CN102776232A - High-efficiency agrobacterium tumefaciens-mediated transformation method of zoysia japonica. - Google Patents
High-efficiency agrobacterium tumefaciens-mediated transformation method of zoysia japonica. Download PDFInfo
- Publication number
- CN102776232A CN102776232A CN2011101197282A CN201110119728A CN102776232A CN 102776232 A CN102776232 A CN 102776232A CN 2011101197282 A CN2011101197282 A CN 2011101197282A CN 201110119728 A CN201110119728 A CN 201110119728A CN 102776232 A CN102776232 A CN 102776232A
- Authority
- CN
- China
- Prior art keywords
- callus
- agrobacterium tumefaciens
- agrobacterium
- substratum
- time
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a high efficiency agrobacterium tumefaciens-mediated transformation method of zoysia japonica. The method comprises the steps of infecting calluses of the zoysia japonica with agrobacterium tumefaciens suspended in a AAM-AS culture medium; putting the infected calluses in a N6D2-AS culture medium and co-culturing for 2-4 days; washing off the agrobacterium tumefaciens; screening and culturing the calluses. Aiming at three important influence factors which influence conversion efficiency of the agrobacterium tumefaciens-mediated genetic transformation system of the zoysia japonica, the best bacterium concentration (OD600 value being 0.5-0.6), infecting time (15 min) and co-culturing time (2-3 days) are found out. Combining an imrovement of the culture medium, final conversion efficiency reaches more than 50%. Besides, by improving washing method of the agrobacterium tumefaciens, pollutions of the agrobacterium tumefaciens can be eliminated in the screening and culturing process. Compared with previous agrobacterium tumefaciens-mediated genetic transformation methods, the method greatly reduces work-load, saves cost and prevents waste of calluses materials.
Description
Technical field
Genetically engineered of the present invention field specifically, relates to a kind of Japanese lawn grass highly effective agrobacterium mediating method for transformation.
Background technology
Japanese lawn grass (Zoysia Japonica Steud.) is the abundant unique turf grass species that does not need import of china natural resources, and it is strong to have flexibility, many good characters such as drought-enduring, heat resistanceheat resistant, cold-resistant, anti-trample and impoverishment tolerant.Its shortcoming mainly is to germinate slowly, and it is slow to emerge into the level ground, causes being prone to occupied by weeds.So general breeding objective all concentrates on and prolongs the green phase, improves its quality and winter hardiness, accelerate aspects such as seedling growth, germination speed, but research in this respect seldom, only some basic researchs have been done on the seed dormancy breaking both at home and abroad.Utilize traditional breeding way to obtain very difficulty of good resistant variety because the complicacy of the complicacy of stress resistance of plant itself and its quantitative trait locus linkage relationship makes, 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.Over nearly 20 years, along with progress, the especially transgenic technology development in the gramineous crop breeding of molecular biology of plants and genetically engineered aspect, in recent years, progress is sent out rapidly in turfgrass transgenic technology aspect.
The agrobacterium mediation converted ratio juris is: agrobacterium tumefaciens (Agrobacterium tumefaciens) is when infecting plant; The section of DNA (T-DNA) that is independent of its extrachromosomal Ti-plasmids can change vegetable cell over to; And stably be retained in the plant cell chromosome; Become a group gene that vegetable cell increases newly, finally can entail filial generation (Matthews etc., 2001) through sexual generation.Both main processes comprise: the recombinant plasmid that contains goal gene imports Agrobacterium → Agrobacterium and transformation receptor is cultivated altogether → and the screening of the processing → transformant of transformation receptor and break up the Molecular Detection and the inheritance stability Journal of Sex Research of plant → plan transformed plant again.The transformation frequency of its mediation is high, and regeneration plant can be educated the rate height, transferable snare sheet segment DNA; Do not need the protoplastis culture technique; The copy number of foreign gene that imports is lower, and required experiment condition is simple, and price is low; Someone thinks that it is the method for transformation of a kind of " natural ", possibly help avoid gene silencing.In addition, the improvement agrobacterium-mediated transformation that adds Syringylethanone, glucose, gallic acid etc. has carried out extensive studies in paddy rice etc. monocotyledons, and has obtained higher transformation frequency.
Previous research thinks that dicotyledons is the natural host of Agrobacterium; Monocotyledons is difficult to infect with Agrobacterium; But along with the agrobacterium mediation converted method at wheat (Deng etc.; 1988) and successful in succession application on the paddy rice (Chan etc., 1993), also in the turfgrass transgenic breeding, succeed in recent years.Be by people such as Rosset (Rosselt etc. in 1998 the earliest; 1998) report; All successfully transformed English ryegrass and Itanlian rye, the hygromycin resistance rate is 1.7%-4.5%, indicates the breakthrough of agrobacterium mediation converted method on the turfgrass genetic transformation.Subsequently, the agrobacterium mediation converted method is in creeping bentgrass (Chai etc., 2000; Luo etc., 2004; Fu etc., 2005; Han etc., 2005), thin and delicate creeping bentgrass (Chai etc., 2000; 2004), orchardgrass (Lee etc., 2000), jielu grass (Chai etc., 2000; Toyama etc., 2003), Festuca Arundinacea (Lee, 2000; Bettany etc., 2003; Lee etc., 2004; Wang etc., 2005), also succeed in the research of English ryegrass (Bettany etc., 2003), English grass multiple turfgrasss such as (Chai etc., 2003).
