CN108531504A - A method of efficiently formulating low content of lignin alfalfa using genetic engineering means - Google Patents
A method of efficiently formulating low content of lignin alfalfa using genetic engineering means Download PDFInfo
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- CN108531504A CN108531504A CN201810299456.0A CN201810299456A CN108531504A CN 108531504 A CN108531504 A CN 108531504A CN 201810299456 A CN201810299456 A CN 201810299456A CN 108531504 A CN108531504 A CN 108531504A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The method that the present invention relates to a kind of efficiently to formulate low content of lignin alfalfa using genetic engineering means.It after alfalfa aseptic seedling cotyledon preculture, is disseminated with the Agrobacterium LBA4404 bacterium solution of the ubi 4fan of p2300 containing antisense expression vector 4CL, is cultivated through co-cultivation, callus induction, differentiation, form adventitious bud；When adventitious bud is grown to 2cm~3cm, it is transferred to root media and takes root；When root long is to 2~3cm, rooted seedling is transplanted in the matrix mixed.Molecular Identification is carried out to regeneration plant, content of lignin measures.Obtaining generation transformation generation using the present invention only needs 55 60d, and 100% is positive plant, content of lignin is normal compared with 10% or more wild type reduction, growth.The present invention provides feasible means for the low lignin alfalfa of high effect culture.
Description
Technical field
The invention belongs to biotechnologies, and in particular to utilize genetic engineering means, efficiently formulate low content of lignin
The method of alfalfa.
Background technology
Alfalfa (Medicago sativa L.) is the maximum artificial grass of China's cultivated area, due to its grass yield
Height, palatability is good, first of nutritive value row herbage, is otherwise known as " herbage is king ".Alfalfa not only contains protein, mineral
The important nutritional ingredient such as matter and vitamin, also containing essential amino acid, trace element and the unknown growth factor needed for animal.
On identical soil, alfalfa is 2.5 times higher than the digestible protein that graminous pasture is harvested, the high 6 times of left sides of minerals
The right side, high 2 times or so of digestible nutrient.Compared with other cereal crops, the yield of alfalfa unit area nutriment also compared with
It is high.In addition, alfalfa also plays important and positive work in soil improvement, water and soil conservation and ecological environmental protection etc.
With.Alfalfa is in the plantation extensively of the country such as China, the U.S., Australia, at present in the cultivated area in China at 2,000 ten thousand mu,
Occupy the world the 4th.The kind for improveing alfalfa, improves its nutritive value, has to the animal husbandry in China or even the world important
Meaning.
After alfalfa enters the stomach of ox and sheep, exists due to the characteristic of herbage and digest incomplete phenomenon, to
Affect absorption of the livestock to nutriment.The main reason for causing this phenomenon is that there are excessive wooden in plant cell wall
Element.Lignin is the important chemical composition of composition plant cell wall can enhance plant between cellulose and hemicellulose
The mechanical strength of body, enhance plant crushing resistance and support force, conducive to the invasion dredged tissue Water Transportation, resist pest and disease damage
Deng being played an important role to the normal growth of plant.But lignin itself is difficult by animal digestion；Moreover, because wooden
The presence of element, also hampers the utilization of other nutritional ingredients such as cellulose, hemicellulose.Studies have shown that livestock is to clover dry
The digestibility of matter has significant negatively correlated with content of lignin, and lignin contained in feed is the limit for influencing feed digestibility
The factor processed.If reducing content of lignin in feed cell wall, so that lignifying cell is easily digested, then can significantly improve feed
Digestibility and utilization rate.
Invention content
The purpose of the present invention is to provide one kind since seed, by plant cell engineering means, obtains and contains antisense
The method of the low content of lignin alfalfa of 4CL1 genes, by this method can 100% obtain content of lignin reduce by 10% with
On alfalfa material.
To achieve the goals above, the technical solution adopted by the present invention is：It is a kind of efficiently to be formulated using genetic engineering means
The method of low content of lignin alfalfa, includes the following steps：
1) alfalfa aseptic seedling is obtained：Alfalfa seed is through 0.1% HgCl2After sterilizing, the nothing in moistening is sowed
On bacterium filter paper, 25 DEG C, light culture, until germination, obtains alfalfa aseptic seedling.
