CN109554329A - A kind of method of target gene conversion protoplast - Google Patents
A kind of method of target gene conversion protoplast Download PDFInfo
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- CN109554329A CN109554329A CN201910010502.5A CN201910010502A CN109554329A CN 109554329 A CN109554329 A CN 109554329A CN 201910010502 A CN201910010502 A CN 201910010502A CN 109554329 A CN109554329 A CN 109554329A
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Abstract
The present invention provides a kind of extracting method of protoplast, and this method comprises the following steps: (1) raw material disposal；(2) enzymatic hydrolysis release protoplast；(3) purifying of protoplast.The protoplast that protoplast extracting method of the invention obtains can be used for target gene conversion protoplast, technical support is provided for important candidate gene approach and genetic transformation, germplasm utilization is carried out for bioengineering and improvement lays the foundation, and is had broad application prospects.
Description
Technical field
The present invention relates to Skill of Plant Protoplasts fields, more particularly to a kind of target gene conversion protoplast
Method.
Background technique
It is Amaryllidaceae that Lycoris (Lycoris spp.) and Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, which belong to (Hippeastrum spp.),
(Amaryllidaceae) napiform root herbaceous plant, flower pattern are abundant, and pattern is gorgeous, and ornamental value is high, may be used as arranging colored border,
Artificial hillock, rock garden and with the doing hayashishita good material of quilt.Also there is medicinal function simultaneously, it is distinctive rich in a variety of amrallids
Alkaloid component.Narciclasine has antitumor activity, and lycorine can induce the Apoptosis of cancerous cell line HL-60, add
Lan Tamin and lycoramine are one of the choice drugs of clinical treatment Alzheimer's disease.Plant slow growth itself plus pair
The predation formula of resource is developed, and reduces plant resources increasingly, therefore there is an urgent need to open using biotechnology existing resource
New germ plasm resource is formulated in hair and protection using the method for genetic transformation.
Plant protoplast can obtain a large amount of genetically cells with homogeney within a short period of time and plant is lost
The good receptor material of conversion is passed, it is significant to gene functional research；Protoplast contains whole hereditary information of individual, and
It can produce important secondary metabolite, separation and Extraction detection carried out to it, new approaches can be provided for production of metabolites；It is primary
Plastid is also extensively used for gene subcellular localization, and it is germplasm innovation that body cell distant hybridization, which can overcome crossing barrier,
Effective way.
Research both at home and abroad has no the separation of amrallid seedling protoplast and transient transformation methods and the report of application.
Summary of the invention
Present invention firstly provides a kind of extracting methods of protoplast, and this method comprises the following steps:
(1) by amrallid seedling, after dark processing 10-12h, seedling leaves raw material disposal: are cut to fragment；
(2) enzymatic hydrolysis release protoplast: fragment obtained in step (1) is put into reagent A, 22-28 DEG C of temperature, is shaken
1-6h is handled, mixed solution is obtained；
(3) purifying of protoplast: the mixed solution that step (2) obtain is filtered with 40-50 μm of strainer, collects filter
Liquid, filtrate are centrifuged 5-10min, and centrifuging temperature is 4 DEG C, collect precipitating；Precipitating is cleaned with reagent B, is centrifuged, and last centrifugation is used
Reagent C suspends, and obtains Protoplast suspension；
The wherein reagent A are as follows: Cellulase R10:0.05-0.5g, Macerozyme R10:0.01-0.5g,
Mannitol:1-10g, MES:0.01-1.0g, CaCl2: 0.01-1.0g, H2O: 20ml volume is supplied；
Reagent B are as follows: NaCl:0.45-4.5g, CaCl2: 0.5-6.0g, KCl:0.01-1.0g, MES:0.02-2.0g, H2O:
Supply 100ml volume；
Reagent C are as follows: Mannitol:0.5-5.0g, MgCl26H2O:0.01-1.0g, MES:0.001-0.1g, H2O: it mends
Sufficient 10ml volume.
The present invention also provides the protoplasts that above-mentioned protoplast extracting method obtains.
The present invention also provides a kind of method that target gene converts above-mentioned protoplast, this method are as follows:
In Protoplast suspension, target gene plasmid vector and reagent D, room temperature dark culture 10-30min are sequentially added
Afterwards, the conversion of target gene is realized；
Wherein reagent D are as follows: PEG4000:0.1-0.5g, Mannitol:0.01-0.15g, CaCl2: 0.0012-0.12g,
H2O: 1ml volume is supplied.
