CN104195170B - A kind of paddy rice vacuum infiltration genetic transforming method - Google Patents
A kind of paddy rice vacuum infiltration genetic transforming method Download PDFInfo
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- CN104195170B CN104195170B CN201410469053.8A CN201410469053A CN104195170B CN 104195170 B CN104195170 B CN 104195170B CN 201410469053 A CN201410469053 A CN 201410469053A CN 104195170 B CN104195170 B CN 104195170B
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Abstract
A kind of paddy rice vacuum infiltration genetic transforming method, a kind of paddy rice vacuum infiltration genetic transformation device of the method carries out.This device establishes through hole in the middle part of supporting table, base plate placed by table, conversion chamber is put on base plate, establish upper and lower cylinder in the middle part of base plate upper and lower surface, on upper and lower cylinder, cover has magnet ring II and I respectively, and lower cylinder and magnet ring I are positioned under desktop, upper cylinder and magnet ring II are positioned at indoor, placing propping up of penetrating fluid container is placed on magnet ring II, and indoor upper inside wall establishes air vent pipe, the vacuum suction adapter of ring-type adapting table and band valve, and bottom nozzle and the top ports of room all establish sealing-ring.This device can after conversion chamber be in vacuum state, then regulates and controls by magnet ring I infiltration that container makes penetrating fluid and plant spike of rice to be transformed contact the goal gene carried out under vacuum state and transform.The inventive method is easy and simple to handle, quick, really can realize paddy rice original position infiltration genetic transformation under vacuum conditions, enhances the invasiveness of Agrobacterium to plant.
Description
Technical field
The present invention relates to a kind of paddy rice vacuum infiltration genetic transforming method, belong to genetically engineered and molecular biology experiment technical field.
Background technology
Paddy rice is the main food crop of global population over half, but Rice Production is subject to the restriction of more and more unfavorable factor (as disease and pest etc.), and therefore, the improvement of rice varieties is very urgent.Transgenic technology can break through obstacle between biological species, realizes the transfer of gene between species, thus provides new way for crop genetic improvement.Utilize transgenic technology to carry out the success or not of genetic improvement to paddy rice, depend on the development and application of effective genetic transforming method to a great extent.Now, known rice transformation method is generally divided into 2 classes: a class is direct gene transformation (comprising particle bombardment, electric shocking method, PEG method, liposome transformation and pollen tube passage method etc.), these class methods are often because cost is high, and operative technique is loaded down with trivial details and seldom apply; Another kind of is by carrier mediated conversion, and widely used is at present agrobacterium-mediated transformation, utilize the method successfully to obtain on paddy rice transgenic line that some have disease and insect resistance, the excellent proterties such as degeneration-resistant.
But there is many deficiencies significantly in traditional agrobacterium-mediated transformation: the realization of conversion have to pass through callus of induce, embryo callus subculture subculture, embryo callus subculture and Agrobacterium Dual culture are carried out to explants such as Mature Embryos Amongs, kanamycin-resistant callus tissue screens and the tissue culture procedures of at least nearly 4 months such as kanamycin-resistant callus tissue differentiation and seedling emergence, its conversion operation is loaded down with trivial details and the cycle is long, person property spends greatly, and high to the technical requirements of operator; Secondly, in the tissue culture procedures of nearly 4 months, the problems such as easy shedder cytometaplasia, form deformity, numerical chromosome change and fertility forfeiture, cause genetic background complicated; Moreover the transformed plant obtained often occurs gene silencing, transformation efficiency is low.Therefore, exploitation is a kind of without the need to passing through group training process, there is the advantages such as easy, quick and efficient, only need namely realize the paddy rice vacuum infiltration conversion method of foreign gene to plant transformation to the of short duration vacuum-treat of plants before blooming, just become another important channel that paddy gene transforms.
