CN106350537A - Method for transforming medicago sativa by aid of galega orientalis gibberellin receptor genes GoGID - Google Patents
Method for transforming medicago sativa by aid of galega orientalis gibberellin receptor genes GoGID Download PDFInfo
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- CN106350537A CN106350537A CN201610798808.8A CN201610798808A CN106350537A CN 106350537 A CN106350537 A CN 106350537A CN 201610798808 A CN201610798808 A CN 201610798808A CN 106350537 A CN106350537 A CN 106350537A
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention discloses a method for transforming medicago sativa by the aid of galega orientalis gibberellin receptor genes GoGID. The method includes particular steps of inserting the genes GoGID into vector plasmid DNA (deoxyribonucleic acid); introducing vectors with the inserted genes GoGID into agrobacterium tumefaciens LBA4404; culturing the agrobacterium tumefaciens; preparing and transforming medicago sativa explants; verifying, propagating and evaluating medicago sativa transgenic plants. The method has the advantages that after the galega orientalis gibberellin receptor genes GoGID are overexpressed in the medicago sativa, the plant heights of transgenic medicago sativa can be increased by 0.2-0.35 time, biomass in the transgenic medicago sativa can be increased by 14.9-46.3%, and the leaf lengths and the leaf widths of the medicago sativa can be obviously larger than the leaf lengths and the leaf widths of wild type medicago sativa; in addition, the flowering time of the medicago sativa is delayed by a week as compared with the wild type medicago sativa, accordingly, theoretical basis can be provided for passing pasture gibberellin signal pathways, and possible candidate genes and theoretical foundations also can be provided for genetically improving yield traits of the medicago sativa.
Description
Technical field
The present invention relates to biology field, more particularly to galega orientalis gibberellins acceptor gene gogid should
With.
Background technology
Gibberellins are class tetracyclic diterpene alkanes materials, belong to one of big hormone of plant five, in the multiple important life of plant
Play a role during long growth.Sprout including seed, vane extension, stem extends, and blooms, and seed development etc..Gibberellins
Receptor is important step in gibberellins signal transduction path, can binding activity ga, formed gid-ga-della complex, through general
Plain e3 combined enzyme agent (scfgid2/sly1) identification, mediate della albumen and form many poly-ubiquitin chains, subsequently dropped by 26s proteasome
Solution, thus cause ga to respond.
Gibberellins acceptor gene (gibberellin insensitive dwarf1, gid1) be 2005 red mould from Oryza sativa L.
Discovery first in the insensitive Dwarf Mutant of element.One solubility egg similar to hormone-sensitive lipase of this gene code
In vain, it is positioned in Cytoplasm and nucleus.Subsequently, also in succession it is found that gibberellins are subject in the species such as arabidopsiss, Cotton Gossypii, willow
Body.Multinomial result of study all shows, gibberellins receptor participates in multiple plant growth and development process, and gid mutation leads to the plant of classics
Thing Dwarfing phenotypes.
Galega orientalis (galegaorientalis) are important Perennial legume forages, and yield is high, nutritious, raises
It is more early than Herba Medicaginiss to feed period of use, green grass or young crops raise ruminant domestic animal must not abdominal tympanites, and have and promote the function such as Lactation of Dairy Cow.
Alfalfa (medicago sativa) is the perennial leguminous plant of Medicago, because its yield height, good palatability,
Best in quality, and extensively plant in the whole world, have the good reputation of " King of Pasture ", global cultivated area is public more than ten million
Hectare.The main of alfalfa is stalk using position, and therefore biological yield is the important breeding objective of alfalfa.Conventional breeding
The method selection-breeding time is long, and effect is also inconspicuous.With the development of genomics and Protocols in Molecular Biology, using genetic engineering skill
Art molecular improvement herbage has become as possibility.Mir156 can regulate and control Biomass and apical dominance and the turning of spending of switchgrass
Become.After the precursor of alfalfa mir156 is separated in alfalfa overexpression, lead to the 3 spl bases being regulated and controled by mir156
Lower because whole.The panel length of transgenic line and stem slightly lower, and branch's number increases, and hairy body density increases, flowering time
Postpone, Biomass obtains and significantly increases.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of galega orientalis gibberellins acceptor gene gogid conversion pale reddish brown
The method of Herba Medicaginiss.