Specifically, the present invention provides a kind of method of target gene conversion protoplast, this method includes following step
It is rapid:
(1) by amrallid seedling, after dark processing 10-12h, seedling leaves raw material disposal: are cut to fragment；
(2) enzymatic hydrolysis release protoplast: fragment obtained in step (1) is put into reagent A, 22-28 DEG C of temperature, is shaken
1-6h is handled, mixed solution is obtained；
(3) purifying of protoplast: the mixed solution that step (2) obtain is filtered with 40-50 μm of strainer, collects filter
Liquid, filtrate are centrifuged 5-10min, and centrifuging temperature is 4 DEG C, collect precipitating；Precipitating is cleaned with reagent B, is centrifuged, and last centrifugation is used
Reagent C suspends, and obtains Protoplast suspension；
(4) conversion of target gene: the Protoplast suspension for taking step (3) to obtain sequentially adds target gene plasmid
Carrier and reagent D after room temperature dark culture 10-30min, realize the conversion of target gene；
The determination of yield for the protoplast being prepared in a kind of method of target gene conversion protoplast of the invention
Method:
The protoplast being suspended in reagent C in above-mentioned steps (3) is gently inhaled with the suction pipette head for removing tip and is beaten
It is allowed to be evenly distributed；Clean one piece of blood counting chamber is taken, 8-10 μ l Protoplast suspension is drawn with pipettor, is allowed to be full of
Count block repeat count 5 times, is averaged；According to the weight for the amrallid seedling fragment being added in step (1), calculate
Every gram of obtained protoplasm somatocyte number (a/gram).
The detection method of destination gene expression in a kind of method of target gene conversion protoplast of the invention are as follows:
Using the expression of target gene in confocal laser scanning microscope protoplast, by observing green fluorescence
The region that signal is concentrated, determines the expression position of gene.
The amount of destination gene expression be by measurement expression fluorescence and different target gene expression product (such as Garland he
Quick or chlorophyllide) whether succeed to embody to convert.
Specifically, a kind of method of target gene conversion amrallid protoplast of the present invention, by following steps group
At:
(1) the short-tube lycoris seedling of diameter 0.5-1cm, dark processing 10- raw material disposal: are chosen in the amrallid Sheng Ye phase
Seedling leaves are cut into the fragment of 0.5-2mm wide by 12h；
(2) enzymatic hydrolysis release protoplast: reagent A is added in the fragment that step (1) obtains, the additional amount of reagent A is with just
Not crossing fragment segment is advisable, and carries out being protected from light enzymatic hydrolysis, 22-28 DEG C of temperature, slowly concussion processing 1-6h, shaking speed are on shaking table
50-200rpm, enzymatic hydrolysis is gently inhaled with the suction pipette head for removing tip after the completion beats mixed solution, sufficiently release protoplast；
(3) purifying of protoplast: the protoplast solution that step (2) obtain is filtered with 40-50 μm of strainer, is received
Collect filtrate, 5-10min is centrifuged at 50-200g, centrifuging temperature is 4 DEG C；Precipitating reagent B repeated washing centrifugation 2-5 times, finally
Centrifugation reagent C suspension protoplast；
(4) conversion of target gene: target gene plasmid vector is added in the Protoplast suspension for taking step (3) to obtain
And reagent D realizes the conversion of target gene at room temperature after dark culture 10-30min；
Reagent B will be added in protoplast solution after conversion, be centrifuged 3-5min, abandon supernatant, 0.5-2ml examination is added in precipitating
Agent B after cultivating 4-16h, detects conversion results.
Specifically reagent A ingredient is shown in Table 1, and reagent B component is shown in Table 2, and reagent C ingredient is shown in Table 3, and reagent D ingredient is shown in Table 4:
1 reagent A ingredient of table
2 reagent B component of table
3 reagent C ingredient of table
4 reagent D ingredient of table
The method of purpose of the present invention genetic transformation amrallid protoplast can be used for:
(1) target gene is expressed in protoplast；
(2) gene subcellular localization and functional verification；
(3) application in target gene Methanogenesis.
A kind of method of target gene conversion amrallid protoplast provided by the invention, is that Lycoris resource is secondary
Methanogenesis, gene function verifying and germplasm innovation establish technical foundation.
Specifically, the method and its application of a kind of target gene instantaneous conversion amrallid protoplast of the invention
It has the following advantages and beneficial effects:
1. the method for extracting method and target gene the conversion protoplast of protoplast of the invention is simple and easy to operate,
It is repeatable high.
2. the present invention may be up to 2.66 × 10 using the protoplast yield that amrallid obtains7A/gram, by purpose
After Gene A conversion, it was demonstrated that the assignment of genes gene mapping is in cytoplasm, and secondary metabolite galanthamine content is up to 550 milligrams per grams；Through
After crossing target gene B conversion, it was demonstrated that the assignment of genes gene mapping is in chloroplaset, and metabolite chlorophyllin ester concentration is up to 158.09 mmoles
You/liter；It can be used to verify gene function and carry out the Subcellular Localization of gene, can be also used for detection cell metabolism and produce
Object accumulation.
The method of purpose of the present invention genetic transformation protoplast, high conversion rate, and establish amrallid plasm
Body transformation system provides technical support for important candidate gene approach and genetic transformation, carries out germplasm utilization for bioengineering
It lays the foundation, has broad application prospects with improvement.
