CN109735562A - A method for constructing an effective root transgenic system for economical plants - Google Patents
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Abstract
The present invention provides a kind of construction methods of the effective root system transgenic system of economic plants, belong to technical field of plant transgene.The bacterium solution of the agrobacterium rhizogenes of recombinant vector containing target is separated by solid-liquid separation, the obtained agrobacterium rhizogenes thallus of recombinant vector containing target is resuspended, the stem of economic plants seedling, 10~12d after injection are injected into obtained agrobacterium rhizogenes suspension, economic plants seedling stem wound grows callus；Seedling continues 28~35d of culture, cuts original feather shaped root system when callus is divided into regeneration hairy.Construction method of the present invention is applicable in diversified economy plant, and hairy regeneration rate and callus regeneration rate are higher.The construction method provides very quickly and effectively genic system functional analysis, secondary metabolites engineering and plant stress reaction research.It is transgenosis rather than whole plant that the root system transgenic system of building, which only has root, this is also the better systems for studying signal transduction between root and stem.
Description
Technical field
The invention belongs to technical field of plant transgene, and in particular to a kind of effective root system transgenic system of economic plants
Construction method.
Background technique
The plant conversion system of mediated by agriculture bacillus be in recent decades in plant genetic engineering most extensively and most successful side
Method (Gelvin, 2003；Vain, 2007；Matveeva and Lutova, 2014).Agrobacterium tumefaciems can be by injuring and inciting somebody to action
Tumor inducing (Ti) plasmid is transferred to plant nucleolus and grows tumour in plant.Based on this theory, by modification and
It removes Ti-plasmids and converts required gene for tumor inducting gene, and carry the dyeing that any required gene enters plant cell
Body.The methods for plant transformation success foreign gene transfer that mediated by agriculture bacillus is widely used for the first time from nineteen eighty-three enters the demonstration of tobacco
Since, the gene functional research and plant genetic of model plant and some crops have been established in more effective transgenic system
Modification (Zhang et al., 2006；Wen Zile et al., 1989；Stone field et al., 1996).
However, more effort have been paid in the exploitation of the Model Plants stable conversion scheme of mediated by agriculture bacillus, so far
Until be only limitted to a small number of plants (Ke Lao and Ben Te, 1998；Hoekema et al., 1989；Mu Lizuowaaiaier, 2014
Year).Due to its unique or long growth cycle, most of medicinal plants and xylophyta plant lack efficient gene function
Quick screening system is analyzed, and significantly limits the development of these Related Research Domains.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of economic plants roots novel and simple, quickly and efficiently
It is the construction method of transgenic system, has the characteristics that hairy root regeneration rate is high.
The present invention provides a kind of construction methods of the effective root system transgenic system of economic plants, comprising the following steps:
1) bacterium solution of the agrobacterium rhizogenes of recombinant vector containing target is separated by solid-liquid separation, the obtained root of hair agriculture of recombinant vector containing target
Bacillus thallus is resuspended, and obtains agrobacterium rhizogenes suspension；
2) stem of economic plants seedling is injected into the agrobacterium rhizogenes suspension, 10~12d after injection is economical
Plant seedlings stem wound grows callus；
3) the economic plants seedling with callus is cultivated into 28~35d, when callus is divided into hairy of regeneration
When cut original feather shaped root system.
Preferably, the bacterial strain of agrobacterium rhizogenes includes K599, MSU440, C58C1 or ArA4 in step 1).
Preferably, in step 1) agrobacterium rhizogenes suspension OD600Value is 0.2~0.6.
Preferably, economic plants includes herbaceous plant, shrub plant and magaphanerophytes in step 2).
Preferably, in step 2) economic plants include pigeonpea, safflower, cassia seed, Radix Isatidis, Golden flower, gumbo, castor-oil plant,
Caragana and agalloch eaglewood.
Preferably, the cultural method of injection front and back described in step 2) is by economic plants seed through tissue cultures 2~5
Then week is planted in soil media obtained tissue culture seedling direct injection.
