CN108949820A - Plant fruit-ripening related genes original position instant expression method - Google Patents
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 42
- 230000004345 fruit ripening Effects 0.000 title claims abstract description 40
- 230000014509 gene expression Effects 0.000 title claims abstract description 34
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 42
- 239000000243 solution Substances 0.000 claims abstract description 36
- 241000196324 Embryophyta Species 0.000 claims abstract description 32
- 238000002347 injection Methods 0.000 claims abstract description 21
- 239000007924 injection Substances 0.000 claims abstract description 21
- 241000589158 Agrobacterium Species 0.000 claims abstract description 10
- 230000005070 ripening Effects 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 33
- 240000008790 Musa x paradisiaca Species 0.000 claims description 11
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 11
- 230000010474 transient expression Effects 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 108700008625 Reporter Genes Proteins 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 6
- 230000003204 osmotic effect Effects 0.000 claims description 6
- 235000011430 Malus pumila Nutrition 0.000 claims description 5
- 235000015103 Malus silvestris Nutrition 0.000 claims description 5
- 241000220324 Pyrus Species 0.000 claims description 5
- 235000021017 pears Nutrition 0.000 claims description 5
- 244000144730 Amygdalus persica Species 0.000 claims description 4
- 235000006040 Prunus persica var persica Nutrition 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 229910052564 epsomite Inorganic materials 0.000 claims description 4
- 239000004615 ingredient Substances 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 244000141359 Malus pumila Species 0.000 claims 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims 1
- 239000013604 expression vector Substances 0.000 abstract description 9
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 abstract description 4
- 239000005977 Ethylene Substances 0.000 abstract description 4
- 238000011065 in-situ storage Methods 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 230000003993 interaction Effects 0.000 abstract description 3
- 230000006916 protein interaction Effects 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 238000005259 measurement Methods 0.000 abstract description 2
- 235000016709 nutrition Nutrition 0.000 abstract description 2
- 240000005561 Musa balbisiana Species 0.000 abstract 1
- 235000021015 bananas Nutrition 0.000 abstract 1
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 12
- 230000008520 organization Effects 0.000 description 5
- 241000220225 Malus Species 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
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- 238000000749 co-immunoprecipitation Methods 0.000 description 3
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- 230000002068 genetic effect Effects 0.000 description 2
- 239000002304 perfume Substances 0.000 description 2
- 235000013599 spices Nutrition 0.000 description 2
- JXCKZXHCJOVIAV-UHFFFAOYSA-N 6-[(5-bromo-4-chloro-1h-indol-3-yl)oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid;cyclohexanamine Chemical compound [NH3+]C1CCCCC1.O1C(C([O-])=O)C(O)C(O)C(O)C1OC1=CNC2=CC=C(Br)C(Cl)=C12 JXCKZXHCJOVIAV-UHFFFAOYSA-N 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
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- 101150039352 can gene Proteins 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 230000035614 depigmentation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 101150054900 gus gene Proteins 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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Abstract
The invention discloses a kind of plant fruit-ripening related genes original position instant expression methods, it uses needleless injector, utilize its high-pressure jet principle, Agrobacterium bacterium solution containing corresponding expression vectors is formed into thinner liquid, moment delivers the target tissue region into fruit, then fruit is cultivated under corresponding temperature and humidity conditions, the target gene on correlative expression vector is made to express in situ in target tissue region and play its function.Later period the means such as can accelerate the ripening by natural maturity or ethylene to observe influence of the related target gene to fruit maturation characteristic, can also analyze it to mature influence by measurement injection areas Mature property, nutritional quality parameter and related gene expression.This method can also be expanded further in the research applied to protein interaction, such as with the interaction of the endogenous verifying GAP-associated protein GAP of COIP method.Plant fruit-ripening related genes original position instant expression method design is rationally, easy to operate, quickly can carry out endogenous verifying to plant fruit-ripening related genes such as bananas.
Description
Technical field
The present invention relates to gene engineering technology fields, more particularly, to a kind of plant fruit-ripening related genes original position transient expression
Method.