Detailed description of the invention
Fig. 1 is Bulbus Lycoridis longitubae Leaves Protoplast microexamination figure (20 times of amplification factor) in embodiment 1.
Fig. 2 is Hipeastrum vittalum's Leaves Protoplast microexamination figure (20 times of amplification factor) in embodiment 2.
Fig. 3 is expression of the target gene A in Bulbus Lycoridis longitubae protoplast in example 1.A is that green fluorescence turns off field
Change the protoplast of target gene A；B is the protoplast that target gene A is converted under light field；C is that purpose base is converted under superimposed field
Because of the protoplast of A.Bar=10 μm.
Fig. 4 is expression of the target gene B in Bulbus Lycoridis longitubae protoplast in example 3.A is that green fluorescence turns off field
Change the protoplast of target gene B；B is the protoplast that target gene B is converted under light field；C is that purpose base is converted under superimposed field
Because of the protoplast of B.Bar=10 μm.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described, and following instance is merely to illustrate the present invention, but not
For limiting the scope of the invention.
Material source:
Bulbus Lycoridis longitubae seedling: originating from Academy of Agricultural Sciences, Shanghai City short-tube lycoris Germplasm Resources, and type is Bulbus Lycoridis longitubae
(Lycoris longituba)
Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait seedling: originating from Academy of Agricultural Sciences, Shanghai City short-tube lycoris Germplasm Resources, and type is that merry christmas
(Hippeastrum rutilum)
Cellulase R10 (cellulase R10): BIOSHARP
Macerozyme R10 (macerozyme R10): BIOSHARP
Mannitol (mannitol): BIOSHARP
MES (2-morpholine ethane sulfonic acid): BIOSHARP
Galanthamine standard items: Sinopharm Chemical Reagent Co., Ltd.
BsaI/Eco31I enzyme: Thermo Scientific
BamHI/XbaI enzyme: Thermo Scientific
PBWA (V) HS-35S-gfp-NOS carrier of green fluorescent protein: BioRun is merged
The pCAMBIA1301-35S-GFP-NOS carrier of green fluorescent protein: Changsha Yingrun is merged
Biotechnology Co.,Ltd
Target gene A: sequence such as SEQ NO.1
Target gene B: sequence such as SEQ NO.2
Remaining reagent is conventional commercial product.
1 target gene A of embodiment converts Bulbus Lycoridis longitubae protoplast
(1) the Bulbus Lycoridis longitubae seedling (1 year raw seedling that Bulbus Lycoridis longitubae cutting propagation generates) of diameter 1.0cm is chosen, it is dark
12h is handled, seedling leaves are cut, 3g blade is weighed, is 1mm or so wide fragment by blade cuts；
(2) chip material obtained in 20ml reagent A processing step (1) is injected, be protected from light enzymatic hydrolysis, 28 DEG C of temperature,
Slowly concussion processing 5h, shaking speed 50rpm, enzymatic hydrolysis are used the suction pipette head for removing tip gently to inhale after the completion and are beaten on shaking table
Mixed solution, sufficiently release protoplast；
Reagent A ingredient described in example 1 is shown in Table 5
5 example of table, 1 reagent A ingredient
(3) protoplast solution that step (2) obtain is filtered with 40 μm of strainers, collects filtrate, is centrifuged at 60g
5min, centrifuging temperature are 4 DEG C, collect precipitating；Precipitating is centrifuged 4 times with 10ml reagent B repeated washing, last centrifugation 1ml
Reagent C suspension protoplast.Fig. 1 is from 3g Bulbus Lycoridis longitubae blade according to said extracted step operation, finally obtained primary
Plastid figure can determine the protoplasm somatocyte number that every gram of blade extracts after blood counting chamber counts.
It states reagent B component and is shown in Table 6, reagent C ingredient is shown in Table 7
6 example of table, 1 reagent B component
7 example of table, 1 reagent C ingredient
The protoplast obtained in step (3) is gently inhaled to beat with the suction pipette head for removing tip and is allowed to be evenly distributed.
Clean one piece of blood counting chamber is taken, draws 10 μ l plasm body fluid with pipettor, is allowed to full of count block, repeat count 5 times,
It is averaged.According to the weight 3g being added in step (1), the protoplasm somatocyte number 2.66 × 10 that every gram of blade obtains is calculated7
A/gram.
(4) 10 μ g target gene A plasmid vectors and 110 μ are added in the Protoplast suspension for taking 100 μ l steps (3) to obtain
L reagent D, after the CDS that the plasmid of target gene A is configured to target gene A removes terminator codon, by BsaI/Eco31I enzyme
(Thermo Scientific) is cut, is connected on pBWA (V) the HS-35S-gfp-NOS carrier for having merged green fluorescent protein
(BioRun).After room temperature dark culture 20min, the conversion of target gene A is realized.