Preferably, the cultural method of injection front and back described in step 2) is to grow economic plants seed in soil media
It is injected at 3~8 weeks, then proceedes to grow in soil media.
Preferably, the position of injection described in step 2) is located at stem at 0~1.5cm above the original feather shaped root system of seedling
Portion.
Preferably, the position of injection described in step 2) is the stem at 0.1~1.0cm above seedling original hairy
Portion.
Preferably, the carrier of target recombinant vector described in step 1) includes pROK2；Target in the target recombinant vector
Gene includes GFP.
The present invention provides a kind of construction methods of the effective root system transgenic system of economic plants, comprising the following steps: 1)
The bacterium solution of the agrobacterium rhizogenes of recombinant vector containing target is separated by solid-liquid separation, the obtained agrobacterium rhizogenes thallus weight of recombinant vector containing target
It is outstanding, obtain agrobacterium rhizogenes suspension；2) stem of plant seedlings is injected into the agrobacterium rhizogenes suspension, 10 after injection
~12d, plant seedlings stem wound grow callus；3) plant seedlings 28~35d of culture of callus will be had,
Original feather shaped root system is cut when callus is divided into regeneration hairy.Experiment shows using building side provided by the invention
The regeneration rate that method is hairy is 15%~45%.The method that the present invention constructs simultaneously, callus regeneration rate is higher, specially
16%~85%.
Meanwhile construction method provided by the invention has the characteristics that have a wide range of application, especially medical nine kinds of selected warps
Ji plant.The phenotype of Four Plants in nine kinds of plants shows that they have higher root regeneration rate.It is anti-through reverse transcriptase polymerase chain
It answers result and GFP signal to confirm that target gene (GFP) is expressed in transgenic hairy root and all confirms that target gene has turned
It moves on in hairy.It is all the result shows that hairy method is widely used, including draft and xylophyta.
Further, the present invention further defines the type of agrobacterium rhizogene strain, has selected agrobacterium rhizogene strain
K599, MSU440, C58C1 and Ar4 measure the transformation efficiency of seedling, the results showed that, agrobacterium rhizogene strain K599 is in callus
High efficiency is all shown in tissue induction (80%) and hairy root induction (30%).Compared with K599, other three kinds of bacterial strain conversions
Efficiency is all relatively low.For callus, the induced efficiency of bacterial strain C58C1 is 15%, followed by bacterial strain MSU440 (10%)
With ArA4 (8%).For hairy, these three bacterial strain inducing efficiency are 5%~8%.This illustrates that K599 induction turns at hairy
Should be more effective in genic system, and pigeonpea may be more suitable for.
Further, the present invention further defines the concentration of agrobacterium suspension, has studied injection concentration to callus group
Knit the influence of inductivity, hairy root induction rate and transgenosis rate.Injection concentration is 0.2,0.3,0.4,0.5 and 0.6OD600Selection
Value is tested.The result shows that OD2 (OD value 0.3) and OD3 (OD value 0.4) are to obtain the optimum value of high callus induction rate
(OD2:58% ± 2%；OD3:60% ± 4%).OD2 still obtain hairy best regeneration rate optimum value (33% ±
4%), followed by OD3 (26% ± 4%).In addition, the transgenosis root that the Agrobacterium solution injection of various concentration obtains is not poor
It is different.In general, it is 0.3 that the concentration of best callus and hairy root induction, which is OD value,.
Further, the present invention further defines injection position, above the original feather shaped root system of seedling at 0~1.0cm
Stem's injection, callus and hairy are 60% or so, and comparatively 1.0~1.5cm has higher efficiency.
Further, the present invention further defines the injection age of tissue culture seedling.After 15 days, 30 days, tissue culture bottle training
Feeding seedling be used to inject, and be then transferred into soil environment.The result shows that the infection rate of seedling (15 days and 30 days) is than old
Height of seedling (45 days).