Though the agrobacterium mediation converted method has obtained transfer-gen plant in above grass seeds, also exist genotypic restriction.The reason that monocotyledons is difficult to agroinfection mainly is (Ye Jianming, 1999; Guo Dianjing, 1997): 1. monocotyledons seldom or fully can not produce the signaling molecule that activates Ti-plasmids Vir district gene; 2. the 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, just can obtain transformed plant and have only those regeneration and integrate the strong competent cell of conversion capability.So far, be that the agrobacterium-mediated transformation transformation system of acceptor still rests on the laboratory study stage with the Japanese lawn grass embryo callus.
It is results of interaction between agrobacterium strains and the vegetable cell that agriculture bacillus mediated foreign gene transforms, and every various factors that can influence vegetable cell conversion responsibility and Agrobacterium infection ability and transformant regenerative power all can exert an influence to changing effect.Therefore, the raising of Agrobacterium-mediated Transformation efficient depends on improve (being prone to rely on oneself 2001) to the optimization of various factors of influence and conversion condition.
Summary of the invention
The object of the invention overcomes the defective that existing agriculture bacillus mediated technology is difficult to be applied to the grass genetic transformation, and a kind of highly effective agrobacterium mediating method for transformation that is applicable to Japanese lawn grass is provided.
In order to realize the object of the invention; A kind of Japanese lawn grass highly effective agrobacterium mediating method for transformation of the present invention; It is to use the agrobacterium tumefaciens (Agrobacterium tumefaciens) that is suspended in the AAM-AS substratum to infect the callus of Japanese lawn grass, and the callus after will infecting places N
6D
2Cultivated altogether in the-AS substratum 2-4 days, preferred 2-3 days, last flush away agrobacterium tumefaciens, screening and cultured calli;
Wherein, the AAM-AS culture medium prescription is: 463mg/L (NH
4)
2SO
4+ 283mg/LKNO
3+ 185mg/L MgSO
47H
2O+400mg/L KH
2PO
4+ 166mg/L CaCl
22H
2O+4.4mg/L MnSO
44H
2O+1.5mg/L ZnSO
47H
2O+1.6mg/LH
3BO
3+ 0.8mg/L KI+0.4mg/L glycocoll+0.02mg/L VITMAIN B1+0.1mg/L Y factor+0.1mg/L nicotinic acid+37.24mg/L Na
2-EDTA+27.84mg/L FeSO
47H
2O+100mg/L inositol+900mg/L L-glutaminate+300mg/L L-aspartic acid+176.7mg/L L-l-arginine+10mg/L thiamine hydrochloride+68.5g/L sucrose+36g/L glucose+100 μ mol/L Syringylethanones, pH5.2;
N
6D
2-AS culture medium prescription is: 463mg/L (NH
4)
2SO
4+ 2830mg/LKNO
3+ 185mg/L MgSO
47H
2O+400mg/L KH
2PO
4+ 166mg/L CaCl
22H
2O+10mg/L MnSO
44H
2O+2mg/L ZnSO
47H
2O+3mg/L H
3BO
3+ 0.75mg/LKI+0.25mg/L Na
2MoO
42H
2O+0.025mg/L CuSO
45H
2O+0.025mg/LCoCl
26H
2O+37.24mg/L Na
2-EDTA+27.84mg/L FeSO
47H
2O+10mg/L vitamin+1mg/L pyridoxine hydrochloride+1mg/L nicotinic acid+100mg/L inositol+500mg/LL-proline(Pro)+300mg/L caseinhydrolysate+500mg/L Stimulina+2mg/L2; 4-D+30g/L sucrose+10g/L glucose+1.0g/L caseinhydrolysate+100 μ mol/L Syringylethanone+2g/L plant gels, pH5.2.
Aforesaid method, the preparation method who is suspended in the agrobacterium tumefaciens in the AAM-AS substratum is: as the bacterium liquid OD of agrobacterium tumefaciens
600When value was 0.5-0.8, when being preferably 0.5-0.6, centrifugal results thalline added AAM-AS substratum suspension thalline then in thalline.
Aforesaid method, time of infection is 10-40min, is preferably 15min.
Aforesaid method, the flush away agrobacterium tumefaciens comprises step: the callus that 1) will cultivate altogether is with sterile distilled water flushing 3-5 time, and the limit side washing is vibrated, the liquid clarification after cleaning; 2) then callus is suspended in the sterilized water that contains 450-550mg/L cephamycin and 150-250mg/L penbritin, 25-28 ℃, 100-120rpm, 1.5-2h.
Aforesaid method, said agrobacterium tumefaciens are the agrobacterium tumefaciens that contains the conversion of goal gene, and its starting strain is preferably EHA105.
Aforesaid method, the preparation method of the callus of Japanese lawn grass is: the Japanese lawn grass seed is dipped among the chlorine bleach liquor on magnetic stirring apparatus, sterilizes, with aseptic washing 3-5 time, 4 ℃ are soaked 3-4d down with the seed after the sterilization; Seed after the immersion with the chlorine bleach liquor 15-25min that sterilizes, after aseptic washing 3-5 time, blots the moisture of seed-coat again, is inoculated on the callus inducing medium callus induction under the 26-30 ℃ of dark condition; Every month subculture of callus is once selected white or yellow, the particulate state callus is transferred on the subculture medium and cultivates during subculture, promptly get; Wherein, the callus inducing medium prescription of use is: NB
0+ 2,4-D2mg/L, pH5.8; Callus succeeding transfer culture based formulas is: NB
0+ 2,4-D2mg/L, pH5.8.