Although the research report at present about vacuum infiltration conversion method successful Application is a lot, but be substantially only confined to the individual less plant of Arabidopis thaliana, leaf mustard, Chinese cabbage, tender flower stalk, swede type rape, radish, tobacco etc. is applied, and transformation efficiency is on the low side, what really obtain has the transgenic plant of using value less.Existing vacuum infiltration genetic transformation is mostly with ready-made, the vacuum drier (as shown in figure 14) of small volume is as vacuum infiltration genetic transformation device, its elementary operation is: pour infiltration conversion fluid into vacuum drier, except Arabidopis thaliana plants transforms, remaining vegetable is (as leaf mustard, Chinese cabbage, tender flower stalk, swede type rape, radish, tobacco etc.) be all that plant to be transformed is directly extracted from culturing pot, after cleaning, first plant complete stool to be transformed or part are immersed in the infiltration conversion fluid in vacuum drier, now vacuum drier is in atmospheric pressure state, but plant be immersed in infiltration conversion fluid in start infiltration genetic transformation, then, vacuum drier is made to reach vacuum state with vacuum pump pressure again, continue to permeate genetic transformation (i.e. vacuum-treat) under vacuum conditions, (infiltration genetic transformation is namely completed) after reaching the scheduled time, close vacuum pump switch, open vacuum drier gas valve, after normal pressure to be restored, open dryer door, taking out plant is planted in culturing pot or field.Arabidopis thaliana vacuum infiltration genetic transformation unlike, due to Arabidopsis plant less (20-30cm), generally in vacuum drier, place the beaker that infiltration conversion fluid is housed, then being inverted together with nutrition pot by the Arabidopis thaliana plants be planted in nutrition pot is immersed in beaker, then carries out vacuum infiltration conversion as mentioned above.Existing vacuum infiltration conversion system has the following disadvantages: the vacuum infiltration that (1) adopts existing vacuum drier to carry out transforms, before vacuumizing decompression, plant has been immersed in the Agrobacterium penetrating fluid (infiltration conversion fluid) in container, although the plant in container and penetrating fluid are also in atmospheric pressure state, but plant is immersed in the Agrobacterium penetrating fluid in container and starts genetic transformation, the infiltration that its conversion process contains infiltration conversion under part normal pressure and partial vacuum transforms, really do not realize vacuum infiltration genetic transformation, its Agrobacterium reduces the invasiveness of plant, transformation efficiency also decreases.This may also be current vacuum infiltration conversion method transformation efficiency major cause on the low side.(2) existing vacuum drier is owing to being subject to small volume, the factor restrictions such as the corresponding matching component that unsuitable live plant is placed and inconvenient operation, make paddy rice, flowers, cotton, infiltration genetic transformation is carried out under the individual larger crop such as wheat class can not realize the condition of living organism (i.e. in-situ condition) of former planting conditions, recipient plant digs out by many employings, it is directly put upside down and to transform in container, the operating method of transplanting again after conversion, be not real converted in-situ on the one hand, on the other hand to the growth conditions transforming rear plant, robustness and setting percentage all have impact, and then cause vacuum infiltration transformation efficiency on the low side.(3) even if the vacuum infiltration genetic transformation operation of plants little as Arabidopis thaliana, also there is many side shoots and be pressed down, cause processing the low problem of changing effect.In sum, existing vacuum infiltration conversion system and method for transformation thereof are not suitable for the vacuum infiltration genetic transformation of the crops such as paddy rice, therefore design and development go out a kind of easy and simple to handle, fast and be suitable for efficiently the crops such as paddy rice vacuum infiltration genetic transformation device and and method for transformation, this greatly will shorten the transformation period, save scientific research cost, improve transformation efficiency, for the genetic improvement of crop varieties and gene functional research provide strong technical guarantee.
Summary of the invention
For overcoming the real deficiency realizing infiltration genetic transformation under vacuum conditions and be unsuitable for the individual larger crops such as paddy rice of existing vacuum infiltration genetic transformation device, the invention provides a kind of real under vacuum conditions, easy and simple to handle, the method that realizes paddy rice original position vacuum infiltration genetic transformation.
Technical scheme of the present invention is a kind of paddy rice vacuum infiltration genetic transforming method, carries out vacuum infiltration genetic transformation, comprise the following steps with a kind of paddy rice vacuum infiltration conversion system to paddy rice:
A. cultivate paddy rice recipient plant to be transformed: according to genetic transformation plan, in greenhouse, rice plant to be transformed is planted in culturing pot 20 in advance;
B. preparation is containing the Agrobacterium infiltration conversion fluid of goal gene: be transformed in Agrobacterium LBA4404 as selection markers containing the carrier electric shocking method to be transformed of goal gene using the Bar gene built; The Agrobacterium bacterium liquid of preservation is inoculated in containing in corresponding antibiotic YEP liquid nutrient medium, in 28 DEG C, 200r/min shaking culture is 0.7 ~ 0.9 to OD600, collected by centrifugation thalline, thalline infiltration nutrient solution resuspended adjustment OD600 to 0.6-0.8 is for subsequent use as the Agrobacterium infiltration conversion fluid containing goal gene, described infiltration nutrient solution is 1/2MS liquid nutrient medium+As19.6mg/L+200ul/LSilwetL-77+ sucrose 50g/L, pH5.8;
C. vacuum infiltration genetic transformation:
1. the base plate being fixedly connected with upper cylinder and lower cylinder is placed on supporting table, makes lower cylinder pass the through hole on supporting table and be positioned under the desktop of supporting table, magnet ring II being enclosed within upper cylinder;
2. vacuum infiltration genetic transformation room is placed on plate upper surface, and makes cylinder be positioned at the indoor of vacuum infiltration genetic transformation room, with sealing-ring II, the bottom nozzle of vacuum infiltration genetic transformation room and the upper surface of base plate are tightly connected;