Then the reagent B of 440 μ l will be added in solution, be centrifuged 5min in 60g, abandon supernatant, 1ml reagent B, training is added in precipitating
After supporting 10h, conversion results are detected.
The reagent D ingredient is shown in Table 8
8 example of table, 1 reagent D ingredient
The expression feelings of target gene in Bulbus Lycoridis longitubae protoplast are observed under laser confocal microscope (Nikon C2-ER)
Condition, as shown in Figure 3.The result shows that conversion target gene A is expressed in protoplast, the protoplast of target gene A is converted
In, green florescent signal concentrates in protoplasm somatocyte matter, shows that gene is expressed in cytoplasm.The method of the present invention can incite somebody to action
Target gene carries out transient expression in Bulbus Lycoridis longitubae protoplast, and can carry out following gene function verifying:
Bulbus Lycoridis longitubae blade galanthamine content analysis:
Target gene A encodes the key enzyme in galanthamine route of synthesis, and 4 '-oxygen transmethylases, it can single-minded catalysis
It drops belladonna lily pyridine contraposition methylation and forms the drop belladonna lily pyridine of 4 '-O- methyl, this product is the key precursor object for synthesizing galanthamine
Matter, therefore target gene A expression quantity will affect galanthamine accumulation.It is primary with the blade that 1ml is transferred to target gene A Bulbus Lycoridis longitubae
The Leaves Protoplast that plastid solution and 1ml are transferred to the Bulbus Lycoridis longitubae of empty vectors is sample to be tested, and 70% ethyl alcohol of 2mL is added,
Ultrasonic extraction 2 times, each 28min, then 12000rpm is centrifuged 10min, takes supernatant, and nitrogen, which blows to be concentrated into, closely to be done, and 1mL methanol is multiple
It is molten, cross 0.22 μm of filter membrane.
Appropriate galanthamine standard items (Sinopharm Chemical Reagent Co., Ltd.) 0.010g is weighed with assay balance precision
(Gal, Galanthamine, C17H21NO3, molecular weight 287.359), with methanol dissolution and constant volume is to 100ml, obtains its standard
Stock solution, Stock concentrations are 100 μ g/mL；Accurately 1mL standard reserving solution is pipetted to be placed in 100mL volumetric flask, it is fixed with methanol
Hold the hybrid standard intermediate fluid for being made into 1.0 μ g/mL；Standard intermediate fluid is pipetted as needed, respectively with methanol dilution at concentration
The standard working solution of 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL.
Galanthamine standard solution is measured using the chromatograph for second order ms instrument of having connected, utilizes the peak face measured
Volume data and galanthamine concentration of standard solution draw standard curve, obtain linear equation, Y=7.7e6X+8.4e3, related coefficient
R2=0.9998.Will sample analytes mass spectrum peak area substitute into the obtained linear equation of step in, obtain solution Garland he
Quick concentration C (unit ng/ml), further according to the methanol volume V (unit ml) of redissolution, extension rate D and sample quality
(unit g) calculates the content m (unit ng/g) of galanthamine in sample, calculation formula are as follows: m=C × V × D/M (its to M
In, galanthamine content in m=sample, unit ng/g；C=sample galanthamine concentration, unit ng/ml；The first that V=redissolves
Alcohol volume, unit ml；D=extension rate；M=sample quality, unit g) are finally calculated and are transferred to target gene A's
Galanthamine content is 550mg/g in Bulbus Lycoridis longitubae Leaves Protoplast, is transferred to the Bulbus Lycoridis longitubae Leaves Protoplast of empty carrier
Middle galanthamine content is 417mg/g.Illustration purpose Gene A is converted to function in protoplast.
2 target gene A of embodiment converts Hipeastrum vittalum's protoplast
(1) the Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait seedling (4 months seedling that the sowing of Hipeastrum vittalum's seed generates) of diameter 0.5cm, dark processing are chosen
12h cuts seedling leaves, weighs 1g blade, is 1mm or so wide fragment by blade cuts；
(2) 10ml reagent A is added in chip material obtained in step (1), carries out being protected from light enzymatic hydrolysis, 28 DEG C of temperature, shake
Processing 4h, shaking speed 50rpm are slowly shaken on bed, are gently inhaled to beat with the suction pipette head for removing tip after the completion of enzymatic hydrolysis and be mixed
Solution is closed, sufficiently release protoplast；
The 2 reagent A ingredients are shown in Table 9
9 example of table, 2 reagent A ingredient
(3) protoplast solution that step (2) obtain is filtered with 50 μm of strainers, collects filtrate, is centrifuged at 60g
10min, centrifuging temperature are 4 DEG C.Precipitating is centrifuged 4 times with 10ml reagent B repeated washing, and last centrifugation is suspended with 1ml reagent C
Protoplast.Fig. 2 is according to said extracted step operation from 1g Hipeastrum vittalum's blade, and finally obtained protoplast figure passes through
After blood counting chamber counts, the protoplasm somatocyte number that every gram of blade extracts can be determined.
