CN103740751B - A kind of agriculture bacillus mediated sugarcane genetically modified method - Google Patents
A kind of agriculture bacillus mediated sugarcane genetically modified method Download PDFInfo
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Abstract
The invention discloses a kind of agriculture bacillus mediated sugarcane genetically modified method, it comprises Agrobacterium-mediated Transformation, super clean bench dries up, contaminate, root induction, hardening, transplanting and other steps, with the Caulis Sacchari sinensis leaf disk after disinfecting for acceptor, by agriculture bacillus mediated, obtain a kind of transgenic sugarcane fast.Method of the present invention does not limit by material, saves time, in three months, just can obtain transgenic sugarcane plant, is applicable to the development of large-scale transgenic sugarcane; Meanwhile, method incubation time is short, avoids in chronic tissue culturing process, and exogenous hormone causes the impact made a variation.
Description
Technical field
The present invention relates to plant transgene culture technique field, be specifically related to a kind of agriculture bacillus mediated sugarcane genetically modified method.
Background technology
Transgenic technology refer to by be manually separated and the channel genes of modified in organism genome, due to the expression of quiding gene, causing the change of organism proterties, is heritable modification.Plant transgenic technology is the importance of phytology research field, is the basic technology means of research specific purpose gene function and exploitation transgenic plant.All the time, plant transgenic technology is the focus that investigator pays close attention to, and along with going deep into gradually of research, transgenic technology is also gradually improved.
Sugarcane is a kind of important sugar crop, and sucrose accounts for more than 90% of China's sugar total amount.Meanwhile, sugarcane is also a kind of biomass energy crop, and sugarcane is as the specular removal C that in current raise crop, photosynthetic efficiency is the highest, output is the highest
4crop is the best energy crop producing ethanol.Sugarcane genetic background is complicated, and the conventional cross-breeding cycle is long, inefficiency.Transgenic technology can carry out orderly improvement to existing kind fast, cane breeding has extensively application prospect.Sugarcane genetically modified technology is compared with other plant, very backward, until last century, the nineties just obtained transfer-gen plant by callus approach.At present, sugarcane genetically modified commonly used acceptor material mostly is callus or embryoid.Both all needs the regular hour to carry out the induction of callus, compares and wastes time and energy, be subject to the restriction of Regeneration System difficulty simultaneously.
Summary of the invention
For overcoming the defect of prior art, of the present invention being provides a kind of agriculture bacillus mediated sugarcane genetically modified method, utilizes the Caulis Sacchari sinensis leaf disk after process to be acceptor, by agriculture bacillus mediated quick acquisition transgenic sugarcane.
The technical solution adopted in the present invention is as follows for achieving the above object:
An agriculture bacillus mediated sugarcane genetically modified method, its step is as follows:
1) Agrobacterium-mediated Transformation: the Agrobacterium containing foreign gene is cultured to logarithmic phase, collects thalline, is resuspended in the MS liquid nutrient medium containing Syringylethanone, obtains transformed bacteria liquid;
2) drought stress: the blade getting sugarcane tip, is cut into the Caulis Sacchari sinensis leaf disk that thickness is 0.3 ~ 0.5cm after alcohol surface sterilization, super clean bench is placed 15 ~ 50 minutes, carries out drought stress;
3) contaminate: by step 2) the Caulis Sacchari sinensis leaf disk that obtains was immersed in transformed bacteria liquid prepared by step 1), is placed on Dual culture substratum again after being blotted by liquid by Caulis Sacchari sinensis leaf disk, in 25 ~ 30 DEG C of light culture 3 ~ 4 days;
4) root induction: above-mentioned sugarcane cells,primordial after contaminating is placed in Selective agar medium and cultivates, regenerate; When height of seedling is 2 ~ 3cm, cut plant and receive root media and carry out root induction, obtain the plant taken root;
5) hardening, transplanting: by take root plant hardening, transplanting, obtain transgenic sugarcane plant.
In aforesaid method, as the preferred scheme of one of the present invention, in the MS liquid nutrient medium of step 1), the content of Syringylethanone is 100 ~ 250 μMs.
In the present invention, the object that Caulis Sacchari sinensis leaf disk processes on super clean bench allows leaves water loss, strengthens the injury to blade, to reach the object improving transformation efficiency.
