CN109593780A - A kind of screening technique and screening plant of resistance to air plant with high salt - Google Patents
A kind of screening technique and screening plant of resistance to air plant with high salt Download PDFInfo
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Abstract
The present invention relates to field of plant variety breeding technology, more particularly to a kind of screening technique and screening plant of resistance to air plant with high salt.Its screening technique includes the 2 years raw healthy and strong cuttage seedings or seedling for selecting several common afforestation shrubs, measures its initial root condition and height of seedling；In the simulation for carrying out saliferous air environment in strand in simulation box, simulation box includes several simulation layers；In different simulation layers, different salinity is set, every layer of simulation layer includes ocean province, sand culture area, sandy soil junctional area and earth culture area, plant is planted respectively in sand culture area, sandy soil junctional area and earth culture area, under 10-25 DEG C of temperature match curing conditions, 10-12h/d is shone with the intensity of illumination simulating sun of 1000-5000lx with fluorescent lamp, measures root condition and height of seedling after planting 14-20d；Scale is expanded numerous after genescreen.The invention can effectively avoid the difference of experimental situation with environment on the spot, to filter out the plant for being more suitable for high saliferous air environment under natural conditions.
Description
Technical field
The present invention relates to screening technique and the screenings of field of plant variety breeding technology, more particularly to a kind of resistance to air plant with high salt
Device.
Background technique
The soil salinization is a global resource and ecological problem, is united according to FAO (Food and Agriculture Organization of the United Nation) and Educational, Scientific and Cultural Organization
Meter, there is about 1,000,000,000 hm of various salinized soil in the whole world2, the 10% of Global land area is accounted for, more than 100 country and ground are distributed widely in
Area.Meanwhile global salt-soda soil is every year with 1.0 × 106-1.5 × 106 hm2Speed increase it is old.In China.Salinized soil face
Product is 3.47 × 10 7hm2.There are about the oceanfronts of 1.8 ten thousand km and island bank for each provinces, municipalities and autonomous regions of China coast, extensively
Various marine solonchaks are dispersed with, the gross area is up to 5 × 106 hm2, main Shandong, Hebei, Liaoning including North of Yangtze River etc. saves
And Along North Jiangsu beach alluvial plain and the province region following the line of the sea such as Zhejiang, Fujian, Guangdong on the south the Changjiang river some areas.Strand
The air of high salinity in area can cause certain negative effect to environment, and it is low to will cause fertility, it is difficult to build
The problem of vertical vegetation, seriously restricts the quality and quantity of these regional afforestations.Meanwhile for beach saline land afforestation
Vegetable material it is few, the seed of forest is single, and tree species are dull, limits the forest development of beach saline land.Therefore, in order to improve ecological ring
Air quality is improved in border, needs to cultivate the plant that a batch adapts to high salinity air environment, coerces plant in air with high salt
Under, it remains to be normally carried out photosynthesis.Meanwhile this improves the landscape and bio-diversity of horn of plenty beach saline land coastal
The ecological environment and ecologic stability of developed regions improve soil salination, improve the safeguard function of coast protection forest, increase society
Meeting benefit and economic benefit provide important guarantee.
Summary of the invention
The invention solves the above problems, and high saliferous air environment under reform of nature state can be filtered out by providing one kind
The screening technique and screening plant of plant.
The technical solution that the present invention solves the problems, such as is to provide a kind of screening technique of resistance to air plant with high salt, including following
Step:
(1) plant sample prepares: selecting the 2 years raw healthy and strong cuttage seedings or seedling of several common afforestation shrubs, measurement
Its initial root condition and height of seedling；
(2) littoral environment is simulated: in the simulation for carrying out saliferous air environment in strand in simulation box, the simulation box includes several moulds
Quasi- layer；
(3) Salt Tolerance: different salinity is arranged in Yu Butong simulation layer, and every layer of simulation layer includes ocean province, sand culture area, sandy soil friendship
Battery limit (BL) and earth culture area plant plant in sand culture area, sandy soil junctional area and earth culture area, respectively in 10-25 DEG C of alternating temperature item
Under part, 10-12h/d is shone with the intensity of illumination simulating sun of 1000-5000lx with fluorescent lamp, measures root condition after planting 14-20d
And height of seedling；
(4) it genescreen: chooses the plant that root condition is good, arboreal growth situation is good and utilizes RNA after extracting its RNA respectively
5 ' Side Template switch technologies synthesize the cDNA of overall length, recycle the large fragment of 500bp or more；By recovery product and pYES2.1 carrier
Connection, electricity consumption robin obtain cDNA transformed saccharomyces cerevisiae competent cell using uracil auxotrophy culture medium culture
Yeast transformant；Yeast transformant is screened again in above-mentioned salinity gradient culture medium, chooses the yeast conversion of high salt tolerant
Son, which is inoculated in YPGal culture medium, expands culture；Plant expression vector is constructed with yeast transformant and is transferred to various plants.
