CN103497970B - Gene transfer method with corn ovule as receptor - Google Patents
Gene transfer method with corn ovule as receptor Download PDFInfo
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- CN103497970B CN103497970B CN201310395028.5A CN201310395028A CN103497970B CN 103497970 B CN103497970 B CN 103497970B CN 201310395028 A CN201310395028 A CN 201310395028A CN 103497970 B CN103497970 B CN 103497970B
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Abstract
The invention relates to a gene transfer method with corn ovule as a receptor. The invention belongs to the technical field of plant genetic engineering. With an agrobacterium-infection-mediated genetic transformation principle, pollinated corn ovules are adopted as receptors; the ovule walls are pieced at placed close to micropyles; agrobacterium infection liquid comprising target gene is dropped into the ovules; through steps such as co-culture, immature embryo induction, differentiation and regeneration, rooting, molecular identification, and the like, transgenic corn plants are finally obtained. With the method, technical difficulties such as limited genotypes, complicated tissue culture operation, long transferring period, and the like of traditional corn transgenic methods can be effectively solved. Also, with the method, transgenic plant fertility is improved, target gene expression efficiency is high, and inheritance is stable. The method has great significance upon corn genetic engineering theoretical research and genetic breeding practices.
Description
Technical field
The present invention is specifically related to a kind of transgenic method being acceptor with corn ovule, belongs to field of plant genetic.
Background technology
Corn is one of main food crop of China.All the time, the growth of corn yield depends on improving constantly of corn breeding level to a great extent, but traditional breeding technology can not meet the demand of current corn production to improved seeds.Along with the development of biotechnology, utilize transgenic method by good character channel genes corn, greatly shorten breeding time, to the genetic improvement of corn variety significant (Maize Sciences, 2011/19 (5) // 64 ~ 67).At present, be that the traditional corn transgenic technology system of acceptor exists two technical barriers with rataria: first, maize genetic conversion is only limitted to H99, A188 etc., and a few can induce the genotype producing embryo callus, and these self-mating system economical characters being suitable for transforming are poor, good character gene is applied to actual production and through many generation hybridization and need backcrosses, this seriously slow down corn gene breeding process (Plant Cell Tiss Organ Cul, 2007/91//201 ~ 214); The second, the group training step that the hormone induction, herbicide screening etc. of embryo callus are loaded down with trivial details can cause high-caliber somatic variation, causes genetically modified low expression level and instability heredity (Mol Breed/2004/13//201 ~ 208).In order to solve above-mentioned technical barrier, carry out the transgenic research using fertilized egg cell as receptor system in recent years.Result of study finds, plant fertilization ovum is in similar " protoplastis " state not forming cell walls, now DNA replication dna, separation and restructuring are comparatively active, genetic transformation is carried out as acceptor, not only be easy to exogenous origin gene integrator, and transfer-gen plant can with development of fertilized ova Direct Regeneration, and then shorten the problem (GM Crops, 2010/1//276 ~ 287) that transfer-gen plant obtains, avoids genotype to limit.
In corn gene, carrying out genetic transformation progress using fertilized egg cell as acceptor larger is pollen tube passage method and Ovary injection, but owing to involving the slightly aobvious complicated plant double fertilization process of mechanism, conversion processing condition is not easily optimized, this makes these two kinds of method for transformation in external source channel genes and integration, have blindness to a certain degree, and repeatability not high (Plant Physiol, 2000/124//1540 ~ 1547).Anatomical research shows that fertilized egg cell is positioned at micropylar end (the In Vitro Cell Dev Biol Plant at corn ovule top, 2003/39//437 ~ 442), on this basis, using the in vitro corn ovule after pollination as transformation receptor, and directly carry out Agrobacterium in position, the hole of bead and infect, foreign gene just accurately can enter ovule and be incorporated into zygote genome, therefore has more practicality and operability.
Summary of the invention
The object of the present invention is to provide a kind of transgenic method being acceptor with corn ovule, which solve the technical barriers such as limited, the tissue culture complex operation of genotype that traditional corn transgenic method exists, transformation period be long, and there is the advantages such as transfer-gen plant fertility improves, target gene high expression and genetic stability, the theoretical investigation of corn gene engineering and genetic breeding practice are all significant.