The present invention is directed to agriculture bacillus mediated Japanese lawn grass genetic conversion system influences the three big material impact factors of transformation efficiency, finds out best bacterial concentration (OD
600Value is 0.5-0.6), time of infection (15min) reaches incubation time (2-3 days) altogether.In conjunction with the improvement of substratum, (AS100 μ mol/L is pH5.2) as Agrobacterium suspension culture base, N to use the AAM substratum
6D
2(AS100 μ mol/L, pH5.2) as being total to culture medium, final transformation efficiency reaches more than 50% substratum.
In addition, the present invention passes through to improve the Agrobacterium purging method, and the callus of cultivating is altogether washed 3～5 times with sterile distilled water earlier; Resuspending in the sterilized water that contains 450-550mg/L cephamycin and 150-250mg/L penbritin, 25-28 ℃, 100-120rpm; 1.5-2h, with 60 order cells sieve callus is leached, blot with aseptic filter paper; Transfer in selecting in the process of screening and culturing, to have got rid of the pollution of Agrobacterium on the substratum, compare with agrobacterium mediation converted method in the past; Significantly reduce workload, practiced thrift cost, avoided the waste of callus material.
Description of drawings
Fig. 1 is the structural representation of carrier pCAMBIA1301.
Fig. 2 is the structural representation of plant expression vector pc1301-ubi.
Fig. 3 is the structural representation of carrier pc1301-ubi-ZjGA20-dsRNAi.
Fig. 4 A-D is selecting 3 months kanamycin-resistant callus tissue of screening on the substratum.
Fig. 5 A-C is the regeneration and the cultivation of transfer-gen plant, and A-C representes differentiation, strong sprout and plantlet of transplant respectively.
Fig. 6 is a resistant plant RT-PCR detected result, ck0: transfer-gen plant not; Ck1: change the empty carrier plant; 1～7: transfer-gen plant.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Reagent that uses in following examples and material like no specified otherwise, are the commercial goods.
The screening of embodiment 1 agriculture bacillus mediated Japanese lawn grass genetic conversion system top condition
1 recipient plant material
With Japanese lawn grass kind ' Zenith ' mature seed (' Zenith ' variety seeds available from U.S. TMI company (TurfMerchants, Inc)) be immersed in the chlorine bleach liquor (available from the Beijing Chemical Plant,＞5% reactive chlorine; Add 1-2 and drip polysorbas20) in, sterilization is 1 hour on magnetic stirring apparatus, with aseptic water washing 3-5 time; 4 ℃ are soaked 3d down; Behind the hypochlorous sodium solution disinfection 20min again, aseptic water washing 3 times, blot excessive moisture with aseptic filter paper; Be inoculated on the callus inducing medium the following 28 ℃ of callus inductions of dark state.Every month subculture of callus is once selected white or yellow, particulate state callus and is transferred on the subculture medium and cultivates, as the acceptor material of gene transformation during subculture.
2 bacterial strains and carrier
Intestinal bacteria (Escherichia coli) competence DH5a, agrobacterium strains EHA105 is available from the full Shi Jin in Beijing biotech firm.Plant expression vector (empty carrier) pCAMBIA1301 (Fig. 1), screening-gene are hph, by the CaMV35S promoter regulation.The building process of plant expression vector pc1301-ubi (Fig. 2) is following:
(1) acquisition of Ubi promotor: with the corn gene group is template amplification Ubi promotor, introduces the HindIII restriction enzyme site at the upper reaches of Ubi promotor, and the BamHI restriction enzyme site is introduced in downstream.Pcr amplification product is cloned into the exactness of order-checking affirmation Ubi sequence on the pMD18-T carrier.Discharge the Ubi promotor with the HindIII+BamHI double digestion from the pMD18 carrier.
(2) introduce terminator at the MCS of pCAMBIA1301: with SacI+EcoRI double digestion pBI121 and pCAMBIA1301 respectively, the big fragment of pCAMBIA1301 that goes up behind small segment (Noster terminator) that enzyme scales off and the double digestion from pBI121 is connected.
(3) the Ubi promotor is introduced the MCS of pCAMBIA1301: HindIII+BamHI double digestion pCAMBIA1301, the Ubi promotor of getting off with the same endonuclease digestion of above-mentioned usefulness is connected.
Behind expression vector conversion Agrobacterium, preserve through detecting also, be ready to use in agriculture bacillus mediated transgenic.
3 substratum
3.1 bacteria culture medium
LB substratum (being used to cultivate intestinal bacteria): peptone 10g/L+ yeast extract 5g/L+NaCl10g/L, pH7.2.
YEB substratum (being used to cultivate Agrobacterium): beef extract 5g/L+ yeast extract 1g/L+ peptone 5g/L+MgSO
47H
2O0.5g/L+ sucrose 5g/L, pH7.0.
3.2 agrobacterium mediation converted improved culture medium
I. acceptor material substratum
(1) callus inducing medium: NB
0+ 2,4-D2mg/L, pH5.8.