In aforesaid method, as the preferred scheme of one of the present invention, the Dual culture substratum of step 3) is the mixed culture medium of MS substratum and 2 ~ 5mg/L2,4-dichlorphenoxyacetic acid, 1 ~ 3mg/L6-benzyladenine and 5.5g/L agar powder, its pH5.5.Preferably, the time of soaking in step 3 is 10 ~ 15min.
In aforesaid method, as the preferred scheme of one of the present invention, Selective agar medium described in step 4) is MS substratum and 2 ~ 5mg/L2, the mixed culture medium of 4-dichlorphenoxyacetic acid, 1 ~ 3mg/L6-benzyladenine, 250mg/L cephamycin, 5.5g/L agar powder and resistance marker screening material, its pH5.8; Described root media is the mixed culture medium that MS substratum and 2-5mg/L naphthylacetic acid, 250mg/L cephamycin, 5.5g/L agar powder and resistance marker screen material, its pH5.8.The object adding resistance marker screening material in the substratum of step 4) in the present invention allows non-transformed necrocytosis, described in cell normal growth containing foreign gene, resistance marker screening material is microbiotic or weedicide, and the add-on of resistance marker screening material is: 3-5mg/L.
In aforesaid method, as the preferred scheme of one of the present invention, in step 1), Agrobacterium adopts YEB liquid nutrient medium to cultivate, concrete steps are: by the Agrobacterium inoculation containing foreign gene in containing in antibiotic YEB liquid nutrient medium, 25 ~ 30 DEG C of incubator overnight, again by volume per-cent 1% inoculum size transfer to cultivate in the same substratum of 50mL 2 ~ 4 little be 0.5 ~ 0.6 up to OD600, collect thalline; Then precipitate containing the MS liquid nutrient medium Eddy diffusion of 100 μMs of AS with 5mL, bacterium liquid is proceeded to 50mL containing in the MS liquid nutrient medium of 100 μMs of AS, 28 DEG C of shaking tables are cultured to OD600 to 0.5 ~ 0.6.
In aforesaid method, as the preferred scheme of one of the present invention, described foreign gene is careless fourth phosphine resistant gene.
Preferably, in step 4), the time of root induction is 4 weeks.
In aforesaid method, as the preferred scheme of one of the present invention, transplanting matrix used in step 5) is garden mould and peat soil 1:1 mixing in mass ratio.
First than prior art, beneficial effect of the present invention is:
1. the present invention is with Sugarcane Leaves directly for transformation receptor is drawn materials easily, can be reduced the time obtaining transfer-gen plant by the direct differentiation-inducing seedling of blade in a large number; Infect early stage Agrobacterium, carry out Osmotic treatment to explant, strengthen the injury of explant, effectively promote the infection ability of Agrobacterium, transformation efficiency improves;
2. method of the present invention does not limit by material, saves time, in three months, just can obtain transgenic sugarcane plant;
3. the present invention compares with the transgenic method of other acceptors such as callus, draws materials easily, also substantially reduces the time obtaining transgenic sugarcane plant, just can obtain sugarcane plant at 2 ~ 3 months, is more suitable for the development of large-scale transgenic sugarcane; Meanwhile, method incubation time is short, avoids in chronic tissue culturing process, and exogenous hormone causes the impact made a variation;
4. present method adopts Caulis Sacchari sinensis leaf disk directly to do a transgene receptor, provides novel method for obtaining transgenic sugarcane;
5. transformation efficiency of the present invention is high, and the mosaic obtained is few; And there is expansibility, go for the development of extensive transgenic sugarcane, greatly reduce workload, reduce research expenditure;
Below in conjunction with concrete embodiment, the present invention is described in further detail.
Accompanying drawing explanation
Fig. 1 is the photo after Caulis Sacchari sinensis leaf disk of the present invention is contaminated;
Fig. 2 is the photo of the plant obtained after root induction of the present invention;
Fig. 3 is the Sugarcane Leaves after method of the present invention is cultivated and the PPT Detached-leaf test of the Sugarcane Leaves normally cultivated; Wherein two test tubes on both sides are normal blades cultivated, and three middle test tubes are the blades after method of the present invention is cultivated;
Fig. 4 is the electrophorogram that the sugarcane plant leaf PCR obtained by method of the present invention is detected, and wherein 1 is mark, and 5,8,9 is sugarcane plant leaf pcr amplification product of the present invention.
