of the plant that changes 35S:pBI121, and the color of its middle period and root is darker.The plant that changes rbcS:pBI121 only observes tangible blueness in leaf portion, and the plant leaf portion histochemical stain of changeing rbcS:pBI121 is changeed the color of the plant leaf GUS of the portion histochemical stain of 35S:pBI121 and is wanted dark, show strong than CaMV35S of ability that tobacco rbcS promotor expresses at tobacco leaf portion promotor gene, and in the not expression of promotor gene substantially of other tissue sites.
6, the transcriptional expression of transfer-gen plant detects
Genetically modified purpose is exactly in order to allow foreign gene express in the purpose plant, PCR detects and Southern blot can only prove that foreign gene has inserted in the purpose Plant Genome, and the position that can foreign gene expression insert with promotor and foreign gene is relevant.We know according to central dogma, and expression of gene is by DNA → RNA → protein, and the transgenic plant expression analysis can carry out the evaluation of rna expression level by the method for RT-PCR, to T
0Extract RNA for the transgenic positive plant, the laggard performing PCR of reverse transcription detects, and the 32-40PCR circulation is the plateau of actin reaction, gets 36 circulations.Dui Zhao RNA no specific band behind reverse transcription amplifies as shown in Figure 14, also not having specific band behind the 1st, 4,5,11 reverse transcriptions of transfer-gen plant amplifies, all the other plant all amplify specificity SLC band, according to the brightness of actin band and SLC specific band as can be seen, the expression amount of transfer- gen plant 2,3,6,7,8,12,14,15,16 is higher.
7, transgene tobacco T
0For the greasy mensuration of plant
According to the T of RT-PCR result to commentaries on classics rbcS:SLC1-1
0Plant (individual plant), adjoining tree (choosing three strain balanced mix) carry out the oleaginousness of tobacco leaf dry weight and measure.The collection of cigarette sample and preparation are with reference to Wang Ruixin chief editor " tobacco chemistry ", and the mensuration of fat content is finished by Inst. of Oil Crops, Chinese Academy of Agriculture.Get the complete stool blade and grind the mixing numbering, carry out the mensuration of crude fat, the crude fat content of sample slc5 than the crude fat content height of ck 1.1 percentage points.
8, transgene tobacco T
2(the primary transfer-gen plant is T for the greasy mensuration of plant
0Generation, T
0For being T under the seed kind that obtains after the plant selfing
1For plant, T
1For being T under the seed kind that obtains after the plant selfing
2For plant)
Select transgenosis T
2Lower and RT-PCR result sample is preferably got the complete stool blade and is ground the mixing numbering for copy number in the transfer-gen plant, carries out the mensuration of crude fat, the crude fat content of sample slc5 than the crude fat content height of ck 5.9 percentage points.
Claims (2)
1. a method that improves the tobacco leaf oleaginousness is characterized in that it comprises the steps:
1), the clone of tobacco rbcS promotor and sequential analysis
With tobacco gene group DNA is that template is carried out pcr amplification, through the agarose gel electrophoresis demonstration of 1% (quality), goes out single band of 1000bp from tobacco gene group DNA cloning; This specific band is reclaimed, be connected to cloning vector pMD-18T vector, after importing host bacterium E.coli bacterial strain DH5 α, choose single bacterium colony and shake bacterium, the pcr amplification of recon pMD-18T-rbcS through screening and enzyme are cut the result and are shown, the purpose band all occurs, illustrate that amplified fragments successfully is connected among the pMD-18T;
Sequencing result shows that expanding fragment length is 979bp, and sequential analysis shows that this sequence is the rbcS promoter sequence;
2), the clone of SLC1-1 gene and point mutation thereof:
1. the clone of SLC1 gene coding region
Obtain barms from DSMZ of Wuhan University, the numbering of this barms: AY92007=AS2.159, source: Institute of Micro-biology of the Chinese Academy of Sciences, with the YM substratum 28 ℃ of overnight incubation, reclaim culture, its genomic dna of extracting uses high-fidelity Pyrobest archaeal dna polymerase and primer as the pcr amplification masterplate, and Primer 120 is 5 ' coding primer: 5 '-TTTCCCGGATCCGCCACCATGAGTGTGATAGGTAGGTTCTT-3 ' of SLC1; Primer 121 is 3 ' coding primer: 5 '-TTTCCCGAGCTCTTAATGCATCTTTTTTACAGATGAAC-3 ' of SLC1, carries out pcr amplification, and amplification condition is: 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 38cycles; 72 ℃ are extended 5min;
The YM medium component:
Add 2U Taq enzyme and 1 μ l 2.5mM dATP in the 25 μ l pcr amplification products and add A 20min in 72 ℃, the clone inserts the pMD18-T carrier, transforms DH5 α and is applied to the Amp flat board; Screen with universal primer M13+ and M13-, program is 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 40cycles; 72 ℃ are extended 5min; In order to verify the direction of insertion sequence, once more with 120/121, M13+/120, M13-/121 verify, two clones of picking forward and oppositely insertion carry out unidirectional order-checking at random, found that 1 its sequence of clone and SLC1 gene order only have the difference of 1 Nucleotide, itself is exactly a genome mutation probably; 1 site so must suddenly change just can obtain and the identical sequence of mutant SLC1-1 gene function;
2. obtain the SLC1-1 gene after the SLC1 point mutation