(2) callus subculture medium: NB
0+ 2,4-D2mg/L, pH5.8.
II. agrobacterium mediation converted is used substratum
(1) Agrobacterium suspension culture base: AAM substratum+AS100 μ mol/L, pH5.2.
(2) be total to culture medium: N
6D
2Substratum+AS100 μ mol/L, pH5.2.
(3) callus screening culture medium: NB-CH substratum, pH5.8.
(4) resistant calli regeneration culture medium: 1/2MS-KT-CH.
(5) resistant plant strong seedling culture base: 1/2MS-C.
Each culture medium prescription is as shown in table 1.
The substratum that uses in table 1 Japanese lawn grass tissue culture and the genetic transformation process
The genetic transformation of 4 agriculture bacillus mediated Japanese lawn grass
4.1 cultivation of Agrobacterium and activation
(1) cultivation of Agrobacterium
Bacterium liquid coated plate is selected on the substratum in the YEB solid that contains 1 times of microbiotic (50mg/L Rif, 50mg/L Kan).Be inverted, 28 ℃, secretly cultivate 1-2d.
(2) activation of Agrobacterium
Activation is for the first time: 2mL bacterium liquid in containing 1 times of antibiotic YEB substratum, 28 ℃, the 200rpm/min overnight cultures.
Activation is for the second time: 2mL bacterium liquid is not in containing antibiotic YEB substratum, and 28 ℃, 200rpm/min cultivates, and 600nm surveys its OD down
600Value is respectively 0.5,0.6,0.7,0.8, prepares to infect callus.
4.2 the optimization of agriculture bacillus mediated Japanese lawn grass genetic conversion system
(1) bacterial concentration: at bacterium liquid OD
600Value is respectively 0.5,0.6, and 0.7,0.8 o'clock, bacterium liquid is changed in the 50mL centrifuge tube, 4 ℃, 4500rpm/min, 8min.Fall supernatant.
(2) infect: every centrifuge tube add isopyknic AAM (AS 100 μ mol/L, pH5.2) Agrobacterium suspension culture base is transferred to behind the mixing in 7 triangular flasks, and callus is chosen in the triangular flask; Carry out the mark of 7 time of infection gradients respectively, 25 ℃, 120rpm infects 10min; 15min, 20min, 25min; 30min, 35min, 40min.
(3) cultivate altogether: with 60 order cells sieve callus is leached, blot with aseptic filter paper.Callus changes N over to
6D
2Altogether in the substratum, cultivated altogether 2 days under 25 ℃, 3 days, 4 days, 2 petridish of each time gradient, 14 callus of every ware inoculation.
(4) cleaning of Agrobacterium: the callus that will cultivate altogether is with sterile distilled water flushing 3～5 times; Limit side washing vibration, the liquid clarification after cleaning is with the whole flush awaies of bacterium; Resuspending is in the sterilized water that contains 500mg/L cephamycin (cefatoxime) and 200mg/L penbritin (amplicillim); 25 ℃, 120rpm, 2h.
(5) select to cultivate: with 60 order cells sieve callus is leached, after blotting with aseptic filter paper, go to and select that (NB-CH) carried out screening and culturing 2 months on the substratum, add up the kanamycin-resistant callus tissue rate.
5 experimental results
5.1 different factors are to the influence of agriculture bacillus mediated Japanese lawn grass genetic transformation efficiency
(1) bacterial concentration is to the influence of transformation efficiency
Can Agrobacterium effectively adhere to and infect explant is the key that transforms success or not.Agrobacterium concentration is too low, and thalline can not effectively infect callus in time of infection.But, arrive through electron microscopic observation in the culturing process altogether, when bacterial concentration was too high, thalline itself was prone to mutual coalescence, and influences its adhering on explant (Zhu Changxiang, 2001).Agrobacterium concentration is too high, because the hypertrophy of bacterium makes the callus excessive damage even suffocates, and the removing of thalline after also being unfavorable for, thereby influence the kanamycin-resistant callus tissue rate.
With the different bacterium liquid of concentration, infect the Japanese lawn grass callus, cultivate 2-4d altogether, the result sees table 2, and is visible, OD
600The processing kanamycin-resistant callus tissue rate of=0.6 o'clock bacterial concentration is higher than OD
600=0.7 and OD
600=0.8, and and OD
600=0.5 processing does not have marked difference, finds OD simultaneously
600=0.7 and OD
600The callus that=0.8 bacterium liquid is infected Agrobacterium in screening process is difficult to suppress, and most of callus is contaminated.Therefore, OD
600Be that 0.5～0.6 bacterial concentration is suitable for the Japanese lawn grass Agrobacterium-mediated Transformation.
Table 2 bacterial concentration is to the influence of Agrobacterium-mediated Transformation kanamycin-resistant callus tissue rate
(2) time of infection is to the influence of transformation efficiency
With OD
600Be about 0.5 bacterium liquid and infect the jielu grass callus, visible by table 3, the kanamycin-resistant callus tissue rate that the processing of 10min obtains is the highest, and with the increase of time of infection, the kanamycin-resistant callus tissue rate also decreases.Find in the experiment simultaneously when selection is subsequently cultivated, to infect 30min, 35min, the Agrobacterium that the processing of 40min is difficult to suppress serious pollutes.
