CN105505956B - A kind of application of alfalfa Single-chip microcomputer dioxygenase gene - Google Patents
A kind of application of alfalfa Single-chip microcomputer dioxygenase gene Download PDFInfo
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Abstract
The invention discloses a kind of alfalfa Single-chip microcomputer dioxygenase gene MsHPPD, SEQ ID NO in its nucleotide sequence such as sequence table:Shown in 1, the invention also discloses a kind of primer pair for being used to clone described alfalfa Single-chip microcomputer dioxygenase gene MsHPPD, nucleotide sequence is respectively such as SEQ ID NO in sequence table:4 and SEQ ID NO:Shown in 5.Application the invention also discloses alfalfa Single-chip microcomputer dioxygenase gene MsHPPD preparation method and its in the content of vitamin E and resistance in improving arabidopsis.The present invention is further studies the function of alfalfa MsHPPD genes, and MsHPPD improvement of genes herbage qualities, and the application of particularly molecular improvement herbage content of vitamin E is laid a good foundation.
Description
Technical field
The present invention relates to biology field, more particularly to a kind of alfalfa Single-chip microcomputer dioxygenase
Gene M sHPPD and its preparation method and application.
Background technology
Vitamin E is a kind of fat-soluble vitamin, and natural products is according to first in the saturation degree and aromatic rings of hydrophobic tails
The difference of base number and location is divided into eight types, is a, β, γ, σ-tocopherol and a, β, γ, σ-tocotrienols respectively, its
The bioactivity highest of middle a- tocopherols.
Vitamin E is trace nutrient needed by human, and human body itself can not be synthesized, and can only be taken in by diet.
Research shows that vitamin E can improve the immunity of animal body.Correlative study shows that its meat of the domestic animal of supplementary feeding vitamin E will
Better than the domestic animal normally raised.And the quality of meat and milk can be improved, prolong storage period.Vitamin E has antioxidation.
The main function of vitamin E is Scavenger of ROS, and protection polyunsaturated fatty acid is not oxidized.There is important work for animals and plants
With.In addition, vitamin E also plays a role in the resistance regulation and control of plant.
Alfalfa is important leguminous forage, is also the whole nation or even plants most herbage variety in the world because it is suitable
The advantages of Ying Xingguang, yield height, quality better, have the good reputation of " King of Pasture ".With the development of China's animal husbandry, to herbage product
The requirement of matter is also improved constantly.Vitamin E can improve the fresh-keeping degree of animal meat, extend shelf life, improve animal
Fecundity, also with anti-oxidant, degeneration-resistant effect, has good value in terms of herbage quality improvement.But it is pale reddish brown
Clover belongs to the tetraploid leguminous forage of cross-pollination, and its genetic transformation is difficult, and growth cycle is long, and the research of gene function is compared
It is delayed, up to the present, to the research of lucerne Vitamin E synthesis related genes also in blank.
The content of the invention
It is an object of the invention to provide a kind of alfalfa Single-chip microcomputer dioxygenase gene MsHPPD, the present invention
For the function of further research alfalfa MsHPPD genes, and MsHPPD improvement of genes herbage qualities, particularly molecule changes
The application of good herbage content of vitamin E lays the foundation.
It is a further object of the present invention to provide a kind of preparation side of alfalfa Single-chip microcomputer dioxygenase gene
Method.
Carried it is a further object of the present invention to provide a kind of above-mentioned alfalfa Single-chip microcomputer dioxygenase gene
The application in content of vitamin E and resistance regulation and control in high arabidopsis.
First technical scheme of the present invention is, a kind of alfalfa Single-chip microcomputer dioxygenase gene,
SEQ ID NO in its nucleotide sequence such as sequence table:Shown in 1.
