CN115178358A - Method and device for rapidly obtaining stably expressed apple transgenic root system - Google Patents
Method and device for rapidly obtaining stably expressed apple transgenic root system Download PDFInfo
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- CN115178358A CN115178358A CN202210726614.2A CN202210726614A CN115178358A CN 115178358 A CN115178358 A CN 115178358A CN 202210726614 A CN202210726614 A CN 202210726614A CN 115178358 A CN115178358 A CN 115178358A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
- B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
- B02C19/00—Other disintegrating devices or methods
- B02C19/08—Pestle and mortar
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/06—Roots
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
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- C12N15/8205—Agrobacterium mediated transformation
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Abstract
The invention relates to the field of modern biotechnology, in particular to a device for rapidly obtaining a stably expressed transgenic apple root system, which comprises a base, wherein a mortar is movably inserted into the upper surface of the base, a jacking plate is sleeved at the upper end of the base, a planetary wheel mechanism is arranged at the front end of the jacking plate, a top plate is placed at the upper end of the mortar, a guide shaft movably penetrates through the middle shaft of the upper end surface of the top plate, a rotating rod movably penetrates through the upper end surface of the top plate, which is close to the edge, and is driven to rotate around the guide shaft through the planetary wheel mechanism and simultaneously rotate, a grinding head extending into the inner side of the mortar is fixedly connected to the lower end of the rotating rod, a connecting rod is fixedly connected to one side of the lower end surface of the top plate, which is far away from the rotating rod, and a method for rapidly obtaining a stably expressed transgenic apple root system is also provided.
Description
Technical Field
The invention relates to the field of modern biotechnology, in particular to a method and a device for rapidly obtaining a stably expressed apple transgenic root system.
Background
Transgenic technology generally refers to the process of altering certain traits in an organism by artificially isolating and recombining a desired target gene and integrating it into the genome of the organism using modern biotechnology means. The process can change the existing genetic characteristics of plants by silencing and inhibiting or knocking out endogenous genes except for introducing new exogenous genes, thereby obtaining new genetic characteristics.
Transgenic technology of plants mainly refers to the insertion of exogenous DNA into recipient cells using cloning and other methods, among which the most representative methods are: vector mediated methods, direct injection methods, and the like. Currently, the most widely used and most effective method is the Agrobacterium-mediated method. The method takes agrobacterium as a vector, introduces a target plasmid into the agrobacterium, and then implants the agrobacterium into a plant cell to achieve the purpose of cell transformation. The transgenic technology has outstanding contribution in the aspects of screening high-quality characters of plants, cultivating excellent offspring, reducing the harm of diseases and pests, improving the yield and the like.
In model plants, such as arabidopsis thaliana, tomato and other plants, a transgenic system is relatively mature, while in apples, in view of complexity of apple genome, obtaining stable apple transgenic plants is relatively difficult, and meanwhile, the traditional apple transgenic method has a low success rate and a series of problems of long transformation time, low survival rate of seedlings and the like. Therefore, the research on how to rapidly obtain the stable transgenic apple plants is particularly important for scientific research on apples.
Disclosure of Invention
The invention aims to solve the defects in the background art, and provides a device for quickly obtaining a transgenic apple root system with stable expression, which comprises a base, wherein a mortar is movably inserted into the upper surface of the base, a jacking plate is sleeved at the upper end of the base, a planetary wheel mechanism is arranged at the front end of the jacking plate, a top plate is placed at the upper end of the mortar, a guide shaft movably penetrates through the middle shaft of the upper end surface of the top plate, a rotating rod movably penetrates through the upper end surface of the top plate, the rotating rod drives the rotating rod to rotate around the guide shaft through the planetary wheel mechanism and simultaneously rotate, a grinding head extending into the inner side of the mortar is fixedly connected to the lower end surface of the rotating rod, a connecting rod is fixedly connected to one side of the top plate, which is far away from the rotating rod, a stirring plate is rotatably connected to the lower end of the connecting rod, and an injection part is movably inserted into the upper end port of the mortar, and close to one side.