The method that galega orientalis gibberellins acceptor gene gogid of the present invention converts alfalfa, including following
Step:
(1) galega orientalis gibberellins acceptor gene gogid is inserted in pbi121 vector plasmid dna, obtains
Pbi121 recombinant vector:
Design 5 ' end comprises the forward primer p1 of the xbai restriction enzyme site and downstream primer p2 of bamhi restriction enzyme site, such as sequence
In list shown in seq id no:5 and seq id no:6；
P1:5'-gctctagaatggtgatcgagaacgacattgag-3'；
P2:5'-cgggatcctcagtgatggccacgcctaagtgatg-3'；
Wherein, underscore part is restriction enzyme site；
With expanding total length reading frame, pbi carrier and the genes of interest of gogid in this primer eastwardly Galega officinalis L cdna template
Full length fragment recovery product after xbai and bamhi double digestion, connects through t4dna ligase, obtains being inserted with gogid gene
Pbi121 carrier, it contains camv35s promoter, gogid gene and kalamycin resistance selection markers；
Wherein, described galega orientalis gibberellins acceptor gene gogid is as shown in seq id no:3 in sequence table；
(2) described pbi121 recombinant vector is imported in Agrobacterium tumefaciems lba4404:
A. add pbi121 transfer vector plasmid dna in Agrobacterium lba4404, mix, ice bath；
B. quick-freezing in liquid nitrogen, is immediately placed in water-bath and incubates；
C. yeb fluid medium, culture are added；
D. thalline is coated yeb and is selected, on flat board, to cultivate two days；
E. picking single bacterium colony, is inoculated in yeb fluid medium, overnight incubation；
Extract and be imported with the plasmid of Agrobacterium lba4404 of gogid gene and be sequenced, result shows the nucleoside of quiding gene
Acid sequence is consistent with the sequence as shown in seq id no:3 in sequence table, then show the expression vector containing genes of interest gogid
Successfully construct；
(3) it is imported with the culture of the Agrobacterium lba4404 of gogid gene；
(4) with alfalfa individual plant as material, it is grown in phjytotron, take bottle green healthy leaves, be cut into explant
Body；
Explant is placed in Agrobacterium re-suspension liquid, is then put on aseptic filter paper, remove surplus liquid；
Common training culture medium grows；Explant is taken out, degerming；Then continue in screening culture medium；Calluss turn
Enter on inducing culture；Embryonic development becomes sprig or root；Plantlet moves into root media and continues culture；
(5) identification of alfalfa transgenic plant, expanding propagation and evaluation:
By the plant of above-mentioned conversion culture, extract dna, with npt gene primer p3, p4, gus gene primer p5, p6 enter
Row detection；Rna transcriptional level detection is carried out with primer p7, p8, wherein, seq idno:7～no:12 in p3～p8 such as sequence table
Shown；
Select the higher transgenic line of expression and tie up to growth in drum, after growing up, copy is obtained by cuttage and expands
Numerous；
Convert Agrobacterium lba4404 with pbi121 carrier, obtain recombinational agrobacterium lba4404, use recombinational agrobacterium
Lba4404 conversion obtains turning the adjoining tree of empty carrier；
Turn the alfalfa of empty carrier, convert the alfalfa of the pbi121 carrier being inserted with gogid gene in artificial gas
Wait after surviving in room, plant division, plant in little basin；After stalwartness, move in big basin, grow in greenhouse, period manages with delicacy, and time control
Pest and disease damage processed；Observe phenotype, measure the indexs such as plant height, fresh weight, dry weight, leaf-stem ratio, florescence.
The method that galega orientalis gibberellins acceptor gene gogid of the present invention converts alfalfa, wherein step (3)
Comprise the following specific steps that: the Agrobacterium of gogod gene will be imported with the solid medium containing kanamycin and streptomycin
Upper stroke of flat board, is put in culture in incubator；
Two days later, picking single bacterium colony from flat board, is inoculated in the yeb fluid medium containing kanamycin and streptomycin
In, culture；
Draw flat board with cultured bacterium solution, culture, after growing single bacterium colony, flat board is put in 4 DEG C of preservations；
Picking single bacterium colony on flat board, is inoculated in the yeb fluid medium containing kanamycin and streptomycin, in constant temperature
Cultivate on shaking table；
Take a small amount of bacterium solution within two days later, be diluted in the yeb fluid medium containing kanamycin and streptomycin, cultivate to
Exponential phase；By microorganism collection in centrifuge tube, centrifugal enrichment thalline, abandon supernatant, then with the resuspended thalline of sh fluid medium,
Make the od of bacterium solution600It is worth for 0.6-0.8, stand-by.
The method that galega orientalis gibberellins acceptor gene gogid of the present invention converts alfalfa, wherein step (1)
Described in galega orientalis gibberellins acceptor gene gogid be to be prepared for raw material by galega orientalis, its preparation method bag
Include following steps:
A. the extraction of the total rna of galega orientalis；
B.race clones gogid gene cdna total length；
C. the escherichia coli conversion of recombiant plasmid, and be sequenced.
The method that galega orientalis gibberellins acceptor gene gogid of the present invention converts alfalfa, wherein, east
Step a of the preparation method of Galega officinalis L gibberellins acceptor gene gogid adopts trizol reagent method, comprises the following specific steps that:
A. galega orientalis organization material is added to trizol reagent, add liquid nitrogen quickly to grind, put to being changed into being homogenized；
B. draw homogenate in centrifuge tube, acutely vibration mixes, and places in room temperature, is then centrifuged for；
C. take supernatant, add chloroform, acutely vibrate, room temperature is placed, and is then centrifuged for；
D., after solution layering, take upper strata to move into another centrifuge tube, add equal-volume chloroform, repeat previous step；
E. take its supernatant, add equal-volume isopropanol, mixing mixes, and room temperature is placed, and is then centrifuged for, obtain white rna and sink
Form sediment；
F. abandon supernatant, add ethanol, precipitation is rushed, centrifugation, outwell ethanol, leave precipitation；
G. repeat previous step, dry, plus depc water dissolution；
H. carry out rna electrophoresis, detection rna extracts quality, measures rna concentration using trace dna protein analyzer.