Second technical scheme of the present invention is, a kind of alfalfa Single-chip microcomputer dioxygenase gene
Preparation method, specifically implements according to following steps：
Step 1, using Trizol reagents method extract alfalfa total serum IgE；
Step 2, alfalfa total serum IgE progress RACE clone's cDNA total lengths to being extracted in step 1, and glue is carried out to PCR
Reclaim；The RACE primers include GSP1 and GSP2, wherein, SEQ ID NO in GSP1 sequence such as sequence table:Shown in 4, tool
Body is：5'-ATGGCACCGTTGTTTACGCTGGTGGTG-3'；SEQ ID NO in GSP2 sequence such as sequence table:Shown in 5, tool
Body is：5'-GACGCCGAACTGGCTTTCACCACC-3'.
Step 3, the recombinant plasmid for building PCR primer, and bacillus coli DH 5 alpha competent cell is converted, obtain bacterium solution；
Step 4, obtained bacterium solution is entered performing PCR detection, select positive bacterium solution, send Invitrogen companies to be sequenced,
Sequencing result is spliced, alfalfa Single-chip microcomputer dioxygenase gene cDNA full length sequences, pale reddish brown lucerne is obtained
SEQ ID NO in the nucleotide sequence of Mu Single-chip microcomputer dioxygenase gene such as sequence table:Shown in 1.
3rd technical scheme of the present invention is, a kind of above-mentioned alfalfa Single-chip microcomputer dioxygenase
Application of the gene in arabidopsis content of vitamin E and resistance regulation and control is improved.
The beneficial effects of the invention are as follows：The present invention based on the research to the biological mechanism of quality responses in alfalfa,
On the one hand alfalfa MsHPPD genes and the albumen of the gene code are provided, is on the other hand additionally provided containing the clover
The expression vector and cell of MsHPPD genes, and the method for cultivating the arabidopsis that content of vitamin E is improved, additionally provide them
Application.
The present invention is to understanding effect of the HPPD genes in herbage quality and resistance regulation and control, to herbage quality molecular improvement
Have very important significance.Enhanced expressing of the alfalfa MsHPPD genes that the present invention is provided in arabidopsis can be notable
Improve the content of arabidopsis vitamin E.The important application prospect of the present invention is：The raising Dominant Species of Forage Grass that is found to be of the gene is planted
Vitamin E provides genetic resources in thing (particularly legume, such as alfalfa), in the research of genetic engineering Improvement
In, particularly played a significant role in the transgenosis new germ plasm initiative of quality-improving.
The invention will be further described below in conjunction with the accompanying drawings.
Brief description of the drawings
Fig. 1 be Medicago sativa is carried out agriculture bacillus mediated MsHPPD DNA homologs conversion it is used inserted with
The structure chart of the ppBI121 carriers of MsHPPD genes；
Fig. 2 is the arabidopsis strain (L1, L2, L3) and vitamin E in empty vector control strain (CK) for turning MsHPPD genes
The comparative analysis result figure of content.
Fig. 3 is the result figure of auxiliary histidine content measure in the embodiment of the present invention 4；
Fig. 4 is the result figure of peroxidase (POD) determination of activity in the embodiment of the present invention 4.
Embodiment
The alfalfa Single-chip microcomputer dioxygenase gene MsHPPD of embodiment 1 preparation
The alfalfa MsHPPD genes that the present invention is provided are screened by the following method to be obtained：
By BLAST comparative analyses, according to the Single-chip microcomputer dioxygenase gene cloned in section shape clover
(Genbank registration numbers are：XM_003617343) guarding domain design primer, (RT-PCR primer is：F：5'-
SEQ ID NO in CCACTTTCTCCGCATCCACTTGTTTC-3', its nucleotide sequence such as sequence table:Shown in 2； R：5'-
SEQ ID NO in CCTCAATGTCCTAAATATATCCTCAG-3', its nucleotide sequence such as sequence table:Shown in 3).From pale reddish brown lucerne
(Medicago sativa L.cv.Zhongmu No.1, middle lucerne one, Beijing Zhong Xu east grass cultivation science and technology Limited Liability is public for Mu
Department) in expanded, fragment (fragment that race electrophoresis after reclaims this length) of the expected size for 600bp is reclaimed, by the piece of recovery
Section is connected into PMD18-T Vector (Takara), and sequencing result carries out BLASTx points in NCBI non-redundant proteins database
Analysis, obtains one section and the cDNA sequence for the nearly source species HPPD gene very high homologies announced.Utilize SMARTerTM RACE
CDNA Amplification Kit (Clontech, USA) carry out 5 ' RACE and 3 ' RACE to the sequence respectively and cloned, finally
Obtain the full length gene sequence (SEQ ID NO in nucleotide sequence such as sequence table:Shown in 1).Wherein, the method for gene cloning
Operated for the normal experiment of biology field, specific method is as follows：
1st, the extraction of alfalfa total serum IgE：
The extraction of alfalfa total serum IgE uses Trizol reagent methods, comprises the following steps that：
(1) 0.3g or so alfalfas organization material is added into about 3000 μ l Trizol reagents, adds liquid nitrogen and quickly grind
It is broken, it is placed in gnotobasis until being changed into homogenate.