2) preculture：The cotyledon for taking alfalfa aseptic seedling, is cut into 0.2cm2~0.3cm2Fritter is inoculated in pre- training culture
On base, 25 DEG C, light culture 1d.
Preferably, the pre- training culture medium is：Using MS culture mediums as minimal medium, 4mg/L 2,4- dichloro-benzenes are added
Fluoroacetic acid (2,4-D) adds 3% sucrose and 0.7% agar by weight percentage, adjusts pH to 5.8.
3) it infects：To include the bacterium solution of the Agrobacterium LBA4404 of antisense expression vector p2300-ubi-4fan-4CL in
3000r/min centrifuges 10min, collects thalline, and thalline is resuspended with culture medium is infected；The cotyledon of step 2) preculture 1d is transferred to bacterium
In body and the mixture for infecting culture medium, cotyledon is infected in 120r/min, concussion processing 20min, acquisition.
The preparation method of the Agrobacterium LBA4404 for including antisense expression vector p2300-ubi-4fan-4CL is：
False indigo total DNA is extracted, using the total DNA of false indigo as template, PCR amplification is carried out with specific primer, obtains false indigo 4CL1
4CL1 genetic fragments are connect by genetic fragment with expression vector p2300-ubi-4fan, structure recombinant expression carrier p2300-
ubi-4fan-4CL.Recombinant expression carrier p2300-ubi-4fan-4CL is transformed into Agrobacterium LBA4404 by freeze-thaw method.
The sequence of the specific primer is：
5 ' end primer sequences (5 ' to 3 ')：TCGCCTATGACTGGGCACAACAGA
3 ' end primer sequences (5 ' to 3 ')：AAGAAGGCGATAGAAGGCGATGCG
The culture medium that infects is：Using MS culture mediums as minimal medium, 2mg/L 2,4- dichlorphenoxyacetic acids are added
(2,4-D), 0.2mg/L 6-benzyladenines (6-BA), 200mg/L acetosyringones (AS), add 3% by weight percentage
Sucrose, adjust pH to 5.8.
4) it co-cultures：Dip dyeing cotyledon is transferred on total training culture medium, 25 DEG C, 12h/ days, illuminance 30umol/m2Under s,
Illumination cultivation 3d, the dip dyeing cotyledon after must co-culturing.
Preferably, the total training culture medium is：Using MS culture mediums as minimal medium, 2mg/L 2,4- dichloro-benzenes are added
Fluoroacetic acid (2,4-D), 0.2mg/L 6-benzyladenines (6-BA), 200mg/L acetosyringones (AS), add by weight percentage
Add 3% sucrose and 0.7% agar, adjusts pH to 5.8.
5) induction of callus：Dip dyeing cotyledon after co-cultivation is inoculated on callus inducing medium, 25 DEG C,
12h/ days, illuminance 30umol/m2Illumination cultivation under s, the formation of evoked callus.
Preferably, the callus inducing medium is：Using MS culture mediums as minimal medium, 2mg/L 2 is added,
4- dichlorphenoxyacetic acids (2,4-D), 0.2mg/L 6-benzyladenines (6-BA), 100mg/L kanamycin sulfates (Kan),
400mg/L cephalosporins (Cef) add 3% sucrose and 0.7% agar by weight percentage, adjust pH to 5.8.
6) differentiation of adventitious bud：The callus of formation is cut into 0.2cm2~0.3cm2, it is transferred to differential medium, 25 DEG C,
12h/ days, illuminance 30umol/m2Illumination cultivation under s, it is primary per 28d subcultures.
The differential medium is：Using MS culture mediums as minimal medium, 0.2mg/L 2,4- Dichlorophenoxy second are added
Sour (2,4-D), 2mg/L 6-Furfurylaminopurines (KT), 100mg/L kanamycin sulfates (Kan), 300mg/L cephalosporins
(Cef), 3% sucrose and 0.7% agar are added by weight percentage, adjust pH to 5.8.
7) rooting induction：When adventitious bud is grown to 2cm~3cm, is cut from base portion, be transferred in root media and take root, 25
DEG C, 12h/ days, illuminance 30umol/m2Illumination cultivation under s.
Preferably, the root media is：Using MS culture mediums as minimal medium, 0.5mg/L α-naphthylacetic acids are added
(NAA), 3% sucrose and 0.7% agar are added by weight percentage, adjust pH to 5.8.