Preferably, the plant sample includes aiphyllium: cypress, podocarpus, Putuo camphor tree, Hong Nan, Neolitsea sericea,
Ligustrum lucidum ait, evergreen Chinese wax, Chinese ilex；It further include deciduous tree: alder, zelkova schneideriana, fragrant flower Chinese scholar tree, Chinese toon, soapberry, jujube tree；Also
Including evergreen shrubs: thick leaf Raphiolepis indica, photinia glabra, Chinese rose, golden-rimmed Chinese littleleaf box, pittosporum tobira, oleander, privet, lotus chrysanthemum；Also
Including machaka: hall crabapple flower, fruit mulberry, crape myrtle.
It is a further object to provide a kind of screening plant of resistance to air plant with high salt, including the simulation box,
The simulation box is equipped with several partitions will simulate box cavity by top-to-bottom and be divided into several simulation layers, the simulation layer top
Equipped with fluorescent lamp；Described in being equipped with inclined water delivery part in the simulation box and being divided into will simulate box cavity by side to the other side
Ocean province and growing area, and make the ocean province of every layer of simulation layer of different sizes, the water delivery part includes passing sequentially through semi-permeable membrane
The ocean is divided into and is planted by the junction of the dentation water pipe portion of connection and interconnecting part, dentation water pipe portion and interconnecting part
The air chamber of area's connection and the water cavity being isolated with growing area, the interconnecting part are connected to the water cavity；The water cavity is also connected with
Quantitative salt pipe.
Preferably, inclined spiral agitator is equipped in the simulation box, axis body one end of the spiral agitator passes through
Universal joint is connect with motor output shaft, and the other end passes through after sequentially passing through the ocean province of every layer of simulation layer with the simulation box bottom
Rolling bearing connection.
Preferably, the simulation layer is equipped with bat wave plate, and described one end for clapping wave plate is connected with movable plate, and the other end passes through
The through-hole that air chamber side wall opens up enters ocean province；One end of the movable plate is connect with simulation box bottom by spring, another
End is equipped with tooth and engages with gear is promoted, and the promotion gear includes the teeth portion being toothed around and smooth arc portion.
Preferably, the promotion geared sleeve, which is set to, promotes axis, and the promotion axis and the motor output shaft pass through umbrella tooth
Wheel construction connection, the umbrella gear structure include being sheathed on the first umbrella gear for promoting axis, connecting shaft and being sheathed on electricity
Second umbrella gear of machine output shaft, one end of the connecting shaft are equipped with third umbrella gear and nibble with first umbrella gear
It closes, the other end is equipped with the 4th umbrella gear and engages with second umbrella gear.
Preferably, the growing area successively includes the sand culture area, sand by the direction close to ocean province to separate ocean province
Native junctional area and earth culture area, the sand culture area are equipped with sand bed, and sandy soil junctional area is equipped with sandy soil mixed layer, and earth culture area is laid with
There is soil layer.
Preferably, the simulation box is equipped with negative-pressure air fan close to the side of growing area.
Preferably, the simulation box is and the consistent inclined-plane of water delivery part gradient close to the side of growing area.
It preferably, further include micro- chamber system, the micro- chamber system includes being set to sand bed, sandy soil mixed layer and soil layer
Mini-rhizotron, be set to miniature CCD camera outside simulation box, regulation handle, Image Acquisition control device and computer graphic
As processing analysis system.
