CN104450778A - Genetic transformation and screening method of transfecting pollen of cotton by agrobacteria - Google Patents
Genetic transformation and screening method of transfecting pollen of cotton by agrobacteria Download PDFInfo
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- CN104450778A CN104450778A CN201410736696.4A CN201410736696A CN104450778A CN 104450778 A CN104450778 A CN 104450778A CN 201410736696 A CN201410736696 A CN 201410736696A CN 104450778 A CN104450778 A CN 104450778A
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Abstract
The invention discloses a genetic transformation and screening method of transfecting pollen of cotton by agrobacteria, belonging to the technical field of genetic transformation methods. The method comprises the following steps: preparing a special agrobacteria-infected pollen germination culture medium; infecting germinal pollen by agrobacteria with binary carrier vectors pCB4-bar by virtue of vacuum infiltration; then, pollinating the infected pollen on stigma; and transferring genetic materials to filial generation by virtue of a pollen tube path to obtain a transgenic plant. By using a herbicide-resistant gene bar as a target gene, cotton is converted by an agrobacteria transfecting pollen method, and meanwhile, a part of factors which affect conversion are optimized and the optimum condition suitable for conversion is screened. The conversion ratio of the optimized agrobacteria-infected pollen can reach 1.3% and resistance segregation of obtained converted subsequent generation Basta meets the separation rule of 3: 1. The method is simple to operate and free of special requirements on equipment and instruments, thereby providing technical supports for transgenic breeding of cotton.
Description
Technical field
The present invention relates to a kind of genetic transforming method, particularly a kind of genetic transformation of Agrobacterium infection cotton pollen and screening method.
Background technology
Genetic engineering technique can break through the genetic block between species, surmounts the incompatibility between species, creates the new variety required for people, greatly widens the approach of cotton genetic improvement.Along with gene transformation method deeply and perfect, numerous method is applied to cotton, and as agrobacterium-mediated transformation, pollen tube passage method, particle bombardment, micro laser method, and Agrobacterium and particle gun are in conjunction with method for transformation etc.Pollen tube passage method is the transgenic technology of Exogenous DNA transfered plant after a set of self-pollination of China period-luminosity Mr. space proposition in 1978, the method avoids somatic embryo carcass that cotton tissue cultivates and occurs and the obstacle of plant regeneration and easy and simple to handle, and required time is short.But deficiency is that its transformation mechanism is uncertain, and transformation efficiency is low and unstable.
Therefore, pollen tube passage method is in conjunction with agrobacterium-mediated transformation, both can not rely on the artificial culture process such as plant tissue culture and regeneration induction plant regeneration, the high efficiency advantage simultaneously utilizing again Agrobacterium to infect plant, enjoys the favor of research worker in genetic plant transformations.Tjokrokusumo etc. use Agrobacterium vacuum infiltration transfection pollen method successful conversion petunia.In cotton, Chen etc. pollinate with after Agrobacterium dipping column cap again, obtain the transformation efficiency of 0.46% ~ 0.93%; Li Xiao etc. and Zhang Yanhong etc. use Agrobacterium vacuum infiltration converting cotton pollen respectively, then pollinate to cotton with process pollen, all obtain transfer-gen plant; Peng Zhenzi carries out live body conversion by Agrobacterium Ovule injection to cotton, transgene efficiency average out to 2.78% in the cotton of different varieties.The transformation efficiency improving Agrobacterium infection cotton pollen is of crucial importance.
Summary of the invention
The object of the invention is to overcome the shortcoming that exists in prior art with not enough, a kind of genetic transformation and screening method of Agrobacterium infection cotton pollen are provided.By greatly improving transformation efficiency to the improvement of transfection method.