Preferably, planet wheel mechanism includes that fixed connection installs ring gear, the fixed mounting of terminal surface under the set casing motor at the set casing upper end at set casing, fixed embedding of top connection board front end, the lower extreme of motor passes through drive shaft fixedly connected with drive gear, drive gear is located the inboard and the isoaxletree of ring gear, be provided with the driven gear who is no less than two around the drive gear, the homogeneous phase meshing between driven gear and ring gear and drive gear, a plurality of driven gear's upper surface is provided with the connecting plate jointly, driven gear passes through pivot and connecting plate normal running fit, just drive shaft activity runs through the connecting plate, the upper end fixed connection of dwang is at one of them driven gear's lower terminal surface, the upper end fixed connection of guide shaft is at drive gear's lower terminal surface.
Preferably, the surface activity cover of guide shaft is equipped with first bowling, the fixed embedding of up end of roof has first arc frame, the inboard of first arc frame is gone into to first bowling activity card, the surface of guide shaft is close to the fixed cover of upper end department and is equipped with last spacing dish.
Preferably, the up end of roof has been seted up and has been passed the mouth, the port department fixedly connected with contact ring that passes the mouth, the below department sliding sleeve that the surface of dwang is close to the roof is equipped with second cover ball, the outside cover of dwang is equipped with the shrouding, fixed embedding has second arc frame on the inner wall of shrouding, the activity of second cover ball imbeds the inboard of second arc frame, the fixed cover in below that the surface of dwang is close to second cover ball is equipped with down the spacing dish.
Preferably, the injection part includes that fixed connection is close to the fixed plate of one side department at the terminal surface before the base, the front end department fixed mounting of fixed plate has the injection pipe, the one end of injection pipe is passed through elastic mechanism sliding sleeve and is equipped with the inserted block, the slot has been seted up to the upper end port department of mortar, the inboard of inserted block sliding insertion slot, elastic mechanism includes that fixed cover establishes at the fixed disk of injection pipe surface, movable sleeve establishes the compression spring in the injection pipe outside, compression spring is located between fixed disk and the inserted block, compression spring's both ends respectively with fixed disk and inserted block fixed connection, the spout has been seted up on the inner wall of inserted block, the protruding conducting bar that is equipped with of surface of injection pipe, conducting bar and spout sliding fit.
Preferably, the up end fixedly connected with sleeve of base, the lower extreme slip of mortar inserts to telescopic inboard, the notch has been seted up to the upper end of base, the lower extreme of top fishplate bar is provided with the embedded groove, the inboard of notch is gone into to the embedded groove slip card.
Preferably, a clamping groove is formed in the lower end of the connecting rod, a connecting block is fixedly connected to the upper end face of the poking plate, the connecting block is movably clamped into the inner side of the clamping groove, a connecting shaft is fixedly connected to the inner side of the clamping groove, the connecting block is rotatably sleeved on the outer surface of the connecting shaft, and two symmetrical limiting faces are formed in the inner wall of the clamping groove
Based on the device, the method for rapidly obtaining the transgenic root system of the apple with stable expression is also provided, and comprises the following steps:
s1, transforming the full-length segment of the apple gene to an agrobacterium expression vector by an escherichia coli transformation method;
s2, transferring the target vector into agrobacterium rhizogenes by an agrobacterium transformation method;
s3, grinding rooting materials by using the device in an agrobacterium rhizogenes mediated mode, and directionally transforming target genes into root systems of apple plants for expression so as to obtain stably inherited transgenic apple root systems;
wherein the vector is pK7GWIWG2-RFP, and the final concentration of the vector transformation is 100 ng/mu l.
Preferably, the agrobacterium is MSU440 rhizogenes agrobacterium competent, and the e.coli is Trans5 alpha e.coli competent.
Preferably, agrobacterium is dissolved in 10mM MES +10mM MgC l2 Infecting in the solution, and scratching the stem of plant material before infecting, wherein the plant material is Malus plant.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention mainly obtains a stably expressed transgenic root system according to the transformation characteristics of the agrobacterium tumefaciens. Compared with the traditional leaf disc method, the method has the advantages of short time consumption, simple operation and high transformation efficiency, and simultaneously avoids plant death in the process of seedling exercising; the transgenic root system obtained by the invention can be used for carrying out a series of related scientific researches, such as the researches on the physicochemical property and the cell signal transduction characteristic of roots; therefore, the method has a great application prospect in the field of plant research.
2. After taking out the plant stem section in ultra-low temperature refrigerator, arrange the inboard of mortar in, make drive gear rotate through the motor for driven gear rotates around drive gear, and the rotation takes place simultaneously, even make the dwang rotate, rotates simultaneously round the guide shaft, and the grinding head rotates, makes the inboard material of mortar smashed, and along with the rotation of grinding head, the board is dialled simultaneously also rotates round the guide shaft, shovels the material of inboard, thereby makes the grinding smash comparatively evenly.