Beneficial effects of the present invention:
Simulation box is set and simulates strand saliferous air environment, can filter out and be more suitable for certainly in progress Salt Tolerance in simulation box
The plant of saliferous air, effectively avoids the otherness of experimental situation with environment on the spot under right state, the plant filtered out for the later period
Plantation provides good basis on the spot.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of the screening plant of resistance to air plant with high salt；
Fig. 2 is a kind of structural schematic diagram of the water delivery part of the screening plant of resistance to air plant with high salt；
In figure: simulation box 100, simulation layer 101, fluorescent lamp 102, spiral agitator 103, negative-pressure air fan 104, ocean province 110, kind
Growing area 120, sand culture area 121, sandy soil junctional area 122, earth culture area 123, water delivery part 200, semi-permeable membrane 201, dentation water pipe portion 210,
Interconnecting part 220, quantitative salt pipe 230 clap wave plate 300, and movable plate 310 promotes gear 320, micro- chamber system 400.
Specific embodiment
It is a specific embodiment of the invention below, and in conjunction with attached drawing, technical solution of the present invention work is further retouched
It states, however, the present invention is not limited to these examples.
A kind of screening technique of resistance to air plant with high salt, comprising the following steps:
(1) plant sample prepares: selecting the 2 years raw healthy and strong cuttage seedings or seedling of several common afforestation shrubs, measurement
Its initial root condition and height of seedling.In order to improve the later period in the diversity of coastal region planting plants on the spot, choose as far as possible more
The plant of type.Cypress, podocarpus, Putuo camphor tree, Hong Nan, Neolitsea sericea, ligustrum lucidum ait, evergreen Chinese wax, winter can be chosen
The aiphylliums such as blueness；The deciduous trees such as alder, zelkova schneideriana, fragrant flower Chinese scholar tree, Chinese toon, soapberry, jujube tree；Thick leaf Raphiolepis indica, red autumnal leaves stone
The evergreen shrubs such as nanmu, Chinese rose, golden-rimmed Chinese littleleaf box, pittosporum tobira, oleander, privet, lotus chrysanthemum；Hall crabapple flower, fruit mulberry, crape myrtle etc. fall
Leaf shrub.
(2) littoral environment is simulated: in the simulation for carrying out saliferous air environment in strand in simulation box 100, simulation box 100 includes
Several simulation layers 101.As shown in Figure 1, simulation box 100 is equipped with several partitions to divide 100 inner cavity of simulation box by top-to-bottom
For several simulation layers 101, fluorescent lamp 102 is equipped at the top of simulation layer 101；Inclined water delivery part 200 is equipped in simulation box 100 to incite somebody to action
100 inner cavity of simulation box is divided into ocean province 110 and growing area 120 by side to the other side, and makes the sea of every layer of simulation layer 101
Oceanic province 110 is of different sizes.As shown in Fig. 2, water delivery part 200 includes 210 He of dentation water pipe portion for passing sequentially through the connection of semi-permeable membrane 201
The sky to be connected to growing area 120 is divided in ocean province 110 by the junction of interconnecting part 220, dentation water pipe portion 210 and interconnecting part 220
Air cavity and the water cavity being isolated with growing area 120, interconnecting part 220 are connected to water cavity；Water cavity is also connected with quantitative salt pipe 230.
Since ocean province 110 is of different sizes, so that its water cavity is of different sizes, then through water delivery part 200 into water cavity
Water delivery simultaneously makes it fill up water cavity, then can be made by quantitative salt pipe 230 to the salt that the water cavity of each simulation layer 101 inputs equivalent
Salinity in each simulation layer 101 is inconsistent, simply obtains experiment salinity gradient.
Mixed uniformly brackish water in order to obtain, simulation box 100 is interior to be equipped with inclined spiral agitator 103, auger
Axis body one end of device 103 is connect by universal joint with motor output shaft, and the other end sequentially passes through the ocean province of every layer of simulation layer 101
It is connect with 100 bottom of simulation box by rolling bearing after 110.Spiral agitator 103 makes salt, water in the stirring action in water cavity
Marine motor is mixed and simulates, and it can also simulate wind-force effect in the stirring action in air chamber.In addition, simulation box 100
Negative-pressure air fan 104 is equipped with close to the side of growing area to cooperate the further simulation nature turbulent flow wind-force of spiral agitator 103.