3. from the top ports of vacuum infiltration genetic transformation room, four of support supporting legs are placed on the upper surface of magnet ring II, the supporting plate of support is placed the penetrating fluid container filling the infiltration of the Agrobacterium containing goal gene conversion fluid prepared by step B;
4. time before the paddy rice be planted in culturing pot when steps A is in and blooms, water in culturing pot is drained and with gauze or foam, the soil in culturing pot is intercepted, soil when making inversion culturing pot in alms bowl does not fall down, then together with paddy rice, culturing pot is upside down on the ring-type adapting table of vacuum infiltration genetic transformation room, live body rice plant spike of rice is naturally drooped, then cover plate is covered the top ports in vacuum infiltration genetic transformation room and be connected with cover plate for sealing by the top ports of sealing-ring I by vacuum infiltration genetic transformation room;
5. the valve on air vent pipe is closed, the valve tube of vacuum pump is taken over the vacuum suction on vacuum infiltration genetic transformation locular wall and is connected, open vacuum pump switch, decompression vacuum pumping, maintain 0.05MPa pressure, vacuum infiltration genetic transformation room is kept to be this vacuum state, and then magnet ring I to be enclosed within lower cylinder and the magnetic pole making magnet ring I adjacent with magnet ring II is homopolarity (namely the magnetic pole of magnet ring I 3 upper surface is identical with the magnetic pole of magnet ring II 6 lower surface), manually upwardly descend the magnet ring I on cylinder, because two magnetic poles adjacent with magnet ring I of the magnet ring II on upper cylinder are homopolarity, according to the principle of homopolar-repulsion, magnet ring II on upper cylinder can move up thereupon, thus the penetrating fluid container promoting to be placed on support is also thereupon to rising, when penetrating fluid container rise to thereupon make inverted live body paddy rice spike of rice be fully immersed in penetrating fluid container containing goal gene Agrobacterium infiltration conversion fluid in time, stop upwards pushing away magnet ring I, and hand rest live magnet ring I motionless and maintain magnet ring I be in make inverted live body paddy rice spike of rice be fully immersed in penetrating fluid container containing goal gene Agrobacterium infiltration conversion fluid in position, now start timing immediately, after live body rice plant soaks and reaches the predetermined infiltration genetic transformation time in penetrating fluid container, take off magnet ring I, magnet ring II thereupon landing downwards on base plate, penetrating fluid container and support also falls downward thereupon, rice plant is separated with the Agrobacterium penetrating fluid containing goal gene in penetrating fluid container, in culturing pot, namely the infiltration genetic transformation of rice plant completes, now close vacuum pump switch, then slowly open valve, treat the indoor recovery atmospheric pressure state in vacuum infiltration genetic transformation room, open cover plate, then from the top mouth of vacuum infiltration genetic transformation room, culturing pot is taken out together with paddy rice,
Described a kind of paddy rice vacuum infiltration genetic transformation device is provided with supporting table, base plate, magnet ring I, magnet ring II, lower cylinder, upper cylinder, vacuum infiltration genetic transformation room, support, penetrating fluid container, sealing-ring I, sealing-ring II parts, concrete structure is:
The structure of vacuum infiltration genetic transformation room: vacuum infiltration genetic transformation room is the cylindrical of hollow, and the top of vacuum infiltration genetic transformation room and bottom are uncovered, cover plate is stamped in the top ports of vacuum infiltration genetic transformation room, the inwall on indoor top, vacuum infiltration genetic transformation room is fixedly connected with ring-type adapting table, locular wall on ring-type adapting table is respectively arranged with air vent pipe and vacuum suction adapter being positioned at, air vent pipe is provided with valve;
The structure of support: support is made up of four vertical supporting legs and supporting plate, the top of four vertical supporting legs is fixedly connected with the lower surface of supporting plate respectively;
Overall structure:
Through hole is provided with in the middle part of the desktop of supporting table, the desktop of supporting table is placed with base plate, lower cylinder is vertically fixedly connected with in the middle part of the lower surface of base plate, on lower cylinder, gap cover has magnet ring I, the diameter D of through hole is greater than the external diameter K of magnet ring I, D > K, lower cylinder and magnet ring I are all positioned under supporting table desktop through through hole, cylinder is fixedly connected with in the middle part of plate upper surface and the position corresponding with lower cylinder is vertical, on upper cylinder, gap cover has magnet ring II, and the magnetic pole that magnet ring I is adjacent with magnet ring II is homopolarity; Vacuum infiltration genetic transformation room is placed on the upper surface middle part of base plate, and upper cylinder and cover thereof have magnet ring II to be positioned at the indoor of vacuum infiltration genetic transformation room; Four supporting legs of support are placed on the upper surface of magnet ring II, and the supporting plate of support is placed with penetrating fluid container, and penetrating fluid container is positioned at the below of the ring-type adapting table of indoor, vacuum infiltration genetic transformation room; The length H of supporting leg and the thickness N sum of magnet ring II are greater than the post height L of cylinder, H+N > L; When magnet ring II is positioned at cylinder lowest part, the distance R between penetrating fluid container mouth and ring-type adapting table lower surface is 80cm ~ 120cm; The bottom nozzle of vacuum infiltration genetic transformation room is tightly connected by the upper surface of sealing-ring II with base plate, and the top ports of vacuum infiltration genetic transformation room is connected with cover plate for sealing by sealing-ring I.
The annular distance aperture M size of the ring-type adapting table that above-mentioned vacuum infiltration genetic transformation chamber interior walls connects is as the criterion so that the culturing pot be upside down on ring-type adapting table can be made not drop from the annular distance of ring-type adapting table.M can be 18 ~ 23cm.