3. Following the traditional process, pour into liquid dry ice into the filling tube in this in-process, liquid dry ice is behind the inboard gasification of mortar, the volume grow, the roof of top is moved to the top, and then make the setting plate shift up, because gaseous outflow is irregular, make the roof take place anomalous swing, because first cover ball can rotate at the inboard of first arc frame, the guide shaft can slide from top to bottom for first cover ball simultaneously, consequently, the jam problem can not take place, the second cover ball can rotate at the inboard of second arc frame, the dwang can slide from top to bottom to the second cover ball relatively, consequently, the jam problem can not take place, and when the dwang rotates around the guide shaft, the border contacts with passing the mouth, and then make the roof rotate, thereby ensured the connecting rod rotates, the setting plate rotates simultaneously, and because the top of gas, the setting plate can the luffing motion, rotate around the guide shaft, therefore the process of free movement has promoted the stirring of inboard material, and then promoted the even degree of grinding.
4. Because the inserted block inserts the inboard to the slot, can ensure the stability of mortar, when promoting the inserted block, after the extrusion compression spring, the upper end of mortar is not by spacingly, can take out the mortar this moment to in the derivation of inboard material, through lifting the top connection board, can make embedded groove and notch part mutually, in order to structure such as grinding head, stirring board take out, thereby be convenient for add the material or take off the mortar to 2 inboards of mortar.
Drawings
FIG. 1 is a schematic structural diagram of an apparatus for rapidly obtaining a stably expressed transgenic root system of apple according to the present invention;
FIG. 2 is a sectional view of the apparatus for rapidly obtaining a transgenic root system of an apple with stable expression in a mortar of the present invention;
FIG. 3 is a partial cross-sectional view of a mortar of an apparatus for rapidly obtaining a stably expressed transgenic root system of apple of the present invention;
FIG. 4 is an enlarged view of the portion A in FIG. 3 of the device for rapidly obtaining a stably expressed transgenic root system of apple according to the present invention;
FIG. 5 is a schematic view of the position of a poke plate of the device for rapidly obtaining a stably expressed transgenic root system of an apple according to the present invention;
FIG. 6 is a schematic view of the injection part of the device for rapidly obtaining a stably expressed transgenic root system of apple according to the present invention;
FIG. 7 is an enlarged view at B in FIG. 6 of a device for rapidly obtaining a stably expressed transgenic root system of apple according to the present invention;
FIG. 8 is a sectional view at the stationary shell of an apparatus for rapidly obtaining a stably expressed transgenic root line of apple of the present invention;
FIG. 9 is an exploded view of the injection part of the device for rapidly obtaining a stably expressed transgenic root system of apple according to the present invention;
FIG. 10 is a partially disassembled view of an apparatus for rapidly obtaining a stably expressed transgenic root system of apple of the present invention.
1. A base; 2. a mortar; 3. a sleeve; 4. a top plate; 5. a connecting rod; 6. clamping the groove; 7. connecting blocks; 8. a connecting shaft; 9. a limiting surface; 10. a poking plate; 11. a guide shaft; 12. a first set of balls; 13. a first arc frame; 14. an upper limiting disc; 15. rotating the rod; 16. a grinding head; 17. a pass-through port; 18. a contact ring; 19. a second set of balls; 20. a second arc frame; 21. closing plates; 22. a lower limiting disc; 23. a ring gear; 24. a driven gear; 25. a drive gear; 26. a connecting plate; 27. a rotating shaft; 28. a drive shaft; 29. a motor; 30. a stationary case; 31. a fixing plate; 32. an injection pipe; 33. fixing the disc; 34. a compression spring; 35. conducting bars; 36. inserting a block; 37. a chute; 38. a slot; 39. a recess; 40. a groove is embedded; 41. and (6) abutting the board.
Detailed Description
The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments described below are by way of example only, and other obvious variations will occur to those skilled in the art.
The following experimental modes are all routine experimental modes unless otherwise specified.