The reagent B component is shown in Table 10, and reagent C ingredient is shown in Table 11
10 example of table, 2 reagent B component
11 example of table, 2 reagent C ingredient
The Protoplast suspension obtained in step (3) is gently inhaled to beat with the suction pipette head for removing tip and is allowed to be distributed
Uniformly.Clean one piece of blood counting chamber is taken, draws 10 μ l plasm body fluid with pipettor, is allowed to repeat to count full of count block
It number 5 times, is averaged.According to the weight 1g being added in step (1), calculating the protoplasm somatocyte number that every gram of blade obtains is
2.1×106A/gram.
(4) 5 μ g target gene A plasmids and 105 μ l reagents are added in the Protoplast suspension for taking 100 μ l steps (3) to obtain
D is converted, (after the CDS that the plasmid of target gene A is configured to target gene A removes terminator codon, by BsaI/
Eco31I digestion is connected on pBWA (V) the HS-35S-gfp-NOS carrier for having merged green fluorescent protein.) room temperature dark culture
After 10min, the reagent B of 420 μ l is added, is centrifuged 5min in 60g, abandons supernatant, 1ml reagent B is added in precipitating, after cultivating 14h, detection
Conversion results.
Reagent D ingredient described in example 2 is shown in Table 12
12 example of table, 2 reagent D ingredient
Hipeastrum vittalum's blade galanthamine content analysis:
Target gene A encodes the key enzyme in galanthamine route of synthesis, and 4 '-oxygen transmethylases, it can single-minded catalysis
It drops belladonna lily pyridine contraposition methylation and forms the drop belladonna lily pyridine of 4 '-O- methyl, this product is the key precursor object for synthesizing galanthamine
Matter, therefore target gene A expression quantity will affect galanthamine accumulation.The blade plasm of target gene A Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait is transferred to 1ml
The Leaves Protoplast that body and 1ml are transferred to the Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait of empty vectors is sample to be tested, and 70% ethyl alcohol of 2mL, ultrasonic extraction is added
2 times, each 28min, then 12000rpm is centrifuged 10min, takes supernatant, and nitrogen, which blows to be concentrated into, closely to be done, and 1mL methanol redissolves, and crosses 0.22 μ
M filter membrane.
Appropriate galanthamine standard items (Sinopharm Chemical Reagent Co., Ltd.) 0.010g is weighed with assay balance precision
(Gal, Galanthamine, C17H21NO3, molecular weight 287.359), with methanol dissolution and constant volume is to 100ml, obtains its standard
Stock solution, Stock concentrations are 100 μ g/mL；Accurately 1mL standard reserving solution is pipetted to be placed in 100mL volumetric flask, it is fixed with methanol
Hold the hybrid standard intermediate fluid for being made into 1.0 μ g/mL；Standard intermediate fluid is pipetted as needed, respectively with methanol dilution at concentration
The standard working solution of 2ng/mL, 6ng/mL, 20ng/mL, 60ng/mL and 100ng/mL.
Galanthamine standard solution is measured using the chromatograph for second order ms instrument of having connected, utilizes the peak face measured
Volume data and galanthamine concentration of standard solution draw standard curve, obtain linear equation, Y=6.41e6X+6.56e6, phase relation
Number R2=0.9989.The mass spectrum peak area of sample analytes is substituted into the obtained linear equation of step, solution Garland is obtained
His quick concentration C (unit ng/ml), further according to the methanol volume V (unit ml) of redissolution, extension rate D and sample matter
Measuring M, (unit g) calculates the content m (unit ng/g) of galanthamine in sample, calculation formula are as follows: m=C × V × D/M (its
In, galanthamine content in m=sample, unit ng/g；C=sample galanthamine concentration, unit ng/ml；The first that V=redissolves
Alcohol volume, unit ml；D=extension rate；M=sample quality, unit g) are finally calculated and are transferred to target gene A's
Galanthamine content is 72mg/g in Hipeastrum vittalum's Leaves Protoplast, is transferred to Garland in Hipeastrum vittalum's Leaves Protoplast of empty carrier
His quick content is 63mg/g.
3 target gene B of embodiment converts Bulbus Lycoridis longitubae protoplast
(1) the Bulbus Lycoridis longitubae seedling (1 year raw seedling that Bulbus Lycoridis longitubae cutting propagation generates) of diameter 1.0cm is chosen, it is dark
12h is handled, seedling leaves are cut, 3g blade is weighed, is 1mm or so wide fragment by blade cuts；
(2) chip material obtained in 20ml reagent A processing step (1) is injected, be protected from light enzymatic hydrolysis, 28 DEG C of temperature,
Slowly concussion processing 5h, shaking speed 50rpm, enzymatic hydrolysis are used the suction pipette head for removing tip gently to inhale after the completion and are beaten on shaking table
Mixed solution, sufficiently release protoplast；
Reagent A ingredient described in example 3 is shown in Table 13
13 example of table, 3 reagent A ingredient
(3) protoplast solution that step (2) obtain is filtered with 40 μm of strainers, collects filtrate, is centrifuged at 60g
5min, centrifuging temperature are 4 DEG C.Precipitating is centrifuged 4 times with 10ml reagent B repeated washing, and last centrifugation is suspended with 1ml reagent C
Protoplast.