(2) about 1mL homogenate is drawn into 1.5mL Eppendorf centrifuge tubes with liquid-transfering gun, acutely vibration is mixed, in room
Temperature places 5min, 4 DEG C, 13,000rpm centrifugation 10min.
(3) supernatant is taken, 200 μ L chloroforms are added, acutely vibration, room temperature places 2-3min, 4 DEG C, 13,000rpm centrifugations
15min。
(4) solution is divided into three layers from top to bottom：Organic phase, protein, colourless aqueous phase；Upper strata is taken to move into new
Eppendorf centrifuge tubes, plus isometric chloroform, repeat the 3rd step.
(5) take and reset and add thereon into isometric isopropanol, mixing is mixed, and room temperature places 10min or so, 4 DEG C, 13,000rpm
10min is centrifuged, white RNA precipitate is obtained.
(6) abandon supernatant, plus the ethanol of 1000 μ L 75% (with DEPC (diethyl pyrocarbonate,Coke acid diethyl Ester) water preparation), precipitation is rushed, in 4 DEG C, 13000rpm centrifugation 10min outwell ethanol, leave precipitation.
(7) the 6th step is repeated, is dried in an aseptic environment.Plus 20~50 μ l sterile DEPC water dissolvings.
RNA electrophoresis is carried out, detection RNA extracts quality, and IMPLEN trace dnas protein analyzer determines RNA concentration.
2nd, RACE clone genes cDNA total lengths
RACE-ready cDNA synthesis
(1) buffer solution is prepared：The chain buffer solutions of 2.0 μ l 5 × the first (First-Strand Buffer), 1.0 μ l DTT
(20mM), 1.0 μ l dNTP Mix (10mM)；
(2) the cDNA reaction solutions for 5 ' RACE are prepared：2.75 μ l RNA, 1.0 μ l 5'-CDS Primer A, prepare and use
In 3 ' RACE cDNA reaction solutions：3.75 μ l RNA, 1.0 μ l 3'-CDS Primer A；
(3) liquid blending that will be prepared in step (2), of short duration centrifugation, 72 DEG C are incubated 3 minutes, then at 42 DEG C of coolings 2
Minute, it is of short duration that reaction solution liquid is collected by centrifugation；
(4) 1 μ l SMARTer IIA oligo are added into 5 ' RACE cDNA reaction solutions, it is of short duration that liquid is collected by centrifugation；
(5) 5 ' RACE and 3 ' RACE-Ready cDNA reaction solutions (Master Mix) are prepared：Obtained in 4.0 μ l steps (1)
Buffer solution, 0.25 μ l RNase inhibitors (40U/ μ l), 1.0 μ l SMARTScribe reverse transcriptases (100U), by more than try
Agent is mixed；
(6) it is separately added into 5 ' the RACE reaction solutions obtained in the 3 ' RACE and step (4) that are obtained into step (3)
The reaction solution obtained in 5.25 μ l steps (5), soft mixing is of short duration that liquid is collected by centrifugation；
(7) by the reaction solution prepared in incubation 90 minutes in 42 DEG C；
(8) 70 DEG C are heated 10 minutes, complete Ready-cDNA synthesis.
The design of RACE primers
(1) gene specific primer (Gene-Specific Primers, GSPs) meets following condition：23-28bp；GC contains
Measure 50%-70%；Tm values are best>70℃；With 3 '-end universal primer (Universal Primer Mix, kit is provided) no
Generation is complementary.