Embodiment
Embodiment 1
An agriculture bacillus mediated sugarcane genetically modified method, its step is as follows:
1) Agrobacterium LBA4404 of preparation containing Bar gene: LBA4404 competent cell is bought in treasured
Biotechnology (Dalian) company limited, Bar gene source, in commercial vector pCAMBIA3301, by the method for pCAMBIA3301 plasmid according to " molecular cloning ", imports in LBA4404 cell;
2) cultivate: picking contains the Agrobacterium LBA4404 list bacterium colony of Bar gene, being inoculated in 5ml contains in the YEB liquid nutrient medium of 50mg/L Rifampin, 28 DEG C of incubator overnight, then 0.5ml bacterium liquid is inoculated in 50mL again and contains in the YEB liquid nutrient medium of 50mg/L Rifampin that to be cultured to OD600 be about 0.5, by bacterium liquid in 4 DEG C, the centrifugal 5min of 5000rpm, collect thalline, precipitate containing the MS liquid nutrient medium Eddy diffusion of 100 μMs of AS with 5mL, bacterium liquid is proceeded to 50mL containing 100 μMs of AS MS liquid nutrient medium in, 28 DEG C of shaking tables being cultured to OD600 is 0.5, obtain Agrobacterium bacterium liquid,
3) drought stress: the blade getting sugarcane (new No. 22, platform sugar, the seed selection of Tai Tang company) tip, after the surface sterilization of volume percent 70% alcohol, being cut into 30 thickness with knife blade is the disk of 0.5cm, and super clean bench is placed 30 minutes;
4) During Agrobacterium: slice Caulis Sacchari sinensis leaf disk of 30 after step 3) process is immersed step 2) acceptor is taken out after 12 in the Agrobacterium bacterium liquid prepared, middlely light and slowly rock for several times; Then acceptor is placed on aseptic filter paper, absorb remaining bacterium liquid and airing, transfer to by MS substratum, 2mg/L2 immediately, 4-dichlorphenoxyacetic acid (2,4-D), 2mg/L6-benzyladenine (6-BA), 250mg/L cephamycin and 5.5g/L agar powder composition, pH was on the Dual culture substratum of 5.5, in 25 DEG C of light culture 3 days;
5) regeneration of Transgenic Resistant Herbicide sugarcane and transplanting: step 4) and the converting material after contaminating are moved on to and selects by MS substratum, 3mg/L2,4-dichlorphenoxyacetic acid (2,4-D) 6-BA1mg/L, 3mg/L grass fourth phosphine, 250mg/L cephamycin, 5.0g/L agar powder composition, pH is 5.8 selects on substratum and regenerate; 2-3 week replaced medium; When height of seedling is 3cm, cut plant and receive and be made up of MS substratum, 3mg/LNAA, 5mg/L grass fourth phosphine, 250mg/L cephamycin, 5.0g/L agar powder, pH is in the root media of 5.8, carries out root induction; After 4 weeks, visible plant height about 6cm, there is the root being about 2cm in bottom;
6) hardening, transplanting: open bottle cap, hardening 1 week, after cleaning the substratum of plant root, plantlet of transplant is that garden mould and peat soil are in mass ratio in 1:1 mixing seedling pan to matrix.
Repeat above-mentioned experiment two groups, often group is 30 Caulis Sacchari sinensis leaf disks, obtains 10 sugarcane plant altogether.