In order to further simulate littoral environment, simulation layer 101, which is equipped with, claps wave plate 300, and the one end for clapping wave plate 300 is connected with
Movable plate 310, the other end enter ocean province 110 by the through-hole that air chamber side wall opens up；One end of movable plate 310 and simulation box
100 bottoms are connected by spring, and the other end is equipped with tooth and engages with gear 320 is promoted, and promoting gear 320 includes being toothed around
Teeth portion and smooth arc portion.When teeth portion is engaged with tooth, promotes gear 320 and drive movable plate 310 and clap wave plate 300
One end is promoted, and is clapped to fall in the other end Yu Haiyang area 110 of wave plate 300 and be patted the salt water in water cavity；When promotion tooth
Wheel 320 continues to rotate when making its arc portion close to tooth, and movable plate 310 is no longer influenced by castering action, and in self gravity and spring
Pulling force effect fall, wait next time teeth portion drive its promotion carry out clap wave.
It is crushed salt water fierceness disturbance in water cavity, stormy waves, sea laps, to generate a large amount of foams, bubble, gas
Small water droplet can be generated when bubble rupture, salt water droplet largely lands due to gravity, is partially in same the Xiamen Bay and keeps
The state of balance and be distributed in above ocean province, it is aerial that they with air-flow rise up into simulation layer 101, through cracking, evaporation, mixes and waited
Journey evolves into disperse system, forms salt fog and diffuses to growing area 120 to the plant progress Salt fog stress planted in it.
Growing area 120 successively includes sand culture area 121, sandy soil friendship by the direction close to ocean province 110 to separate ocean province 110
Battery limit (BL) 122 and earth culture area 123, sand culture area 121 are equipped with sand bed, and sandy soil junctional area 122 is equipped with sandy soil mixed layer, earth culture area
123 are equipped with soil layer, to simulate the various planting environments of littoral environment.Meanwhile in order to guarantee each 101 growing area of layer simulation layer
120 it is in the same size, simulation box 100 close to the side of growing area be and the consistent inclined-plane of 200 gradient of water delivery part.
In addition, will promote gear 320 in order to reach energy-conserving action and be sheathed on promotion axis, promotes axis and motor output shaft is logical
The connection of umbrella gear structure is crossed, umbrella gear structure includes being sheathed on the first umbrella gear for promoting axis, connecting shaft and being arranged
In the second umbrella gear of motor output shaft, one end of connecting shaft be equipped with third umbrella gear and engaged with the first umbrella gear,
The other end is equipped with the 4th umbrella gear and engages with the second umbrella gear.When motor output shaft is rotated around vertical axis, with second
Umbrella gear drives the 4th umbrella gear to rotate around first level axis, and the 4th umbrella gear drives connecting shaft and third umbrella tooth
Wheel is also rotated around first level axis, so that third umbrella gear drives the first umbrella gear and promotes gear 320 around the
The rotation of two trunnion axis, first level axis and the second trunnion axis are set in same plane, and are mutually perpendicular to, thus with same motor
Achieve the purpose that linkage.
(3) Salt Tolerance: plant being planted respectively in sand culture area 121, sandy soil junctional area 122 and earth culture area 123, in
Under 10-25 DEG C of temperature match curing conditions, 10-12h/d, plantation are shone with the intensity of illumination simulating sun of 1000-5000lx with fluorescent lamp 102
Root condition and height of seedling are measured after 14-20d.
Measurement root condition can be used micro- chamber system 400, micro- chamber system include be set to sand bed, sandy soil mixed layer and
The mini-rhizotron of soil layer, miniature CCD camera, regulation handle, Image Acquisition control device and the calculating being set to outside simulation box
Machine image processing and analyzing system, this detection mode not only avoid destructive, time and effort consuming sampling process and can
Original position nondestructively directly carries out dynamic automatic measurement and analysis to the root system of plant being grown under sandy soil.