Object of the present invention is achieved through the following technical solutions: a kind of genetic transformation of Agrobacterium infection cotton pollen and screening method, comprise the following steps:
1) the pollen germination substratum of agroinfection is prepared: the component that pollen germination substratum comprises is 0.5mMKNO
3, 2.7mM MnSO
4, 1.7mM H
3bO
3, 0.5mM MgSO
47H
2o, 1.5 μMs of GA
3, pH 7.5, mass concentration is the sucrose of 40% ~ 45%; Autoclaving after the preparation of pollen germination substratum, then packing obtains liquid nutrient medium, and sealed membrane (parafilm) seals, and 4 DEG C save backup;
2) method for transformation: picking, with the Agrobacterium of binary vector plasmid pCB4-bar, is cultivated in YEP liquid nutrient medium, culture condition is shaking speed 180 ~ 220rpm, and 28 DEG C, shaking culture is spent the night; As the OD of bacterium liquid
600when value is 0.6 ~ 0.8, the centrifugal 10min of 3000r/min collects thalline, is resuspended in step 2) in the pollen germination substratum for preparing; Collect fresh cotton pollen to be soaked in this bacterium liquid, pollen concentration is 100g/L, adds the Tween-20 (tensio-active agent) of 1 ‰ volume ratios and vacuumizes, pressure-80Pa, continues 20min; The pollen writing brush of dipped Agrobacterium is applied in the cotton column cap of emasculation, on column cap, then overlaps the straw that one end is closed; After cotton seeds maturation, collect cotton seeds;
3) screening of transgenic cotton seedling: by step 2) cotton seeds that obtains is after planting, when cotton seedling enters three leaf periods, the Basta solution of 0.3ml/L concentration is adopted to carry out smearing screening, observe the plant of smearing blade and not occurring necrotic spot after 3d, confirm as and transform successful transgenic cotton seedling.
Step 1) described in autoclaving to be preferably under 121 DEG C of conditions sterilizing 20 minutes;
Step 2) described in being prepared as of YEP liquid nutrient medium: get Tryptones 10g; Yeast extract 1g; MgSO
40.5g and sucrose 5g is dissolved in 1L water, adjusts pH to 7.0, autoclaving with 5mmol/L NaOH.
Step 2) described in resuspended, adopt 100mL bacterium liquid (OD600=0.6 ~ 0.8) after the centrifugal 10min of 3000r/min Eddy diffusion in 100mL pollen cultures base.
Step 2) described in binary vector plasmid pCB4-bar for comprising herbicide resistant bar gene, the pCB4 carrier of CaMV35S promotor and NOS terminator.Foreign gene bar gene, derives from streptomyces hygroscopicus (streptomyces hy hygroscopieus), has resistance to herbicide PPT (phosphinothricin).The specifying information of plasmid is see document: Tang Wei etc. " turn the cultivation of Bar gene Herbicide Resistant Rice ", hubei agricultural science, and 2007,46 (4): 488-490.
Step 2) described in Agrobacterium be preferably agrobacterium strains LBA4404;
Step 2) described in fresh cotton pollen be preferably the pollen of upland cotton ' Coker312 ' inbred strains; Described fresh cotton pollen refers to the bud of will be bloomed the next day that the previous day gathers at dusk, puts in 4 DEG C of refrigerators and preserves, and gets the pollen of natural loose powder for transforming in next day 7 ~ 8 time; Or the pollen of fresh acceptor material that 12 ~ 14 hours same day opened.
Step 2) described in pollen smear emasculation cotton column cap preferably fair weather the morning 10:00 ~ 10:30 time smear.
Step 3) described in the commodity of Basta be called Finale, effective ingredient is the careless ammonium phosphine (glufosinate-ammonium) of 5.78%.
The present invention has following advantage and effect relative to prior art:
Pollen granule Agrobacterium infection method of the present invention, can make use of the reproductive system of plant self dexterously---and pollen tube channel as carrier, thus overcomes most conversion system transformation receptor technology barrier difficult for regeneration.Result shows, the transformation efficiency of the agroinfection pollen of optimization of the present invention can reach 1.3%, and the transformation generation plant Basta Resistant segregation of acquisition meets 3:1 law of segregation, and the method is simple to operate, without the particular requirement of equipment and instrument.Pollen granule Agrobacterium infection method avoids the not bery clear and definite shortcoming of naked DNA integrated mechanism that traditional cotton Ovary injection causes, required transformation time is roughly the same with Ovule injection DNA method, repeatability is strong, is a kind of practical approach of cost-effective cotton gene transformation.