Detailed description of the invention
Fig. 1 is hairy transgenic system figure of pigeonpea；Wherein agrobacterium rhizogenes of the Fig. 1 (a) containing recombinant vector
(Agrobacterium rhizogenes) solution is injected into the stem of a month big passage Cajanus cajan seeds；Fig. 1 (b)~Fig. 1
(e) process of callus and hairy root regeneration；The left side is the schematic diagram in each stage, and the right is the picture in this stage；Fig. 1
(f) original can be cut off after being shown in transgenic hairy root well-grown；
Fig. 2 is the OD value and transgenic line identification for selecting bacterium solution；OD1 to OD5 respectively indicate 0.2,0.3,0.4,0.5 and
0.6 OD value；Wherein Fig. 2 (a) is the regeneration rate of callus after the Agrobacterium K599 for injecting different OD values；Fig. 2 (b) is not
With hairy in OD value Agrobacterium solution regeneration rate；Fig. 2 (c) is the transgenosis rate that hairy is regenerated under different OD values；Fig. 2
(d)~Fig. 2 (e) is original picture of transgenic hairy root；Fig. 2 (g) is stalk Callus Regeneration after pigeonpea injection
Picture；Fig. 2 (f)~Fig. 2 (h) is the transgenic hairy root cut off after original；Fig. 2 (d)~Fig. 2 (h) scale bar is 1cm；
Fig. 2 (i) is the transgenosis rate that RT-PCR analyzes hairy；Fig. 2 (j) is the GFP signal in transgenosis root of hair, and Control is agriculture
Bacillus injection compares, and the scale bar in Fig. 2 (J) is 50 μm；
Fig. 3 is the optimal injection condition test in seedling stage and injection position, and wherein Fig. 3 (a) is the pigeonpea figure of sowing in 15 days
Piece；From the bottom of stem, three positions is selected to be injected, is named as each position width about 0.5cm of position C to A.；Fig. 3 (b)
For the callus and hairy regeneration rate for injecting different location；The phenotype of the different sowing seedling ages of Fig. 3 (c)；Fig. 3 (d) is used
Callus and hairy regeneration rate after different seedling age seeds；Wherein the scale bar in Fig. 3 (a) and Fig. 3 (c) is 0.5cm；
Fig. 4 is the hairy transgenic method used in four kinds of typical economic plants；Fig. 4 (a)~Fig. 4 (d) four choosings
Fixed economic plants, regeneration rate with higher, come from 9 economic plants, respectively illustrate Golden flower, gumbo, Radix Isatidis,
The hairy phenotype of pigeonpea；Right lateral position point lists the RT-PCR and western blot analysis (T1 of each transgenic hairy root system
Two transgenosis systems are indicated with T2), scale bar 1cm；Callus and hairy regeneration rate in Fig. 4 (e) Four Plants；
Fig. 5 is hairy herbaceous plant, shrub and trees transgenic method flow chart；I type is first trained in MS culture medium
Seedling is supported, injection is then taken out from culture bottle.II type is planted in soil culture medium before the injection after taking out in culture bottle
Plant squamous subculture seedling about 3~5d.Type III directly plants vegetable seeds in the soil before injection；Text box shows hairy
The possible application range of transgenic system.
Specific embodiment
The present invention provides a kind of construction methods of the effective root system transgenic system of economic plants, comprising the following steps:
1) bacterium solution of the agrobacterium rhizogenes of recombinant vector containing target is separated by solid-liquid separation, the obtained root of hair agriculture of recombinant vector containing target
Bacillus thallus is resuspended, and obtains agrobacterium rhizogenes suspension；
2) stem of economic plants seedling is injected into the agrobacterium rhizogenes suspension, 10~12d after injection is economical
Plant seedlings stem wound grows callus；
3) the economic plants seedling with callus is cultivated into 28~35d, when callus is divided into hairy of regeneration
When cut original feather shaped root system.
The bacterium solution of the agrobacterium rhizogenes of recombinant vector containing target is separated by solid-liquid separation by the present invention, the obtained hair of recombinant vector containing target
Root Agrobacterium thallus is resuspended, and obtains agrobacterium rhizogenes suspension.