1. Cultivation of materials
Carrying out vernalization treatment on Malus hupehensis seeds:
selecting Malus hupehensis seeds harvested in the current year, soaking and sterilizing the Malus hupehensis seeds for 24 hours by using 3% urea, cleaning the seeds, draining, burying the seeds in wet sand, covering the surface of the sand with gauze, and standing the sand in a low-temperature environment (4 ℃) for 30-40 days for layering. When the hypocotyl of the seed begins to extend, the seed is taken out from the low-temperature environment, sowed in nutrient soil uniformly mixed with a seedling substrate and vermiculite 1:1, and cultured under the long-day condition (16 h light/8 h dark).
The formula of the nutrient solution is as follows:
wherein the formula of the inorganic mixed solution (without inositol) is (500 mL): glycine (0.1 g) + vitamin B1 (0.02 g) + vitamin B6 (0.025 g) + Niacin (0.025 g)
The macroelement mixture formula is (1L), KNO3 (38 g) + NH4NO3 (33 g) + MgSO4 (7.4 g) + KH2PO4 (3.4 g)
The formula of the mixed solution of the trace elements is (500 mL): mnSO4 (1.69 g) + ZnSO4 (0.86 g) + H3BO3 (0.62 g) + KI (0.083 g) + CuSO4 (0.0025 g) + CoCl2 (0.0025 g) + Na2MnSO4 (0.25 g)
The formula of 200 Xiron salt is (500 mL): na2EDTA (3.73 g) + FeSO4 (2.78 g)
The formulation of 40 × CaCl2 was (500 mL): 6.64gCaCl2-500 mL
The formula of 1000 XKH 2PO4 is (1L): 151g KH2PO4-1L
The formula of 1000 XMgSO 4 is (1L): 121g MgSO4-1L
2. Transformation of plasmids
HiFi Taq DNA polymerase from Takara (Japan) was selected for gene cloning in the following reaction system:
the amplification procedure was 90 deg.C, 3min pretreatment, 94 deg.C 30s,56 deg.C 30s,72 deg.C 1min,30 cycles, 72 deg.C, 5min post-extension, 10 deg.C storage.
The amplified product was subjected to Agarose Gel electrophoresis, and a fragment corresponding to the size of the desired band was excised and recovered using Mini BEST Agarose Gel DNA Extraction Kit from Takara (Japan) by the following method:
1. about 500. Mu.L of colloidal lysate buffer GM was added to the colloidal block, and the colloidal block was dissolved at room temperature or 37 ℃.
2. The spin column was removed and placed on the collection tube. The solution from step 1 was added, centrifuged at 12000rpm for 1 minute, and the waste stream was discarded. Can be centrifuged again, and the recovery rate of DNA can be improved.
3. Buffer WB was added and centrifuged at room temperature for 30 seconds, and the filtrate was discarded.
4. The DNA was eluted at 12000 rpm/separation for 1min at room temperature, and the eluted DNA was stored at-20 ℃.
The recovered DNA fragment was ligated to a PMD19-T (Clontech, takara, japan) cloning vector at 16 ℃ overnight in the following manner:
and transforming the connecting product into escherichia coli, and then carrying out plasmid extraction to obtain a large amount of expressed enriched target DNA. The extraction method of the Plasmid selected from the TIANPrep Mini Plasmid Kit (Tiangen, beijing, china) is as follows:
1. the overnight cultured broth was added to a centrifuge tube, centrifuged at 12000rpm for 1min, and the supernatant was decanted.
2. And (5) repeating the step 1.
Adding 250 mu L of the solution P1 and mixing uniformly.
4. Adding 250 μ L of solution P2, turning upside down and mixing uniformly for 6-8 times.
5. Adding 350 μ L of solution P3, turning upside down, mixing well for 6-8 times, generating white flocculent precipitate, and centrifuging at 12000rpm for 10min.
6.500. Mu.L of the equilibrium solution BL was put on a CP3 column, centrifuged at 12000rpm for 1min, the supernatant of 5 was transferred to an adsorption column, centrifuged at 12000rpm for 1min, and the waste liquid was discarded.
7.500 mul of rinsing solution PW was added, centrifuged at 12000rpm for 1min, and the waste solution was discarded.
8. The column was emptied for 2min.
9. 100 mul elution buffer was added dropwise, left for two minutes, centrifuged for 2min and the plasmid solution was collected.
And adding the corresponding restriction enzyme sites to the full length of the cloned gene for amplification to obtain plasmids added with the corresponding restriction enzyme sites, and then carrying out restriction enzyme digestion by using the corresponding restriction enzymes. The Enzyme digestion reaction adopts Quick Cut Enzyme of Takara company (Japan), and the reaction system is as follows:
placing the mixture in a test tube for 1 hour at 37 ℃, detecting the mixture by agarose gel electrophoresis, cutting a target fragment, and recovering the target fragment by a gel recovery kit.