(2) two GSP primers need to be designed, reverse primer (GSP1) is used for 5 ' RACE, and forward primer (GSP2) is used for 3 '
RACE。
According to above principle, design GSP1 (5'-ATGGCACCGTTGTTTACGCTGGTGGTG-3', its nucleotide sequence
Such as SEQ ID NO in sequence table:Shown in 4) and GSP2 (5'-GACGCCGAACTGGCTTTCACCACC-3', its nucleotide sequence
Such as SEQ ID NO in sequence table:Shown in 5) two primers.
The Fast back-projection algorithm of cDNA ends
(1) enough PCR mixed liquors (Master Mix) are prepared:Following reagent is added in every 50 μ l PCR reaction systems：
34.5 μ l PCR water, 5.0 μ l 10 × Advantage 2PCR buffer solutions, 1.0 μ l dNTP Mix (10mM),
The polymerase Mix of 1.0 50 × Advantage of μ l 2.Liquid is gently mixed, is careful not to produce bubble, it is of short duration that liquid is collected by centrifugation
Body；
(2) the PCR reaction solutions for 5 ' RACE are prepared：2.5 μ l 5 ' RACE-ready cDNA, 5.0 μ l UPM (10 ×),
1.0μl GSP1；The PCR mixed liquors prepared in the step of 41.5 μ l (1)；
Prepare and reacted for 3 ' RACE PCR：2.5 μ l 3 ' RACE-ready cDNA, 5.0 μ l UPM (10 ×), 1.0 μ l
GSP2；The PCR mixed liquors prepared in the step of 41.5 μ l (1)；
(3) RACE is expanded：Reaction system is：94 DEG C 30 seconds, 72 DEG C 3 minutes, totally 5 circulation：94 DEG C 30 seconds, 70 DEG C 3 points
Clock, 72 DEG C 3 minutes, totally 5 circulation；94 DEG C 30 seconds, 68 DEG C 30 seconds, 72 DEG C 3 minutes, totally 20 circulation.
Take PCR primer in carrying out electrophoresis detection on 1.0 weight % Ago-Gel, reclaim purpose band, use T4 DNA
Ligase (Takara) connects recovery product on PMD18-T (Takara) carrier, converts bacillus coli DH 5 alpha competent cell.
3rd, the Escherichia coli conversion of recombinant plasmid
(1) DH5 α competent cells melt on ice, draw 50 μ l, add 5 μ l DNA connection products, after being well mixed
Ice bath 30min.
(2) 42 DEG C of thermal shock 60sec, immediately ice bath 3-5min.
(3) add on 700 μ l LB fluid nutrient mediums, 37 DEG C of constant temperature oscillators (150r/min) and cultivate 60min.
(4) 5000rmp centrifuges 3min and collects thalline.
(5) (Amp) is coated with X-gal (the chloro- 3- indoles-β-D- galactosides of the bromo- 4- of l5-, 2%) and IPTG on LB flat boards
(isopropylthio-β-D-galactoside, 50mg/ml), makes its even spread planar surface, and normal temperature dries.
(6) 100 μ l bacterium solutions are taken to be spread evenly across on Amp containing antibiotic (100mg/L) LB flat boards, 37 DEG C are fallen in incubator
Put overnight incubation；Flat board is put into 4 DEG C of placements and is allowed to fully colour developing by next day.
(7) select white single bacterium to fall within 1ml LB fluid nutrient mediums (Amp final concentration 100mg/L), 37 DEG C of 225r/min
Overnight incubation.
Enter performing PCR detection to bacterium solution, select positive bacterium solution, send Invitrogen companies to be sequenced.Sequence is carried out to result
Row splicing, as a result shows that the gene has such as SEQ ID NO in sequence table:Nucleotide sequence (2064bp) shown in 1.
The agriculture bacillus mediated MsHPPD genetic transformation Arabidopsis plants of embodiment 2
1.1st, material and reagent
1.1.1, vegetable material
It is alfalfa (Medicago sativa L.cv.Zhongmu No.1, middle lucerne one) for examination alfalfa variety.