Although in prior art, the vacuum infiltration of plant to be transformed transforms is carry out in vacuum drier, but when plant to be transformed has just started to be immersed in the penetrating fluid in container, it is also atmospheric pressure state in vacuum drier, be fully immersed in when plant to be transformed after in penetrating fluid in container, vacuum drier could be made to be vacuum state with vacuum pump evacuation, therefore, although prior art treats transformed plant to carry out vacuum infiltration genetic transformation in vacuum drier, be soaked in penetrating fluid carry out permeating the plant of genetic transformation actual be part-time at ambient pressure, part-time under vacuum conditions, really treating transformed plant does not under vacuum conditions carry out infiltration genetic transformation.
The principle of work of a kind of paddy rice vacuum infiltration genetic transformation device described in the inventive method is:
The present invention is after vacuum infiltration genetic transformation room is in vacuum state, utilize the principle of magnet homopolar-repulsion, drive indoor magnet by desired trajectory motion (rising) by the magnet outside OPS, reach the object of carrying out permeating genetic transformation under real vacuum state to paddy rice.The present invention does not bore a hole on base plate, be homopolarity by being arranged on two magnetic poles adjacent with the magnet ring I on the lower cylinder of base plate lower surface of the magnet ring II on the upper cylinder of plate upper surface, utilize the principle of magnet homopolar-repulsion, during magnet ring I below upwardly base plate, on magnet ring II on the upper cylinder of vacuum infiltration genetic transformation indoor also with on move, thus hold magnet ring I motionless maintenance magnet ring I present position till the penetrating fluid container of vacuum infiltration genetic transformation indoor also rises in the infiltration of the Agrobacterium containing the goal gene genetic transformation liquid that live body rice plant spike of rice can be made to be fully immersed in penetrating fluid container thereupon position, subsequent operations is carried out after treating vacuum-treat.The present invention does not bore a hole on base plate, vacuum infiltration genetic transformation room top port and bottom port are again respectively by sealing-ring I, sealing-ring II seals, and the valve 11 of closing on air vent pipe, then vacuumize, make vacuum infiltration genetic transformation room live body rice plant also do not permeate with the Agrobacterium containing goal gene genetic transformation liquid contact time, vacuum infiltration genetic transformation room is just in vacuum state, it is exactly contact under vacuum conditions that live body rice plant is permeated when genetic transformation liquid contacts with the Agrobacterium containing goal gene from the beginning, achieve the paddy rice infiltration genetic transformation under real vacuum state, enhance the invasiveness of Agrobacterium to plant, therefore, the present invention compared with prior art, there is following beneficial effect:
1, apparatus of the present invention can make plant to be transformed contact at the beginning when infiltration genetic transformation liquid carries out genetic transformation just to carry out under vacuum conditions, really achieve and under vacuum conditions genetic transformation (namely plant to be transformed carries out to terminating whole process at the beginning all under vacuum conditions from infiltration genetic transformation) is carried out to paddy rice, enhance the invasiveness of Agrobacterium to plant.Overcome after in prior art is immersed in container infiltration genetic transformation liquid plant, just draws air, be soaked in penetrating fluid carry out permeating the plant of genetic transformation actual be part-time at ambient pressure, part-time under vacuum conditions, really do not realize vacuum infiltration to transform, Agrobacterium reduces the invasiveness of plant, the defect that transformation efficiency also decreases.
2, present invention achieves the original position vacuum infiltration genetic transformation of paddy rice, and do not damage rice plant.In apparatus of the present invention, the setting of ring-type adapting table can make to be upside down in easily on the ring-type adapting table of vacuum infiltration genetic transformation room together with the rice plant under the in-situ condition of culturing pot, live body rice plant spike of rice is naturally drooped and injury-free.
3, equipment therefor design of the present invention is ingenious, and each block construction is reasonable, connects easy.The ring-type adapting table of vacuum infiltration genetic transformation indoor can be accepted heavier inverted culturing pot, achieve the live body original position of larger bion infiltration genetic transformation, and plant can not be pressed down or fracture.Equipment therefor of the present invention is not by the restriction of crop Individual Size, and be not only applicable to the less crops of individuality such as leaf mustard, Chinese cabbage, tender flower stalk, swede type rape, radish, tobacco, the larger crops of individuality such as paddy rice, flowers, cotton, wheat class are applicable equally.
4, the inventive method and equipment therefor easy and simple to handle, substantially increase working efficiency.
Accompanying drawing explanation
Fig. 1 is the structural representation of apparatus of the present invention.
Fig. 2 is the main diagram of apparatus of the present invention.
Fig. 3 is A-A sectional view in Fig. 2.
Fig. 4 is the structural representation of vacuum infiltration genetic transformation room.
Fig. 5 is the structural representation of base plate.
Fig. 6 is the structural representation of supporting table.
Fig. 7 is the structural representation that vacuum infiltration genetic transformation room is connected with base plate.
The structural representation that Fig. 8 is base plate, upper cylinder, magnet ring II connect.
The structural representation that Fig. 9 is base plate, upper cylinder, magnet ring II, lower cylinder, magnet ring I connect.
Figure 10 is the structural representation of support.
The structural representation that Figure 11 is penetrating fluid container, support, upper cylinder, magnet ring II, base plate, lower cylinder, magnet ring I connect.