The present invention is not particularly limited the bacterial strain of agrobacterium rhizogenes, using agrobacterium rhizogene strain known in the art
?.The bacterial strain of the agrobacterium rhizogenes preferably includes K599, MSU440, C58C1 or ArA4, more preferably K599.Root of hair agriculture
Bacillus strain K599, MSU440, C58C1 and ArA4 are purchased from Shanghai Wei Di Bioisystech Co., Ltd.
In the present invention, carrier preferably includes pROK2 in the target recombinant vector.The pROK2 carrier is purchased from
BioVector plasmid vector bacterium cell gene collection.Kind of the present invention to target gene in the target recombinant vector
Class do not do it is specifically limited, using any target gene.In order to illustrate being successfully established for effective root system transgenic system, this hair
In bright embodiment, the target gene includes GFP, is transferred to GFP gene and expresses target protein in system, by detection architecture
The luminous signal of fluorescin detects target gene successful conversion.The preparation method of the agrobacterium rhizogenes of recombinant vector containing target is preferred
It is carried out using electroporated method.
In the present invention, the bacterium solution of the agrobacterium rhizogenes of recombinant vector containing target is will to contain target recombinant vector root of hair agriculture
12~14h of shaken cultivation is obtained bacillus at 180rpm, 28 DEG C in liquid medium.The fluid nutrient medium is containing 20mg/L
The YEP fluid nutrient medium of rifampin and 50mg/L kanamycins.It cultivates to the OD of bacterium solution600Value terminates to cultivate when being 0.2~0.6.
It is separated by solid-liquid separation and is preferably centrifuged.The revolving speed of the centrifugation is preferably 8,000rpm.The time of the centrifugation is preferably 10min.It takes
Supernatant collects thallus, is resuspended with the buffer of supernatant same volume.The buffer is preferably MES buffer；It is described
MES buffer includes the component of following content: 10mmol/L MES-KOH, 10mmol/L MgCl2With 100 μm of ol/L acetyl fourths
The aqueous solution of ketone musk, pH value 5.2.The OD of agrobacterium rhizogenes suspension600Value preferably 0.2~0.6, more preferably 0.3~
0.5。
After obtaining agrobacterium rhizogenes suspension, the present invention is injected into economic plants seedling with the agrobacterium rhizogenes suspension
Stem, 10~12d after injection, economic plants seedling stem wound grows callus.
In the present invention, economic plants preferably includes herbaceous plant, shrub plant and magaphanerophytes.It is economical in the present invention
Plant includes medical nine kinds of selected economic plants, more preferably include pigeonpea, safflower, cassia seed, Radix Isatidis, Golden flower, gumbo,
Castor-oil plant, caragana and agalloch eaglewood.All nine kinds of economic plants can successfully induce generation callus.In addition to cassia seed, castor-oil plant and
Suspension culture of Aquilaria sinensis, other six only need all can successfully induce hairy by this method with minor modifications.
In the present invention, it in order to optimize influence of the rearing condition to callus induction and hairy root regeneration rate, provides
Two kinds of method for culturing seedlings: first method is as follows: by economic plants seed through tissue cultures 2~5 weeks, to obtained tissue culture seedling
Then direct injection is planted in soil media.The tissue cultures are preferably MS culture medium with culture medium.It is excellent before tissue cultures
It selects economic plants seed disinfection.The present invention is not particularly limited the method for the disinfection, is disappeared using known in the art
Malicious method.The temperature of tissue cultures is preferably 24~26 DEG C, and more preferably 25 DEG C.The method for culturing seedlings is to Golden flower and pigeonpea
Carrying out the test period will be shorter, can obtain hairy root induction rate more higher than other two methods.
Second method is as follows: injecting, then proceedes to when preferably growing vegetable seeds in soil media 3~8 weeks
It is grown in soil media.The soil media is preferably the mixture of soil and sand.The mixture of the soil and sand
The volume ratio of middle soil and sand is preferably 3:1.The partial size of the sand is preferably 0.35~0.5mm.The soil media is excellent
Diameter 10cm × high 9cm basin is selected to contain.The temperature of the growth is preferably 25 DEG C.The humidity of the growing environment is preferably
85%~90%.The intensity of illumination of the growth period is preferably 50 μm of ol photon m-2s-1.The light week of the growth period
Phase is 16h.The resulting hairy root induction rate of second method is lower than first method, but includes high viability.