Agrobacterium infection pollen method of the present invention infects with Agrobacterium vacuum infiltration the pollen just sprouted, then pollinate in column cap by metainfective pollen, by pollen tube passage, genetic material passed to filial generation, obtain transfer-gen plant.This experiment, using herbicide resistant bar gene as goal gene, by Agrobacterium infection pollen method converting cotton, is optimized the factor that some effects transform simultaneously, is filtered out the optimum condition being applicable to transforming, for cotton transgenic breeding provides technical support.
Accompanying drawing explanation
Fig. 1 is the physical map of plasmid pCB4-bar;
Fig. 2 is the effect diagram of different sucrose to pollen viability;
Fig. 3 is that antiweed (Basta) smears conversion and non transformed plants blade resistance and sensitivity response figure; Wherein a is cotton for turning bar gene; B is contrast (Basta smears the rear 7d of process);
Fig. 4 is that the PCR turning bar gene plant detects electrophorogram; Wherein: swimming lane M:DL2000marker; Swimming lane P: positive control; Swimming lane CK: negative control; Swimming lane 1-22: the transformed plant that pollen granule Agrobacterium infection method obtains.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Agrobacterium strains and acceptor material:
The bacterial strain of this institute is the agrobacterium strains LBA4404 with binary vector plasmid pCB4-bar, and this plasmid comprises herbicide resistant bar gene, CaMV 35S promoter and NOS terminator (plasmid construct figure is shown in Fig. 1).Acceptor material is upland cotton ' Coker312 ' inbred strains.
The foreign gene bar gene source of above-mentioned binary vector plasmid pCB4-bar, in streptomyces hygroscopicus (streptomyces hy hygroscopieus), has resistance to herbicide PPT (phosphinothricin).The specifying information of plasmid is see document: Tang Wei etc. turn the cultivation of Bar gene Herbicide Resistant Rice, hubei agricultural science, and 2007,46 (4): 488-490.
BLOCK M D,BOTTERMAN J,VANDEWIELE M,et al.Engineeringherbicide resistance in plants by expression of adetoxifying enzyme[J].EMBO J.1987,6:2513-2518.
THOMPSON C J,MOVVA N R,CRAMERI R,et al.Characterization of theherbicide-resistance gene Bar from streptomyces hygroscopicus[J].EMBO J.1987,6:2519-2523.
Embodiment 1
The genetic transformation of Agrobacterium infection cotton pollen and a screening method, comprise the following steps:
1) the pollen germination substratum of agroinfection is prepared: the component that pollen germination substratum comprises is 0.5mMKNO
3, 2.7mM MnSO
4, 1.7mM H
3bO
3, 0.5mM MgSO
47H
2o, 1.5 μMs of GA
3, pH 7.5, adds the saccharose treatment of 7 different concns respectively, and namely mass concentration is the sucrose of 0%, 10%, 25%, 35%, 40%, 45% and 50%; Autoclaving after the preparation of pollen germination substratum, then packing obtains liquid nutrient medium, and sealed membrane (parafilm) seals, and 4 DEG C save backup;
2) method for transformation: picking, with the Agrobacterium of binary vector plasmid pCB4-bar, is cultivated in YEP liquid nutrient medium, culture condition is shaking speed 180 ~ 220rpm, and 28 DEG C, shaking culture is spent the night; ; As the OD of bacterium liquid
600when value is 0.6 ~ 0.8, the centrifugal 10min of 3000r/min collects thalline, is resuspended in step 2) in the pollen germination substratum for preparing; Collect fresh cotton pollen to be soaked in this bacterium liquid, pollen concentration is 100g/L, adds the Tween-20 (tensio-active agent) of 1 ‰ volume ratios and vacuumizes, pressure-80Pa, continues 20min; At the 10-10:30 in the morning of fair weather, the pollen writing brush of dipped Agrobacterium is applied in the cotton column cap of emasculation, on column cap, then overlaps the straw that one end is closed, each process 300 flowers, repeat 3 times, listing mark;
3) screening of transgenic cotton seedling: by step 2) cotton seeds that obtains is after planting, when cotton seedling enters three leaf periods, the basta solution of 0.3ml/L concentration is adopted to carry out smearing screening, observe the plant of smearing blade and not occurring necrotic spot after 3d, confirm as and transform successful transgenic cotton seedling.