Table 3OD
600=0.5 time of infection is to the influence of Agrobacterium-mediated Transformation kanamycin-resistant callus tissue rate
With OD
600Be about 0.6 bacterium liquid and infect the jielu grass callus, visible by table 4,10min, 15min, the kanamycin-resistant callus tissue rate that the processing of 20min obtains is higher relatively, behind 25min, obviously descends.This shows that time of infection is long, the Agrobacterium that adheres on the explant is too much, causes the over-drastic infringement to callus, thereby influences the acquisition of kanamycin-resistant callus tissue.
Table 4OD
600=0.6 time of infection is to the influence of Agrobacterium-mediated Transformation kanamycin-resistant callus tissue rate
(3) be total to the influence of incubation time to transformation efficiency
Altogether incubation time to the influence of Japanese lawn grass kanamycin-resistant callus tissue pick-up rate and bacterial concentration and time of infection to influence mechanism similar, three kinds of factors all are to find out suitable " degree ".Incubation time is too short altogether, and Agrobacterium is infected deficiency, and T-DNA can not shift and expression to vegetable cell effectively; Prolonging altogether, incubation time can guarantee that T-DNA shifts to vegetable cell effectively; But along with the prolongation of incubation time altogether, the Agrobacterium of callus absorption is too many, to the damage increase of callus; And later antibacterial difficulty is increased, so can not prolong incubation time altogether by over-drastic.
With OD
600Be about 0.5 bacterium liquid and infect the Japanese lawn grass callus, inquire into the influence of incubation time antagonism callus pick-up rate altogether.Visible by table 5 and table 6, the result infers like expection, cultivates 2d and 3d kanamycin-resistant callus tissue pick-up rate altogether and all is higher than common cultivation 4d, explains that the incubation time of 4d is long, and 2-3d is more suitable.Find that in the screening and culturing process in later stage bacterium appears in the periphery of cultivating most callus of 4d altogether, the Agrobacterium that is difficult to suppress serious pollutes, if prolong incubation time altogether again, Agrobacterium can be covered with whole callus, even pollutes.
Table 5OD
600=0.5 is total to the influence of incubation time to Agrobacterium-mediated Transformation kanamycin-resistant callus tissue rate
Table 6OD
600=0.6 is total to the influence of incubation time to Agrobacterium-mediated Transformation kanamycin-resistant callus tissue rate
5.2 the improvement of Agrobacterium purging method
The present invention is through improving the Agrobacterium purging method, and earlier with sterile distilled water flushing 3～5 times, resuspending is in the sterilized water that contains 500mg/L cephamycin (cefatoxime) and 200mg/L penbritin (amplicillim) with the callus of cultivating altogether; 25 ℃, 120rpm, 2h; With 60 order cells sieve callus is leached, blot, transfer in selecting on the substratum with aseptic filter paper; In the process of screening and culturing, got rid of the pollution of Agrobacterium, compared, significantly reduced workload with agrobacterium mediation converted experiment in the past; Practice thrift cost, avoided the waste of callus material.
1 recipient plant material
Japanese lawn grass kind ' Zenith ' mature seed hypochlorous sodium solution (available from the Beijing Chemical Plant,＞5% reactive chlorine adds 1-2 and drips polysorbas20) was sterilized 1 hour on magnetic stirring apparatus; With aseptic water washing 3-5 time, 4 ℃ are soaked 3d down, and the seed after the immersion is hypochlorous sodium solution disinfection 20min again; Behind the aseptic water washing 3 times; Blot excessive moisture with aseptic filter paper, be inoculated on the callus inducing medium, the following 28 ℃ of callus inductions of dark state.Every month subculture of callus is once selected white or yellow, particulate state callus and is transferred on the subculture medium and cultivates, as the acceptor material of gene transformation during subculture.
2 bacterial strains and carrier
Intestinal bacteria (Escherichia coli) competence DH5a, agrobacterium strains EHA105 is available from the full Shi Jin in Beijing biotech firm.RNA disturbs (RNAi) plant expression vector pc1301-ubi-ZjGA20-dsRNAi (Fig. 3); Comprise 283bp positive-sense strand and 283bp antisense strand in the Japanese lawn grass GA20 oxidase gene ORFs; By the ubi promoters driven, screening-gene is hph, by the CaMV35S promoter regulation.
The structure flow process of this expression vector is: select the fragment of the interior 283bp of ORFs of Japanese lawn grass GA20-oxidase oxidase gene to make up palindromic sequence; For ease this fragment is connected on the carrier with specific direction, before two pairs of primers, adds to be used for directed restriction enzyme site.PEASY-T1 (Transgene) carrier to be connected with the GA20-oxidase gene is template PCR, obtains forward and reverse fragment respectively.