Figure 12 is apparatus of the present invention using state schematic diagram.Described using state refers to the state of will plant when the culturing pot being implanted with paddy rice is upside down on the ring-type adapting table of vacuum infiltration genetic transformation indoor.
Figure 13 is structural representation when magnet ring II rises to extreme higher position in Fig. 3.Described magnet ring II refers to when rising to extreme higher position that upwardly magnet ring I makes magnet ring II rise to thereupon can not again to the position of rising.
Figure 14 is the vacuum drier schematic diagram that prior art carries out the use of floral vacuum infiltration genetic transformation.
In figure, each mark represents successively: 1-supporting table, 2-lower cylinder, 3-magnet ring I, 4-base plate, 5-upper cylinder, 6-magnet ring II, 7-vacuum infiltration genetic transformation room, 8-support, 9-penetrating fluid container, 10-air vent pipe, 11-valve, 12-cover plate, 13-sealing-ring I, 14-vacuum suction is taken over, 15-ring-type adapting table, 16-sealing-ring II, 17-supporting leg, 18-supporting plate, 19-through hole, 20-culturing pot, D is the diameter of through hole, K is the external diameter of magnet ring I, H is the length of supporting leg, N is the thickness of magnet ring II, L is that the post of upper cylinder is high, R is the distance of magnet ring II when being positioned at cylinder lowest part between (when magnet ring II is on base plate) penetrating fluid container mouth and ring-type adapting table lower surface, Q is the distance between ring-type adapting table lower surface to plate upper surface, E is that magnet ring II is when rising to extreme higher position, distance between penetrating fluid container mouth to plate upper surface (refer to when described magnet ring II rises to extreme higher position upwardly magnet ring I magnet ring II is risen to thereupon can not again to the position of rising), M is the annular distance aperture of ring-type adapting table.
Embodiment
Following examples are ordinary method without specified otherwise.
embodiment 1
See Fig. 1-Figure 13, a kind of paddy rice vacuum infiltration genetic transforming method provided by the present invention, a kind of paddy rice vacuum infiltration conversion system of the method carries out vacuum infiltration genetic transformation to paddy rice, comprises the following steps:
A. cultivate paddy rice recipient plant to be transformed: according to genetic transformation plan, in greenhouse, rice plant to be transformed is planted in culturing pot 20 in advance; Culturing pot 20 size selected is upside down in the vacuum infiltration genetic transformation room 7 in described a kind of paddy rice vacuum infiltration conversion system ring-type adapting table 15 with culturing pot 20 can not drop from the annular distance of ring-type adapting table 15 and be as the criterion;
B. preparation is containing the Agrobacterium infiltration conversion fluid of goal gene: be transformed in Agrobacterium LBA4404 as selection markers containing the carrier electric shocking method to be transformed of goal gene using the Bar gene built; The Agrobacterium bacterium liquid of preservation is inoculated in containing in corresponding antibiotic YEP liquid nutrient medium, in 28 DEG C, 200r/min shaking culture is 0.7 to OD600, collected by centrifugation thalline, thalline infiltration nutrient solution resuspended adjustment OD600 to 0.6 is for subsequent use as the Agrobacterium infiltration conversion fluid containing goal gene, described infiltration nutrient solution is 1/2MS liquid nutrient medium+As19.6mg/L+200ul/LSilwetL-77+ sucrose 50g/L, pH5.8;
C. vacuum infiltration genetic transformation:
1. the base plate 4 being fixedly connected with upper cylinder 5 and lower cylinder 2 is placed on supporting table 1, makes lower cylinder 2 pass the through hole 19 on supporting table 1 and be positioned under the desktop of supporting table 1, magnet ring II 6 is enclosed within upper cylinder 5;
2. vacuum infiltration genetic transformation room 7 is placed on base plate 4 upper surface, and makes cylinder 5 be positioned at the indoor of vacuum infiltration genetic transformation room 7, with sealing-ring II, the bottom nozzle of vacuum infiltration genetic transformation room 7 and the upper surface of base plate are tightly connected;
3. from the top ports of vacuum infiltration genetic transformation room 7, four of support 8 supporting legs 17 are placed on the upper surface of magnet ring II 6, the supporting plate 18 of support 8 is placed the penetrating fluid container 9 filling the infiltration of the Agrobacterium containing goal gene conversion fluid prepared by step B;
4. time before the paddy rice be planted in culturing pot when steps A is in and blooms, water in culturing pot is drained and with gauze or foam, the soil in culturing pot is intercepted, then together with paddy rice, culturing pot is upside down on the ring-type adapting table 15 of vacuum infiltration genetic transformation room 7, live body rice plant spike of rice is naturally drooped, then cover plate 12 is covered the top ports in vacuum infiltration genetic transformation room 7 and by sealing-ring I 13, the top ports of vacuum infiltration genetic transformation room 7 and cover plate 12 be tightly connected;