Reagent B component described in example 3 is shown in Table 14, and reagent C ingredient is shown in Table 15
14 example of table, 3 reagent B component
15 example of table, 3 reagent C ingredient
The protoplast obtained in step (3) is gently inhaled to beat with the suction pipette head for removing tip and is allowed to be evenly distributed.
Clean one piece of blood counting chamber is taken, draws 10 μ l plasm body fluid with pipettor, is allowed to full of count block, repeat count 5 times,
It is averaged.According to the weight 3g being added in step (1), the protoplasm somatocyte number 1.85 × 10 that every gram of blade obtains is calculated7
A/gram.
(4) 10 μ g target gene B plasmid vectors and 110 μ l reagents are added in the protoplast for taking 100 μ l steps (3) to obtain
D, after the CDS that the plasmid of target gene B is configured to target gene B removes terminator codon, by BamHI/XbaI digestion
(Thermo Scientific), is connected on the pCAMBIA1301-35S-GFP-NOS carrier for having merged green fluorescent protein
(Changsha Yingrun Biotechnology Co.,Ltd).After room temperature dark culture 20min, the reagent B of 440 μ l is added,
It is centrifuged 5min in 60g, abandons supernatant, 1ml reagent B is added in precipitating, after cultivating 12h, detects conversion results.
Reagent D ingredient described in example 3 is shown in Table 16
16 example of table, 3 reagent D ingredient
The expression feelings of target gene in Bulbus Lycoridis longitubae protoplast are observed under laser confocal microscope (Nikon C2-ER)
Condition, as shown in Figure 4.The result shows that conversion target gene B is expressed in protoplast, the protoplast of target gene B is converted
In, green florescent signal concentrates in the chloroplaset of protoplast, shows that gene is expressed in chloroplaset.The method of the present invention can be with
Target gene is subjected to transient expression in Bulbus Lycoridis longitubae protoplast, and following gene function verifying can be carried out:
The analysis of Bulbus Lycoridis longitubae leaf chlorophyll acid and esters content:
Target gene B is protochlorophyllide oxidoreducing enzyme B, is the key enzyme in chlcrophyll biosynthesis approach, can
Chlorophyllide is generated to be catalyzed protochlorophyllide, expression quantity will affect the accumulation of chlorophyllide.Purpose is transferred to 1ml
The Leaves Protoplast that the Leaves Protoplast and 1ml of gene B Bulbus Lycoridis longitubae are transferred to the Bulbus Lycoridis longitubae of empty vectors is to test sample
80% acetone of 1mL and 2mL n-hexane is added in product, and concussion mixes, and 1500g is centrifuged 3 minutes, takes lower layer's acetone layer extract, benefit
Fluorescent value is recorded at 667nm with sepectrophotofluorometer (Beckman DTX-800), measures the content of chlorophyllide.Root
According to Beer-Lambert law, calculation formula is extrapolated are as follows: C=A667/ 74.9 (wherein, C=sample Determination of Chlorophyll acid esters concentration, it is single
Position is mM；A667Fluorescent value at=667nm, 74.9 be molar absorption coefficient), the length for being transferred to target gene B is finally calculated
Cylinder short-tube lycoris Leaves Protoplast Determination of Chlorophyll acid esters concentration is 158.09mM, is transferred to the Bulbus Lycoridis longitubae Leaves Protoplast of empty carrier
Determination of Chlorophyll acid esters concentration is 132.27mM.