The method that galega orientalis gibberellins acceptor gene gogid of the present invention converts alfalfa, wherein, east
Step b of the preparation method of Galega officinalis L gibberellins acceptor gene gogid comprises the following specific steps that:
(1) synthesis of race-ready cdna
A. prepare buffer: 5 × the first chain buffer, dtt, dntp mix；
B. the cdna reactant liquor for 5 ' race and the cdna reactant liquor for 3 ' race are prepared；
C. by the liquid blending preparing in step b, centrifugation, incubation, then cool down, reactant liquor liquid is collected by centrifugation；
D. add smarter iia oligo in the cdna reactant liquor of 5 ' race, reactant liquor liquid is collected by centrifugation；
E. 5 ' race and 3 ' race-ready cdna reactant liquors are prepared: by the buffer obtaining in step a, rna enzyme level
Agent, reverse transcriptase mix；
F. it is separately added in 5 ' the race reactant liquors obtaining in the 3 ' race obtaining in step c and step d in step e and obtain
The reactant liquor arriving, mixing, liquid is collected by centrifugation；
G. the reactant liquor preparing is incubated；
H. heat, complete the synthesis of ready-cdna；
(2) design of race primer
Design principle includes:
A. gene specific primer gsps meets following condition: 23-28bp；Gc content 50%-70%；Tm value > 70 DEG C；With
There is not complementation in 3 '-end universal primer；
B. need to design two gsp primers, reverse primer gsp1 is used for 5 ' race, forward primer gsp2 is used for 3 ' race；
According to above design principle, design two primers of gsp1 and gsp2；
(3) Fast back-projection algorithm of cdna end
A. prepare pcr mixed liquor: in pcr reaction system, add following reagent: pcr water, 10 × advantage 2pcr
Buffer, dntp mix, 50 × advantage 2 polymerase mix；Mixing, is collected by centrifugation liquid；
B. prepare the pcr reactant liquor for 5 ' race: make in 5 ' race ready cdna, 10 × upm, gsp1, step a
Standby pcr mixed liquor；
Prepare the pcr reactant liquor for 3 ' race: prepare in 3 ' race ready cdna, 10 × upm, gsp2, step a
Pcr mixed liquor；
C.race expands:
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 minutes, 72 DEG C 3 minutes,
Totally 5 circulations；94 DEG C 30 seconds, 68 DEG C 30 seconds, 72 DEG C 3 minutes, totally 20 circulation；
Take pcr product that electrophoresis detection is carried out on agarose gel, reclaim purpose band, connected back to t4dna ligase
Receive product on pmd18-t carrier, convert escherichia coli dh5 α competent cell.
The method that galega orientalis gibberellins acceptor gene gogid of the present invention converts alfalfa, wherein, east
Seq in primer gsp1 and gsp2 described in step b of preparation method of Galega officinalis L gibberellins acceptor gene gogid such as sequence table
Shown in idno:1 and seq id no:2.
The method that galega orientalis gibberellins acceptor gene gogid of the present invention converts alfalfa, wherein, east
Step c of the preparation method of Galega officinalis L gibberellins acceptor gene gogid comprises the following specific steps that:
A.dh5 α competent cell melts, and adds dna connection product, ice bath after mix homogeneously；
B. thermal shock, ice bath immediately；
C. add lb fluid medium, cultivate under constant temperature oscillator；
D. it is centrifuged, collects thalline；
On e.lb flat board, so as to even spread planar surface, room temperature dries coating x-gal and iptg；
F. bacterium solution is taken to be spread evenly across on lb flat board, overnight incubation in incubator；Flat board is placed and is allowed to fully show by next day
Color；
G. select white single bacterium colony in lb fluid medium, overnight incubation；
Pcr detection is carried out to bacterium solution, selects positive bacterium solution, be sequenced.
The prominent effect of the conversion alfalfa that the present invention is finally obtained is:
(1) after galega orientalis gibberellins acceptor gene gogid overexpression in alfalfa, transgenic alfalfa
Plant height increased 0.2-0.35 times, and Biomass increased 14.9-46.3%, and the long leaf width of leaf of alfalfa is also significantly greater than wild
Type, additionally, flowering time is late one week than wild type.And, the index of quality of transgenic alfalfa is not significantly affected.
Result shows gogid as a gibberellins acceptor gene, has regulated and controled plant, especially multiple growth and development processes of herbage,
In particular improve the length and width of blade, and regulate and control the florescence of transgenic alfalfa.This be before for red
Had no report in the research of mycin acceptor gene.
(2) present invention is that parsing herbage gibberellins signal pathway is provided fundamental basis, also for Alfalfa Output character
Genetic improvement provides possible candidate gene and theoretical foundation.
Explanation and specific embodiment turn to the galega orientalis gibberellins acceptor gene gogid of the present invention below in conjunction with the accompanying drawings
The method changing alfalfa is described further.
Brief description
Fig. 1 is plant expression vector construction schematic diagram in the embodiment of the present invention.