The target fragment to be used is reacted to the corresponding expression vector through connection, and the connection reaction system is as follows:
a freeze-thaw method is selected for agrobacterium transformation:
10 uL plasmid +20 uL MSU440 Agrobacterium rhizogenes competent state is stood for 30min on ice, is frozen for 5min in liquid nitrogen, is bathed for 5min at 37 ℃, is shaken for 3h at 28 ℃ and 100rpm, and is coated on a corresponding culture medium.
3. Infestation of hairy root material
And (3) carrying out amplification culture on the obtained agrobacterium tumefaciens bacterial liquid until OD600=1.0-1.5, dissolving the bacterial liquid by using an MMA solution, diluting the bacterial liquid until OD600=0.5-0.6, and adding acetosyringone into the mixed liquid in a ratio of 1. Scratching the stem of the Malus hupehensis Rehd seedling growing to 3-4 true leaves, immersing the stem together with roots into a bacterial solution, carrying out vacuum treatment for 30min by using a vacuum pump, wherein the pressure is 0.6MPA, and taking out the plant after vacuum treatment, and planting the plant and vermiculite: nutrient soil =1:1, the scratched stem sections were exposed to air and the plug surface was covered with a film. After about two weeks, new roots will grow at the wound, old roots are cut off, new roots are planted in a new culture pot, and identification is performed after the new roots grow.
Detecting the expression quantity of the related gene of the obtained transgenic root system by using real-time fluorescent quantitative PCR to determine whether the target gene is transferred into the root system, wherein the specific detection method comprises the following steps:
extraction of plant Total RNA
1. The cleaned mortar was wrapped with tin foil and placed in an oven at 200 ℃ for 3 hours to remove rnase from the surface of the mortar and stainless steel spoon as required for the experimental materials.
2. Taking out the material stored in the ultra-low temperature refrigerator, grinding the material into powder in liquid nitrogen by using a grinding rod, and gently transferring the powder into a centrifuge tube. Add 500. Mu.L buffer RLS to the tube and shake by hand for 1min.
3.12000rpm, 4 ℃, 2min of centrifugation.
4. Preparing DNAse I DNase removing liquid, adding 10% Reaction Buffer and 30% DNase I into 52 mu L RNase-Free Water, shaking and uniformly mixing, adding into an adsorption column RM, and standing for 20min at room temperature.
5. Add 500. Mu.L of buffer RW2, centrifuge for 1min, discard waste.
6. Step 8 was repeated twice.
7. Placing the adsorption column RM in a centrifugal tube without RNase, dripping 30 μ L of precipitation Buffer, centrifuging for 1min, and placing the non-standby sample in an ultra-low temperature refrigerator for preservation.
In foretell 2's the grinding process, adopt following device to go on, including base 1, base 1's upper surface activity cartridge has mortar 2, base 1's upper end cover is equipped with top board 41, the front end of top board 41 is provided with the planet wheel mechanism, roof 4 has been placed to mortar 2's upper end, epaxial activity of up end of roof 4 runs through has guide shaft 11, the up end of roof 4 is close to the activity of border department and runs through dwang 15, dwang 15 orders about around guide shaft 11 through the planet wheel mechanism and rotates simultaneously, the lower extreme fixedly connected with of dwang 15 stretches into grinding head 16 to mortar 2 inboard, one side fixedly connected with connecting rod 5 of dwang 15 is kept away from to the lower terminal surface of roof 4, the lower extreme of connecting rod 5 rotates and is connected with stirring board 10, mortar 2's upper end port is close to one side department activity cartridge and has the injection portion. Planet wheel mechanism is including fixed connection at the set casing 30 of top connection board 41 front end, fixed embedding installs ring gear 23 at the terminal surface under set casing 30, fixed mounting is at the motor 29 of set casing 30 up end, the lower extreme of motor 29 passes through drive shaft 28 fixedly connected with drive gear 25, drive gear 25 is located the inboard and the isoaxletree of ring gear 23, be provided with no less than two driven gear 24 around drive gear 25, homogeneous phase meshing between driven gear 24 and ring gear 23 and drive gear 25, a plurality of driven gear 24's upper surface is provided with connecting plate 26 jointly, driven gear 24 is through pivot 27 and connecting plate 26 normal running fit, and drive shaft 28 activity runs through connecting plate 26, the upper end fixed connection of dwang 15 is at the lower terminal surface of one of them driven gear 24, the upper end fixed connection of guide shaft 11 is at the lower terminal surface of drive gear 25.