Background technique
The general growth cycle of fruit is long, thus hope verifies phase with the method that conventional genetic converts as model plant
The period of the function of correlation gene is very long, and the fruit having is to all there are no establish corresponding genetic conversion system at present.This
Outside, relatively other gene function analysis that can analyze Relevant phenotype in seedling stage, plant fruit-ripening related genes functional analysis period
It is longer.Therefore, the function that mature related gene is verified using the method for transient expression is particularly important in fruit.At present
More mature instant expression method has been established in model plant blade, but carries out the method for transient expression in situ on fruit
Be not much and see, in tomato it is paid try out traditional injector for medical purpose containing syringe needle by hand forcibly will be containing corresponding expression vectors
Bacterium solution is injected into pulp organization to study the example of related gene function.However, since the pulp organizations such as banana, apple, pears cause
Close, conventional method is difficult for bacterium solution to be equably injected into pulp organization, bacterium solution four often occurs and dissipates the case where spraying full whole body, behaviour
There is certain difficulty on work, need repeatedly a large amount of injections, then therefrom selects injection and preferably carry out relevant experimental study.
It is difficult to quantify each bacterium solution in this way, repeatability is not strong, and effect is general.In banana, someone cuts banana
It is soaked in after piece in the bacterium solution containing corresponding expression vectors, helps bacterial invasion pulp group in conjunction with ultrasound and the processing vacuumized
It knits, then pulp organization is placed in relevant culture medium and is cultivated, transient expression related gene is come with this.With immersion on banana
Method, process is very cumbersome, and need cut into pieces, be not belonging to transient expression in situ, and be normally only used for studying relevant
Can gene express in pulp organization, can not verify influence of the mature related gene to Mature property in this approach.
Summary of the invention
Based on this, it is necessary to provide a kind of plant fruit-ripening related genes original position transient expression that can be quantified and repeatability is strong
Method.
The technical solution that the present invention solves above-mentioned technical problem is as follows.
A kind of plant fruit-ripening related genes original position instant expression method, includes the following steps:
Conversion containing destination gene expression carrier bacterial solution is packed into needleless injector；
The target tissue region that the bacterium solution is injected to fruit to be studied by the way of needleless injection, in the target group
Target gene described in the transient expression of tissue region original position.
The target gene obtains in the following way in one of the embodiments:
The RNA of fruit or after-ripening fruit is synthesized by way of reverse transcription cDNA to get.
Also contain reporter gene on the expression vector in one of the embodiments,.
The conversion is Agrobacterium with bacterium in one of the embodiments,.
The culture medium that the bacterium solution uses in one of the embodiments, is osmotic medium, and ingredient is: 10mM
MgSO4·7H2O, MES and 100 μM of acetosyringone (acetosyringone) that 9mM pH is 5.6.
The fruit to be studied is but not limited to banana, apple, pears or peach in one of the embodiments,.
The fruit to be studied is green mature banana in one of the embodiments,.
Multiple target tissue regions, each mesh are selected on each fruit to be studied in one of the embodiments,
Mark tissue regions inject the bacterium solution each independently.
Plant fruit-ripening related genes original position instant expression method further includes will be described in one of the embodiments,
After bacterium solution injects the target tissue region of fruit to be studied, the step of sealing corresponding injection position.
Plant fruit-ripening related genes original position instant expression method in one of the embodiments, further include: by institute
It states after bacterium solution injects the target tissue region of fruit to be studied, makes in such a way that natural maturity or ripener are accelerated the ripening described wait grind
Fruit maturation is studied carefully, to study influence of the target gene to the fruit maturation characteristic to be studied.
In one of the embodiments, when the bacterium solution to be injected to the target tissue region of fruit to be studied, the bacterium
The bolus injection amount of liquid is up to 300 μ l.
Plant fruit-ripening related genes original position instant expression method of the invention uses needleless injector, utilizes its high-pressure jet
Agrobacterium bacterium solution containing corresponding expression vectors is formed thinner liquid by principle, and moment delivers the target tissue region into fruit,
The bacterium solution of 300 μ l can be delivered every time, and fruit is cultivated under corresponding temperature and humidity conditions then, is made on correlative expression vector
Target gene expresses in situ in target tissue region and plays its function.Later period can be accelerated the ripening by natural maturity or ethylene waits hands
Section observes influence of the related target gene to fruit maturation characteristic, can also by measurement injection areas Mature property (such as color,
Hardness), nutritional quality parameter and related gene expression analyze its to mature influence.
Plant fruit-ripening related genes original position instant expression method of the invention can rapidly and efficiently and equably use conversion
Bacterial solution is delivered to the target tissue region of fruit, cooperates relevant osmotic medium, is more advantageous to bacterial invasion mesh in this way
It marks tissue regions and expresses corresponding target gene, to reach the mesh for quickly identifying that related target gene influences fruit maturation
's.The fruit of plant fruit-ripening related genes original position instant expression method of the invention after injection is substantially seamless, can continue into
Row relevant processing verifies influence of the relevant target gene to After-ripening such as ethylene induced maturation, with this.In addition, this method
It can further expand in the research applied to protein interaction, such as use the endogenous verifying of co-immunoprecipitation (COIP) method
The interaction of GAP-associated protein GAP.