Sequence table
<110>Academy of Agricultural Sciences, Shanghai City
<120>a kind of method of target gene conversion protoplast
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 720
<212> DNA
<213>Bulbus Lycoridis longitubae (Lycoris longituba)
<400> 1
atgggtgcta gccaagatga ttatgcacta atccacaaga atattttgca tagtgaagat 60
cttcttaagt acatattgga gactagtgtt tatccaagag agcatgaaca gctcaagggg 120
ttgagggagg tgactgagaa acatgaatgg agtatggcgc ttgtcgcagc cgatgaagga 180
ttatttcttt ctatgttgtt aaagctcatg aatgccaaga gaaccattga gattggtgta 240
tacaccggtt attctctgct cacaaccgct ttggctttac cagaagatgg aaagataacg 300
gcaattgacg tcaacaagtc ctactttgag ataggactgc catttattca gaaagcagga 360
gttgagcata agatcaattt tattgaatca gaagcacttc ctgttcttga tcaaatgctt 420
caagagatga aggaagaaga cctctacgac tttgcatttg tcgatgcaga caaaccaaac 480
tatgctaatt accacgagcg attagtgaag cttgtcaggg ttggaggagc aatcgtttac 540
gacaacacgc tctggttcgg aactgtagca tttccagaat atccaggcct tcatccggaa 600
gaggaggagt gtagggtctc tttcagaaac ctgaataagc tcttggcagc tgatccccgt 660
gtcgagatat cccaagtctc aatcggcgat ggactgacta tttgccgacg tctttattga 720
<210> 2
<211> 1209
<212> DNA
<213>rice (Oryza sativa)
<400> 2
atggctctcc aggcggccac caccacctcc ttcctcccct ccgcgctctc cgcccgcaag 60
gagggagcgg tgaaggactc ggcgttcttg ggcgttcgtc tcggcgacgg gctcaagctg 120
gagaccagtg ctctcggcct tcgcaccaag agggtgagca cgtcgtcggt ggccatccgc 180
gcgcaggcgt cggcggcggt gtcgtccccg acggtgacgc cggcgtcgcc gtcgggcaag 240
cagacgctgc gcaagggcac ggcggtcatc accggcgcgt cgtccgggct tggcctcgcg 300
acggcgaagg cgctggcgga gacgggcagg tggcacgtcg tcatggggtg ccgcgacttc 360
ctcaaggcgt cgcgcgccgc caaggccgcc ggcatggaga agggcagcta caccatcgtc 420
cacctcgacc tggcgtcgct cgacagcgtc aggcagttcg tcgccaacgt ccggcggctg 480
gagatgcccg tcgacgtggt ggtgtgcaac gccgccgtgt accagcccac cgccaagcag 540
ccgagcttca ccgccgacgg cttcgagatg agcgtcggcg tcaaccacct cgggcacttc 600
ctcctcgccc gcgagctcct cgccgacctc acctcctccg actacccctc caagcgcctc 660
atcatcgtcg gctccatcac cgggaacacg aacacgctgg cggggaacgt gccgccgaag 720
gcgaacctgg gggacctccg ggggctcgcc tcgggcctcg acggcgtgtc gagctccgcc 780
atgatcgacg gcggcgagtt cgacggcgcc aaggcctaca aggacagcaa ggtgtgcaac 840
atgctgacga tgcaggagtt ccaccgccgg taccacggcg agaccggggt gacgttcgcg 900
tcgctctacc ccgggtgcat cgccaccacg ggcctcttcc gggagcacgt cccgctgttc 960
cgcctcctct tcccgccctt ccagaagtac atcaccaagg gctacgtctc cgaggaggag 1020
gccggcaagc ggctggccca ggtcgtcagt gaccccagcc tcaccaagtc cggggtgtac 1080
tggagctgga acaacaactc ggcctcgttc gagaaccagc tctccgagga ggcctccgat 1140
ccggagaagg ccaagaaggt ctgggagctc agcgagaagc tcgtcggctt ggccgatcac 1200
gatcagtga 1209
Claims (4)
1. a kind of extracting method of protoplast, it is characterised in that this method comprises the following steps:
(1) by amrallid seedling, after dark processing 10-12h, seedling leaves raw material disposal: are cut to fragment；
(2) enzymatic hydrolysis release protoplast: fragment obtained in step (1) being put into reagent A, 22-28 DEG C of temperature, concussion processing
1-6h obtains mixed solution；
(3) purifying of protoplast: the mixed solution that step (2) obtain is filtered with 40-50 μm of strainer, collects filtrate,
Filtrate is centrifuged 5-10min, and centrifuging temperature is 4 DEG C, collects precipitating；Precipitating is cleaned with reagent B, is centrifuged, last centrifugation examination
Agent C suspends, and obtains Protoplast suspension；
The wherein reagent A are as follows: Cellulase R10:0.05-0.5g, Macerozyme R10:0.01-0.5g,
Mannitol:1-10g, MES:0.01-1.0g, CaCl2: 0.01-1.0g, H2O: 20ml volume is supplied；
Reagent B are as follows: NaCl:0.45-4.5g, CaCl2: 0.5-6.0g, KCl:0.01-1.0g, MES:0.02-2.0g, H2O: it supplies
100ml volume；
Reagent C are as follows: Mannitol:0.5-5.0g, MgCl26H2O:0.01-1.0g, MES:0.001-0.1g, H2O: it supplies
10ml volume.
2. the protoplast that extracting method described in claim 1 obtains.
3. a kind of method of protoplast described in target gene conversion claim 2, it is characterised in that this method are as follows:
In Protoplast suspension, target gene plasmid vector and reagent D are sequentially added, after room temperature dark culture 10-30min,
Realize the conversion of target gene；
Wherein reagent D are as follows: PEG4000:0.1-0.5g, Mannitol:0.01-0.15g, CaCl2: 0.0012-0.12g, H2O: it mends
Sufficient 1ml volume.