The movable cover of surface of guide shaft 11 is equipped with first cover ball 12, and the fixed embedding of the up end of roof 4 has first arc frame 13, and the inboard of first arc frame 13 is gone into to first cover ball 12 activity card, and the fixed cover of the surface of guide shaft 11 near upper end department is equipped with spacing dish 14 on, and spacing dish 14 on can preventing first cover ball 12 excessive that moves up promptly prevents roof 4 excessive that moves up.
The up end of roof 4 has been seted up and has been passed a mouthful 17, the port department fixedly connected with contact ring 18 that passes a mouthful 17, the below department sliding sleeve that the surface of dwang 15 is close to roof 4 is equipped with second cover ball 19, the outside cover of dwang 15 is equipped with shrouding 21, fixed embedding has second arc frame 20 on the inner wall of shrouding 21, the inboard of second cover ball 19 activity embedding second arc frame 20, the fixed cover in below that the surface of dwang 15 is close to second cover ball 19 is equipped with down spacing dish 22, take place the card problem of dying when can preventing dwang 15 from removing or the rotation.
The injection part includes fixed connection and is close to the fixed plate 31 of one side department at base 1 preceding terminal surface, the front end department fixed mounting of fixed plate 31 has injection pipe 32, the one end of injection pipe 32 is passed through elastic mechanism sliding sleeve and is equipped with inserted block 36, slot 38 has been seted up to mortar 2's upper end port department, inserted block 36 slides and inserts the inboard of slot 38, elastic mechanism includes that fixed cover establishes fixed disk 33 at injection pipe 32 surface, the activity cover establishes compression spring 34 in the injection pipe 32 outside, compression spring 34 is located between fixed disk 33 and inserted block 36, compression spring 34's both ends respectively with fixed disk 33 and inserted block 36 fixed connection, spout 37 has been seted up on the inner wall of inserted block 36, the protruding guide strip 35 that is equipped with of injection pipe 32 surface, guide strip 35 and spout 37 sliding fit, can ensure the translation of inserted block 26, and can not take place the rotation.
The snap-in groove 6 has been seted up to the lower extreme of connecting rod 5, and the up end fixedly connected with connecting block 7 of stirring board 10, the inboard of snap-in groove 6 is gone into to 7 movable cards of connecting block, 6 inboard fixedly connected with connecting axles 8 of snap-in groove, and connecting block 7 rotates the cover and establishes the surface at connecting axle 8, sets up the spacing face 9 of two symmetries on the inner wall of snap-in groove 6, and spacing face 9 can prevent that connecting block 7 from rotating excessively.
In the implementation process, after the plant stem segments are taken out of the ultra-low temperature refrigerator, the plant stem segments are placed inside the mortar 2, the driving gear 25 is rotated through the motor 29, the driven gear 24 rotates around the driving gear 25 and simultaneously rotates, namely, the rotating rod 15 rotates and simultaneously rotates around the guide shaft 11, the grinding head 16 rotates, so that the material inside the mortar 2 is crushed, and the poking plate 10 also rotates around the guide shaft 11 along with the rotation of the grinding head 16 to scoop up the material inside, so that the grinding and crushing are more uniform; according to the traditional process, liquid dry ice is injected into the injection pipe 32 in the process, the liquid dry ice is gasified on the inner side of the mortar 2, the size of the liquid dry ice is increased, the top plate 4 above the injection pipe is jacked to move upwards, and then the stirring plate 10 moves upwards; because the inserted block 36 inserts the inboard to slot 38, can ensure mortar 2's stability, when promoting inserted block 36, after extrusion compression spring 34, mortar 2's upper end is not by spacing, can take out mortar 2 this moment to in the derivation of inboard material, through lifting up the top connection board 41, can make embedded groove 40 and notch 39 separately mutually, so that grinding head 16, stirring board 10 isotructure take out, thereby be convenient for add the material to mortar 2 inboard or take off mortar 2.
Reverse transcription of cDNA
Reverse transcription of cDNA (PrimeScript RT reagent Kit) and removal of genomic DNA were performed using the cDNA reverse transcription Kit from Takala (Takala, dalian).
