CN1715416A

CN1715416A - Agrobacterium mediated transformation system for obtaining perennial rye-grass transgenic plant in high efficiently and its use

Info

Publication number: CN1715416A
Application number: CN 200510085470
Authority: CN
Inventors: 韩烈保; 李雪; 刘君; 曾会明; 信金娜
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2005-07-21
Filing date: 2005-07-21
Publication date: 2006-01-04
Anticipated expiration: 2025-07-21
Also published as: CN1291024C

Abstract

The present invention includes the following steps: obtaining embryonic callus acceptor material via high frequency regeneration system, culturing donor strain, determining concentration of ablastin, optimizing agrobacterium transforming condition, screening and culturing transformed callus and molecular detection of plant regeneration and resisting plant. By means of optimizing the conditions for transforming agrobacterium, including bacterium liquid concentration, infection time, infection condition, co-culture time, ablastin concentration, etc., the present invention provides agrobacterium mediated transformation system suitable for perennial ryegrass. The present invention solves the technological problem of applying agrobacterium mediating technology in genetic transformation of gramineous plant and establishes efficient agrobacterium mediated transformation system with embryonic callus as acceptor material for perennial ryegrass.

Description

A kind of agrobacterium mediation converted system and application thereof of efficient acquisition English ryegrass transfer-gen plant

Technical field:

The invention belongs to plant genetic engineering field or crop biotechnology breeding field.Specifically, relate to the molecular detection technology of tissue culture technique, agrobacterium mediation converted technology and the transformed plant of English ryegrass, thereby obtain the English ryegrass transfer-gen plant efficiently.

Background technology:

Turf grass species is second largest in the world transaction seed, and the main dependence on import of the present used grass seeds of China, breeding work is especially weak.English ryegrass is southern and northern widespread use of China and the very strong turf grass species of adaptability, and turf speed is fast, and disease-resistant worm and tillering ability are strong, the important grass seeds in Chang Zuowei lawn planting, long-term rotation pasture, permanent pasture.But it still exist water loss big, be impatient at extremum hot and cold, these weathers of arid, and sprouting and seedling stage are to the bigger deficiency that waits of water demand.Because the complicacy of the complicacy of stress resistance of plant itself and its quantitative trait locus linkage relationship makes that to utilize traditional breeding way to obtain good resistant variety very difficult, utilize genetic engineering technique to obtain to adapt to or the lawn grass variety of resisting these adverse environmental factors will be to address these problems one of quick and effective means.In improving the molecular breeding of plant, improve or the transcription factor that increases a key can obviously improve the many-sided resistance capacity of plant environment stress.

Agriculture bacillus mediated plant gene conversion system is present known unique naturally occurring gene swapping factor, has been widely used in plant genetic engineering research.A kind of special Ti-plasmids is arranged in the agrobacterium tumefaciens cell, and the part dna fragmentation of this plasmid can be incorporated in the host plant gene group, with host genome heredity and expression.Carry genes such as growth hormone, cell fission and synthetic opine owing to be integrated into the Ti-plasmids dna fragmentation of Plant Genome, thereby make the plant tissue that was infected by agrobacterium tumefaciens produce crown-gall nodule, and synthetic in a large number opine.Opine promotes the breeding of agrobacterium tumefaciens and the transfer of Ti-plasmids conversely again, enlarges the scope that infects.The agrobacterium mediation converted method has the following advantages compared with other method for transformation:

A transformation frequency height, changing effect is good, transferable bigger dna fragmentation;

The foreign gene Chang Weidan copy that b shifts is integrated, and seldom can methylate and gene silencing;

The c inheritance stability, and majority meets mendelian inheritance, and transfer-gen plant can be used as the intermediate materials of breeding preferably;

The foreign gene that d integrates has clearer and more definite border sequence, normally circle, T-DNA left and right sides sequence;

E is cheap, and is not high to requirement for experiment condition.