Specific embodiment
The preparation of embodiment 1 galega orientalis gibberellins acceptor gene gogid
According to the aobvious Hybrid Library data message of galega orientalis difference, design race amplimer gsp1 and gsp2, such as sequence
In table shown in seq id no:1 and seq id no:2, using smartertmrace cdna amplification kit
(clontech, usa) carries out 5 ' race and 3 ' race clone respectively to this sequence, finally obtains this full length gene sequence.Tool
Body method is as follows:
1st, the extraction of the total rna of galega orientalis:
The extraction of the total rna of galega orientalis adopts trizol reagent method, specifically comprises the following steps that
(1) 0.3g galega orientalis organization material is added to 3000 μ l trizol reagent, adds liquid nitrogen quickly to grind,
It is placed in gnotobasiss until being changed into being homogenized；
(2) draw 1ml with liquid-transfering gun to be homogenized in 1.5ml centrifuge tube, acutely vibration mixes, and places 5min in room temperature, so
13000rpm centrifugation 10min at 4 DEG C；
(3) take supernatant, add 200 μ l chloroforms, acutely vibrate, room temperature places 2-3min, then at 4 DEG C 13000rpm from
Heart 15min；
(4) solution is divided into three layers from top to bottom: organic faciess, protein, colourless aqueous phase；Upper strata is taken to move into another fragmented
Heart pipe, adds equal-volume chloroform, repeats previous step；
(5) take and reset and add into equal-volume isopropanol thereon, mixing mixes, and room temperature places 10min, then at 4 DEG C
13000rpm is centrifuged 10min, obtains white rna precipitation；
(6) abandon supernatant, add 75% ethanol that 1000 μ l are prepared by depc water, precipitation is rushed, 13000rpm at 4 DEG C
Centrifugation 10min, outwells ethanol, leaves precipitation；
(7) repeat previous step, dry in an aseptic environment, plus 20-50 μ l aseptic depc water dissolution；
(8) carry out rna electrophoresis, detection rna extracts quality, and trace dna protein analyzer measures rna concentration.
2nd, race clone gene cdna total length
(1) synthesis of race-ready cdna
A. prepare buffer: 2.0 μ l 5 × the first chain buffer, the dtt of 1.0 μ l, the dntp mix of 1.0 μ l；Wherein,
The concentration of dtt is 20mm, and the concentration of dntp mix is 10mm；
B. the cdna reactant liquor for 5 ' race: 2.75 μ l rna, 1.0 μ l 5'-cds primer a are prepared；Preparation is used
Cdna reactant liquor in 3 ' race: 3.75 μ l rna, 1.0 μ l 3'-cds primer a；
C. by the liquid blending preparing in step b, of short duration centrifugation, 72 DEG C are incubated 3 minutes, cool down 2 minutes then at 42 DEG C,
Of short duration reactant liquor liquid is collected by centrifugation；
D. add the smarter iia oligo of 1 μ l in the cdna reactant liquor of 5 ' race, of short duration reactant liquor is collected by centrifugation
Liquid；
E. 5 ' race and 3 ' race-ready cdna reactant liquors: the buffer obtaining in 4.0 μ l steps a, 0.25 μ l are prepared
Rna enzyme inhibitor, the reverse transcriptase of 1.0 μ l, by above reagent mix；Wherein, the concentration of rna enzyme inhibitor is 40u/ μ l；
The concentration of reverse transcriptase is 100u/ μ l；
F. it is separately added into 5.25 μ l steps in 5 ' the race reactant liquors obtaining in the 3 ' race obtaining in step c and step d
The reactant liquor obtaining in rapid e, soft mix, of short duration liquid is collected by centrifugation；
G. the reactant liquor preparing is incubated 90 minutes in 42 DEG C；
H.70 DEG C heating 10 minutes, complete the synthesis of ready-cdna；
(2) design of race primer
Design principle includes:
A. gene specific primer gsps meets following condition: 23-28bp；Gc content 50%-70%；Tm value > 70 DEG C；With
There is not complementation in 3 '-end universal primer；
B. need to design two gsp primers, reverse primer gsp1 is used for 5 ' race, forward primer gsp2 is used for 3 ' race；
According to above design principle, design two primers of gsp1 and gsp2, such as seq id no:1 and seq in sequence table
Shown in idno:2；
(3) Fast back-projection algorithm of cdna end
A. prepare pcr mixed liquor: in every 50 μ l pcr reaction systems, add following reagent: 34.5 μ l pcr water, 5.0 μ l
10 × advantage 2pcr buffer, the dntp mix of 1.0 μ l, 1.0 μ l 50 × advantage 2 polymerase mix；Gently
Mixing liquid, can not produce bubble, of short duration liquid is collected by centrifugation；Wherein, the concentration of dntp mix is 10mm；
B. prepare the pcr reactant liquor for 5 ' race: 2.5 μ l 5 ' race ready cdna, 5.0 μ l 10 × upm, 1.0
μlgsp1；The pcr mixed liquor of preparation in step a of 41.5 μ l；
Prepare the pcr reactant liquor for 3 ' race: 2.5 μ l 3 ' race ready cdna, 5.0 μ l 10 × upm, 1.0 μ
lgsp2；The pcr mixed liquor of preparation in step a of 41.5 μ l；
C.race expands:
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 minutes, 72 DEG C 3 minutes,
Totally 5 circulations；94 DEG C 30 seconds, 68 DEG C 30 seconds, 72 DEG C 3 minutes, totally 20 circulation；
Take pcr product in percentage by weight be 1.0% agarose gel on carry out electrophoresis detection, reclaim purpose band,
Connect recovery product on pmd18-t carrier with t4dna ligase, convert escherichia coli dh5 α competent cell.
