CN105087642B - The transgenic method of Nandina domestica'Fire power' - Google Patents
The transgenic method of Nandina domestica'Fire power' Download PDFInfo
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- CN105087642B CN105087642B CN201510629324.6A CN201510629324A CN105087642B CN 105087642 B CN105087642 B CN 105087642B CN 201510629324 A CN201510629324 A CN 201510629324A CN 105087642 B CN105087642 B CN 105087642B
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Abstract
The present invention discloses a kind of transgenic method of Nandina domestica'Fire power', comprising: exogenous plasmid carrier is transferred in Agrobacterium LBA4404, obtains recombinational agrobacterium；Recombinational agrobacterium described in activation culture is 0.4~0.6 to OD600, then infects 50~70min of blade explant of Nandina domestica'Fire power'；Blade explant after infecting successively is lured bud culture, culture of rootage to obtain Nandina domestica'Fire power' plant, and screening obtains transgenosis Nandina domestica'Fire power' from Nandina domestica'Fire power' plant.The present invention establishes an efficient, quick, stable Nandina domestica'Fire power' genetic conversion system, conversion ratio can reach 10% or more, practical basis is provided further to apply genetic transforming method to improve Nandina domestica'Fire power' kind, is conducive to the development of Nandina domestica'Fire power' molecular breeding work from now on.
Description
Technical field
The present invention relates to gene engineering technology field more particularly to a kind of transgenic methods of Nandina domestica'Fire power'.
Background technique
Nandina domestica'Fire power' (Nandina domestica Fire power) is a kind of emerging Landscape Trees, now
It is largely used in garden landscape.Nandina domestica'Fire power' resists cold evergreen, and blade is oval or oval, has obvious area with original seed lanceolate leaf
Not, this important character for being an advantage over original seed.Nandina domestica'Fire power' encounters cold air to annual autumn, and leaf color is just switched to by green scarlet
Color, leaf color can continue to the second year early spring, therefore have splendid appreciation effect in southern area.In afforestation
In, Nandina domestica'Fire power' can be cultivated simultaneously with camellia in tree altar, and also suitable planting is in phytobiocoenose lower layer, enhancing landscape level.
Meanwhile it is a kind of potted plant with good development prospect again, and has higher medical value.Nandina domestica'Fire power' color with
Temperature reduction gradually reddens, and the kind to take on a red color throughout the year has not found, and the kind that the culture four seasons take on a red color has
Better market prospects.With the rapid development of molecular biology and genetic engineering in recent years, carried out by transgenic technology
Molecular improvement breeding is unusual conveniently breeding technique.
Also do not report mainly there is agriculture bar in the transgenic method of other species about the transgenosis of Nandina domestica'Fire power' at present
Bacterium mediated method, particle bombardment and electric shocking method, wherein the leaf disk method of mediated by agriculture bacillus is ground in plant transgene research so far
Study carefully most common a kind of transgenic method the most mature.Although the transformation system for having comparative maturity in other plants,
It is that conventional method is bothersome laborious, time-consuming is long, and cumbersome and efficiency is very low, and the transgenic plant of acquisition is very little, to turning base
Because the later period identification work of plant brings obstacle.And since species difference transgene method is also different, other plants are used for reference
The transgenic method success rate of object is very low, therefore how to obtain an efficiently quick Nandina domestica'Fire power' stable conversion system, is
A problem urgently to be resolved in Nandina domestica'Fire power' transgenosis.
Summary of the invention
A kind of flame Nan Tian of efficient fast and stable is provided the purpose of the invention is to overcome the deficiencies of existing technologies
The transgenic method of bamboo, conversion ratio is up to 10% or more.
In order to achieve the above objectives, the present invention is achieved by the following technical solutions:
The transgenic method of Nandina domestica'Fire power' of the present invention, comprising:
(1) exogenous plasmid carrier is transferred in Agrobacterium LBA4404, obtains recombinational agrobacterium；
(2) recombinational agrobacterium described in activation culture is 0.4~0.6 to OD600, then infects the blade of Nandina domestica'Fire power'
50~70min of explant；
(3) the blade explant after infecting successively is lured bud culture, culture of rootage to obtain Nandina domestica'Fire power' plant, from flame
Screening obtains transgenosis Nandina domestica'Fire power' in nandina plant.
The present invention selects Agrobacterium LBA4404 as bacterial strain is infected, using conjunction using the blade of Nandina domestica'Fire power' as explant
Suitable bacterial concentration and time of infection can successfully obtain transgenosis Nandina domestica'Fire power', and conversion ratio is up to 10% or more.Into one
Step is preferred, and in step (2), recombinational agrobacterium described in activation culture to OD600 is 0.4, then infects Nandina domestica'Fire power'
Blade explant 60min；
Preferably, the exogenous plasmid carrier is the pCAMBIA2301 of pCAMBIA2301 or foreign gene-carrying.