In the present invention, the position of the injection is preferably placed at the stem of 0~1.5cm above the original feather shaped root system of seedling
Portion position, specifically point three regions, the height in each region are 0.5cm, such as (0 above the original feather shaped root system of seedling
~0.5) the stem position of cm be the area C, above the original feather shaped root system of seedling [0.5~1.0) cm stem position be the area B；
Above the original feather shaped root system of seedling [1.0~1.5) cm stem position is the area A, the more preferably area B and the area C.
After evoked callus, the economic plants seedling with callus is cultivated 28~35d by the present invention, when callus group
It knits and cuts original feather shaped root system when being divided into regeneration hairy.
In the present invention, over time, callus gradually increases dimmed.After approximately three weeks, similar to adventitious root
Small hairy grows from callus.Hairy increases after 1~2 month, it is sufficient to support the nutrition of entire plant, and help to plant
The development of object.
In the present invention, transgenosis rate is tested using the method for RT-PCR and fluorescence microscope, detects hair regeneration
Whether target gene expresses in hairy in shape root.After confirmation is transgenic hairy root, original feather shaped root system can be cut
Disconnected, transgenosis, which regenerates hairy and can play a role, drives root system (Fig. 1 f).The root system transgenic system that the present invention constructs is one
It is a to be very suitable to quickly and effectively genic system functional analysis, secondary metabolites engineering and plant stress reaction research.At this
In system, only root is transgenosis, rather than whole plant.This is also the good of signal transduction between a research root and stem
System.
Below with reference to embodiment to a kind of construction method of the effective root system transgenic system of economic plants provided by the invention
It is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Illustrate effective root system transgenic system construction method by taking economic plants green soy bean as an example
Cajanus cajan seeds key lab, the Forestry University forest plants ecology Ministry of Education northeast.The seed of pigeonpea is table
Face sterilizes 5~10min with 0.1% mercury chloride, then with sterile water washing five times.Cajanus cajan seeds after disinfection are seeded in MS training
It supports and is cultivated about 1~2 month in base.For pigeonpea, bacterium solution can be injected into squamous subculture plant after growth about 30 days.For other
Plant, Ying Yizhi soil (10 centimetres of (diameter) × 9 centimetre (height) basins, soil and sand, volume ratio 3:1), and in 25 DEG C of height
It is grown in humidity environment.Before carrying out injection experiment, in 50 μm of ol photon m in 16 hour photoperiod-2·s-1Place shines.
In order to screen one in pigeonpea with agrobacterium rhizogenes (A. rhizogenes) bacterial strain of higher transformation efficiency, hair has been selected
Root agrobacterium strains K599, MSU440, C58C1 and Ar4 measure the transformation efficiency of seedling.By 4 kinds of weights containing GFP gene
Agrobacterium solution (the OD of group plasmid (pROK2-GFP)600=0.3) it is injected into the pigeonpea seedling of 15 ages in days, grows hair therewith
Shape root.It is injected into the pigeonpea seedling of 15 ages in days using 4 kinds of Agrobacterium solutions containing pROK2 empty carrier as control experiment.System
Count the regeneration rate of four kinds of agrobacterium rhizogenes injection pigeonpea root transgenic system, wherein Callus induction rate=induce callus seedling number/
Total seedling number × 100%, root regeneration rate=rooted seedling number/total seedling number × 100%, the results are shown in Table 1.
The regeneration rate of 1 four kinds of agrobacterium rhizogenes injection pigeonpea root transgenic systems of table
As shown in table 1, root of hair bacterial strain K599 shows high efficiency in callus (80%) and root induction (30%).