Instant expression method design in plant fruit-ripening related genes original position of the invention is rationally, easy to operate, can be quickly to perfume (or spice)
Any of several broadleaf plants plant fruit-ripening related genes carry out endogenous verifying, and can further be applied to other fruit such as apple, pears, peach etc., at
The verifying of ripe correlation target gene function provides fast and convenient method.
Detailed description of the invention
Fig. 1 is that GUS report carrier delivers poststaining result.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
The present invention provides a kind of plant fruit-ripening related genes original position instant expression methods comprising following steps:
Step 1: the bacterial solution of the conversion containing destination gene expression carrier is packed into needleless injector；
Step 2: bacterium solution is injected to the target tissue region of fruit to be studied by the way of needleless injection, in target group
Tissue region original position transient expression target gene.
Target gene can be obtained through but not limited to such as under type: the RNA of fruit or after-ripening fruit is passed through reverse transcription
Mode synthesize cDNA to get.
Also contain reporter gene, such as antibiotic reporter gene, fluorescent staining reporter gene on expression vector, such as at one
In specific example, expression vector can be the report carrier containing GUS (beta-glucuronidase).
Conversion can be with bacterium but be not limited to Agrobacterium.In a specific example, Agrobacterium is EHA105 root
Cancer Agrobacterium.
The culture medium that bacterium solution uses is preferably osmotic medium, and ingredient is: 10mM MgSO4·7H2O, 9mM pH is 5.6
MES and 100 μM of acetosyringone (acetosyringone).
Fruit to be studied can be but not limited to perfume (or spice) in plant fruit-ripening related genes original position instant expression method of the invention
Any of several broadleaf plants, apple, pears or peach, such as in a specific example, fruit to be studied is green mature banana.
Preferably, multiple target tissue regions are selected on each fruit to be studied, each target tissue region is each independently
Inject bacterium solution.
Further, plant fruit-ripening related genes original position instant expression method further includes that bacterium solution is being injected fruit to be studied
After target tissue region, the step of sealing corresponding injection position.
Further, plant fruit-ripening related genes original position instant expression method further includes that bacterium solution is being injected fruit to be studied
Target tissue region after, make fruit maturation to be studied in such a way that natural maturity or ripener are accelerated the ripening, with research purpose base
The step of because of influence to fruit maturation characteristic to be studied.
When bacterium solution to be injected to the target tissue region of fruit to be studied, the bolus injection amount of bacterium solution is up to 300 μ l, injection
Rapidly and efficiently and uniformity is high, cooperates relevant osmotic medium, can be more advantageous to the expression of bacterial invasion target tissue region
Corresponding target gene, to achieve the purpose that quickly to identify that related target gene influences fruit maturation.Fruit of the invention
The fruit of real maturation related gene original position instant expression method after injection is substantially seamless, can continue relevant processing such as
Ethylene induced maturation verifies influence of the relevant target gene to After-ripening with this.In addition, this method can be opened up further
Exhibition is applied in the research of protein interaction, such as with the interaction of the endogenous verifying GAP-associated protein GAP of COIP method.
The following are specific embodiment parts.
Embodiment 1
The present embodiment is tested using the report carrier containing GUS, the banana pulp tissue GUS dye liquor after injection
(contain X-Gluc) dyeing is displayed in blue, illustrate gus gene can in pulp organization specifically expressing.Steps are as follows for specific experiment.
1. the Agrobacterium single bacterium for choosing the report carrier containing GUS drops down onto LB liquid medium of the 1ml containing corresponding antibiotic,
200rpm cultivates 48h.
2. being forwarded to (25 μM of acetosyringones of supplement) the LB culture medium of 25ml containing antibiotic (such as kanamycins) by 1:100
In, 28 DEG C of 150rpm are incubated overnight to OD600 about 0.7.
3.3000g is centrifuged 10min and collects thallus, and with 10ml osmotic medium, (ingredient is: 10mM MgSO4·7H2O、9mM
MES and 100 μM of acetosyringone (acetosyringone) cleaning thallus that pH is 5.6, repeated washing 1 time.Finally with infiltration
Saturating culture medium resuspension thallus makes final OD600 1.It is stand-by after low speed culture 3h at room temperature.
5. loading 0.3ml bacterium solution to needleless injector (such as Injex-30TM needle-free injection system)
In, it selects intact green mature banana and is injected in opposed flattened position, every banana injects 2-3 needle, is then sealed with adhesive tape
Firmly injection position.It is subsequently placed in 20 DEG C of cultures.