Preferably, before infecting, preculture is carried out to the blade explant.The temperature of the preculture is 22~26
DEG C, the time is 7~9 hours, and illumination is 2000~3000LX.Further, the temperature of the preculture is 25 DEG C, the time 8
Hour.Generally, the culture medium of preculture is MS.
Preferably, the blade explant is 0.2~0.6cm2Leaf block.
Preferably, the method infected are as follows: it is immersed in the blade explant in recombinational agrobacterium bacterium solution, every
3~7min shakes bacterium solution.
Preferably, it after infecting, carries out luring bud culture after blotting the bacterium solution on the blade explant surface.
Preferably, the culture medium for luring bud culture is addition antibiotic, sucrose, agar, 2~3mg/L of TDZ and 2,4-D
The MS culture medium of 0.1~0.3mg/L.It is further preferred that luring in the culture medium of bud culture, the concentration of sucrose is 30g/L, agar
Concentration be 5.5g/L, the concentration of TDZ is 2.5mg/L, and 2,4-D concentration is 0.2mg/L.
Preferably, the culture medium of the culture of rootage is addition antibiotic, sucrose, agar and NAA0.3~0.5mg/L
1/2MS culture medium.It is further preferred that the concentration of sucrose is 30g/L, and the concentration of agar is in the culture medium of culture of rootage
The concentration of 5.5g/L, NAA are 0.4mg/L.
Those skilled in the art could be aware that, in the culture medium for luring bud culture and culture of rootage, the type and concentration of antibiotic
It is determined according to resistant gene entrained by exogenous plasmid carrier.
Preferably, in step (3), blade explant obtains unrooted plant after being lured bud culture, to the unrooted plant
It is identified, the transgenosis unrooted plant filtered out, then culture of rootage is carried out to transgenosis unrooted plant.
Compared with prior art, the invention has the benefit that
The present invention, which uses, lower infects Agrobacterium bacterial concentration (OD600 value for 0.4~0.6), longer time of infection
(50~70min) can reduce Agrobacterium and other living contaminants, while improve infect efficiency again.
The present invention, which infects, does not add the drug medication that any auxiliary infects in Agrobacterium bacterium solution, reduce to Nandina domestica'Fire power'
The injury of blade explant, improves the survival rate and regeneration rate of the culture of blade explant, and acetyl fourth is often added in conventional method
The medicament that the auxiliary such as ketone musk infect can damage blade explant, reduce the power of regeneration of blade explant.
The present invention can directly screen to obtain using the carrier pCAMBIA2301 for carrying strongly expressed reporter gene by observation
Transgenic plant avoids work cumbersome in traditional detection, shortens the period of transgenosis.
The present invention establishes an efficient, quick, stable Nandina domestica'Fire power' genetic conversion system, and conversion ratio can reach
10% or more, practical basis is provided further to apply genetic transforming method to improve Nandina domestica'Fire power' kind, is conducive to fiery from now on
The development of flame nandina molecular breeding work.
Specific embodiment
Combined with specific embodiments below, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention, after the present invention has been read, those skilled in the art are to various equivalences of the invention
The modification of form falls within the application range as defined in the appended claims.
The material that the specific embodiment of the invention uses is as follows:
1, vegetable material
Nandina domestica'Fire power' tissue-cultured seedling.
2, culture medium
The formula of culture medium is as follows:
Nandina domestica'Fire power' subculture medium (code name S1): MS+ZT 0.6mg/L+IBA 0.2mg/L+ glucose 15g/L+ fine jade
Cosmetics 6g/L, pH5.7；
Nandina domestica'Fire power' transgenic seedling culture medium (code name S2): MS+ sucrose 30g/L+ agar 5.5g/L+TDZ2.5mg/L+
2,4-D 0.2mg/L+ kanamycins 20mg/L+ carbenicillin 400mg/L, pH5.7；
Nandina domestica'Fire power' transgenic seedling root media (code name S3): 1/2MS culture medium+sucrose 30g/L+ agar 5.5g/L
+ NAA0.4mg/L+ kanamycins 20mg/L+ carbenicillin 400mg/L, pH5.7；
YEB solid medium: beef extract 5g/L, yeast extract 1g/L, peptone 5g/L, sucrose 5g/L, MgSO4·7H2O
0.4g/100ml and agar 1.5g/100ml, PH7.4；
YEB fluid nutrient medium: beef extract 5g/L, yeast extract 1g/L, peptone 5g/L, sucrose 5g/L and MgSO4
7H2O 0.4g/100ml, PH7.4；
The equal high pressure sterilization of all culture mediums, cooling are spare.