8) when the root long of rooted seedling is to 2~3cm, bottleneck hardening is opened, is then transplanted in the matrix mixed.
Preferably, the matrix is：By weight, cultivation soil：Turf：Vermiculite=1：2：1 mixing.
The beneficial effects of the invention are as follows：
1, of the invention, agrobacterium strains used are LBA4404, include the expression vector p2300- built by this laboratory
Ubi-4fan-4CL, structure are as shown in Figure 1.Target gene 4CL1 derives from false indigo in carrier, will amplify the 4CL1 bases come
It, will with freeze-thaw method because segment is reversely inserted into carrier p2300-ubi-4fan, construction recombination plasmid p2300-ubi-4fan-4CL
P2300-ubi-4fan-4CL is transferred to Agrobacterium LBA4404, identifies that correct Agrobacterium is sub-packed in cryopreservation tube, in -80 DEG C of ice
Case preserves.Inoculation after activation is to containing 125mg/L streptomycin sulphates (Str) and 100mg/L kanamycin sulfates (Kan)
YEP fluid nutrient mediums in, 28 DEG C, 200r/min is enlarged culture, waits for bacterium solution OD600For soaking when value is 0.5~0.6
Dye.
2, method using the present invention, to regeneration plant carry out Molecular Identification, content of lignin measure, with appearance, plant height,
Weight per plant is index, compares transgenosis and difference of the wild type material in terms of growth potential, with growth potential with wild type without notable
The transfer-gen plant of difference is the growth that finally obtains normally low content of lignin plant.
3, can be in 55d-60d using this method, the transgenosis that 100% acquisition content of lignin reduces by 10% or more is pale reddish brown
Clover, the normal material of growth and development therefrom selected can be used as alfalfa quality breeding.The present invention is quick, high effect culture
Quality alfalfa germ plasm resource provides feasible means.
Description of the drawings
Fig. 1 is the structure chart of recombinant expression carrier p2300-ubi-4fan-4CL.
Specific implementation mode
A kind of method that efficiently formulating low content of lignin alfalfa using genetic engineering means of embodiment 1
(1) method is as follows：
1) preparation of bacterium solution
1. the structure of p2300-ubi-4fan-4CL and the method for being transferred to Agrobacterium
False indigo total DNA is extracted, using the total DNA of false indigo as template, PCR amplification is carried out with specific primer, is amplified
False indigo 4CL1 genetic fragments are connect structure recombination table by false indigo 4CL1 genetic fragments with expression vector p2300-ubi-4fan
Up to carrier p2300-ubi-4fan-4CL, recombinant expression carrier p2300-ubi-4fan-4CL is transformed into Agrobacterium with freeze-thaw method
It in LBA4404, will identify that correct Agrobacterium is sub-packed in cryopreservation tube, preserved in -80 DEG C of refrigerators.The specific primer
Sequence is：
5 ' end primer sequences (5 ' to 3 ')：TCGCCTATGACTGGGCACAACAGA
3 ' end primer sequences (5 ' to 3 ')：AAGAAGGCGATAGAAGGCGATGCG
2. by the Agrobacterium LBA4404 inoculation containing p2300-ubi-4fan-4CL to contain 125mg/L sulfate chains
The YEP fluid nutrient mediums of mycin (Str) and 100mg/L kanamycin sulfates (Kan), 28 DEG C, 200r/min is enlarged training
It supports, waits for bacterium solution OD600For disseminating when value is 0.5.
2) acquisition of aseptic seedling
Select the alfalfa (kind of full grains：" sweet agriculture 4 ") seed, with 0.1% HgCl2Sterilize 10min, nothing
Bacterium is washed 3 times, sows on the aseptic filter paper of moistening, 25 DEG C, light culture, until germination, obtains alfalfa aseptic seedling.
3) preculture
The cotyledon for taking alfalfa aseptic seedling, is cut into 0.2cm2~0.3cm2Fritter is inoculated on pre- training culture medium, 25 DEG C,
Light culture 1d.
Training culture medium is in advance：+ 0.7% agar of MS+4mg/L 2,4-D+3% sucrose adjusts pH to 5.8.