Embodiment 2
An agriculture bacillus mediated sugarcane genetically modified method, its step is as follows:
1) Agrobacterium LBA4404 of preparation containing Bar gene: LBA4404 competent cell is bought in precious biotechnology (Dalian) company limited, Bar gene source is in commercial vector pCAMBIA3301, by the method for pCAMBIA3301 plasmid according to " molecular cloning ", import in LBA4404 cell;
2) cultivate: picking contains the Agrobacterium LBA4404 list bacterium colony of Bar gene, being inoculated in 5ml contains in the YEB liquid nutrient medium of 50mg/L Rifampin, 25 DEG C of incubator overnight, then 0.5ml bacterium liquid is inoculated in 50mL again and contains in the YEB liquid nutrient medium of 50mg/L Rifampin that to be cultured to OD600 be 0.6, by bacterium liquid in 4 DEG C, the centrifugal 5min of 5000rpm, collect thalline, precipitate containing the MS liquid nutrient medium Eddy diffusion of 100 μMs of AS with 5mL, bacterium liquid is proceeded to 50mL containing 100 μMs of AS MS liquid nutrient medium in, 28 DEG C of shaking tables being cultured to OD600 is 0.6, obtain Agrobacterium bacterium liquid,
3) drought stress: the blade getting sugarcane (new No. 20, platform sugar, the seed selection of Tai Tang company) tip, after the surface sterilization of volume percent 70% alcohol, being cut into 30 thickness with knife blade is the disk of 0.5cm, and super clean bench is placed 15 minutes;
4) During Agrobacterium: slice Caulis Sacchari sinensis leaf disk of 30 after step 3) process is immersed step 2) acceptor is taken out after 10 in the Agrobacterium bacterium liquid prepared, middlely light and slowly rock for several times; Then acceptor is placed on aseptic filter paper, absorb remaining bacterium liquid and airing, transfer to by MS substratum, 2mg/L2 immediately, 4-dichlorphenoxyacetic acid (2,4-D), 2mg/L6-benzyladenine (6-BA) and 5.5g/L agar powder composition, pH was on the Dual culture substratum of 5.5, in 25 DEG C of light culture 2 days;
5) regeneration of Transgenic Resistant Herbicide sugarcane with transplant: by step 4) with contaminate after converting material move on to select and be made up of MS substratum, the careless fourth phosphine of 6-BA3mg/L, 5mg/L, 250mg/L cephamycin, 5.0g/L agar powder, pH is 5.8 selects on substratum and regenerate; When height of seedling is 2cm, cut plant and receive and be made up of MS substratum, 3mg/LNAA, 5mg/L grass fourth phosphine, 250mg/L cephamycin, 5.0g/L agar powder, pH is in the root media of 5.8, carries out root induction; After 4 weeks, visible plant height about 6cm, there is the root being about 2cm in bottom;
6) hardening, transplanting: open bottle cap, hardening 6 days, after cleaning the substratum of plant root, plantlet of transplant is that garden mould and peat soil are in mass ratio in 1:1 mixing seedling pan to matrix.
Adopt 30 Caulis Sacchari sinensis leaf disks, obtain 2 sugarcane plant altogether.
Embodiment 3
An agriculture bacillus mediated sugarcane genetically modified method, its step is as follows:
1) Agrobacterium LBA4404 of preparation containing Bar gene: LBA4404 competent cell is bought in precious biotechnology (Dalian) company limited, Bar gene source is in commercial vector pCAMBIA3301, by the method for pCAMBIA3301 plasmid according to " molecular cloning ", import in LBA4404 cell;
2) cultivate: picking contains the Agrobacterium LBA4404 list bacterium colony of Bar gene, being inoculated in 5ml contains in the YEB liquid nutrient medium of 50mg/L Rifampin, 28 DEG C of incubator overnight, then 0.5ml bacterium liquid is inoculated in 50mL again and contains in the YEB liquid nutrient medium of 50mg/L Rifampin that to be cultured to OD600 be about 0.5, by bacterium liquid in 4 DEG C, the centrifugal 5min of 5000rpm, collect thalline, precipitate containing the MS liquid nutrient medium Eddy diffusion of 200 μMs of AS with 5mL, bacterium liquid is proceeded to 50mL containing 200 μMs of AS MS liquid nutrient medium in, 28 DEG C of shaking tables being cultured to OD600 is 0.5, obtain Agrobacterium bacterium liquid,
3) drought stress: get sugarcane (Guangdong agriculture 91-600, the seed selection of Guangdong Agricultural science crop investigations institute) blade of tip, after the surface sterilization of volume percent 70% alcohol, being cut into 30 thickness with knife blade is the disk of 0.5cm, and super clean bench is placed 50 minutes;
4) During Agrobacterium: the explant step 3) prepared immerses step 2) after 15min, acceptor is taken out in the Agrobacterium bacterium liquid prepared, middlely light and slowly rock for several times; Then acceptor is placed on aseptic filter paper, absorb remaining bacterium liquid and airing, transfer to by MS substratum, 3mg/L2 immediately, 4-dichlorphenoxyacetic acid (2,4-D), 1mg/L6-benzyladenine (6-BA) and 5.5g/L agar powder composition, pH was on the Dual culture substratum of 5.5, in 25 DEG C of light culture 4 days;
5) regeneration of Transgenic Resistant Herbicide sugarcane with transplant: by step 4) with contaminate after converting material move on to select and be made up of MS substratum, the careless fourth phosphine of 6-BA3mg/L, 5mg/L, 250mg/L cephamycin, 5.0g/L agar powder, pH is 5.8 selects on substratum and regenerate; When height of seedling is 3cm, cut plant and receive and be made up of MS substratum, 3mg/LNAA, 5mg/L grass fourth phosphine, 250mg/L cephamycin, 5.0g/L agar powder, pH is in the root media of 5.8, carries out root induction; After 4 weeks, visible plant height about 6cm, there is the root being about 2cm in bottom;
6) hardening, transplanting: open bottle cap, hardening 5 days, after cleaning the substratum of plant root, plantlet of transplant is that garden mould and peat soil are in mass ratio in 1:1 mixing seedling pan to matrix.