(4) genescreen: choosing the plant that root condition is good, arboreal growth situation is good, after extracting its RNA respectively, utilizes
5 ' Side Template switch technology of RNA synthesizes the cDNA of overall length, recycles the large fragment of 500bp or more；By recovery product and pYES2.1
Carrier connection, electricity consumption robin is by cDNA transformed saccharomyces cerevisiae competent cell, using uracil auxotrophy culture medium culture,
Obtain yeast transformant；Yeast transformant is screened again in above-mentioned salinity gradient culture medium, chooses the yeast conversion of high salt tolerant
Son, which is inoculated in YPGal culture medium, expands culture；Plant expression vector is constructed with yeast transformant and is transferred to various plants.
Wherein, 5 ' Side Template switch technology method of RNA are as follows: the first chain cDNA synthesis one modified olig(dT)
Primer, oligonucleotides hold the template extended out as mRNA5 ', and when reverse transcription, which reaches mRNA 5 ', to be held, reverse transcription has mould
Plate is not dependent to add several C at the end the first chain cDNA 3 ', matches with several G that oligonucleotides 3 ' is held, to make few nucleosides
Acid is connected to the end mRNA5 '.Reverse transcriptase jumps and using oligonucleotides as template, the end of reverse transcription to oligonucleotides, and this
It jumps to move and occurs often in Eukaryotic mRNA cap sequence-m7GpppX, the cDNA that such reverse transcription goes out include mRNA complete
5 ' and oligonucleotides complementary series, then with PCR synthesize the second chain of cDNA.So that the cDNA clone contains phase to the full extent
Complete 5 ' the non-coding area sequence of mRNA molecule is answered, to construct the cDNA library of salt-tolerant plant.
Using simplest eukaryon model organism-saccharomycete as the host of plant foreign gene, due to yeast expression system
With plant expression system more closely, also there is glycosylation, disulfide bond formation and protein folding stool post translational processing to wait
Journey, therefore have the advantages that growth is fast, be easy culture, being easy to genetic manipulation, being easy to screen after converting, at the same cost be it is cheap,
Period is shorter, more efficient.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Claims (10)
1. a kind of screening technique of resistance to air plant with high salt, it is characterised in that: the following steps are included:
Plant sample prepares: the 2 years raw healthy and strong cuttage seedings or seedling of several common afforestation shrubs is selected, at the beginning of measuring it
Beginning root condition and height of seedling；
Littoral environment simulation: in the simulation for carrying out saliferous air environment in strand in simulation box (100), simulation box (100) packet
Include several simulation layers (101)；
Salt Tolerance: Yu Butong simulation layer (101) be arranged different salinity, every layer of simulation layer (101) include ocean province (110),
Sand culture area (121), sandy soil junctional area (122) and earth culture area (123) plant plant respectively in sand culture area (121), sandy soil friendship
Battery limit (BL) (122) and earth culture area (123), under 10-25 DEG C of temperature match curing conditions, with fluorescent lamp (102) with the light of 1000-5000lx
According to strength simulation sunshine 10-12h/d, root condition and height of seedling are measured after planting 14-20d；
Genescreen: choosing the plant that root condition is good, arboreal growth situation is good, after extracting its RNA respectively, is held using RNA 5 '
Template switching technology synthesizes the cDNA of overall length, recycles the large fragment of 500bp or more；Recovery product is connect with pYES2.1 carrier,
CDNA transformed saccharomyces cerevisiae competent cell is obtained yeast using uracil auxotrophy culture medium culture by electricity consumption robin
Transformant；Yeast transformant is screened again in above-mentioned salinity gradient culture medium, the yeast transformant for choosing high salt tolerant is inoculated in
Expand culture in YPGal culture medium；Plant expression vector is constructed with yeast transformant and is transferred to various plants.
2. the screening technique of the resistance to air plant with high salt of one kind according to claim 1, it is characterised in that: the plant sample
Including aiphyllium: cypress, podocarpus, Putuo camphor tree, Hong Nan, Neolitsea sericea, ligustrum lucidum ait, evergreen Chinese wax, Chinese ilex；Also wrap
Include deciduous tree: alder, zelkova schneideriana, fragrant flower Chinese scholar tree, Chinese toon, soapberry, jujube tree；It further include evergreen shrubs: thick leaf Raphiolepis indica, red autumnal leaves
Chinese photinia, Chinese rose, golden-rimmed Chinese littleleaf box, pittosporum tobira, oleander, privet, lotus chrysanthemum；Further include machaka: hall crabapple flower, fruit mulberry,
Crape myrtle.