Because monocotyledons is not the natural host of Agrobacterium, thinks once that agrobacterium-mediated transformation was unsuitable for unifacial leaf plant genetic transformations (Narasimhulu et al.1996) such as cereal class.The reason that monocotyledons is difficult to carry out Agrobacterium-mediated Transformation mainly contains 2 points: (1) monocotyledons seldom or fully can not produce the signaling molecule that activates Ti-plasmids Vir district gene; (2) target tissue or the cell of monocotyledons conversion can not carry out effective bacterial adhesion, there is not tangible response to traume, can not induce near the cell dedifferentiation of wound to form a large amount of competent cells, and be that those regeneration and the strong competent cell of integration conversion capability just can obtain transformed plant.So far, be that the agrobacterium-mediated transformation transformation system of acceptor still rests on the laboratory study stage with the English ryegrass embryo callus.

Summary of the invention:

The agrobacterium mediation converted system and the application thereof of a kind of efficient acquisition English ryegrass transfer-gen plant of the present invention, purpose is to overcome the defective that existing agriculture bacillus mediated technology is difficult to be applied to the grass genetic transformation, adjust the interpolation concentration of bacterial concentration, time of infection, infection condition, common incubation time, ablastins by experiment, a kind of agrobacterium mediation converted system that is applicable to English ryegrass is provided.

Description of drawings:

Accompanying drawing 1 changes English ryegrass Cupid spy (Accent) regrowth of DREB over to; The special regrowth of English ryegrass Cupid that accompanying drawing 2 changes DREB over to is taken root; Accompanying drawing 3 changes the special transfer-gen plant of English ryegrass Cupid of DREB over to; Accompanying drawing 4 changes the special transfer-gen plant root system of English ryegrass Cupid of DREB over to; Accompanying drawing 5gus gene transient expression; Accompanying drawing 6gus stable gene is expressed; Accompanying drawing 7DREB gene PCR amplification (1-7 is a transformed plant, 8 negative contrasts, and 9 positive contrasts, 10 is marker); Accompanying drawing 8DREB gene Southern results of hybridization (1-6 is a transformed plant, 7 positive contrasts, and 8 positive contrasts, 9 is marker).

Embodiment:

Acceptor material is prepared: with the English ryegrass mature embryo is the explant induction callus, and callus of induce and subculture medium are MS+4-6mg/L2,4-D+0.2-0.3mg/L 6-BA+0.1g/L caseinhydrolysate.Use the Parafilm closed petridish, 25 ℃ of dark cultivations.Picking age 2-3 month embryo callus is cultivated 3d in advance and is waited to infect.

Determining of ablastins concentration: agrobacterium strains LBA4404 is in containing the YEP liquid nutrient medium of different concns ablastins, and 28 ℃ of 200-220rmp secretly cultivate, and surveys bacterium liquid OD ₆₀₀Value.Treat that Agrobacterium grows to logarithmic phase,, make OD with 1/2MS nutrient solution dilution bacterium liquid ₆₀₀Value infects callus 10-15min for 0.5-0.8, and constantly concussion makes Agrobacterium fully contact with callus in the infection processs.Take out vegetable material, blot residual bacterium liquid, change in the callus of induce substratum that contains the different concns ablastins with filter paper.25 ℃ of dark cultivations.Carry out the record of pollution condition every day.

F+strain is cultivated: picking list bacterium colony from the flat board is inoculated in the additional antibiotic bacterial liquid substratum.28 ℃, be cultured to OD on the 200-220rmp constant temperature shaking table ₆₀₀Be 0.8-1.0, be diluted to about 0.1 and transform, add 100 μ mol/L Syringylethanones (AS) simultaneously.

Infect: bacterium liquid is poured in the sterile petri dish that fills embryo callus, jiggled bacterium liquid is fully contacted with callus, vacuumize and soak 10min.Callus after will infecting places bottom to be covered with the culture dish of some sterilization filter paper, and the upper strata covers fritter filter paper once more, and gently exerts pressure with tweezers, and bacterium liquid unnecessary on the callus can be blotted fully.Callus is changed over to the upper strata again and be covered with for one to the containing on the 100 μ mol/L AS callus of induce substratum of two-layer sterilization filter paper, the callus of avoiding being stained with bacterium liquid contacts with the direct of substratum, has avoided the excessive multiplication of Agrobacterium on the substratum that does not contain ablastins.25 ℃ of dark cultivations are cultivated 4d altogether.With the sterilized water washing part callus of 500mg/L saitomycin (cef), carry out gus chemistry tissue staining, observe the transient expression of gene through the alcohol decolouring