Adopt 30 Caulis Sacchari sinensis leaf disks, obtain 3 sugarcane plant altogether.
Embodiment 4
An agriculture bacillus mediated sugarcane genetically modified method, its step is as follows:
1) preparation this laboratory of Agrobacterium EHA105:EHA105 competent cell containing Bar gene is preserved, and Bar gene source, in commercial vector pCAMBIA3301, by the method for pCAMBIA3301 plasmid according to " molecular cloning ", imports in EHA105 cell;
2) cultivate: picking contains the Agrobacterium EHA105 bacterium colony of Bar gene, being inoculated in 5ml contains in the YEB liquid nutrient medium of 50mg/L Rifampin, 28 DEG C of incubator overnight, then 0.5ml bacterium liquid is inoculated in 50mL again and contains in the YEB liquid nutrient medium of 50mg/L Rifampin that to be cultured to OD600 be about 0.5, by bacterium liquid in 4 DEG C, the centrifugal 5min of 5000rpm, collect thalline, precipitate containing the MS liquid nutrient medium Eddy diffusion of 100 μMs of AS with 50mL, it is 0.5 that 28 DEG C of shaking tables are cultured to OD600, obtains Agrobacterium bacterium liquid;
3) drought stress: the blade getting sugarcane (Guangdong sugar 00-236, Guangzhou Inst of Cane Sugar's seed selection) tip, after the surface sterilization of volume percent 70% alcohol, being cut into 30 thickness with knife blade is the disk of 0.5cm, and super clean bench is placed 30 minutes;
4) During Agrobacterium: the explant step 3) prepared immerses step 2) acceptor is taken out after 12 in the Agrobacterium bacterium liquid prepared, middlely light and slowly rock for several times; Then acceptor is placed on aseptic filter paper, absorb remaining bacterium liquid and airing, transfer to by MS substratum, 2mg/L2 immediately, 4-dichlorphenoxyacetic acid (2,4-D), 2mg/L6-benzyladenine (6-BA) and 5.5g/L agar powder composition, pH was on the Dual culture substratum of 5.5, in 25 DEG C of light culture 3 ~ 5 days;
5) regeneration of Transgenic Resistant Herbicide sugarcane with transplant: by step 4) with contaminate after converting material move on to select and be made up of MS substratum, the careless fourth phosphine of 6-BA3mg/L, 5mg/L, 250mg/L cephamycin, 5.0g/L agar powder, pH is 5.8 selects on substratum and regenerate; When height of seedling is 3cm, cut plant and receive and be made up of MS substratum, 3mg/LNAA, 5mg/L grass fourth phosphine, 250mg/L cephamycin, 5.0g/L agar powder, pH is in the root media of 5.8, carries out root induction; After 4 weeks, visible plant height about 6cm, there is the root being about 2cm in bottom;
6) hardening, transplanting: open bottle cap, hardening 1 week, after cleaning the substratum of plant root, plantlet of transplant is that garden mould and peat soil are in mass ratio in 1:1 mixing seedling pan to matrix.
Adopt 30 Caulis Sacchari sinensis leaf disks, obtain 5 sugarcane plant altogether.