Use Pyrobest archaeal dna polymerase and primer 120/126,121/127 and 1,20/,121 5 ' fragment, 3 ' fragment and the full length sequence of the above-mentioned SLC1 gene of pcr amplification in 25 μ l systems respectively earlier, the PCR program is: 94 ℃ of 2min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 35cycles; 72 ℃ are extended 5min; Primer 126 is the 5 ' mutant primer of SLC1: 5 '-CGCGCAGTAATCCACAGAGCCAAATGTTG-3 '; Primer 127 is the 3 ' mutant primer of SLC1: 5 '-TTGGCCTTGACGTCAAGGTCGTTGGCG-3 ';
5 ' fragment, 3 ' fragment and the full length sequence of SLC1 gene are mixed, carry out pcr amplification with the Pyrobest archaeal dna polymerase in 50 μ l systems, the PCR program is 94 ℃ of 1min; 94 ℃ of 30sec, 68 ℃ of 90sec, 20cycles; 72 ℃ are extended 5min; The PCR system is composed as follows:
Total length PCR product in the electrophoretic separation Pyrobest amplification system is cut glue recovery and order-checking; Order-checking shows that one of them clone has completed successfully 1 target amino acid site mutation; Downcut the purpose fragment and insert the PBI121 carrier from SLC-3-31 with BamHI and SacI, promptly obtained the SLC1-1 gene after the point mutation;
3), the structure of purpose expression vector rbcS:SLC1-1
With HindIII and Xba I restriction enzymes double zyme cutting plasmid 35S:pBI121,35S:SLC1-1 and pMD-18T-rbcS, reclaim enzyme after finishing respectively and cut big fragment of carrier and promoter sequence fragment product, enzyme is cut the big fragment of carrier and promoter sequence fragment product to be mixed according to 4: 1 mass ratio, under T4DNA ligase effect more than 16 ℃ of incubation 12h, to connect product and import host bacterium E.coli DH5 α, cut evaluation by PCR evaluation and enzyme and show, obtain plant expression vector rbcS:pBI121 and rbcS:SLC1-1;
4), the PCR of the foundation of tobacco transformation system and positive seedling detects
Purpose expression vector rbcS:SLC1-1 is transformed Agrobacterium LBA4404, obtain containing the Agrobacterium of purpose expression vector rbcS:SLC1-1; Get the tender aseptic blade of tobacco children, produce the leaf dish with punch tool, and infect with containing purpose expression vector rbcS:SLC1-1 Agrobacterium;
With the leaf dish directly place tobacco leaf disc pre-, altogether on the substratum MS1, under the low light level pre-cultivate and cultivate 3-4 days altogether after; Cultivate altogether after 3-4 days the leaf dish is transferred to the enterprising row filter cultivation of screening culture medium MS2, every carrying out succeeding transfer culture 2-3 week one time; With carrying out root culture on the budlet cutting-out placement tobacco root media MS3, treat that the slightly bigger DNA of extraction of young plant growth carries out PCR and detects when waiting to occur the 1cm budlet;
The tobacco transgenosis adopts the During Agrobacterium method to transform, and agriculture bacillus mediated tobacco leaf disc conversion method is with reference to Agrobacterium protocols I; Minimum medium MS with additional AS
0Resuspended bacterium liquid, the OD value reaches 0.5-1.0, infect through pre-cultivation or without pre-incubated tobacco leaf disc, infected 20-30 minute, cultivated altogether under the low light level 3 days, contain the 100mg/L card that, 400mg/L carboxylic benzyl substratum screening, per two weeks are changed a fresh culture, when growing to 1cm, resistant buds moves into tobacco root media MS3, additional that screening of 75mg/L card; The plant of growing way stalwartness is adopted NPTII to carry out PCR and detects;
Budlet is transplanted, grown to maturation, obtain T
0For the tobacco transfer-gen plant.
2. a kind of method that improves the tobacco leaf oleaginousness according to claim 1 is characterized in that: described minimum medium MS
0: macroelement+trace element+organic composition+molysite composition+3% sucrose+0.7% agar;
Pre-, the common substratum MS1:MS of described tobacco leaf disc
0+ 2mg/L 6-BA+0.2mg/L IAA;
Described screening culture medium MS2:MS
0+ 2mg/L 6-BA+0.2mg/L IAA+500mg/L Carb+100mg/L Kan;
Described tobacco root media MS3:MS
0+ 0.2mg/L IAA+200mg/L Carb+75mg/L Kan;
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| CN107586880A (en) * | 2017-10-31 | 2018-01-16 | 云南省烟草农业科学研究院 | A kind of molecular labeling and application for differentiating tobacco cadmium transporter gene NtHMA2 wild types and mutant |
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| CN1186519A (en) * | 1995-05-31 | 1998-07-01 | 加拿大国家研究委员会 | Modification of plant lipids and seed oils utilizing yeast SLC genes |
| US20070006346A1 (en) * | 2004-06-30 | 2007-01-04 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring modulated plant growth rate and biomass in plants |
| CN101265479A (en) * | 2007-12-05 | 2008-09-17 | 昆明理工大学 | Plant expression vector of citric acid synthesis gene and application thereof |
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|---|---|---|---|---|
| CN1186519A (en) * | 1995-05-31 | 1998-07-01 | 加拿大国家研究委员会 | Modification of plant lipids and seed oils utilizing yeast SLC genes |
| US20070006346A1 (en) * | 2004-06-30 | 2007-01-04 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring modulated plant growth rate and biomass in plants |
| CN101265479A (en) * | 2007-12-05 | 2008-09-17 | 昆明理工大学 | Plant expression vector of citric acid synthesis gene and application thereof |
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| CN107586880A (en) * | 2017-10-31 | 2018-01-16 | 云南省烟草农业科学研究院 | A kind of molecular labeling and application for differentiating tobacco cadmium transporter gene NtHMA2 wild types and mutant |
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