3rd, the escherichia coli conversion of recombiant plasmid
(1) dh5 α competent cell, in thawed on ice, draws 50 μ l, adds the dna connection product of 5 μ l, after mix homogeneously
Ice bath 30min；
(2) 42 DEG C of thermal shock 60sec, ice bath 3-5min immediately；
(3) add 700 μ l lb fluid mediums, in 37 DEG C, under constant temperature oscillator 150r/min, cultivate 60min；
(4) 5000rpm centrifugation 3min, collects thalline；
(5) contain on the lb flat board of antibiotic amp100mg/l coating x-gal and iptg so as to even spread planar surface,
Room temperature dries；Wherein, the concentration for 2%, iptg for the concentration of x-gal is 50mg/ml；
(6) 100 μ l bacterium solution are taken to be spread evenly across on the lb flat board containing antibiotic amp100mg/l, 37 DEG C of inversions in incubator
Overnight incubation；Flat board is allowed to fully develop the color by next day in 4 DEG C of placements；
(7) select white single bacterium colony in the lb fluid medium of the final concentration of 100mg/l of 1ml amp, under 225r/min,
In 37 DEG C of overnight incubation；
Pcr detection is carried out to bacterium solution, selects positive bacterium solution, send invitrogen company to be sequenced.Sequence is carried out to result
Row splicing, result shows that this gene has the nucleotide sequence as shown in seq id no:3 in sequence table, the aminoacid of its coding
Sequence is as shown in seq id no:4 in sequence table.
The agriculture bacillus mediated gogid gene transformation Medicago sativa of embodiment 2
1 material and reagent
1.1 vegetable material
It is alfalfa (medicago sativa l.cv.zhongmu no.1, middle lucerne one) for examination alfalfa variety.
1.2 agrobacterium strains and plasmid vector
Agrobacterium strains used are Agrobacterium tumefaciems: lba4404
Agrobacterium culture medium:
Table 1
121 DEG C of high pressure steam sterilization 20min；Carrier: pbi121.
2 experimental techniques
2.1 gogid gene is inserted in pbi121 vector plasmid dna, concretely comprises the following steps: design 5 ' end comprises xbai enzyme
The forward primer of enzyme site and the downstream primer of bamhi restriction enzyme site, as seq id no:5 and seq idno:6 institute in sequence table
Show；
(p1:5'-gctctagaAtggtgatcgagaacgacattgag-3',
P2:5'-cgggatccTcagtgatggccacgcctaagtgatg-3',
Wherein, underscore part is restriction enzyme site)；
With expanding total length reading frame, pbi carrier and the genes of interest of gogid in this primer eastwardly Galega officinalis L cdna template
Full length fragment recovery product after xbai and bamhi double digestion, connects through t4dna ligase, obtains being inserted with gogid gene
Pbi121 carrier, it contains camv35s promoter, gogid gene and kalamycin resistance selection markers, carries out heredity
The transfer-gen plant that Preliminary Identification obtains, the structure of the plasmid vector containing purpose fragment can be screened by kanamycin during conversion
As shown in Figure 1.
2.2 will be inserted with the pbi121 vector introduction of gogid gene in Agrobacterium tumefaciems lba4404, and concrete steps are such as
Under:
A. add about 1 μ g plasmid dna in 100 μ l Agrobacterium competent cell lba4404, gently mix, ice bath
30min；
B. quick-freezing 1min in liquid nitrogen, is immediately placed on incubation 5min in 37 DEG C of water-baths；
C. 800 μ l yeb fluid mediums are added, 28 DEG C of 150rpm cultivate 4-6h；
D. thalline is coated on the selection flat board of the yeb containing 50mg/l kanamycin and 100mg/l streptomycin, 28 DEG C of inversions
Culture two days.
E. picking single bacterium colony, is inoculated in yeb fluid medium (kanamycin containing 50mg/l and 100mg/l streptomycin),
28 DEG C of concussion and cultivates are overnight.
Extract and be imported with the plasmid of Agrobacterium of gogid gene and be sequenced, result shows the nucleotide sequence of quiding gene
Consistent with the sequence as shown in seq id no:3 in sequence table, show that the expression vector establishment containing genes of interest gogid becomes
Work(.
2.3 the culture of Agrobacterium
The Agrobacterium being imported with gogod gene is trained in the solid containing 50mg/l kanamycin and 100mg/l streptomycin
Draw flat board on foster base, be put in incubator, 28 DEG C of cultures.Two days later, picking single bacterium colony from flat board, is inoculated in containing 50mg/l
In the 20ml yeb fluid medium of kanamycin and 100mg/l streptomycin, 180rpm, 28 DEG C of cultures.Use cultured bacterium solution
Draw flat board, 28 DEG C of cultures, after growing single bacterium colony, flat board is put in 4 DEG C of preservations.