1.1.2, agrobacterium strains and plasmid vector
Agrobacterium strains used are Agrobacterium tumefaciems：LBA4404 (Beijing day bounties Gene Tech. Company Limited)
Agrobacterium culture medium：
| Reagent | Content (1L) |
| MgSO4 .7H2O | 1g/L |
| Peptone | 10g/L |
| Yeast extract | 1g/L |
| Sucrose | 5g/L |
| Agar (solid medium) | 15g/L |
| pH | 7.0 |
121 DEG C of high pressure steam sterilization 20min；Carrier：PBI 121
1.2nd, experimental method
1.2.1 MsHPPD genes are inserted into pBI121 vector plasmid DNAs, concretely comprised the following steps：The end of design 5 ' is included
The sense primer of XbaI restriction enzyme sites and the anti-sense primer (P1 of BamHI restriction enzyme sites： 5'-
GCTCTAGASEQ ID NO in ATGGGGACAAATTCTAACAACAACAC-3'(nucleotide sequences such as sequence table:Shown in 6),
P2：5'-CGGGATCCSEQ ID NO in TCATGCAGTTCTTCTAGTTTCCAAAG-3'(nucleotide sequences such as sequence table:7 institutes
Show), wherein, underscore part is restriction enzyme site；The total length that MsHPPD is expanded from alfalfa cDNA templates with the primer is read
Frame, PBI carriers and target gene the full length fragment recovery product after XbaI and BamHI double digestions, connect through T4 DNA ligases
Connect, obtain the pBI121 carriers inserted with MsHPPD genes, it contains CaMV35s promoters, (such as sequence table of MsHPPD genes
Middle SEQ ID NO:Shown in 1) and a kalamycin resistance selection markers, can be sieved during genetic transformation by kanamycins
The transfer-gen plant for selecting Preliminary Identification to obtain.The structure of plasmid vector containing purpose fragment is as shown in Figure 1.
1.2.2 by the pBI121 vector introductions inserted with MsHPPD genes into agrobacterium tumefaciens lba4404, specific steps
It is as follows：
A. about 1 μ g DNAs are added into 100 μ l Agrobacterium competent cells LBA4404, gently mixed, ice bath 30
min；
B. the quick-frozen 1min in liquid nitrogen, is immediately placed in 37 DEG C of water-baths and incubates 5min；
C. 800 μ l YEB fluid nutrient mediums are added, 28 DEG C of 150rpm cultivate 4-6h；
D. thalline is coated on containing 50mg/L kanamycins (Kanamycin Sulfate) and 100mg/L streptomysins
(Streptomycin) on YEB selection flat boards, 28 DEG C are inverted culture two days.
E. picking single bacterium colony, is inoculated in YEB fluid nutrient mediums (Kan containing 50mg/L and 100mg/L Str), 28 DEG C of shakes
Swing overnight incubation.
Plasmid and the sequencing for the Agrobacterium for being imported with MsHPPD genes are extracted, the nucleotide sequence of quiding gene is as a result shown
With SEQ ID NO in such as sequence table:Sequence shown in 1 is consistent, show the expression vector establishment containing target gene MsHPPD into
Work(.
1.2.3, the culture of Agrobacterium
The Agrobacterium of MsHPPD genes will be imported with the solid medium containing 50mg/LKan and 100mg/L Str
Flat board is drawn, is put in incubator, 28 DEG C of cultures.Two days later, the picking single bacterium colony from flat board, is inoculated in containing 50mg/L Kan
In 100mg/L Str 20ml YEB fluid nutrient mediums, 180rpm, 28 DEG C of cultures.Flat board, 28 DEG C of trainings are drawn with the bacterium solution shaken
Support, after single bacterium colony is grown, flat board is put in 4 DEG C of preservations.