6. after injection 3 days, taking cross section sample, GUS dyeing liquor is added, dehydrated alcohol: vinegar is used in 37 DEG C of 12h processing later
Sour (volume ratio 3:1) solution removes depigmentation, is finally placed in 70% ethyl alcohol and saves, as a result as shown in Figure 1.
The result shows that: leftmost figure illustrates the side trace after injection, shows that bacterium solution can enter the center of fruit；
Intermediate figure be more can uniformly be expressed after the carrier with gus reporter gene enters fruit under the mediation of Agrobacterium and by
The colouring of GUS dyes；The figure of rightmost be without gus reporter gene carrier under the mediation of Agrobacterium enter fruit after cannot
It is coloured by dyestuff.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of plant fruit-ripening related genes original position instant expression method, which comprises the steps of:
Conversion containing destination gene expression carrier bacterial solution is packed into needleless injector；
The target tissue region that the bacterium solution is injected to fruit to be studied by the way of needleless injection, in the targeted tissue areas
Target gene described in the transient expression of domain original position.
2. plant fruit-ripening related genes original position as described in claim 1 instant expression method, which is characterized in that the purpose base
Because obtaining in the following way:
The RNA of fruit or after-ripening fruit is synthesized by way of reverse transcription cDNA to get.
3. plant fruit-ripening related genes original position as described in claim 1 instant expression method, which is characterized in that the expression carries
Also contain reporter gene on body.
4. plant fruit-ripening related genes original position as described in claim 1 instant expression method, which is characterized in that the conversion is used
Bacterium is Agrobacterium.
5. plant fruit-ripening related genes original position as described in claim 1 instant expression method, which is characterized in that the bacterium solution makes
Culture medium is osmotic medium, and ingredient is: 10mM MgSO4·7H2O, MES and 100 μM of acetyl that 9mM pH is 5.6
Syringone.
6. such as plant fruit-ripening related genes original position according to any one of claims 1 to 5 instant expression method, feature exists
In the fruit to be studied is but not limited to banana, apple, pears or peach.
7. such as plant fruit-ripening related genes original position according to any one of claims 1 to 5 instant expression method, feature exists
In selecting multiple target tissue regions on each fruit to be studied, each target tissue region injects each independently
The bacterium solution.
8. such as plant fruit-ripening related genes original position according to any one of claims 1 to 5 instant expression method, feature exists
In further including the step of sealing corresponding injection position after the target tissue region that the bacterium solution is injected to fruit to be studied.
9. such as plant fruit-ripening related genes original position according to any one of claims 1 to 5 instant expression method, feature exists
In, further includes: after the target tissue region that the bacterium solution is injected to fruit to be studied, accelerated the ripening using natural maturity or ripener
Mode make the fruit maturation to be studied, to study influence of the target gene to the fruit maturation characteristic to be studied.
10. such as plant fruit-ripening related genes original position according to any one of claims 1 to 5 instant expression method, feature exists
In when the bacterium solution to be injected to the target tissue region of fruit to be studied, the bolus injection amount of the bacterium solution is up to 300 μ l.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621516A (en) * | 2020-06-01 | 2020-09-04 | 河北农业大学 | Gene transient expression method using in-vivo jujube fruit as material |
| CN112481293A (en) * | 2020-11-13 | 2021-03-12 | 北京农学院 | Method for constructing genetic transformation system by adopting non-isolated grape fruits |
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| DIEGO ORZAEZ等: "Agroinjection of Tomato Fruits. A Tool for Rapid Functional Analysis of Transgenes Directly in Fruit", 《PLANT PHYSIOLOGY》 * |
| STEPHEN BUAH: "REGULATION OF CAROTENOID BIOSYNTHESIS IN BANANA FRUIT", 《DOCTORAL THESIS CENTRE FOR TROPICAL CROPS AND BIOCOMMODITIES INSTITUTE FOR FUTURE ENVIRONMENTS QUEENSLAND UNIVERSITY OF TECHNOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621516A (en) * | 2020-06-01 | 2020-09-04 | 河北农业大学 | Gene transient expression method using in-vivo jujube fruit as material |
| CN111621516B (en) * | 2020-06-01 | 2022-05-31 | 河北农业大学 | A kind of gene transient expression method using in vivo jujube fruit as material |
| CN112481293A (en) * | 2020-11-13 | 2021-03-12 | 北京农学院 | Method for constructing genetic transformation system by adopting non-isolated grape fruits |
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