Compared with K599, other three kinds of bacterial strain transformation efficiencies are all relatively low.For callus, the induced efficiency of bacterial strain C58C1 is only
It is 15%, followed by bacterial strain MSU440 (10%) and Ar A4 (8%).For hairy, these three bacterial strain inducing efficiency only have
5%~8%.The result shows that K599 induction should be more effective in hairy transgenic system, and pigeonpea may be more suitable for.
In terms of the induction of pigeonpea transgenic hairy root
The present invention establishes an effective pigeonpea seedling Agrobacterium injecting systems.As shown in Figure 1, the carrier of building is turned
Agrobacterium rhizogenes is dissolved into, is then injected into 15 -day-old of pigeonpea seedling (Fig. 1 (a)).After growth about 7~14 days, injecting
Occur callus (Fig. 1 (b) and 1 (c)) around position.Over time, callus gradually increases dimmed.Approximately three weeks
Afterwards, similar to the hairy small of adventitious root, (Fig. 1 (d)) is grown from callus.Hairy increases after one or two moon, it is sufficient to
It supports the nutrition of entire plant, and facilitates the development (Fig. 1 (e)) of plant.
Whether the present invention is imported by RT-PCR or successfully these hairs using immunoblotting test target gene
In shape root.Transgenosis root RNA, DNA are extracted and PCR verifying analysis method is specific as follows:
The RNA (Meng etc., 2018) of regeneration induction root is extracted using CTAB method.Pass through NanoDrop spectrophotometer
(NanoDrop Technologies, Inc) carries out quantifying for RNA.After being handled with the DNA enzymatic I of no RNase, use
Invitrogen TM SuperScript TM (Invitrogen, USA) is by 1 μ g total serum IgE reverse transcription at cDNA.For RT-
The reaction system of PCR is shown in Table 2, and amplification program is shown in Table 3.Primer sequence for RT-PCR is in table 4.
The reaction system of 2 RT-PCR of table
The amplification condition of 3 RT-PCR of table
The primer sequence of 4 RT-PCR of table
After confirmation is transgenic hairy root, original is cut off, and transgenic hairy root can play a role and drive root system
(Fig. 1 (f)).This is very quickly and effectively genic system functional analysis, secondary metabolites engineering and a plant stress reaction
Research.In this system, only root is transgenosis, rather than whole plant.Construction method can provide a research root and
The better systems of signal transduction between stem.
Embodiment 2
Agrobacterium solution injection concentration is tested to the shadow of callus induction rate, hairy root induction rate and transgenosis rate
It rings.With K599 bacterial strain be infect bacterial strain, in order to select the optium concentration of K599 bacterial strain suspension, be provided with concentration be 0.2,0.3,
0.4,0.5 and 0.6OD600Value, using pigeonpea as object, implements according to the method for embodiment 1 and calculates hairy root regeneration rate.
Using the transfer case of RT-PCR (operating method is with embodiment 1) and detection GFP signal experimental verification target gene.
The detection GFP signal experiment is specific as follows: taking pigeonpea to regenerate hairy radixin, by PAGE (polyacrylamide gel electrophoresis)
Isolated protein example is transferred on solid phase carrier (NC film), be added rabbit-anti GFP serum rise immune response, then with horseradish mistake
Oxide enzyme reacts, and detects the expression of GFP albumen, and blank control group is arranged.
OD1 to OD5 respectively indicates the OD value of 0.2,0.3,0.4,0.5 and 0.6.The result shows that OD2 (OD value 0.3) and
OD3 (OD value 0.4) is optimum value (OD2:58% ± 2% for obtaining high callus induction rate；OD3:60% ± 4%) (Fig. 2
(a)).OD2 still obtains the optimum value (33% ± 4%) of hairy best regeneration rate, followed by OD3 (26% ± 4%) (Fig. 2
(b)).In addition, the transgenosis root that the Agrobacterium solution injection of various concentration obtains does not have difference (Fig. 2 (c)).In short, best be cured
The concentration of injured tissue and hairy root induction is OD value 0.3.The transgenic hairy root and pigeonpea original phenotype for cutting front and back are such as
Shown in Fig. 2 (d)~Fig. 2 (h).RT-PCR (operating method is with embodiment 1) result and GFP signal all confirm this point target base
Because being transferred into hairy (Fig. 2 (I) and 2 (j)).