3, bacterial strain and plant expression vector
It is agrobacterium strains LBA4404 that bacterial strain is infected used in the present invention, which is suitble to Nandina domestica'Fire power' transgenosis
It uses, the bacterial strain infection ability is stronger.The bacterial strain is bought in precious bioengineering Dalian Co., Ltd.
Plant expression vector selects pCAMBIA2301 plant expression vector, plant expression vector reporter gene gus
(beta-glucosiduronatase gene) shortens detection time convenient for the detection of genetically modified plants.The plant expression vector is bought in English
Pretty (Invitrogen) biotech company.
Embodiment 1
The transgenic method of Nandina domestica'Fire power' includes the following steps:
1, actication of culture
1. plasmid pCAMBIA2301 is transformed into Agrobacterium LBA4404, the recombination of the pCAMBIA2301 containing plasmid is obtained
Agrobacterium LBA4404.
2. by the recombinational agrobacterium LBA4404 of the pCAMBIA2301 containing plasmid addition Km 50mg/L (kanamycins) and
It crosses on the YEB solid medium of Rif 50mg/L (rifampin), 28 DEG C are cultivated 2 days.
2. the single colonie of picking YEB cultured on solid medium 2~3 is inoculated into 50mL addition Km 50mg/L and Rif
In the YEB fluid nutrient medium of 50mg/L, 28 DEG C, 220rpm OD600 value of shaken cultivation about 12-16h to bacterium solution in shaking table is
0.8。
3. under room temperature, bacterium solution is centrifuged 8 minutes in 5000rpm, removes supernatant, centrifuge tube is buckled on aseptic filter paper, to the greatest extent
Amount removes supernatant, and thallus is resuspended with 100mLMS liquid, and (revolving speed is 8000 turns/min to shaken cultivation, and temperature is in shaking table
15 DEG C) about 1 hour measurement OD600 value be 0.4 to be, best (OD600 value value is critically important, cannot be below 0.4, not above
0.6, it controls between 0.4~0.6,0.4 is best, should be as far as possible close to 0.4).Activated bacterium solution can be used to do to plant in next step
Object material infects.
2, Nandina domestica'Fire power' transgenosis
1. vegetable material prepares
The blade of Nandina domestica'Fire power' tissue-cultured seedling is cut into 0.4cm2Fritter, put them on 100mlMS fluid nutrient medium
Middle carry out preculture, the temperature of preculture are 25 DEG C, and the time is 8 hours, illumination 2000-3000LX.
2. the transgeneic procedure method of Nandina domestica'Fire power'
It infects: after the completion of preculture, after the MS liquid of preculture is poured out, 100ml suspension bacteria liquid being poured into the leaf sheared
It in piece, slightly shakes up, disseminates 60 minutes, shook primary, the contact surface of increase bacterium solution and blade in infection processs every 5 minutes
Product.Bacterium solution is poured out after the completion of infecting, blade is blotted to the bacterium solution on surface on aseptic filter paper.
Screen induced bud culture: the blade inoculation after infecting makes blade on Nandina domestica'Fire power' transgenic seedling culture medium S2
Wound come into full contact with culture medium, 25 ± 2 DEG C in culturing room, illumination cultivation (intensity of illumination 2000-3000LX, photoperiod 8
Hour dark culture, illumination in 16 hours).Culture 40 days or so, Nandina domestica'Fire power' blade edge has white callus and grows, and resists
Property callus can differentiate resistant plant after the culture of about 40d or so.Carry out a GUS tissue staining detection again at this time,
Colouring method are as follows: resistant plant is taken out from tissue culture bottle, appoints and cuts one piece of plant leaf blade, it is ensured that blade surrounding has wound,
It is placed into GUS dyeing liquor, 37 DEG C, dyes overnight.The chlorophyll in blade is sloughed with 70% alcohol after dyeing, after decoloration
Blue is transgenic plant, and not having coloured is nontransgenic plants, and nontransgenic plants are removed.
The subculture of the callus of Nandina domestica'Fire power' uses subculture medium S1.
Screening induction root culture: the transgenic plant for taking previous step to select is put into Nandina domestica'Fire power' transgenic seedling culture of rootage
On base S3,25 ± 2 DEG C in culturing room, (intensity of illumination 2000-3000LX, 8 hours photoperiods dark culture, 16 is small for illumination cultivation
Shi Guangzhao), it takes root after culture 40d.Carry out the detection of GUS tissue staining, colouring method again at this time are as follows: by resistant plant from
It is taken out in tissue culture bottle, appoints and cut one piece of plant leaf blade and one section of root system, it is ensured that blade surrounding has wound, they are placed into
In GUS dyeing liquor, 37 DEG C, dye overnight.The chlorophyll in blade is sloughed with 70% alcohol after dyeing, after root system and decoloration
Blade is blue for transgenic plant, in blade, root system it is any either with or without blue for nontransgenic plants, by non-turn
Gene plant removal.