Picking single bacterium colony on flat board, is inoculated in the yeb liquid that 20ml contains 50mg/l kanamycin and 100mg/l streptomycin
In body culture medium, in 28 DEG C on constant-temperature table, 180rpm cultivates.Take a small amount of bacterium solution within two days, with the ratio of 1:50-1:100 later
Example is diluted in kanamycin containing 50mg/l and the yeb fluid medium of 100mg/l streptomycin, and 28 DEG C of culture 6-12h are to logarithm
Trophophase.By microorganism collection in centrifuge tube, 4000rpm centrifugation 10min enrichment thalline, abandon supernatant, then do not contained with about 20ml anti-
The resuspended thalline of sh fluid medium of the improvement of raw element, makes the od of bacterium solution600It is worth for 0.6-0.8, stand-by.
The preparation of 2.4 explants:
With alfalfa individual plant as material, it is grown in phjytotron, after 3-4 week, take blade (bottle green health leaf
Piece), under aseptic condition, it is cut into explant.
2.5 conversion process
Explant prepares, sterilizing.Explant is big, thick, bottle green blade, visually seems in the pink of condition being preferred.Will be outer
Implant 15-30 in the Agrobacterium re-suspension liquid (yep of sterilizing) of a600 0.6-0.8 divides, and is then put on aseptic filter paper, removes
Surplus liquid；Common training culture medium grows 7-8 days；Explant is taken out, puts in aquesterilisa, reverse 10-20 time, to remove
Bacterium.Repeat above procedure 2-3 time.Then continue in screening culture medium；After 2-3 week, calluss proceed on inducing culture；3
Zhou Hou, embryo will develop into sprig；Plantlet moves into and continues culture on root media.
2.6 transgenic line plant identification and expanding propagation
By the plant of above-mentioned conversion, extract dna, with npt gene primer p3, p4, gus gene primer p5, p6, with primer
P7, p8 carry out rna transcriptional level detection；Wherein, p3～p8 is as shown in seq id no:7～no:12 in sequence table；
Select expression higher transgenic line l9, l38, l39, grow in drum, after growing up, obtained by cuttage
Must copy and expanding propagation.
The pbi121 carrier conversion alfalfa evaluation of 2.7gogid gene:
Convert Agrobacterium with pbi121 carrier, obtain recombinational agrobacterium, with recombinational agrobacterium conversion, obtain turning empty carrier
Adjoining tree, method such as step 2.2～2.6, obtain and turn empty vector control plant (ck), as a comparison case 1.
The items evaluated the alfalfa turning empty carrier and convert the pbi121 carrier alfalfa being inserted with gogid gene
Index, concrete grammar is as follows:
Comparison and transgenic cuttage plant in the controlled environment chamber in survive after, plant division, plant in little basin.After stalwartness, move in
Big basin, grows in greenhouse, and period manages with delicacy, timely disease and pest control.Observe phenotype, measure plant height, fresh weight, dry weight, leaf
The indexs such as stem ratio, florescence.Statistical result is shown in Table 2.
Table 2
Note: * * represents has significant difference between transgenic alfalfa and the alfalfa turning empty carrier.
After the expression of gogid genes amplification, compared with comparative example alfalfa, the plant height of transgenic alfalfa increased
0.2-0.35 times, Biomass increased 14.9-46.3%, and length of blade and width are significantly higher than comparative example.Additionally, transgenic is purple
The flowering time of Herba Trigonellae Ruthenicae has also postponed one week.Result shows that gogid, as a gibberellins acceptor gene, has regulated and controled plant,
Multiple growth and development processes of especially herbage, in particular improve the length and width of blade, and it is purple to have regulated and controled transgenic
The florescence of Herba Trigonellae Ruthenicae.
Embodiment described above is only that the preferred embodiment of the present invention is described, the not model to the present invention
Enclose and be defined, on the premise of without departing from design spirit of the present invention, the technical side to the present invention for the those of ordinary skill in the art
Various modifications and improvement that case is made, all should fall in the protection domain of claims of the present invention determination.