5. the valve 11 on air vent pipe 10 is closed, the valve tube of vacuum pump is connected with the vacuum suction adapter 14 on the wall of vacuum infiltration genetic transformation room 7, open vacuum pump switch, decompression vacuum pumping, maintain 0.05MPa pressure, vacuum infiltration genetic transformation room 7 is kept to be this vacuum state, and then magnet ring I 3 to be enclosed within lower cylinder 2 and the magnetic pole making magnet ring I 3 adjacent with magnet ring II 6 is homopolarity (namely the magnetic pole above magnet ring I 3 is identical with the magnetic pole below magnet ring II 6), manually upwardly descend the magnet ring I 3 on cylinder 2, because two magnetic poles adjacent with magnet ring I 3 of the magnet ring II 6 on upper cylinder 5 are homopolarity, according to the principle of homopolar-repulsion, magnet ring II 6 on upper cylinder 5 can move up thereupon, thus the penetrating fluid container 9 promoting to be placed on support 8 is also thereupon to rising, when penetrating fluid container 9 rise to make inverted live body paddy rice spike of rice be fully immersed in penetrating fluid container 9 containing goal gene Agrobacterium infiltration conversion fluid in time, stop upwards pushing away magnet ring I 3, and hand rest live magnet ring I 3 motionless and maintain magnet ring I 3 be in make inverted live body paddy rice spike of rice be fully immersed in penetrating fluid container 9 containing goal gene Agrobacterium infiltration conversion fluid in position, now start timing immediately, after live body rice plant soaks and reaches the predetermined infiltration genetic transformation time in penetrating fluid container 9, take off magnet ring I 3, magnet ring II 6 thereupon landing downwards on base plate 4, penetrating fluid container 9 and support 8 also falls downward thereupon, rice plant is separated with the Agrobacterium penetrating fluid containing goal gene in penetrating fluid container 9, in culturing pot, namely the infiltration genetic transformation of rice plant completes, now close vacuum pump switch immediately, then slowly open valve 11, after the indoor recovery atmospheric pressure state in vacuum infiltration genetic transformation room 7, open cover plate 12, then from the top ports of vacuum infiltration genetic transformation room 7, culturing pot 20 is taken out together with paddy rice.
After taking out culturing pot 20, carry out rice plant infiltration genetic transformation complete after follow-up work, as the rice plant in culturing pot all covered upper plastic film moisturizing after 24 hours, its culturing pot is placed in greenhouse, cultivating method cultivates plant to ripe routinely, results seed can carry out the screening of transformant, obtains transformant.
See Fig. 1-Figure 13, a kind of paddy rice vacuum infiltration genetic transformation device of the present invention is provided with supporting table 1, base plate 4, magnet ring I 3, magnet ring II 6, lower cylinder 2, upper cylinder 5, vacuum infiltration genetic transformation room 7, support 8, penetrating fluid container 9, sealing-ring I 13, sealing-ring II 16 parts, concrete structure is:
A. the structure of vacuum infiltration genetic transformation room 7: vacuum infiltration genetic transformation room 7 is the cylindrical of hollow, and the top of vacuum infiltration genetic transformation room 7 and bottom are uncovered, cover plate 12 is stamped in the top ports of vacuum infiltration genetic transformation room 7, the top ports of vacuum infiltration genetic transformation room 7 all contacts with the lower surface of cover plate 12, the inwall on indoor top, vacuum infiltration genetic transformation room 7 is fixedly connected with ring-type adapting table 15, the size of the annular distance aperture M of ring-type adapting table 15 is as the criterion so that the paddy rice culturing pot 20 be upside down on ring-type adapting table 15 can be made not drop from the annular distance of ring-type adapting table 15.Ring-type adapting table 15 annular distance aperture M can be 18 ~ 23cm, the internal diameter usually cultivating the culturing pot of paddy rice is 21cm ~ 25cm, and therefore, the culturing pot being upside down in ring-type adapting table 15 can not leak down from the annular distance of ring-type adapting table 15, can reach test objective.Be respectively arranged with air vent pipe 10 and vacuum suction adapter 14 being positioned on the locular wall on ring-type adapting table 15, air vent pipe 10 be provided with valve 11;
B. the structure of support 8: support 8 is made up of four vertical supporting legs 17 and supporting plate 18, the top of four vertical supporting legs 17 is fixedly connected with the lower surface of supporting plate 18 respectively;
C. overall structure:
Through hole 19 is provided with in the middle part of the desktop of supporting table 1, the desktop of supporting table 1 is placed with base plate 4, the desktop of supporting table 1 all covers by base plate 4, lower cylinder 2 is vertically fixedly connected with in the middle part of the lower surface of base plate 4, on lower cylinder 2, gap cover has magnet ring I 3(to be namely enclosed within magnet ring on lower cylinder 2 I 3 and lower cylinder 2 running fit), the diameter D of through hole 19 is greater than the external diameter K of magnet ring I 3, D > K, lower cylinder 2 and magnet ring I 3 are all positioned under supporting table 1 desktop through through hole 19, vertically in base plate 4 upper surface middle part and in the position corresponding with lower cylinder 2 be fixedly connected with the medullary ray that namely cylinder 5(go up cylinder 5 and overlap with the medullary ray of lower cylinder 2), on upper cylinder 5, gap cover has magnet ring II 6(to be namely enclosed within magnet ring on cylinder 5 II 6 and upper cylinder 5 running fit), the magnetic pole that magnet ring I 3 is adjacent with magnet ring II 6 is homopolarity (namely the magnetic pole of magnet ring I 3 upper surface is identical with the magnetic pole of magnet ring II 6 lower surface), vacuum infiltration genetic