1. Removal of genomic DNA
Reaction system:
the amount of Extracted RNA was calculated based on the measured concentration of RNA, extracted RNA (μ L) =1000/RNA concentration.
Reacting at 42 deg.C for 2min, and storing on ice.
2. Low fidelity cDNA fragment reverse transcription (SYBR GreenTM-047A)
Reaction system:
reacting at 37 ℃ for 15min, reacting at 85 ℃ for 5s, and storing the non-standby sample in a refrigerator at-20 ℃.
Fluorescence-calibrated polymerase chain reaction (real-time quantitative PCR, qRT-PCR)
Three biological replicates and three technical replicates were performed per target sample using MdACTIN (GenBank accession number: CN 938024) as the reference gene using a BIO-RAD IQ5 quantifier. The data were quantitatively analyzed by 2-. DELTA.CT, and the results were analyzed for the significance of the differences by SPSS22 (ver.22.0), and the data were plotted by Prism7 (ver.7.0.2).
The reaction system of the real-time fluorescent quantitative PCR is as follows:
the reaction procedure of the real-time fluorescent quantitative PCR is as follows:
summarizing the steps, the method for quickly obtaining the transgenic root system of the apple with stable expression comprises the following steps:
s1, transforming the full-length segment of the apple gene to an agrobacterium expression vector by an escherichia coli transformation method;
s2, transferring the target vector into agrobacterium rhizogenes by an agrobacterium transformation method;
s3, directionally transforming the target gene into the root system of the apple plant for expression by using an agrobacterium rhizogenes mediated mode so as to obtain a transgenic apple root system with stable inheritance;
wherein the vector is pK7GWIWG2-RFP, and the final concentration of the vector transformation is 100 ng/. Mu.l.
The agrobacterium is MSU440 rhizogenic agrobacterium competent, and the escherichia coli is Trans5 alpha escherichia coli competent.
Agrobacterium was dissolved in 10mM MES +, 10mM MgC l2 Infecting in the solution, and scratching the stem of plant material before infecting, wherein the plant material is Malus plant.
The invention obtains the stably expressed transgenic root system mainly according to the transformation characteristics of the plant agrobacterium. Compared with the traditional leaf disc method, the method has the advantages of short time consumption, simple operation and high transformation efficiency, and simultaneously avoids plant death in the process of seedling exercising; the transgenic root system obtained by the invention can be used for carrying out a series of related scientific researches, such as the researches on the physicochemical property and the cell signal transduction characteristic of roots; therefore, the method has a wide application prospect in the field of plant research.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The utility model provides a device that is used for obtaining transgenic apple root system of stable expression fast, includes base (1), its characterized in that: the upper surface activity cartridge of base (1) has mortar (2), the upper end cover of base (1) is equipped with top connection board (41), the front end of top connection board (41) is provided with planetary gear mechanism, roof (4) have been placed to the upper end of mortar (2), epaxial activity runs through has guide shaft (11) in the up end of roof (4), the up end of roof (4) is close to border department activity and runs through dwang (15), dwang (15) order about round guide shaft (11) through planet wheel mechanism and rotate and rotation simultaneously, the lower extreme fixedly connected with of dwang (15) stretches into to mortar (2) inboard cartridge (16), one side fixedly connected with connecting rod (5) of dwang (15) are kept away from to the lower terminal surface of roof (4), the lower extreme rotation of connecting rod (5) is connected with stirring board (10), the upper end port of mortar (2) is close to one side department activity and has the injection portion.
2. The device for rapidly obtaining the transgenic root system of the stably expressed apple as claimed in claim 1, wherein: planetary gear mechanism includes that fixed casing (30), the fixed embedding of fixed connection at top board (41) front end install ring gear (23), the motor (29) of fixed casing (30) up end of terminal surface under fixed casing (30), fixed mounting, drive shaft (28) fixedly connected with drive gear (25) are passed through to the lower extreme of motor (29), drive gear (25) are located the inboard and the isoaxle of ring gear (23), be provided with around drive gear (25) and be no less than two driven gear (24), homogeneous phase meshing between driven gear (24) and ring gear (23) and drive gear (25), a plurality of the upper surface of driven gear (24) is provided with connecting plate (26) jointly, driven gear (24) are through pivot (27) and connecting plate (26) normal running fit, just drive shaft (28) activity runs through connecting plate (26), the lower terminal surface at one of them driven gear (24) is connected to the upper end fixed connection of dwang (15), the lower terminal surface of the upper end fixed connection of guide shaft (11) is at drive gear (25).