Embodiment 3
The optimal injection position of pigeonpea small plants is tested respectively.
Firstly, being located at the three parts that stem at the original 0.1~1.0cm of hairy top of seedling is divided into 0.5cm high；Respectively
It is named as position A, B and C (Fig. 3 (a)).It is to infect bacterial strain with K599 bacterial strain, implements according to the method for embodiment 1.
The result shows that: the callus and hairy inductivity of B and location of C are all for 60% or so, opposite location A
There is higher efficiency (Fig. 3 (b)).
Embodiment 4
The best age of pigeonpea small plants is tested respectively
The pigeonpea seedling of three all ages and classes is used to the optimal injection age of selection seedling, respectively through tissue cultures 15
It, 30 days and 45 days, inoculation position be the area B, other operation according to the embodiment of the method in embodiment 3.As shown in Fig. 3 (c), point
Not after 15 days, 30 days and 45 days, the seedling of tissue culture bottle culture be used to inject, and be then transferred into soil environment.As a result table
Bright, the infection rate of seedling (15 days and 30 days) is than old height of seedling (45 days) (see Fig. 3 (d)).
Embodiment 5
Applicability of the hairy transgenic method to other economic plants
In order to expand this method, in other economic plants, especially medical nine kinds of selected economic plants, including Cassia
Son, gumbo, Golden flower and pigeonpea are selected to test and optimize this root transgenic system, the specific structure of every kind of economic plants
Construction method is shown in Table 5.Hairy root regeneration rate is calculated using the method in embodiment 1.
Whether expressed in transgenic hairy root using western blot test experience verifying target gene (GFP).Specific side
Method is as follows:
The protein that each economic plants regenerates hairy is extracted, by PAGE (polyacrylamide gel electrophoresis) separation
Protein example is transferred on solid phase carrier (NC film), using economic plants albumen as antigen, rabbit-anti GFP serum is added and rises and exempts from
Epidemic disease reaction, then react with horseradish peroxidase secondary antibody, detect the expression of GFP albumen.
59 kinds of economic plants building transgenosis root systems system lists of table
Note: None expression does not induce hairy.
As shown in table 5, all nine kinds of plants can succeed evoked callus.In addition to cassia seed, castor-oil plant and suspension culture of Aquilaria sinensis,
Its six need all can successfully induce hairy by this method with minor modifications.At the same time, root regeneration rate is become mildewed 15%
It is 16%~85% relative to callus regeneration rate between~45%.The phenotype of Four Plants in nine kinds of plants shows it
Have higher root regeneration rate, as shown in Figure 4.RT-polymerase chain reaction and Western blotting result confirm target gene
(GFP) it is expressed in transgenic hairy root.It is all the result shows that hairy method is widely used, including draft and woody plant
Object.
Embodiment 6
In order to obtain the accurate method (herbaceous plant, shrub or trees) to variety classes plant, rearing condition is tested
Influence.It is as follows to assess three kinds of condition of culture: I type, then tissue culture seedling direct injection is planted in soil media within 2~5 weeks.II
Type, after tissue culture culture in 2~5 weeks by seedling cultivation in soil culture medium.Restore to increase after about 3~5 days, then infuse
It penetrates.Seed is directly planted in soil culture medium by type III, is then injected after 3~8 weeks.
As shown in figure 5, summarized hairy root induction flow chart and possible application.Two kinds of typical plants, golden flower
Certain herbaceous plants with big flowers, pigeonpea verify the above method.
6 Golden flower of table, pigeonpea verify three kinds of method for culturing seedlings situations
Will be shorter the result shows that carrying out the test period to Golden flower and pigeonpea with I type method, can obtain than other two
The kind higher hairy root induction rate of method.However, the survival rate of I type is very low.When using type III method, hairy root induction rate
Lower than I type, but there is high viability.If necessary to one kind in pigeonpea and Golden flower simpler, faster method, III
Type method is best.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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