transformation room 7 is placed on the upper surface middle part of base plate 4, and vacuum infiltration genetic transformation room 7 bottom nozzle all contacts with the upper surface of base plate 4, and upper cylinder 5 and cover thereof have magnet ring II 6 to be positioned at the indoor of vacuum infiltration genetic transformation room 7, four supporting legs 17 of support 8 are placed on the upper surface of magnet ring II 6, the supporting plate 18 of support 8 is placed with penetrating fluid container 9, support 8 and penetrating fluid container 9 are all positioned at the indoor being located at vacuum infiltration genetic transformation room 7, and penetrating fluid container 9 is positioned at the below of the ring-type adapting table 15 of vacuum infiltration genetic transformation room 7, the length H of supporting leg 17 and the thickness N sum of magnet ring II 6 are greater than the post height L of cylinder 5, H+N > L, when magnet ring II 6 is positioned at cylinder 5 lowest part (magnet ring II 6 is at the upper surface of base plate 4), the distance R between penetrating fluid container 9 mouthfuls and ring-type adapting table 15 lower surface is 80cm ~ 120cm, 80cm ~ 120cm is that to be usually suitable for carrying out the paddy rice whole plant of rice transformation high, this R value can meet the demand of conventional rice transformation, when making also not to be in vacuum state in vacuum infiltration genetic transformation room 7 (also not carrying out vacuum pumping), the Agrobacterium that inverted live body rice plant spike of rice and penetrating fluid container 9 include goal gene is permeated conversion fluid and does not contact.R size also can adjust according to the plant height of plant to be transformed, its objective is when making also not to be in vacuum state in vacuum infiltration genetic transformation room 7, and inverted plants permeates conversion fluid with the Agrobacterium containing goal gene and do not contact.The bottom nozzle of vacuum infiltration genetic transformation room 7 is tightly connected by the upper surface of sealing-ring II 16 with base plate 4, air tight to make the bottom nozzle of vacuum infiltration genetic transformation room 7 seal, the top ports of vacuum infiltration genetic transformation room 7 is tightly connected by sealing-ring I 13 and cover plate 12, air tight to make the top ports of vacuum infiltration genetic transformation room 7 seal, make whole vacuum infiltration genetic transformation room 7 reach sealed state.
Claims (2)
1. a paddy rice vacuum infiltration genetic transforming method, a kind of paddy rice vacuum infiltration conversion system of the method carries out vacuum infiltration genetic transformation to paddy rice, comprises the following steps:
A. cultivate paddy rice recipient plant to be transformed: according to genetic transformation plan, rice plant to be transformed is planted in culturing pot (20) in greenhouse in advance;
B. preparation is containing the Agrobacterium infiltration conversion fluid of goal gene: be transformed in Agrobacterium LBA4404 as selection markers containing the carrier electric shocking method to be transformed of goal gene using the Bar gene built; The Agrobacterium bacterium liquid of preservation is inoculated in containing in corresponding antibiotic YEP liquid nutrient medium, in 28 DEG C, 200r/min shaking culture is 0.7 ~ 0.9 to OD600, collected by centrifugation thalline, thalline infiltration nutrient solution resuspended adjustment OD600 to 0.6-0.8 is for subsequent use as the Agrobacterium infiltration conversion fluid containing goal gene, described infiltration nutrient solution is 1/2MS liquid nutrient medium+As19.6mg/L+200ul/LSilwetL-77+ sucrose 50g/L, pH5.8;
C. vacuum infiltration genetic transformation:
1. the base plate (4) being fixedly connected with cylinder (5) and lower cylinder (2) is placed in supporting table (1), make lower cylinder (2) pass the through hole (19) in supporting table (1) and be positioned under the desktop of supporting table (1), magnet ring II (6) being enclosed within upper cylinder (5);
2. vacuum infiltration genetic transformation room (7) is placed on base plate (4) upper surface, and make cylinder (5) be positioned at the indoor of vacuum infiltration genetic transformation room (7), with sealing-ring II, the bottom nozzle of vacuum infiltration genetic transformation room (7) and the upper surface of base plate are tightly connected;
3. be placed on the upper surface of magnet ring II (6) from the top ports of vacuum infiltration genetic transformation room (7) by four supporting legs (17) of support (8), the supporting plate (18) in support (8) is upper places the penetrating fluid container (9) filling the infiltration of the Agrobacterium containing goal gene conversion fluid prepared by step B;
4. time before the paddy rice be planted in culturing pot when steps A is in and blooms, water in culturing pot is drained and with gauze or foam, the soil in culturing pot is intercepted, then together with paddy rice, culturing pot is upside down on the ring-type adapting table (15) of vacuum infiltration genetic transformation room (7), live body rice plant spike of rice is naturally drooped, then cover plate (12) is covered the top ports in vacuum infiltration genetic transformation room (7) and by sealing-ring I (13), the top ports of vacuum infiltration genetic transformation room (7) and cover plate (12) be tightly connected;