Forward fragment primer PS 1:5 '-
GAATTCGCACCACGGAGCTGTTTAC-3 '
EcoR?I
Reverse fragment primer PS2:5 '-
AAGCTTCCATCATCTCCAGAGAGAGACG-3 '
HindIII
Forward fragment primer PA1:5 '
CTCGAGCCATCATCTCCAGAGAGAGACG-3 '
Xho?I
Reverse fragment primer PA2:5 '-
GGTACCGCACCACGGAGCTGTTTAC-3 '
Kpn?I
The PCR product is called after forward fragment Fga20 and reverse fragment Rga20 respectively; And be connected into pEASY-T1simple carrier (pEASY-T1simple is available from the full Shi Jin in Beijing biotech firm) respectively; Obtain recombinant vectors pEASY-T1-F and pEASY-T1-R, with EcoR I and HindIII hit a carrier pBluescript SK plus (available from the full Shi Jin in Beijing biotech firm) and recombinant vectors pEASY-T1-F of enzyme simultaneously, the enzyme of recovery pEASY-T1-F is cut small segment; Be connected among the pBluescript SKplus; Constitute intermediate carrier pBluescript SK plus-F, with Xho I and Kpn I hit a carrier pBluescript SK plus-F and a recombinant vectors pEASY-T1-R of enzyme simultaneously, the enzyme of recovery pEASY-T1-R is cut small segment again; Be connected among the pBluescript SK plus-F, constitute intermediate carrier pBluescript SK plus-FR.
With BamH I and Kpn I hit a carrier pBluescript SK plus-FR and a plant expression vector pc1301-ubi of enzyme simultaneously; Reclaim the enzyme of pBluescript SK plus-FR and cut small segment; Be connected in the big fragment of pc1301-ubi, make up plant expression vector pc1301-ubi-ZjGA20-dsRNAi (Fig. 3).After expression vector transforms Agrobacterium,, be ready to use in agriculture bacillus mediated transgenic through detecting and preserving.
3 substratum
3.1 bacteria culture medium
LB substratum (being used to cultivate intestinal bacteria): peptone 10g/L+ yeast extract 5g/L+NaCl10g/L, pH7.2.
YEB substratum (being used to cultivate Agrobacterium): beef extract 5g/L+ yeast extract 1g/L+ peptone 5g/L+MgSO
47H
2O0.5g/L+ sucrose 5g/L, pH7.0.
3.2 agrobacterium mediation converted improved culture medium
I. acceptor material substratum
(1) callus inducing medium: NB
0+ 2,4-D2mg/L, pH5.8.
(2) callus subculture medium: NB
0+ 2,4-D2mg/L, pH5.8.
II. agrobacterium mediation converted is used substratum
(1) Agrobacterium suspension culture base: AAM substratum+AS100 μ mol/L, pH5.2.
(2) be total to culture medium: N
6D
2Culture medium A S100 μ mol/L, pH5.2.
(3) callus screening culture medium: NB-CH substratum, pH5.8.
(4) resistant calli regeneration culture medium: 1/2MS-KT-CH.
(5) resistant plant strong seedling culture base: 1/2MS-C.
Each culture medium prescription is as shown in table 1.
The genetic transformation of 4 agriculture bacillus mediated Japanese lawn grass
4.1 cultivation of Agrobacterium and activation
(1) cultivation of Agrobacterium
Bacterium liquid coated plate is selected on the substratum in the YEB solid that contains 1 times of microbiotic (50mg/L Rif, 50mg/L Kan).Be inverted, 28 ℃, secretly cultivate 1-2d.
(2) activation of Agrobacterium
Activation is for the first time: 2mL bacterium liquid in containing 1 times of antibiotic YEB substratum, 28 ℃, the 200rpm/min overnight cultures.
Activation is for the second time: 2mL bacterium liquid is not in containing antibiotic YEB substratum, and 28 ℃, 200rpm/min cultivates, and 600nm surveys its OD down
600Value is respectively 0.5,0.6,0.7,0.8, prepares to infect callus.
4.2 agriculture bacillus mediated Japanese lawn grass genetic transformation
Best bacterial concentration (the OD that obtains according to preliminary experiment
600Value is 0.5～0.6), time of infection (15min) reaches incubation time (2～3 days) altogether, restarts large batch of conversion.
(1) bacterial concentration: at bacterium liquid OD
600Value is 0.5～0.6 o'clock, bacterium liquid changed in the 50mL centrifuge tube, and 4 ℃, 4500rpm/min, 8min.
(2) infect: fall supernatant, every pipe adds isopyknic AAM, and (AS100 μ mol/L, pH5.2) Agrobacterium suspension culture base is transferred to behind the mixing in the 250mL triangular flask.Callus is chosen in the triangular flask, and 25 ℃, 120rpm infects 15min.
(3) cultivate altogether: with 60 order cells sieve callus is leached, blot with aseptic filter paper.Callus changes N over to
6D
2(AS100 μ mol/L pH5.2) altogether in the substratum, cultivated 2～3 days under 25 ℃ altogether.
(4) cleaning of Agrobacterium: the callus that will cultivate altogether is with sterile distilled water flushing 3～5 times; Limit side washing vibration, the liquid clarification after cleaning is with the whole flush awaies of bacterium; Resuspending is in the sterilized water that contains 500mg/L cephamycin (cefatoxime) and 200mg/L penbritin (amplicillim); 25 ℃, 120rpm, 2h.
(5) select to cultivate: with 60 order cells sieve callus is leached, after blotting with aseptic filter paper, go to that (NB-CH) carries out screening and culturing on the selection substratum.
4.3 the screening of transformant and cultivation
Callus is transferred to the resistant calli that newly grows on the new selection substratum selecting dark the cultivation for 8 weeks on the substratum, continues 4 weeks of screening.