Step 1) described in autoclaving to be preferably under 121 DEG C of conditions sterilizing 20 minutes;
Step 2) described in being prepared as of YEP liquid nutrient medium: get Tryptones 10g; Yeast extract 1g; MgSO
40.5g; Sucrose 5g; Be dissolved to 1L, adjust pH to 7.0 with 5mmol/L NaOH, autoclaving.
Step 2) described in resuspended, adopt 100mL bacterium liquid (OD600=0.6 ~ 0.8) after the centrifugal 10min of 3000r/min Eddy diffusion in 100mL pollen cultures base.
Step 2) described in binary vector plasmid pCB4-bar for comprising herbicide resistant bar gene, the pCB4 carrier of CaMV35S promotor and NOS terminator, structure is as shown in Figure 1.
Step 2) described in Agrobacterium be preferably agrobacterium strains LBA4404;
Step 2) described in binary vector plasmid pCB4-bar for comprising herbicide resistant bar gene, the pCB4 carrier of CaMV35S promotor and NOS terminator.Foreign gene bar gene, derives from streptomyces hygroscopicus (streptomyces hy hygroscopieus), has resistance to herbicide PPT (phosphinothricin).The specifying information of plasmid is see document: Tang Wei etc. " turn the cultivation of Bar gene Herbicide Resistant Rice ", hubei agricultural science, and 2007,46 (4): 488-490.
To step 1) in the pollen germination substratum of 7 kinds of different sucrose, p-diaminodiphenyl-methyl naphthol reaction method is adopted to measure pollen viability in substratum, concrete detecting step is: (1) takes 0.2g xenol and is dissolved in 100mL50% alcohol, and brown bottle keeps in Dark Place; (2) 0.15g methyl naphthol is claimed to be dissolved in 100mL 50% alcohol,
Brown bottle keeps in Dark Place; (3) 0.25g Na is claimed
2cO
3be dissolved in 100mL distilled water.Then rear use is shaken up, by above-mentioned solution and H by (1) (2) (3)=1:1:1 mixing
2o
2mix with the ratio of 2:1, after pollen being immersed in this solution 5min, examine under a microscope color.Color is dark and the normal pollen envelop of form is considered as viable pollen, and of light color, colourless or paramorph pollen is weak vitality and previable pollen.
Then pollen germination rate and breakage rate is examined under a microscope, determine best sucrose concentration, detected result as shown in Figure 2, in sucrose-free pollen germination nutrient solution, the breakage rate of cotton pollen is 48.6%, and along with sucrose concentration increases gradually, the breakage rate of pollen is in downtrending gradually, until at 50% sucrose concentration, breakage rate drops to 5.8%.And pollen germination rate contrast without sucrose nutrient solution in be 2.3%, along with the increase of sucrose concentration, it is 65.7% that pollen germination takes the lead in reaching maximum value when sucrose concentration is 40%, after decline gradually.Result shows, the sucrose solution of 40% and 45% can keep the running balance of pollen osmotic pressure, can preserve pollen to greatest extent.
Choose the pollen germination substratum of 40% and 45% sucrose according to step 2) carry out agroinfection pollen, detect the effect of different pollen transfection process, as can be seen from Table 1, seed number, bolling rate and transformation efficiency that 1. process obtains are all higher than processing 2. mode, process transformation efficiency 1. reaches 1.3%, but difference is not remarkable compared with the transformation efficiency 1.1% processed 2..