Select to cultivate: the explant transfer through cultivating is altogether contained on the callus of induce substratum of 300mg/Lcef and 50mg/L Totomycin 25 ℃ of dark 20d that cultivate.The callus of surviving is transferred to the division culture medium that contains 250mg/Lcef and 50mg/L Totomycin, and (division culture medium is meant MS+0.1-0.2mg/L 6-BA+0.4-0.6mg/L NAA+0.3-0.5mg/L ZT+6.4mg/L Cu ²⁺) on, picking part callus carries out gus gene chemistry tissue staining.The budlet that grows is transferred to treats in the division culture medium that does not contain selective agent that seedling is long and change in the root media (root media is meant MS+0.4mg/L NAA+0.1mg/L ZT) strengthening seedling and rooting over to during to 3-4 sheet leaf.

The transplanting of resistant plant: the root leaf completely behind the room temperature lower refining seedling 3d in strong sprout, is washed the substratum of base portion, and (matrix is for husky: soil: the peat composed of rotten mosses=1: 1: 1), natural light is growth down directly to plant flowerpot.

Embodiment:

Embodiment 1

Agrobacterium-mediated transformation changes the DREB1A gene over to the transfer-gen plant that obtains drought resisting, salt tolerant alkali in the English ryegrass

The acquisition of acceptor material: with the special mature embryo of English ryegrass Cupid is the explant induction callus, and callus of induce and subculture medium are MS+6mg/L 2,4-D+0.2mg/L 6-BA+0.1g/L caseinhydrolysate.25 ℃ of dark cultivations.The embryo callus that picking was cultivated 2-3 month is cultivated 3d in advance and is waited to infect.

Determining of rhzomorph concentration: agrobacterium strains LBA4404 is in the YEP liquid nutrient medium that contains different concns ablastins head saitomycin, and 28 ℃ of 220rmp secretly cultivate, and surveys bacterium liquid OD ₆₀₀Value (table 1).Saitomycin can be restrained the growth of Agrobacterium LBA4404 substantially more than 250mg/L.Treat that Agrobacterium grows to logarithmic phase,, make OD with 1/2MS nutrient solution dilution bacterium liquid ₆₀₀Value infects callus 10-15min for 0.5-0.8, and constantly concussion makes Agrobacterium fully contact with callus in the infection processs.Take out vegetable material, blot residual bacterium liquid, change in the callus of induce substratum that contains the different concns ablastins with filter paper.25 ℃ of dark cultivations.Note down pollution condition (table 2) every day.But a saitomycin can cause the pollution of Agrobacterium fully more than 300mg/L.

Table 1 cephamycin is to the influence of Agrobacterium LBA4404 growth

cef mg/L	OD ₆₀₀Value			Mean value
	OD ₆₀₀Value				I	II	III
	0 30 50 80 100 150 200 250 300 350 400	2.013 1.245 0.947 0.781 0.427 0.39 0.183 0.065 0.035 0.025 0.016	1.982 1.375 0.929 0.603 0.497 0.438 0.092 0.029 0.02 0.034 0.019		I	II	III	1.873 1.508 0.899 0.716 0.348 0.402 0.11 0.052 0.035 0.041 0.024	1.956 1.376 0.925 0.700 0.424 0.410 0.128 0.049 0.030 0.033 0.020

F+strain is cultivated: picking list bacterium colony from the flat board is inoculated in the bacterial liquid substratum of additional 100mg/L kantlex and 125mg/L Streptomycin sulphate.Be cultured to OD on 28 ℃ of 220rmp constant temperature shaking tables ₆₀₀Be 0.8-1.0, be diluted to four concentration shown in the table 3 respectively and transform, add 100 μ mol/L Syringylethanones (AS) simultaneously.