4) it infects
By OD600The bacterium for the Agrobacterium LBA4404 for including antisense expression vector p2300-ubi-4fan-4CL that value is 0.5
Liquid is transferred in the centrifuge tube of the 50ml of sterilizing, is centrifuged 10min in 3000r/min, is collected thalline, and bacterium is resuspended with culture medium is infected
Body.The cotyledon of step 3) preculture is transferred to thalline and is infected in the mixture of culture medium, 25 DEG C, 120r/min concussions make son
Leaf comes into full contact with Agrobacterium.It is taken out after concussion processing 20min and infects cotyledon, be placed in the culture dish for being covered with aseptic filter paper, inhale
The extra bacterium solution of dry cotyledon surface.
Infecting culture medium is：MS+2mg/L 2,4-D+0.2mg/L 6-BA+200mg/L AS+3% sucrose, adjust pH to
5.8。
5) it co-cultures
Cotyledon will be disseminated, will be transferred on the not total training culture medium of added with antibiotic, 25 DEG C, 12h/ days, illuminance 30umol/m2·
Under s, illumination cultivation 3d, the dip dyeing cotyledon after must co-culturing.
Training culture medium is altogether：+ 0.7% fine jade of MS+2mg/L 2,4-D+0.2mg/L 6-BA+200mg/L AS+3% sucrose
Fat adjusts pH to 5.8.
6) induction of callus
Dip dyeing cotyledon after co-cultivation is inoculated on callus inducing medium, 25 DEG C, 12h/ days, illuminance
30umol/m2Illumination cultivation under s, the formation of evoked callus form the callus of yellow green after cultivating 15d.
Callus inducing medium is：MS+2mg/L 2,4-D+0.2mg/L 6-BA+100mg/L Kan+400mg/L
+ 0.7% agar of Cef+3% sucrose adjusts pH to 5.8.
7) differentiation of adventitious bud
After callus is formed, callus is cut into 0.2cm2~0.3cm2, it is transferred to differential medium, 25 DEG C, 12h/
Day, illuminance 30umol/m2Illumination cultivation under s.It is primary per 28d subcultures, initially form green bud point after cultivating 20d；30d
Elongation of adventitious bud is to 2cm or so afterwards.
Differential medium is：MS+0.2mg/L 2,4-D+2mg/L KT+100mg/L Kan+300mg/L Cef+3% sugarcanes
Sugared+0.7% agar, adjusts pH to 5.8.
8) rooting induction
It when adventitious bud is grown to 2cm~3cm, is cut from its base portion, is transferred to root media and takes root, 25 DEG C, 12h/ days, illumination
Spend 30umol/m2Illumination cultivation under s.Preferably, the root media is：MS+0.5mg/L NAA+3% sucrose+
0.7% agar adjusts pH to 5.8.
9) hardening and transplanting
When the root long of rooted seedling is to 2~3cm, bottleneck 2~3d of hardening is opened, the matrix mixed is then transplanted to.
Matrix is：By weight, cultivation soil：Turf：Vermiculite=1：2：1 mixing.
(2) Molecular Detection of regeneration plant
Transgenic alfalfa plant genomic DNA, wild-type alfalfa genomic DNA are obtained with above-mentioned (one) respectively
It is that template carries out PCR reactions with plasmid p2300-ubi-4fan-4CL.
PCR detections and purpose band sequencing are carried out to the alfalfa of conversion, the results showed that, using transplant survival in (one)
10 plants of transgenic alfalfa plant in amplify antisense 4CL1 segments, survive plant 100% be transformant.
Content of lignin detection shows that the content of lignin of 10 plants of transfer-gen plants lowers 10% or more.