Embodiment 5
An agriculture bacillus mediated sugarcane genetically modified method, its step is as follows:
1) Agrobacterium LBA4404 of preparation containing Bar gene: LBA4404 competent cell is bought in precious biotechnology (Dalian) company limited, Bar gene source is in commercial vector pCAMBIA3301, by the method for pCAMBIA3301 plasmid according to " molecular cloning ", import in LBA4404 cell;
2) cultivate: picking contains the Agrobacterium LBA4404 list bacterium colony of Bar gene, being inoculated in 5ml contains in the YEB liquid nutrient medium of 50mg/L Rifampin, 28 DEG C of incubator overnight, then 0.5ml bacterium liquid is inoculated in 50mL again and contains in the YEB liquid nutrient medium of 50mg/L Rifampin that to be cultured to OD600 be about 0.5, by bacterium liquid in 4 DEG C, the centrifugal 5min of 5000rpm, collect thalline, precipitate containing the MS liquid nutrient medium Eddy diffusion of 200 μMs of AS with 5mL, bacterium liquid is proceeded to 50mL containing 200 μMs of AS MS liquid nutrient medium in, 27 DEG C of shaking tables being cultured to OD600 is 0.5, obtain Agrobacterium bacterium liquid,
3) drought stress: get sugarcane (Guangdong agriculture 91-600, the seed selection of crop investigations institute of Guangdong Agricultural section) sugarcane callus that induces of the blade of tip, be cut into the callus particle of 0.2*0.2cm size with the knife blade after sterilization, super clean bench placed 50 minutes;
4) During Agrobacterium: the explant step 3) prepared immerses step 2) after 15min, acceptor is taken out in the Agrobacterium bacterium liquid prepared, middlely light and slowly rock for several times; Then acceptor is placed on aseptic filter paper, absorb remaining bacterium liquid and airing, transfer to by MS substratum, 1mg/L2 immediately, 4-dichlorphenoxyacetic acid (2,4-D), 3mg/L6-benzyladenine (6-BA) and 5.5g/L agar powder composition, pH was on the Dual culture substratum of 5.5, in 25 DEG C of light culture 4 days;
5) regeneration of Transgenic Resistant Herbicide sugarcane with transplant: by step 4) with contaminate after converting material move on to select and be made up of MS substratum, the careless fourth phosphine of 6-BA3mg/L, 5mg/L, 500mg/L cephamycin, 5.0g/L agar powder, pH is 5.8 selects on substratum and regenerate; When height of seedling is 3cm, cut plant and receive and be made up of MS substratum, 3mg/LNAA, 5mg/L grass fourth phosphine, 250mg/L cephamycin, 5.0g/L agar powder, pH is in the root media of 5.8, carries out root induction; After 4 weeks, visible plant height about 6cm, there is the root being about 2cm in bottom;
6) hardening, transplanting: open bottle cap, hardening 5 days, after cleaning the substratum of plant root, plantlet of transplant is that garden mould and peat soil are in mass ratio in 1:1 mixing seedling pan to matrix.
Adopt the callus of 30 Caulis Sacchari sinensis leaf disk inductions, obtain 3 sugarcane plant altogether.
Comparative example 1:
According to the conditional operation that above-described embodiment is same, difference is to be cut into after 30 thickness are the disk of 0.5cm with knife blade, does not carry out arid and injures, directly contaminate.Repeat experiment two groups, often group is 30 Caulis Sacchari sinensis leaf disks, and three groups of experiments obtain 2 sugarcane plant altogether.
See Fig. 3, two test tubes on both sides are normal blades cultivated, and three middle test tubes are the blades after method of the present invention is cultivated; The photo display of Fig. 3, transgenic sugarcane blade obtains careless fourth phosphine resistance, still keep green, and contrast does not have resistance, comes to harm, turn yellow in careless fourth phosphine solution.
See Fig. 4, Fig. 4, in 1 be mark, 5,8,9 is sugarcane plant leaf pcr amplification product of the present invention; Can show that the clip size amplified is consistent with carrier by result in figure.
transgenic sugarcane detects
Regeneration sugarcane in above-described embodiment 1-3 seedling pan is detected.
A) get and be planted in regeneration plant blade in seedling pan, adopt micromethod to extract STb gene and carry out pcr amplification.