3. a kind of screening plant for the resistance to air plant with high salt of -2 any one according to claim 1, it is characterised in that: including
The simulation box (100), the simulation box (100) are equipped with several partitions to divide simulation box (100) inner cavity by top-to-bottom
For several simulation layers (101), simulation layer (101) top is equipped with fluorescent lamp (102)；It is equipped with and inclines in the simulation box (100)
Oblique water delivery part (200) is to be divided into the ocean province (110) and growing area by side to the other side for simulation box (100) inner cavity
(120), and make the ocean province (110) of every layer of simulation layer (101) of different sizes, the water delivery part (200) includes passing sequentially through
The dentation water pipe portion (210) of semi-permeable membrane (201) connection and interconnecting part (220), dentation water pipe portion (210) and interconnecting part
(220) junction by the ocean province (110) be divided into the air chamber being connected to growing area (120) and with growing area (120) every
From water cavity, the interconnecting part (220) is connected to the water cavity；The water cavity is also connected with quantitative salt pipe (230).
4. the screening plant of the resistance to air plant with high salt of one kind according to claim 3, it is characterised in that: the simulation box
(100) inclined spiral agitator (103) are equipped in, axis body one end of the spiral agitator (103) passes through universal joint and electricity
Machine exports axis connection, the other end sequentially pass through the ocean provinces (110) of every layer of simulation layer (101) afterwards with the simulation box (100) bottom
Portion is connected by rolling bearing.
5. the screening plant of the resistance to air plant with high salt of one kind according to claim 4, it is characterised in that: the simulation layer
(101) it is equipped with and claps wave plate (300), described one end for clapping wave plate (300) is connected with movable plate (310), and the other end passes through air
The through-hole that chamber side wall opens up enters ocean province (110)；One end and simulation box (100) bottom of the movable plate (310) pass through bullet
Spring connection, the other end are equipped with tooth and engage with gear (320) are promoted, and the promotion gear (320) includes the tooth being toothed around
Portion and smooth arc portion.
6. the screening plant of the resistance to air plant with high salt of one kind according to claim 5, it is characterised in that: the promotion gear
(320) it is sheathed on promotion axis, the promotion axis is connect with the motor output shaft by umbrella gear structure, the umbrella gear
Structure includes being sheathed on the first umbrella gear, connecting shaft and the second umbrella gear for being sheathed on motor output shaft that promote axis,
One end of the connecting shaft be equipped with third umbrella gear and engage with first umbrella gear, the other end is equipped with the 4th umbrella tooth
It takes turns and is engaged with second umbrella gear.
7. the screening plant of the resistance to air plant with high salt of one kind according to claim 3, it is characterised in that: the growing area
It (120) successively include the sand culture area (121), sandy soil boundary by the direction close to ocean province (110) to separate ocean province (110)
Area (122) and earth culture area (123), the sand culture area (121) are equipped with sand bed, and it is mixed that sandy soil junctional area (122) are equipped with sandy soil
Layer is closed, earth culture area (123) are equipped with soil layer.
8. the screening plant of the resistance to air plant with high salt of one kind according to claim 3, it is characterised in that: the simulation box
(100) negative-pressure air fan (104) are equipped with close to the side of growing area.
9. the screening plant of the resistance to air plant with high salt of one kind according to claim 3, it is characterised in that: the simulation box
(100) it is and the consistent inclined-plane of water delivery part (200) gradient close to the side of growing area.
10. the screening plant of the resistance to air plant with high salt of one kind according to claim 7, it is characterised in that: further include micro-
Chamber system (400), the micro- chamber system includes the mini-rhizotron for being set to sand bed, sandy soil mixed layer and soil layer, is set to simulation
Miniature CCD camera, regulation handle, Image Acquisition control device and Computer Image Processing analysis system outside case.
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