3. The device for rapidly obtaining the transgenic root system of the stably expressed apple as claimed in claim 1, wherein: the surface activity cover of guide shaft (11) is equipped with first cover ball (12), the fixed embedding of up end of roof (4) has first arc frame (13), the inboard of first arc frame (13) is gone into to first cover ball (12) activity card, the surface of guide shaft (11) is close to the fixed cover of upper end department and is equipped with spacing dish (14) on.
4. The device for rapidly obtaining the transgenic root system of the stably expressed apple as claimed in claim 1, wherein: the up end of roof (4) has been seted up and has been passed mouth (17), the port department fixedly connected with contact ring (18) that passes mouth (17), the below department sliding sleeve that the surface of dwang (15) is close to roof (4) is equipped with second cover ball (19), the outside cover of dwang (15) is equipped with shrouding (21), fixed embedding has second arc frame (20) on the inner wall of shrouding (21), the inboard of second cover ball (19) activity embedding second arc frame (20), the fixed cover in below that the surface of dwang (15) is close to second cover ball (19) is equipped with down spacing dish (22).
5. The device for rapidly obtaining the transgenic root system of the stably expressed apple as claimed in claim 1, wherein: injection portion includes fixed connection and is close to fixed plate (31) of one side department at base (1) preceding terminal surface, the front end department fixed mounting of fixed plate (31) has injection pipe (32), the one end of injection pipe (32) is passed through elastic mechanism sliding sleeve and is equipped with inserted block (36), slot (38) have been seted up to the upper end port department of mortar (2), inserted block (36) slide insert the inboard of slot (38), elastic mechanism establishes fixed disk (33), the activity cover in injection pipe (32) surface and establishes compression spring (34) in the injection pipe (32) outside including fixed cover, compression spring (34) are located between fixed disk (33) and inserted block (36), the both ends of compression spring (34) respectively with fixed disk (33) and inserted block (36) fixed connection, spout (37) have been seted up on the inner wall of inserted block (36), the surface protruding conducting bar (35) of injection pipe (32), conducting bar (35) and spout (37) sliding fit.
6. The device for rapidly obtaining the transgenic root system of the stably expressed apple as claimed in claim 1, wherein: the up end fixedly connected with sleeve (3) of base (1), the lower extreme of mortar (2) slides and inserts the inboard to sleeve (3), notch (39) have been seted up to the upper end of base (1), the lower extreme of top connection board (41) is provided with embedded groove (40), embedded groove (40) slide card goes into the inboard of notch (39).
7. The device for rapidly obtaining the transgenic root system of the stably expressed apples according to the claim 1, wherein: the lower extreme of connecting rod (5) has been seted up the jack-in groove (6), the up end fixedly connected with connecting block (7) of stirring board (10), the inboard of jack-in groove (6) is gone into to connecting block (7) activity card, inboard fixedly connected with connecting axle (8) of jack-in groove (6), connecting block (7) are rotated the cover and are established the surface at connecting axle (8), spacing face (9) of two symmetries are seted up on the inner wall of jack-in groove (6).
8. A method for rapidly obtaining a stably expressed apple transgenic root system is characterized by comprising the following steps: the method comprises the following steps:
s1, transforming the full-length segment of the apple gene to an agrobacterium expression vector by an escherichia coli transformation method;
s2, transferring the target vector into agrobacterium rhizogenes by an agrobacterium transformation method;
s3, grinding rooting materials by using the device of any one of claims 1 to 7 in an agrobacterium rhizogenes mediated mode, and directionally transforming a target gene into a root system of an apple plant for expression so as to obtain a transgenic apple root system with stable inheritance;
wherein the vector is pK7GWIWG2-RFP, and the final concentration of the vector transformation is 100 ng/. Mu.l.
9. The method for rapidly obtaining the transgenic root system of the stably expressed apple according to the claim 1, which is characterized in that: the agrobacterium is infected by MSU440 rooting agrobacterium, and the escherichia coli is infected by Trans5 alpha escherichia coli.
10. The method for rapidly obtaining the transgenic root system of the stably expressed apple according to the claim 1, which is characterized in that: agrobacterium thalli is dissolved in 10mM MES +10mM MgC l2 Infecting in the solution, and scratching the stem of plant material before infecting, wherein the plant material is Malus plant.
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