Claims (7)
1. galega orientalis gibberellins acceptor gene gogid convert alfalfa method it is characterised in that: include following walking
Rapid:
(1) galega orientalis gibberellins acceptor gene gogid is inserted in pbi121 vector plasmid dna, obtains pbi121 weight
Group carrier:
Design 5 ' end comprises the forward primer p1 of the xbai restriction enzyme site and downstream primer p2 of bamhi restriction enzyme site, such as sequence table
Shown in middle seq id no:5 and seq id no:6；
P1:5'-gctctagaatggtgatcgagaacgacattgag-3'；
P2:5'-cgggatcctcagtgatggccacgcctaagtgatg-3'；
Wherein, underscore part is restriction enzyme site；
With expanding total length reading frame, pbi carrier and the genes of interest total length of gogid in this primer eastwardly Galega officinalis L cdna template
Fragment recovery product after xbai and bamhi double digestion, connects through t4dna ligase, obtains being inserted with gogid gene
Pbi121 carrier, it contains camv35s promoter, gogid gene and kalamycin resistance selection markers；
Wherein, described galega orientalis gibberellins acceptor gene gogid is as shown in seq id no:3 in sequence table；
(2) described pbi121 recombinant vector is imported in Agrobacterium tumefaciems lba4404:
A. add pbi121 transfer vector plasmid dna in Agrobacterium lba4404, mix, ice bath；
B. quick-freezing in liquid nitrogen, is immediately placed in water-bath and incubates；
C. yeb fluid medium, culture are added；
D. thalline is coated yeb and is selected, on flat board, to cultivate two days；
E. picking single bacterium colony, is inoculated in yeb fluid medium, overnight incubation；
Extract and be imported with the plasmid of Agrobacterium lba4404 of gogid gene and be sequenced, result shows the nucleotides sequence of quiding gene
Row are consistent with the sequence as shown in seq id no:3 in sequence table, then show the expression vector establishment containing genes of interest gogid
Success；
(3) it is imported with the culture of the Agrobacterium lba4404 of gogid gene；
(4) with alfalfa individual plant as material, it is grown in phjytotron, take bottle green healthy leaves, be cut into explant；
Explant is placed in Agrobacterium re-suspension liquid, is then put on aseptic filter paper, remove surplus liquid；
Common training culture medium grows；Explant is taken out, degerming；Then continue in screening culture medium；Calluss proceed to and lure
Lead in culture medium；Embryonic development becomes sprig or root；Plantlet moves into root media and continues culture；
(5) identification of alfalfa transgenic plant, expanding propagation and evaluation:
By the plant of above-mentioned conversion culture, extract dna, with npt gene primer p3, p4, gus gene primer p5, p6 are examined
Survey；Rna transcriptional level detection is carried out with primer p7, p8, wherein, seq id no:7～no:12 institute in p3～p8 such as sequence table
Show；
Select the higher transgenic line of expression and tie up to growth in drum, after growing up, copy and expanding propagation are obtained by cuttage；
Convert Agrobacterium lba4404 with pbi121 carrier, obtain recombinational agrobacterium lba4404, turned with recombinational agrobacterium lba4404
Change the adjoining tree obtaining turning empty carrier；
Turn empty carrier alfalfa, conversion be inserted with gogid gene pbi121 carrier alfalfa in the controlled environment chamber
In survive after, plant division, plant in little basin；After stalwartness, move in big basin, grow in greenhouse, period manages with delicacy, control disease in time
Insect pest；Observe phenotype, measure the indexs such as plant height, fresh weight, dry weight, leaf-stem ratio, florescence.
2. the method that galega orientalis gibberellins acceptor gene gogid converts alfalfa according to claim 1, its feature
It is: step (3) comprises the following specific steps that: the Agrobacterium of gogod gene will be imported with containing kanamycin and streptomycin
Solid medium on draw flat board, be put in culture in incubator；
Two days later, picking single bacterium colony from flat board, is inoculated in the yeb fluid medium containing kanamycin and streptomycin, training
Support；
Draw flat board with cultured bacterium solution, culture, after growing single bacterium colony, flat board is put in 4 DEG C of preservations；
Picking single bacterium colony on flat board, is inoculated in the yeb fluid medium containing kanamycin and streptomycin, in constant-temperature table
Upper culture；
Take a small amount of bacterium solution within two days later, be diluted in the yeb fluid medium containing kanamycin and streptomycin, cultivate to logarithm
Trophophase；By microorganism collection in centrifuge tube, centrifugal enrichment thalline, abandon supernatant, then with the resuspended thalline of sh fluid medium, make bacterium
The od of liquid600It is worth for 0.6-0.8, stand-by.
3. the method that galega orientalis gibberellins acceptor gene gogid converts alfalfa according to claim 2, its feature
It is: galega orientalis gibberellins acceptor gene gogid described in step (1) are to be prepared for raw material by galega orientalis,
Its preparation method comprises the following steps:
A. the extraction of the total rna of galega orientalis；
B.race clones gogid gene cdna total length；
C. the escherichia coli conversion of recombiant plasmid, and be sequenced.
4. the method that galega orientalis gibberellins acceptor gene gogid according to claim 3 converts alfalfa, it is special
Levy and be: preparation method step a of galega orientalis gibberellins acceptor gene gogid adopts trizol reagent method, including as follows
Concrete steps:
A. galega orientalis organization material is added to trizol reagent, add liquid nitrogen quickly to grind, put to being changed into being homogenized；
B. draw homogenate in centrifuge tube, acutely vibration mixes, and places in room temperature, is then centrifuged for；
C. take supernatant, add chloroform, acutely vibrate, room temperature is placed, and is then centrifuged for；
D., after solution layering, take upper strata to move into another centrifuge tube, add equal-volume chloroform, repeat previous step；
E. take its supernatant, add equal-volume isopropanol, mixing mixes, and room temperature is placed, and is then centrifuged for, obtain white rna precipitation；
F. abandon supernatant, add ethanol, precipitation is rushed, centrifugation, outwell ethanol, leave precipitation；
G. repeat previous step, dry, plus depc water dissolution；
H. carry out rna electrophoresis, detection rna extracts quality, measures rna concentration using trace dna protein analyzer.