Claims (9)
1. a kind of method for efficiently formulating low content of lignin alfalfa using genetic engineering means, which is characterized in that including
Following steps：
1) alfalfa aseptic seedling is obtained：Alfalfa seed is through 0.1% HgCl2After sterilizing, the aseptic filter paper in moistening is sowed
On, 25 DEG C, light culture, until germination, obtains alfalfa aseptic seedling；
2) preculture：The cotyledon for taking alfalfa aseptic seedling, is cut into 0.2cm2~0.3cm2Fritter is inoculated on pre- training culture medium,
25 DEG C, light culture 1d；
3) it infects：The bacterium solution of the Agrobacterium LBA4404 of antisense expression vector p2300-ubi-4fan-4CL will be included in 3000r/
Min centrifuges 10min, collects thalline, and thalline is resuspended with culture medium is infected；The cotyledon of step 2) preculture 1d is transferred to thalline and invaded
In the mixture for contaminating culture medium, cotyledon is infected in 120r/min, concussion processing 20min, acquisition；
4) it co-cultures：Cotyledon will be disseminated, will be transferred on total training culture medium, 25 DEG C, 12h/ days, illuminance 30umol/m2Under s, illumination
Cultivate 3d, the dip dyeing cotyledon after must co-culturing；
5) induction of callus：Dip dyeing cotyledon after co-cultivation is inoculated on callus inducing medium, 25 DEG C, 12h/
Day, illuminance 30umol/m2Illumination cultivation under s, the formation of evoked callus；
6) differentiation of adventitious bud：The callus of formation is cut into 0.2cm2~0.3cm2, it is transferred to differential medium, 25 DEG C, 12h/
Day, illuminance 30umol/m2Illumination cultivation under s, it is primary per 28d subcultures；
7) rooting induction：When adventitious bud is grown to 2cm~3cm, is cut from base portion, be transferred in root media and take root, 25 DEG C,
12h/ days, illuminance 30umol/m2Illumination cultivation under s；
8) when the root long of rooted seedling is to 2~3cm, bottleneck hardening is opened, is then transplanted in the matrix mixed.
2. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 2), the pre- training culture medium is：Using MS culture mediums as minimal medium, 4mg/L2 is added,
4- dichlorphenoxyacetic acids add 3% sucrose and 0.7% agar by weight percentage, adjust pH to 5.8.
3. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 3), include the system of the Agrobacterium LBA4404 of antisense expression vector p2300-ubi-4fan-4CL
Preparation Method is：The total DNA for obtaining false indigo carries out PCR amplification with specific primer, obtains using the total DNA of false indigo as template
False indigo 4CL1 genetic fragments；False indigo 4CL1 genetic fragments are connect with carrier p2300-ubi-4fan, structure recombinant expression
Carrier p2300-ubi-4fan-4CL；Recombinant expression carrier p2300-ubi-4fan-4CL is transformed into Agrobacterium with freeze-thaw method
In LBA4404.The sequence of the specific primer is：
5 ' end primer sequences (5 ' to 3 ')：TCGCCTATGACTGGGCACAACAGA
3 ' end primer sequences (5 ' to 3 ')：AAGAAGGCGATAGAAGGCGATGCG.
4. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 3), the culture medium that infects is：Using MS culture mediums as minimal medium, 2mg/L 2 is added,
4- dichlorphenoxyacetic acids, 0.2mg/L 6-benzyladenines, 200mg/L acetosyringones add 3% sugarcane by weight percentage
Sugar adjusts pH to 5.8.
5. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 4), the total training culture medium is：Using MS culture mediums as minimal medium, 2mg/L2 is added,
4- dichlorphenoxyacetic acids, 0.2mg/L 6-benzyladenines, 200mg/L acetosyringones add 3% sugarcane by weight percentage
Sugar and 0.7% agar, adjust pH to 5.8.
6. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 5), the callus inducing medium is：Using MS culture mediums as minimal medium, addition
2mg/L 2,4 dichlorophenoxyacetic acids, 0.2mg/L 6-benzyladenines, 100mg/L kanamycin sulfates, 400mg/L cephalos are mould
Element adds 3% sucrose and 0.7% agar by weight percentage, adjusts pH to 5.8.
7. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 6), the differential medium is：Using MS culture mediums as minimal medium, 0.2mg/ is added
L2,4- dichlorphenoxyacetic acid, 2mg/L 6-Furfurylaminopurines, 100mg/L kanamycin sulfates, 300mg/L cephalosporins, by weight
It measures percentage and adds 3% sucrose and 0.7% agar, adjust pH to 5.8.
8. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 7), the root media is：Using MS culture mediums as minimal medium, 0.5mg/L is added
α-naphthylacetic acid adds 3% sucrose and 0.7% agar by weight percentage, adjusts pH to 5.8.
9. a kind of side efficiently formulating low content of lignin alfalfa using genetic engineering means according to claim 1
Method, which is characterized in that in step 8), the matrix is：By weight, cultivation soil：Turf：Vermiculite=1：2：1 mixing.
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