Select well-grown; The resistant calli of compact structure (Fig. 4) is transferred to resistant calli regeneration culture medium (1/2MS-KT-CH) and is gone up cultivation; The inductor embryo takes place and plant regeneration, after about 1 month, treats that (Fig. 5) appears in plantlet; Change it over to nothing screening pressure, but contain the middle strong sprout of 1/2MS substratum (1/2MS-C) of 300mg/L cephamycin (Cef).
4.4 the transplanting of resistant plant
After root system is strong, refining seedling 3 days, careful then flush away plant root agar, (matrix is sand: soil: the peat composed of rotten mosses=1: 1: 1), natural light is growth down directly to plant flowerpot.
The Molecular Detection of 5 transformed plants
Extract resistant plant RNA and carry out the RT-PCR detection, detected result is as shown in Figure 6.
Through the RT-PCR experiment, detect resistant plant 7 strains altogether, wherein positive plant 6 strains, transformation efficiency 85.7%.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (10)
1. Japanese lawn grass highly effective agrobacterium mediating method for transformation; It is characterized in that; It is the callus that agrobacterium tumefaciens (Agrobacterium tumefaciens) in 5.2 the AAM-AS substratum is infected Japanese lawn grass that use is suspended in the pH value, and it is 5.2 N that the callus after will infecting places the pH value
6D
2Cultivated last flush away agrobacterium tumefaciens, screening and cultured calli in-the AS substratum altogether 2-4 days.
2. method according to claim 1 is characterized in that, the preparation method who is suspended in the agrobacterium tumefaciens in the AAM-AS substratum is: as the bacterium liquid OD of agrobacterium tumefaciens
600When value was 0.5-0.8, centrifugal results thalline added AAM-AS substratum suspension thalline then in thalline.
3. method according to claim 2 is characterized in that, the preparation method who is suspended in the agrobacterium tumefaciens in the AAM-AS substratum is: as the bacterium liquid OD of agrobacterium tumefaciens
600When value was 0.5-0.6, centrifugal results thalline added AAM-AS substratum suspension thalline then in thalline.
4. according to each described method of claim 1-3, it is characterized in that time of infection is 10-40min.
5. method according to claim 4 is characterized in that, time of infection is 15min.
6. according to each described method of claim 1-3, it is characterized in that incubation time is 2-3 days altogether.
7. according to each described method of claim 1-3, it is characterized in that the flush away agrobacterium tumefaciens comprises step:
1) callus that will cultivate altogether is with sterile distilled water flushing 3-5 time, and the limit side washing is vibrated, the liquid clarification after cleaning;
2) then callus is suspended in the sterilized water that contains 450-550mg/L cephamycin and 150-250mg/L penbritin, 25-28 ℃, 100-120rpm, 1.5-2h.
8. according to each described method of claim 1-3, it is characterized in that said agrobacterium tumefaciens is the agrobacterium tumefaciens that contains the conversion of goal gene.
9. method according to claim 8 is characterized in that, the agrobacterium tumefaciens of said conversion, and its starting strain is EHA105.
10. according to each described method of claim 1-3; It is characterized in that; The preparation method of the callus of Japanese lawn grass is: the Japanese lawn grass seed is dipped among the chlorine bleach liquor on magnetic stirring apparatus, sterilizes, with aseptic washing 3-5 time, 4 ℃ are soaked 3-4d down with the seed after the sterilization; Seed after the immersion with the chlorine bleach liquor 15-25min that sterilizes, after aseptic washing 3-5 time, blots the moisture of seed-coat again, is inoculated on the callus inducing medium callus induction under the 26-30 ℃ of dark condition; Every month subculture of callus is once selected white or yellow, the particulate state callus is transferred on the subculture medium and cultivates during subculture, promptly get;
Wherein, the callus inducing medium prescription of use is: NB
0+ 2,4-D2mg/L, pH5.8; Callus succeeding transfer culture based formulas is: NB
0+ 2,4-D2mg/L, pH5.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101197282A CN102776232A (en) | 2011-05-10 | 2011-05-10 | High-efficiency agrobacterium tumefaciens-mediated transformation method of zoysia japonica. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101197282A CN102776232A (en) | 2011-05-10 | 2011-05-10 | High-efficiency agrobacterium tumefaciens-mediated transformation method of zoysia japonica. |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102776232A true CN102776232A (en) | 2012-11-14 |
Family
ID=47121339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101197282A Pending CN102776232A (en) | 2011-05-10 | 2011-05-10 | High-efficiency agrobacterium tumefaciens-mediated transformation method of zoysia japonica. |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102776232A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030106108A1 (en) * | 2001-11-15 | 2003-06-05 | Tohyama Kohichi | Genetic transformation method for zoysiagrass |