The different pollen of table 1 infects the changing effect of process
Basta smears qualification result:
Step 3) the middle cotton seedling smearing tri-leaf period with 0.30ml/L Basta, blade causing harm by weedicide just can be shown after contrast seedling (not carrying out the seedling of transfection experiment) 3d, there is necrotic spot in blade, this symptom aggravation after 1 week, visible cotton seedling is for Basta ten points of sensitivities, the transgene cotton seedling turning bar gene is then reactionless (to be the results are shown in Figure in 3 a and b), illustrates and smear qualification by Basta to turn bar gene cotton very effective.Basta is carried out to the transformed plant obtained and smears qualification, obtain and turn totally 42 strains of bar gene cotton plants.
PCR Molecular Identification is verified:
From conversion and non transformed plants, get young leaflet tablet respectively, extract STb gene by improved method of CTAB and carry out PCR detection.Pcr amplification Bar upstream region of gene primer 5 ＇-CAGGAACCGCAGGAGTGGA-3 ＇ and downstream primer 5 ＇-CCAGAAACCCACGTCATGCC-3 ＇.Response procedures is: 94 DEG C of 5min; Then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 circulations; 72 DEG C of 10min; 4 DEG C of preservations.Pcr amplification product is carried out to the agarose electrophoresis of 1%, analyze and take pictures.
To the transformed plant DNA obtained, carry out the pcr amplification of bar gene primer, as shown in Figure 4, result shows that all Basta identify that negative plant (anti-Basta) plant has all amplified the special object band of 430bp to result, illustrates that Basta smears authentication method reliable and stable.
The genetic analysis of the anti-Basta proterties of transfer-gen plant:
Through selfing, T is obtained to acquisition transfer-gen plant
1for seed, use Basta resistance screening after sowing seedling, add up resistance and non-resistance T respectively
1for plant.The resistance T obtained
1plant selfing obtains T
2for seed, T
2for selfed seed sowing, smear screening with Basta, investigation statistics Resistant segregation situation.
To transgenosis cotton plant T
1and T
2the basta Resistant segregation situation statistics in generation is in table 2, and result shows the T that pollen granule Agrobacterium infection obtains
1show as resistance for 45 strains in plant, 16 strains show as perception, be greater than 0.05 through chi square test P (3:1) value, show the T obtained by the method
1meet the Mendel's regulavity of segregation of 3:1 for plant Basta resistance and non-resistance segregation ratio, this be single-gene as single proterties at T
1the segregation ratio in generation, the bar gene describing conversion copies genetic stability to offspring with list.T
2resistant segregation situation for plant shows (table 2): the T obtained from pollen granule Agrobacterium infection
2the ratio of the crossbred of the unseparated homozygote of plant resistance and Resistant segregation is 1:2, is greater than 0.05 through chi square test P (1:2) value, shows as bar gene as the genetic stability of single dominant character and expression.
Offspring's Resistant segregation situation of table 2 transfer-gen plant
Pollen granule Agrobacterium infection method, can make use of the reproductive system of plant self dexterously---and pollen tube channel as carrier, thus overcomes most conversion system transformation receptor technology barrier difficult for regeneration.Result shows, the transformation efficiency of the agroinfection pollen of optimization of the present invention can reach 1.3%, the transformation generation plant Basta Resistant segregation obtained meets 3:1 law of segregation, the method is simple to operate, without the particular requirement of equipment and instrument, but need to do corresponding variation according to the weather temperature light of every day on pollen collecting and pollination time.Pollen granule Agrobacterium infection method avoids the not bery clear and definite shortcoming of naked DNA integrated mechanism that cotton Ovary injection causes, required transformation time is roughly the same with Ovule injection DNA method, repeatability is strong, is a kind of practical approach of cost-effective cotton gene transformation.