4. a kind of method of target gene conversion protoplast, it is characterised in that this method comprises the following steps:
(1) by amrallid seedling, after dark processing 10-12h, seedling leaves raw material disposal: are cut to fragment；
(2) enzymatic hydrolysis release protoplast: fragment obtained in step (1) being put into reagent A, 22-28 DEG C of temperature, concussion processing
1-6h obtains mixed solution；
(3) purifying of protoplast: the mixed solution that step (2) obtain is filtered with 40-50 μm of strainer, collects filtrate,
Filtrate is centrifuged 5-10min, and centrifuging temperature is 4 DEG C, collects precipitating；Precipitating is cleaned with reagent B, is centrifuged, last centrifugation examination
Agent C suspends, and obtains Protoplast suspension；
(4) conversion of target gene: the Protoplast suspension for taking step (3) to obtain sequentially adds target gene plasmid vector
And reagent D, after room temperature dark culture 10-30min, realize the conversion of target gene；
The wherein reagent A are as follows: Cellulase R10:0.05-0.5g, Macerozyme R10:0.01-0.5g,
Mannitol:1-10g, MES:0.01-1.0g, CaCl2: 0.01-1.0g, H2O: 20ml volume is supplied；
Reagent B are as follows: NaCl:0.45-4.5g, CaCl2: 0.5-6.0g, KCl:0.01-1.0g, MES:0.02-2.0g, H2O: it supplies
100ml volume；
Reagent C are as follows: Mannitol:0.5-5.0g, MgCl26H2O:0.01-1.0g, MES:0.001-0.1g, H2O: it supplies
10ml volume；
Reagent D are as follows: PEG4000:0.1-0.5g, Mannitol:0.01-0.15g, CaCl2: 0.0012-0.12g, H2O: it supplies
1ml volume.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343717A (en) * | 2019-07-13 | 2019-10-18 | 福建农林大学 | The method for building up of China fir exogenous gene high-efficient instantaneous conversion system |
| CN112753410A (en) * | 2021-01-13 | 2021-05-07 | 上海科立特农科（集团）有限公司 | Method for improving alkaloid content in lycoris by using exogenous substances |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402938A (en) * | 2008-11-17 | 2009-04-08 | 中国科学院植物研究所 | Method for producing protoplast |
| US20120042409A1 (en) * | 2009-01-15 | 2012-02-16 | Anthony Conner | Plant transformation using dna minicircles |
| CN107267549A (en) * | 2017-07-06 | 2017-10-20 | 江苏省中国科学院植物研究所 | A kind of method of the middle mesophyll protoplast of mountain China fir kind 406 separation, purifying and Efficient Conversion |
| CN107462656A (en) * | 2017-07-04 | 2017-12-12 | 上海市农业科学院 | A kind of method of galanthamine content in quick detection amrallid |
| CN108315292A (en) * | 2018-04-25 | 2018-07-24 | 上海市农业科学院 | A kind of extracting method of lycoris plants protoplast |
-
2019
- 2019-01-07 CN CN201910010502.5A patent/CN109554329A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402938A (en) * | 2008-11-17 | 2009-04-08 | 中国科学院植物研究所 | Method for producing protoplast |
| US20120042409A1 (en) * | 2009-01-15 | 2012-02-16 | Anthony Conner | Plant transformation using dna minicircles |
| CN107462656A (en) * | 2017-07-04 | 2017-12-12 | 上海市农业科学院 | A kind of method of galanthamine content in quick detection amrallid |
| CN107267549A (en) * | 2017-07-06 | 2017-10-20 | 江苏省中国科学院植物研究所 | A kind of method of the middle mesophyll protoplast of mountain China fir kind 406 separation, purifying and Efficient Conversion |
| CN108315292A (en) * | 2018-04-25 | 2018-07-24 | 上海市农业科学院 | A kind of extracting method of lycoris plants protoplast |
Non-Patent Citations (3)
| Title |
|---|
| MATTHEW B.KILGORE ET AL.: "Cloning and Characterization of a Norbelladine 4’-O-Methyltransferase Involved in the Biosynthesis of the Alzheimer’s Drug Galanthamine in Narcissus sp. aff. Pseudonarcissus", 《PLOS ONE》 * |
| 孙剑秋等: "《微生物原生质体技术》", 30 April 2008, 东北林业大学出版社 * |
| 左伟勇等: "《细胞工程技术》", 31 August 2014, 重庆大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343717A (en) * | 2019-07-13 | 2019-10-18 | 福建农林大学 | The method for building up of China fir exogenous gene high-efficient instantaneous conversion system |
| CN112753410A (en) * | 2021-01-13 | 2021-05-07 | 上海科立特农科（集团）有限公司 | Method for improving alkaloid content in lycoris by using exogenous substances |
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