| US20040072353A1 (en) * | 2002-10-11 | 2004-04-15 | Tohyama Kohichi | Transgenic zoysiagrass with reduced shade avoidance |
| KR100490281B1 (en) * | 2002-11-27 | 2005-05-17 | 금호석유화학 주식회사 | Transgenic zoysiagrass with reduced shade avoidance |
| CN1736173A (en) * | 2005-08-17 | 2006-02-22 | 中国林业科学研究院林业研究所 | Method for improving genetic transformation efficiency of Japanese lawngrass |
| JP4228044B2 (en) * | 2002-12-20 | 2009-02-25 | 独立行政法人農業・食品産業技術総合研究機構 | Redifferentiated plant and transformed plant of Shiba spp. |
-
2011
- 2011-05-10 CN CN2011101197282A patent/CN102776232A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030106108A1 (en) * | 2001-11-15 | 2003-06-05 | Tohyama Kohichi | Genetic transformation method for zoysiagrass |
| US20040072353A1 (en) * | 2002-10-11 | 2004-04-15 | Tohyama Kohichi | Transgenic zoysiagrass with reduced shade avoidance |
| KR100490281B1 (en) * | 2002-11-27 | 2005-05-17 | 금호석유화학 주식회사 | Transgenic zoysiagrass with reduced shade avoidance |
| JP4228044B2 (en) * | 2002-12-20 | 2009-02-25 | 独立行政法人農業・食品産業技術総合研究機構 | Redifferentiated plant and transformed plant of Shiba spp. |
| CN1736173A (en) * | 2005-08-17 | 2006-02-22 | 中国林业科学研究院林业研究所 | Method for improving genetic transformation efficiency of Japanese lawngrass |
Non-Patent Citations (6)
| Title |
|---|
| 《Mol Genet Genomics》 20070403 Chunxiao Xu，Chaozu He The rice OsloL2 gene encodes a zinc finger protein involved in rice growth and disease resistance 85－94 1－10 第278卷, * |
| 《中国博士学位论文全文数据库 农业科技辑》 20050815 齐春辉 日本结缕草（Zoysia japonica Steud.）植株再生体系建立与DREB1A基因转化研究 D048-5 1－10 , 第4期 * |
| CHUNXIAO XU，CHAOZU HE: "The rice OsloL2 gene encodes a zinc finger protein involved in rice growth and disease resistance", 《MOL GENET GENOMICS》, vol. 278, 3 April 2007 (2007-04-03), pages 85 - 94, XP019517845, DOI: doi:10.1007/s00438-007-0232-2 * |
| HU ZHANG-HUA, ET AL: "Highly efficient transformation and plant regeneration of Tall Fescue mediated by Agrobacterium tumefaciens", 《JOURNAL OF PLANT PHYSIOLOGY AND MOLECULAR BIOLOLGY》, vol. 31, no. 2, 31 December 2005 (2005-12-31), pages 149 - 159 * |
| 齐春辉: "日本结缕草（Zoysia japonica Steud.）植株再生体系建立与DREB1A基因转化研究", 《中国博士学位论文全文数据库 农业科技辑》, no. 4, 15 August 2005 (2005-08-15), pages 048 - 5 * |
| 齐春辉等: "几种因素对日本结缕草愈伤组织诱导与生长影响的研究", 《四川草原》, no. 4, 31 December 2005 (2005-12-31), pages 1 - 3 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7928292B2 (en) | Refined plant transformation | |
| WO2022135246A1 (en) | R gene for controlling matching of soybean-rhizobium, protein and use thereof | |
| CN108300735B (en) | Efficient lilium tenuifolia genetic transformation system based on somatic embryogenesis | |
| CN102296086B (en) | Agrobacterium tumefaciens-mediated barley mature embryo callus transformation method | |
| CN104031936A (en) | Method of promoting generation of lateral buds of Heveabrasiliensis by trans-AtWUS (Arabidopsisthaliana WUSCHEL) gene | |
| CN100486994C (en) | Nitrate transport protein of diatom and its coding gene and application | |
| CN104450711A (en) | Application of OsmiR156f gene in rice effective tillering increasing | |
| CN106148390A (en) | CHY zinc finger protein transcriptional activation cofactor and application thereof | |
| CN102776231A (en) | Agrobacterium tumefaciens-mediated genetic transformation method of zoysia japonica | |
| CN105296529A (en) | Genetic transformation method for panicum virgatum L. | |
| Yaqoob et al. | Development of an efficient protocol for Agrobacterium mediated transformation of some recalcitrant indica rice varieties | |
| CN102586282A (en) | Application of lea-like gene in deinococcus radiodurans R1 to cultivating drought-resistant plants | |
| CN104017823B (en) | Screening method for fast and effectively reducing false positive rate of peanut genetic transformation plant | |
| CN102776232A (en) | High-efficiency agrobacterium tumefaciens-mediated transformation method of zoysia japonica. | |
| CN102206662B (en) | miR399 fusion gene, construction method thereof and application thereof in plant breeding | |
| CN103255141B (en) | Paddy rice nitrogen deficiency specific induced expression promoter Y5 and application thereof | |
| CN1291024C (en) | Agrobacterium mediated transformation system for obtaining perennial rye-grass transgenic plant in high efficiently and its use | |
| Shukla et al. | Genotypic variability in soybean [Glycine max (L.) Merrill] through agrobacterium-mediated transformation | |
| Xu et al. | High-efficiency agrobacterium-mediated transformation of chrysanthemum via vacuum infiltration of internode | |
| Sharma et al. | Agrobacterium-mediated transformation and regeneration of transformants of Brassica juncea | |
| CN116376933B (en) | Rice germination rate regulating gene OsSCYL2 and application thereof | |
| Bhattacharyya et al. | Agrobacterium mediated transfer of nptII and gus genes in Camellia assamica | |
| CN117844849B (en) | Genetic transformation method of Mongolian agropyron | |
| CN107267513A (en) | A kind of promoter HLP2 by pathogenic bacterium inducing | |
| Cheng et al. | Establishment of Sorghum BTx623 Immature Embryos Genetic Transformation and Regeneration System |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121114 |