The picking single bacterium colony on flat board, is inoculated in the YEB Liquid Cultures that 20ml contains 50mg/L Kan and 100mg/L Str
In base, in 28 DEG C on constant-temperature table, 180rpm cultures.A small amount of bacterium solution is taken after two days, with 1:50-1:100 dilution proportion
Into the YEB fluid nutrient mediums containing 50 μ g/m1Kan and 100 μ g/m1Str, 28 DEG C of culture 6-12h to exponential phase.By thalline
It is collected into centrifuge tube, 4,000rpm centrifugation 10min enrichment thalline abandon supernatant, then be free of with about 20ml the improvement of antibiotic
Thalline is resuspended in SH fluid nutrient mediums, makes the OD of bacterium solution600It is worth for 0.6-0.8, it is stand-by.
1.2.4, the preparation of arabidopsis：
After Col Arabidopsis thaliana ecotype Seed sterilizations, on the culture dish that seed is layered on to 1/2MS solid mediums, 4 DEG C are placed on
Lower vernalization 48 hours.
Seed flat board after vernalization is placed on culture in arabidopsis incubator (24 DEG C of 16h/22 DEG C of 8h), after 7 days, flower is moved on to
(vermiculite in basin：Nutrition Soil：Perlite is about 1：1：1).Arabidopsis, about three weeks or so are cultivated as stated above, are cut off
Stem through blooming, suppresses apical dominance.In or so about four weeks, the side shoot of extraction is largely bloomed, and is now to turn base with infusion method
The best opportunity of cause.
1.2.5, Agrobacterium culture and harvest
Take the μ l of original bacteria liquid 20, add 5ml YEP (Yeast extract 10g/L, Tryptone10g/L, Nacl 5g/L,
After sterilizing, antibiotic is added), 30 DEG C of constant temperature shakings are stayed overnight, and this 5ml bacterium solution is poured into 250ml YEP (addition antibiotic) cultures
Base, 30 DEG C of constant temperature shakings are stayed overnight, and now, the concentration of Agrobacterium should reach OD600=1.8.4000rpm is centrifuged 15 minutes, is discarded
Supernatant, adds infusion method culture medium (a large amount of+1 × MS micro+1 × MS molysite+1 × MS inositol+1 × MS vitamins+MES of 1/2MS
0.5g/L+Sucrose 5%, pH5.7 autoclaving).Make Agrobacterium with 1：1 ratio is resuspended in infiltration medium, and adds
Surfactant Silwet77, makes its final concentration reach 0.02% (every liter plus 200 μ l).
1.2.6, infusion method transgenosis
Arabidopsis flowerpot is coverd with into gauze, tightened with rubber band, in order to avoid leaked during flowerpot back-off under culture matrix.Flowerpot is fallen
It is buckled on the infiltration tank equipped with 250ml bacteria suspensions, the inflorescence of plant is immersed in bacterium solution, infiltration continues 2min, is used after conversion
Blotting paper sucks excessive bacterium solution, but need not inhale very dry.Plant after conversion, film is taken off after being stayed overnight with preservative film covering.Just
It is frequently grown, collects seed.
1.2.7 the screening of transgenic Arabidopsis plants
The arabidopsis seed 70% alcohol 2min of harvest, 5% sodium hypochlorite 5min carry out disinfection.Arabidopsis seed is multicast to
In the culture dish of the 1/2MS solid mediums of the Kan containing 50mg/L, vernalization 2 days under 4 DEG C of low temperature are put into.On resistance culture base
The Arabidopsis thaliana Seedlings of normal growth is convert successfully arabidopsis, and conversion failure Arabidopsis thaliana Seedlings will be unable to normal growth.Can be just
The Arabidopsis thaliana Seedlings being frequently grown are transplanted to flowerpot, in the controlled environment chamber under the conditions of grow.With the gene of transgenic Arabidopsis plants
Group DNA is template, with the gene primer P of npt II3/P4(amino acid sequence is respectively such as SEQ ID NO in sequence table:8 and SEQ ID
NO:Shown in 9), gus gene primers P5/P6(amino acid sequence is respectively such as SEQ ID NO in sequence table:10 and SEQ ID NO:11
It is shown) the detection plant of vector transgene containing pBI121-MsHPPD.Positive plant retains (obtains 12 plants altogether：L1-L12), individually receive
Kind.T1In generation, is through same antibiotic-screening, its segregation ratio (3：1) through Chi-square Test, 43 are obtained altogether：The strain of 1 separation：L1, L7,
L8, L9.These strains reservation sowing is obtained into T2Generation.From T2Homozygote, independent sowing are screened in generation.