B) pcr amplification primer is according to Bar sequences Design in plasmid expression vector, upstream sequence: 5' – ACCATCGTCAACCACTACAT-3', downstream sequence: 5' – AGTCCAGCTGCCAGAAACCC-3'.PCR reaction system: template DNA 50 ~ 100ng, 10 × Buffer1.5 μ L, 10mmol/LdNTP0.35 μ L, 10mmol/LZG30.35 μ L, 10mmol/LZG40.35 μ L, 25mmol/LMgCl
21.0 μ L, Taq enzyme (5U/ μ L) 0.2 μ L, mend ddH
2o to 15 μ L.Reaction conditions: 95 DEG C of denaturation 3min; 95 DEG C of 30sec, 60 DEG C of 40sec, 72 DEG C of 40sec, 35 circulations; 72 DEG C extend 10min, 4 DEG C of preservations;
C) mass volume ratio 1% agarose gel electrophoresis detects.
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (4)
1. an agriculture bacillus mediated sugarcane genetically modified method, its step is as follows:
1) Agrobacterium-mediated Transformation: the Agrobacterium containing foreign gene is cultured to logarithmic phase, collects thalline, is resuspended in the MS liquid nutrient medium containing Syringylethanone, obtains transformed bacteria liquid;
2) drought stress: be cut into the Caulis Sacchari sinensis leaf disk that thickness is 0.3 ~ 0.5cm after alcohol surface sterilization, super clean bench is placed 15 ~ 50 minutes;
3) contaminate: by step 2) Sugarcane Leaves after process is immersed in step 1) in the transformed bacteria liquid prepared, again Caulis Sacchari sinensis leaf disk is placed on Dual culture substratum after liquid is blotted, in 25 ~ 30 DEG C of light culture 3 ~ 4 days;
4) root induction: above-mentioned Caulis Sacchari sinensis leaf disk after contaminating is placed in Selective agar medium and cultivates, regenerate; When height of seedling is 2 ~ 3cm, cut plant and receive root media and carry out root induction, obtain the plant taken root;
5) hardening, transplanting: by take root plant hardening, transplanting, obtain transgenic sugarcane plant;
Wherein, step 1) cultivation of middle Agrobacterium employing YEB liquid nutrient medium, concrete steps are: by the Agrobacterium inoculation containing foreign gene in containing in antibiotic YEB liquid nutrient medium, 25 ~ 30 DEG C of incubator overnight, again by volume per-cent 1% inoculum size transfer to cultivate in the same substratum of 50mL 2 ~ 4 little be 0.5 ~ 0.6 up to OD600, collect thalline; Then precipitate containing the MS liquid nutrient medium Eddy diffusion of 100 μMs of Syringylethanones with 5mL, bacterium liquid is proceeded to 50mL containing in the MS liquid nutrient medium of 100 μMs of Syringylethanones, 28 DEG C of shaking tables are cultured to OD600 to 0.5 ~ 0.6;
Step 3) Dual culture substratum be the mixed culture medium of MS substratum and 2-5mg/L2,4-dichlorphenoxyacetic acid, 1-3mg/L6-benzyladenine and 5.5g/L agar powder, its pH5.5;
Step 4) described in Selective agar medium be by MS substratum and 2 ~ 5mg/L2, the mixed culture medium of 4-dichlorphenoxyacetic acid, 1 ~ 3mg/L6-benzyladenine, 250mg/L cephamycin, 5.5g/L agar powder and resistance marker screening material composition, its pH5.8; Described root media is the mixed culture medium that MS substratum and 2 ~ 5mg/L naphthylacetic acid, 250 ~ 500mg/L cephamycin, 5.5g/L agar powder and resistance marker screen material, its pH5.8.
2. method according to claim 1, is characterized in that: described resistance marker screening material is microbiotic or weedicide.
3. the method according to any one of claim 1-2, is characterized in that: described foreign gene is careless fourth phosphine resistant gene.
4. method according to claim 1, is characterized in that: step 5) in transplant matrix used be the 1:1 mixing in mass ratio of garden mould and peat soil.
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| CN116904507B (en) * | 2023-08-31 | 2024-02-13 | 中国科学院华南植物园 | Genetic transformation method based on sugarcane tissue culture seedlings |
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