5. the valve (11) on air vent pipe (10) is closed, the valve tube of vacuum pump is taken over (14) with the vacuum suction on vacuum infiltration genetic transformation room (7) wall be connected, open vacuum pump switch, decompression vacuum pumping, maintain 0.05MPa pressure, vacuum infiltration genetic transformation room (7) is kept to be this vacuum state, and then magnet ring I (3) is enclosed within lower cylinder (2) is upper and the magnetic pole making magnet ring I (3) adjacent with magnet ring II (6) is homopolarity, manually upwardly descend the magnet ring I (3) on cylinder (2), when penetrating fluid container (9) rise to thereupon make inverted live body paddy rice spike of rice be fully immersed in penetrating fluid container (9) containing goal gene Agrobacterium infiltration conversion fluid in time, stop upwards pushing away magnet ring I 3, and hand rest live magnet ring I (3) motionless and maintain magnet ring I (3) be in make inverted live body paddy rice spike of rice be fully immersed in penetrating fluid container 9 containing goal gene Agrobacterium infiltration conversion fluid in position, now start timing immediately, after live body rice plant soaks and reaches the predetermined infiltration genetic transformation time in penetrating fluid container 9, take off magnet ring I (3), magnet ring II (6) thereupon landing downwards on base plate (4), penetrating fluid container (9) and support (8) also falls downward thereupon, rice plant is separated with the Agrobacterium penetrating fluid containing goal gene in penetrating fluid container 9, in culturing pot, namely the infiltration genetic transformation of rice plant completes, now close vacuum pump switch, then valve (11) is slowly opened, after the indoor recovery atmospheric pressure state in vacuum infiltration genetic transformation room (7), open cover plate (12), then from the top ports of vacuum infiltration genetic transformation room (7), culturing pot (20) is taken out together with paddy rice,
Described a kind of paddy rice vacuum infiltration genetic transformation device, it is characterized in that: this device is provided with supporting table (1), base plate (4), magnet ring I (3), magnet ring II (6), lower cylinder (2), upper cylinder (5), vacuum infiltration genetic transformation room (7), support (8), penetrating fluid container (9), sealing-ring I (13), sealing-ring II (16) parts, concrete structure is:
The structure of vacuum infiltration genetic transformation room (7): what vacuum infiltration genetic transformation room (7) was hollow is cylindrical, and the top of vacuum infiltration genetic transformation room (7) and bottom are uncovered, cover plate (12) is stamped in the top ports of vacuum infiltration genetic transformation room (7), the inwall on vacuum infiltration genetic transformation room (7) indoor top is fixedly connected with ring-type adapting table (15), locular wall on ring-type adapting table (15) is respectively arranged with air vent pipe (10) and vacuum suction adapter (14) being positioned at, air vent pipe (10) is provided with valve (11);
The structure of support (8): support (8) is made up of four vertical supporting legs (17) and supporting plate (18), is fixedly connected with the lower surface of supporting plate (18) respectively on the top of four vertical supporting legs (17);
Overall structure:
Through hole (19) is provided with in the middle part of the desktop of supporting table (1), the desktop of supporting table (1) is placed with base plate (4), lower cylinder (2) is vertically fixedly connected with in the middle part of the lower surface of base plate (4), lower cylinder (2) upper gap cover has magnet ring I (3), the diameter D of through hole (19) is greater than the external diameter K of magnet ring I (3), D > K, lower cylinder (2) and magnet ring I (3) are all positioned under supporting table (1) desktop through through hole (19), upper cylinder (5) is fixedly connected with in base plate (4) upper surface middle part and the position corresponding with lower cylinder (2) is vertical, upper cylinder (5) upper gap cover has magnet ring II (6), the magnetic pole that magnet ring I (3) is adjacent with magnet ring II (6) is homopolarity, vacuum infiltration genetic transformation room (7) is placed on the upper surface middle part of base plate (4), and upper cylinder (5) and cover thereof have magnet ring II (6) to be positioned at the indoor of vacuum infiltration genetic transformation room (7), four supporting legs (17) of support (8) are placed on the upper surface of magnet ring II (6), the supporting plate (18) of support (8) is placed with penetrating fluid container (9), and penetrating fluid container (9) is positioned at the below of the indoor ring-type adapting table (15) in vacuum infiltration genetic transformation room (7), the length H of supporting leg (17) and the thickness N sum of magnet ring II (6) are greater than the post height L of cylinder (5), H+N > L, when magnet ring II (6) is positioned at cylinder (5) lowest part, the distance R between penetrating fluid container (9) mouth and ring-type adapting table (15) lower surface is 80cm ~ 120cm, the bottom nozzle of vacuum infiltration genetic transformation room (7) is tightly connected by the upper surface of sealing-ring II (16) with base plate (4), and the top ports of vacuum infiltration genetic transformation room (7) is tightly connected by sealing-ring I (13) and cover plate (12).
2. paddy rice vacuum infiltration genetic transforming method according to claim 1, is characterized in that: the annular distance aperture M of the ring-type adapting table (15) that vacuum infiltration genetic transformation room (7) chamber interior walls connects is 18 ~ 23cm.
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| WO2001037645A1 (en) * | 1999-11-16 | 2001-05-31 | The United States Of America, As Represented By The Secretary Of Agriculture | Transformation of ricinus communis the castor plant |
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