The pBI121 carrier arabidopsis content of vitamin E of the MsHPPD genes of embodiment 3 is evaluated
Agrobacterium is converted with plasmid pBI121, recombinational agrobacterium is obtained, uses recombinational agrobacterium arabidopsis thaliana transformation, obtains turning sky
The adjoining tree of carrier, method such as embodiment 1, acquisition turns empty vector control plant (CK), and as a comparison case 1.
The obtained arabidopsis for turning empty carrier and the conversion that is obtained in embodiment 1 are evaluated in comparative example 1 inserted with MsHPPD
The content of vitamin E of the pBI121 carrier arabidopsis of gene, specific method is as follows：
It is mixed to take 3g alfalfa blades, 0.03~0.05g is sampled after grinding uniformly again, 1mL methanol is added:Chloroform (2:1/
V:V BHT (BHT) 0.01%) is contained, lucifuge stands 20min.Add 0.33mL chloroforms and 0.6mL water, whirlpool
Rotation vibration, 14000g centrifuges 10min after mixing, takes organic phase (lower floor), is evaporated.Use dichloromethane:Methanol (1:5/V:V it is) molten
Solution takes 20 μ L to be determined in the upper machine of liquid phase bottle to 400 μ L.
External standard method and quantitative analysis condition are high performance liquid chromatograph (model：Agilent 1260)；Double pump system
(G1312B)；Fluorescence detector (G1321B)；Chromatographic column is LiCHrospher 100Diol 250mm × 4mm, 5 μm of particle diameter；Stream
Dynamic phase, methanol:Water (97:3/V:V)；Flow velocity 1.2mlmin-1；Fluorescence excitation 292nm, launches 330nm.Testing result is carried out
Statistical analysis.As a result it is as shown in Figure 2：α-VE (alpha-tocopherol) content of transgenic line (L1, L8, L9) is than control (CK)
Balanced growth 13 times.
The pBI121 carriers arabidopsis thaliana salt-tolerance of the MsHPPD genes of embodiment 4 is evaluated
The obtained arabidopsis for turning empty carrier and the conversion that is obtained in embodiment 1 are evaluated in comparative example 1 inserted with MsHPPD
Auxiliary histidine content and peroxidase activity in the pBI121 carrier Arabidopsis plants of gene, specific method are as follows：
1st, auxiliary histidine content is determined
With 0mM, the Arabidopsis plant in 62.5mM and 125mM NaCl processing comparative examples 1 and embodiment 12 weeks, accurate title
Take each 0.2g of Arabidopsis leaf (every kind of three repetitions of processing) of different disposal.With 3% sulfosalicylic acid milling and extracting, turn respectively
Move in test tube, 5ml 3% sulfosalisylic acid solution be then separately added into each test tube, 10 min are extracted in boiling water bath,
Cooled and filtered is in clean test tube, and filtrate is settled to 5mL with 3% sulfosalicylic acid；2ml extract solutions are drawn in another clean
Band glass plug test tube in, add 2ml glacial acetic acid and 2ml acid ninhydrine reagents, 30min heated in boiling water bath；Add after cooling
Enter 4ml toluene, sway 30s, stand 2h；Draw the red toluene solution of upper strata proline in cuvette with suction pipe, using toluene as
Blank control, colorimetric at 520nm wavelength, tries to achieve absorbance on spectrophotometer.2ml samples are calculated according to standard curve
The concentration of proline in liquid, then converses the content (μ g proline/gFW) of proline in sample.Calculation formula is as follows：
Proline content (μ g.g-1)=c × (v/a)/w
C is that extract solution cumulative volume ml, a are that measure liquid product ml, w are sample to check in proline content μ g, v by standard curve
Quality g.Testing result carries out statistical analysis.As a result it is as shown in Figure 3：Under the conditions of untreated, transgenic line proline
Content is slightly less than after control, salt treatment, and the proline content in transgenic line (L1, L7, L9) is significantly higher than control (CK),
And salinity is higher, proline content difference is bigger.