The conversion ratio of statistics Nandina domestica'Fire power' transgenosis, conversion ratio=(transgenic plant sum/inoculation explant is total
Number) × 100%.The conversion ratio of the present embodiment Nandina domestica'Fire power' is 15%.It screening in induced bud incubation, pollution rate is extremely low,
Only 1.2%.
Embodiment 2
Changing time of infection, time of infection is respectively set to 10 minutes, and 20 minutes, 30 minutes, 40 minutes, 50 minutes, 70
Minute, 80 minutes, remaining step investigated influence of the time of infection to Nandina domestica'Fire power' conversion ratio with embodiment 1.
The results are shown in Table 1.
Influence of 1 time of infection of table to Nandina domestica'Fire power' conversion ratio
| Processing | Time of infection (min) | Conversion ratio |
| 1 | 10 | 4% |
| 2 | 20 | 6% |
| 3 | 30 | 8% |
| 4 | 40 | 10.5% |
| 5 | 50 | 13% |
| 6 | 70 | 14% |
| 7 | 80 | 9% |
As shown in table 1, when the time is shorter, the conversion ratio of Nandina domestica'Fire power' is lower, with the growth of time of infection, conversion ratio
It is increased.When time of infection reaches 80 minutes, conversion ratio declines instead, and the pollution of Agrobacterium and other miscellaneous bacterias also compared with
Seriously, pollution rate is up to 7.8%.
Claims (7)
1. a kind of transgenic method of Nandina domestica'Fire power' characterized by comprising
(1) exogenous plasmid carrier is transferred in Agrobacterium LBA4404, obtains recombinational agrobacterium；
(2) recombinational agrobacterium described in activation culture is 0.4 to OD600, then infects the blade explant of Nandina domestica'Fire power'
60min；The method infected are as follows: be immersed in the blade explant in recombinational agrobacterium bacterium solution, shaken every 3~7min
Dynamic bacterium solution；
(3) the blade explant after infecting successively is lured bud culture, culture of rootage to obtain Nandina domestica'Fire power' plant, from flame Nan Tian
Screening obtains transgenosis Nandina domestica'Fire power' in bamboo plant；The exogenous plasmid carrier is pCAMBIA2301 or carries external source base
The pCAMBIA2301 of cause.
2. the transgenic method of Nandina domestica'Fire power' as described in claim 1, which is characterized in that before infecting, to the blade
Explant carries out preculture.
3. the transgenic method of Nandina domestica'Fire power' as described in claim 1, which is characterized in that the blade explant is
0.2~0.6cm2Leaf block.
4. the transgenic method of Nandina domestica'Fire power' as described in claim 1, which is characterized in that after infecting, blot the leaf
It carries out luring bud culture after the bacterium solution on piece explant surface.
5. the transgenic method of Nandina domestica'Fire power' as described in claim 1, which is characterized in that the culture medium for luring bud culture
For the MS culture medium for adding antibiotic, sucrose, agar, 0.1~0.3mg/L of TDZ 2~3mg/L and 2,4-D.
6. the transgenic method of Nandina domestica'Fire power' as described in claim 1, which is characterized in that the culture medium of the culture of rootage
For addition antibiotic, the 1/2MS culture medium of 0.3~0.5mg/L of sucrose, agar and NAA.
7. the transgenic method of Nandina domestica'Fire power' as described in claim 1, which is characterized in that in step (3), blade explant
After being lured bud culture obtain unrooted plant, the unrooted plant is identified, by the transgenosis unrooted plant filtered out into
Row culture of rootage.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1882686A (en) * | 2003-09-17 | 2006-12-20 | 国立大学法人京都大学 | Process for producing intermediate of useful alkaloid biosynthesis according to RNAi method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1882686A (en) * | 2003-09-17 | 2006-12-20 | 国立大学法人京都大学 | Process for producing intermediate of useful alkaloid biosynthesis according to RNAi method |
Non-Patent Citations (3)
| Title |
|---|
| Chemical composition and inhibitory parameters of essential oil and extracts of Nandina domestica Thunb. to control food-borne pathogenic and spoilage bacteria;Vivek K.Bajpai等;《International Journal of Food Microbiology》;20081231;第125卷;第117-122页 |
| Evaluating North and South Florida Landscape Performance and Fruiting of Ten Cultivars and a Wild-type Selection of Nandina domestica, a Potentially Invasive Shrub;Gary W. Knox等;《J.Environ.Hort》;20060930;第24卷(第3期);第137-142页 |
| 火焰南天竹的开发应用;喻杰等;《中国园艺文摘》;20091231(第8期);第153页 |
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