Table 2 a saitomycin The selection result

Saitomycin mg/L	The fate d that pollution occurs	Pollution rate %
Saitomycin mg/L	The fate d that pollution occurs	Pollution rate %	0 100 200 250 300 350 400	2 9 11 15 0 0 0	100 89.48 38.7 8.3 0 0 0

8 processing of table 3 bacterial concentration and time of infection

Infect: respectively the bacterium liquid of different concns is poured in the sterile petri dish that fills embryo callus, jiggled bacterium liquid is fully contacted with callus, and vacuumize respectively and soak 10 or 20min, form 8 processing shown in the table 3.The taking-up callus places to inhale on the aseptic filter paper removes unnecessary bacterium liquid, cover aseptic filter paper with tweezers and on callus, blot clean bacterium liquid as far as possible, callus changed over to be covered with the containing on the 100 μ mol/L AS callus of induce substratum of one deck sterilization filter paper, 25 ℃ of dark cultivations cultivate 2,3 respectively, 4d altogether.With the sterilized water washing part callus of 500mg/L saitomycin (cef), carry out gus chemistry tissue staining, through the transient expression rate (table 4) of alcohol decolouring observation gus gene.The gus colour developing ratio of processing 1 when cultivating 4d altogether is the highest, can reach 85%, so OD ₆₀₀Be diluted to about 0.1, time of infection is 10min, and cultivating 4d altogether is the best of breed that Agrobacterium is infected.

8 in table 4 is handled the gus colour developing ratio of different incubation times altogether

Select to cultivate: the explant transfer through cultivating is altogether contained on the callus of induce substratum of 300mg/Lcef and 50mg/L Totomycin 25 ℃ of dark 20d that cultivate.The callus of survival is transferred on the division culture medium that contains 250mg/Lcef and 50mg/L Totomycin, and picking part callus carries out gusA chemistry tissue staining.The instantaneous colour developing ratio of processing 1 and processing 7gus gene is higher, also has only these two processing having the resistance seedling to grow after screening through selection.The seedling that grows is transferred to treats in the division culture medium that does not contain selective agent that seedling is long and change in the root media (root media is meant MS+0.4mg/L NAA+0.1mg/L ZT) strengthening seedling and rooting over to during to 3-4 sheet leaf.

The Molecular Detection of transformed plant: extract resistant plant DNA and carry out the PCR detection, the DREB1A primer sequence is:

Primers F: 5 '＞AAA GGA TCC TTA CCC GGG TTC TGA TCA ATG AAC TCA TTT TCT G＜3 ',

Primer R:5 '＞AAA GGT ACC AAT CCC GGG GTT TTA ATA ACT CCA TAA CGA TAC G＜3 ' DNA that gets the PCR positive plant carries out the Southern blot hybridization to be identified, makees probe with the PCR product of goal gene.Detect resistant plant 10 strains altogether, wherein positive plant 8 strains, transformation efficiency 80%.

Claims

The agrobacterium mediation converted system and the application thereof of a kind of efficient acquisition English ryegrass transfer-gen plant of 1 the present invention comprise following technical system:

(1) obtains the embryo callus subculture acceptor material by high frequency regenerating system;

(2) cultivation of F+strain;

(3) ablastins concentration determines;

(4) optimizing transforming condition of agricillin;

(5) screening and culturing and the plant regeneration of conversion callus;

(6) Molecular Detection of resistant plant.
2 in accordance with the method for claim 1, it is characterized in that, it is a kind of highly effective agrobacterium mediating transformation technology that is applied to English ryegrass.
3 in accordance with the method for claim 1, it is characterized in that, it is to be the agrobacterium mediation converted technology of acceptor material with the embryo callus.
4 in accordance with the method for claim 1, it is characterized in that, the concentration screening of ablastins is 300mg/L when cultivating, and regeneration is 250mg/L when cultivating.
5 in accordance with the method for claim 1, it is characterized in that, bacterial concentration is at OD ₆₀₀When value is 0.1 left and right sides, vacuumizes and infect 10min, cultivate 4d altogether.
6 in accordance with the method for claim 1, it is characterized in that, places bottom to be covered with the culture dish of some sterilization filter paper callus after infecting, and the upper strata covers fritter filter paper once more, and gently exerts pressure with tweezers, and bacterium liquid unnecessary on the callus can be blotted fully.
7 in accordance with the method for claim 1, it is characterized in that, the upper strata of containing the common substratum of Syringylethanone is covered with one to two-layer sterilization filter paper, and the callus of avoiding being stained with bacterium liquid contacts with the direct of substratum.