2nd, peroxidase (POD) determination of activity
The Arabidopsis plant 2 in comparative example 1 and embodiment 1 is handled with 0mM, 62.5mM, 125mM and 187.5mM NaCl
In week, the Arabidopsis leaf 1g of different disposal is weighed, add 20mM KH2PO4 solution 5ml, homogenate is ground to form in mortar,
10min is centrifuged under 33000r/min, supernatant is transferred in 25m1 volumetric flasks, residue is extracted once with 5ml KH2PO4 solution again,
Merge supernatant, constant volume is mixed, and stores in the place that cools standby.Take in optical path 1cm cuvettes 2,1 and add the reaction mixed
Mixed liquor 3m1, KH2PO4Solution 1ml, is used as reference liquid；Reaction mixture 3ml, enzyme liquid 1ml are added in another, is clocked immediately
It is placed in spectrophotometer.Optical density is determined under 470nm, 30min is continuously surveyed every 1min readings once, determined every time
It is preceding to be calibrated again with control.
Using the time as abscissa, optical density is mapped for ordinate.React early stage catalase activity straight with the reaction time
Line rises, and is raised to after maximum, and turning point occurs in its correlation curve, and the morning and evening that turnover occurs depends on temperature.Before curve
Phase, part found the part of one section of near linear, and straight line starting time t1 and optical density A1, straight line terminal time t2 and light are close
Spend A2.It is 1 peroxidase activity unit (μ) with A470 per minute changes 0.01, calculates its vigor and Rate activity.
Enzyme activity (0.01A470min-1)=A2-A1/ (t2-t1) × 0.01 × D.
The Rate activity (ug-1) of enzyme=enzyme activity (μ)/sample weight (g).
A2-A1：The change of absorbance, t2-t1：Time change, D：Extension rate, that is, the enzyme liquid total amount extracted is reaction system
The multiple of enzyme liquid in system.U：Enzyme activity unit (i.e. 0.01A470min-1).Testing result carries out statistical analysis.As a result such as
Shown in Fig. 4：In transgenic line (L1, L8, L9) POD activity before treatment with control (CK) is all remarkably higher than after processing, and
The rise of POD activity is more notable after salt treatment.There are some researches prove proline content and peroxidase are all degeneration-resistant with plant
The significantly correlated material of property.Proline can be as a kind of osmotic adjustment in plant, the content under Adversity-stressed Plant
It can dramatically increase, the osmotic adjustment ability of plant be improved, so as to improve the resistance of plant.And peroxidase is the weight of plant
One of enzyme system is protected, the height and reaction speed of peroxidase activity determine that the resistance of plant is strong and weak, and activity is higher,
Often resistance is stronger.Testing result more than, illustrates that the resistance of transgenic line, especially salt tolerance will be significantly higher than
Adjoining tree.
Single-chip microcomputer dioxygenase in the gene code alfalfa in vitamin E route of synthesis.Conversion is intended
After southern mustard, the content of vitamin E in arabidopsis is improved, is contained in addition, transgenic line ties up to internal auxiliary propylhomoserin under condition of salt stress
Amount increase, peroxidase POD activity is improved, so that salt tolerance is improved.The gene, which is enriched, ties up life in alfalfa
The research of plain E biosynthesis pathways.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
Enclose and be defined, on the premise of design spirit of the present invention is not departed from, technical side of the those of ordinary skill in the art to the present invention
In various modifications and improvement that case is made, the protection domain that claims of the present invention determination all should be fallen into.
Claims (1)
1. a kind of alfalfa Single-chip microcomputer dioxygenase gene MsHPPD application, it is characterised in that：It is described pale reddish brown
SEQ ID NO in clover Single-chip microcomputer dioxygenase gene MsHPPD nucleotide sequence such as sequence table:Shown in 1；Institute
State applications of the alfalfa Single-chip microcomputer dioxygenase gene MsHPPD in the salt tolerance regulation and control of arabidopsis.
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