Agrobacterium infection pollen method of the present invention infects with Agrobacterium vacuum infiltration the pollen just sprouted, then pollinate in column cap by metainfective pollen, by pollen tube passage, genetic material passed to filial generation, obtain transfer-gen plant.This experiment, using herbicide resistant bar gene as goal gene, by Agrobacterium infection pollen method converting cotton, is optimized the factor that some effects transform simultaneously, is filtered out the optimum condition being applicable to transforming, for cotton transgenic breeding provides technical support.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1., with genetic transformation and the screening method of Agrobacterium infection cotton pollen, it is characterized in that: comprise the following steps:
1) the pollen germination substratum of agroinfection is prepared: the component that pollen germination substratum comprises is 0.5mMKNO
3, 2.7mM MnSO
4, 1.7mM H
3bO
3, 0.5mM MgSO
47H
2o, 1.5 μMs of GA
3, pH 7.5, mass concentration is the sucrose of 40% ~ 45%; Autoclaving after the preparation of pollen germination substratum, then packing obtains liquid nutrient medium, and sealed membrane seals, and 4 DEG C save backup;
2) method for transformation: picking, with the Agrobacterium of binary vector plasmid pCB4-bar, is cultivated in YEP liquid nutrient medium, culture condition is shaking speed 180 ~ 220rpm, and 28 DEG C, shaking culture is spent the night; As the OD of bacterium liquid
600when value is 0.6 ~ 0.8, the centrifugal 10min of 3000r/min collects thalline, is resuspended in step 2) in the pollen germination substratum for preparing; Collect fresh cotton pollen to be soaked in this bacterium liquid, pollen concentration is 100g/L, adds the Tween-20 of 1 ‰ volume ratios and vacuumizes, pressure-80Pa, continues 20min; The pollen writing brush of dipped Agrobacterium is applied in the cotton column cap of emasculation, on column cap, then overlaps the straw that one end is closed; After cotton seeds maturation, collect cotton seeds;
3) screening of transgenic cotton seedling: by step 2) cotton seeds that obtains is after planting, when cotton seedling enters three leaf periods, the Basta solution of 0.3ml/L concentration is adopted to carry out smearing screening, observe the plant of smearing blade and not occurring necrotic spot after 3d, confirm as and transform successful transgenic cotton seedling.
2. the genetic transformation of Agrobacterium infection cotton pollen according to claim 1 and screening method, is characterized in that: step 1) described in autoclaving be sterilizing 20 minutes under 121 DEG C of conditions.
3. the genetic transformation of Agrobacterium infection cotton pollen according to claim 1 and screening method, is characterized in that: step 2) described in being prepared as of YEP liquid nutrient medium: get Tryptones 10g; Yeast extract 1g; MgSO
40.5g and sucrose 5g is dissolved in 1L water, adjusts pH to 7.0, autoclaving with 5mmol/L NaOH.
4. the genetic transformation of Agrobacterium infection cotton pollen according to claim 1 and screening method, is characterized in that: step 2) described in Agrobacterium be agrobacterium strains LBA4404.
5. the genetic transformation of Agrobacterium infection cotton pollen according to claim 1 and screening method, is characterized in that: step 2) described in fresh cotton pollen be the pollen of upland cotton ' Coker312 ' inbred strains.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109652455A (en) * | 2019-02-19 | 2019-04-19 | 南京农业大学 | The Chinese cabbage high-efficiency genetic transforming method and its application that a kind of magnetic nano-carrier mediates |
| CN110938583A (en) * | 2019-12-25 | 2020-03-31 | 石河子大学 | Solid culture medium for cotton pollen germination and germination method |
| CN113564201A (en) * | 2021-08-03 | 2021-10-29 | 南京农业大学 | Cruciferae crop pollen electric shock genetic transformation method |
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2014
- 2014-12-05 CN CN201410736696.4A patent/CN104450778A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652455A (en) * | 2019-02-19 | 2019-04-19 | 南京农业大学 | The Chinese cabbage high-efficiency genetic transforming method and its application that a kind of magnetic nano-carrier mediates |
| CN110938583A (en) * | 2019-12-25 | 2020-03-31 | 石河子大学 | Solid culture medium for cotton pollen germination and germination method |
| CN113564201A (en) * | 2021-08-03 | 2021-10-29 | 南京农业大学 | Cruciferae crop pollen electric shock genetic transformation method |
| CN113564201B (en) * | 2021-08-03 | 2024-06-07 | 南京农业大学 | Electric shock genetic transformation method for crucifer crop pollen |
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