5. the method that galega orientalis gibberellins acceptor gene gogid according to claim 3 converts alfalfa, it is special
Levy and be: preparation method step b of galega orientalis gibberellins acceptor gene gogid comprises the following specific steps that:
(1) synthesis of race-ready cdna
A. prepare buffer: 5 × the first chain buffer, dtt, dntp mix；
B. the cdna reactant liquor for 5 ' race and the cdna reactant liquor for 3 ' race are prepared；
C. by the liquid blending preparing in step b, centrifugation, incubation, then cool down, reactant liquor liquid is collected by centrifugation；
D. add smarter iia oligo in the cdna reactant liquor of 5 ' race, reactant liquor liquid is collected by centrifugation；
E. prepare 5 ' race and 3 ' race-ready cdna reactant liquors: by the buffer obtaining in step a, rna enzyme inhibitor,
Reverse transcriptase mixes；
F. it is separately added in 5 ' the race reactant liquors obtaining in the 3 ' race obtaining in step c and step d and to obtain in step e
Reactant liquor, mixing, liquid is collected by centrifugation；
G. the reactant liquor preparing is incubated；
H. heat, complete the synthesis of ready-cdna；
(2) design of race primer
Design principle includes:
A. gene specific primer gsps meets following condition: 23-28bp；Gc content 50%-70%；Tm value > 70 DEG C；With 3 '-end
There is not complementation in universal primer；
B. need to design two gsp primers, reverse primer gsp1 is used for 5 ' race, forward primer gsp2 is used for 3 ' race；
According to above design principle, design two primers of gsp1 and gsp2；
(3) Fast back-projection algorithm of cdna end
A. prepare pcr mixed liquor: in pcr reaction system, add following reagent: pcr water, 10 × advantage 2 pcr buffering
Liquid, dntp mix, 50 × advantage 2 polymerase mix；Mixing, is collected by centrifugation liquid；
B. prepare the pcr reactant liquor for 5 ' race: preparation in 5 ' race ready cdna, 10 × upm, gsp1, step a
Pcr mixed liquor；
Prepare the pcr reactant liquor for 3 ' race: the pcr of preparation in 3 ' race ready cdna, 10 × upm, gsp2, step a
Mixed liquor；
C.race expands:
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 minutes, 72 DEG C 3 minutes, totally 5
Individual circulation；94 DEG C 30 seconds, 68 DEG C 30 seconds, 72 DEG C 3 minutes, totally 20 circulation；
Take pcr product that electrophoresis detection is carried out on agarose gel, reclaim purpose band, connect to reclaim with t4dna ligase and produce
Thing, on pmd18-t carrier, converts escherichia coli dh5 α competent cell.
6. the method that galega orientalis gibberellins acceptor gene gogid according to claim 5 converts alfalfa, it is special
Levy and be: the primer gsp1 and gsp2 such as sequence table described in preparation method step b of galega orientalis gibberellins acceptor gene gogid
Shown in middle seq id no:1 and seq id no:2.
7. the method that galega orientalis gibberellins acceptor gene gogid according to claim 3 converts alfalfa, it is special
Levy and be: preparation method step c of galega orientalis gibberellins acceptor gene gogid comprises the following specific steps that:
A.dh5 α competent cell melts, and adds dna connection product, ice bath after mix homogeneously；
B. thermal shock, ice bath immediately；
C. add lb fluid medium, cultivate under constant temperature oscillator；
D. it is centrifuged, collects thalline；
On e.lb flat board, so as to even spread planar surface, room temperature dries coating x-gal and iptg；
F. bacterium solution is taken to be spread evenly across on lb flat board, overnight incubation in incubator；Flat board is placed and is allowed to fully develop the color by next day；
G. select white single bacterium colony in lb fluid medium, overnight incubation；
Pcr detection is carried out to bacterium solution, selects positive bacterium solution, be sequenced.
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| CN108085321A (en) * | 2018-02-05 | 2018-05-29 | 北京农学院 | Gibberellin acceptor gene LsGID1 and its application |
| CN117384950A (en) * | 2023-12-12 | 2024-01-12 | 中国农业科学院北京畜牧兽医研究所 | Application of MsSPL17 gene |
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| CN106591360A (en) * | 2017-03-03 | 2017-04-26 | 中国农业科学院北京畜牧兽医研究所 | High-efficiency stable wide-spectrum alfalfa genetic transformation method, and transformation system obtained by using method |
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| CN108085321A (en) * | 2018-02-05 | 2018-05-29 | 北京农学院 | Gibberellin acceptor gene LsGID1 and its application |
| CN117384950A (en) * | 2023-12-12 | 2024-01-12 | 中国农业科学院北京畜牧兽医研究所 | Application of MsSPL17 gene |
| CN117384950B (en) * | 2023-12-12 | 2024-03-12 | 中国农业科学院北京畜牧兽医研究所 | Application of MsSPL17 gene |
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