For achieving the above object, the present invention is by the following technical solutions: the transgenic method being acceptor with corn ovule, is characterized in that concrete steps are as follows:
1) Agrobacterium infects the preparation of liquid: picking contains the single colony inoculation of Agrobacterium of target gene in YEP liquid nutrient medium, and at 28 DEG C, 180rpm shaking culture is spent the night, 4000rpm, collect thalline after 4 DEG C of centrifugal 10min, what be resuspended in pH 5.2 infects in substratum, regulates bacterial concentration to OD
600be stored in after=0.3-0.4 4 DEG C for subsequent use;
2) separation of corn ovule acceptor: bagging before maize ear reels off raw silk from cocoons, the self-pollination when 5-10cm extracted out by filigree, after filigree pollination, 24h takes off female fringe, first 70% alcohol wipe bract is used, and the inner bract of careful strip off, then female fringe is soaked 15min in 1.5% clorox, sterilized water at least washes 3 times; Female fringe is taken off middle part ovule with scalper by Bechtop in maltose solution (90 gl), is that the entry needle of 0.4mm pierces through ovule wall at ovule wall by admicropylar position with diameter;
3) Agrobacterium is infected and transforms and cultivate: 10 μ l Agrobacteriums are infected liquid and is placed in ovule surface, after infecting 5min, absorbs excess surface bacterium liquid; Agrobacterium is infected the Dual culture substratum that the ovule after conversion proceeds to pH 5.8,25 ovules about inoculated by each culture dish, 23 DEG C of light culture 3 days; After the Dual culture stage terminates, transferred to by ovule in the inducing culture of pH 5.8,25 DEG C of illumination cultivation 1-2 weeks, light/dark cycle is 16/8h, until stem appears in seedling; Seedling is transferred to the root media of pH 5.8, through 25 DEG C of illumination cultivation 2-4 weeks, light/dark cycle is 16/8h, when seedling grows to 10cm, transplants in the flowerpot of band soil;
4) Molecular Identification of transfer-gen plant: adopt PCR or Southern hybridization check transfer-gen plant, positive plant is cultured to maturation in greenhouse.
The described component infecting substratum is as follows: 1/2 N6 is a large amount of, 1/2 B5 trace, MS VITAMIN, 0.001 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 68.4 g/l sucrose, 36 g/l glucose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.01962 g/l Syringylethanone, 0.1% plant surface promoting agent Silwet-L77.
The component of described Dual culture substratum is as follows: 1/2 N6 is a large amount of, 1/2 B5 trace, MS VITAMIN, 0.001 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 150 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.03924 g/l Syringylethanone, 3g/l plant gel.
The component of described inducing culture is as follows: N6 is a large amount of, B5 trace, MS VITAMIN, 1 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 150 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.25 g/l Pyocianil, 0.0015 g/l bialaphos, 3 g/l plant gels.
The component of described regeneration culture medium is as follows: N6 is a large amount of, B5 trace, MS VITAMIN, 0.25 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 60 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.003 g/l bialaphos, 3 g/l plant gels.
The raw material of described root media is composed as follows: N6 is a large amount of, B5 trace, MS VITAMIN, 0.25 g/l caseinhydrolysate, 30 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.003 g/l bialaphos, 3g/l plant gel.
Positively effect of the present invention is 1, wider by genotype restriction, suitability as the corn gene method of acceptor using ovule after pollinating, solve with rataria is that the corn gene method of acceptor is only limitted to a few genotypic technical barrier, in the remarkable shortening corn gene cycle, accelerate corn gene breeding process;
2, transformation receptor corn ovule directly can form rataria and regenerate and differentiate high quality transfer-gen plant, effectively avoiding with rataria is tissue culture cycle of recipient corn transgeneic procedure longer, problem such as somatic variation level is higher, has that transfer-gen plant fertility improves, an advantage such as target gene high expression and genetic stability;
3, because the hole of bead of corn is positioned at ovule top, and ovum is exactly positioned at micropylar end, therefore directly carry out Agrobacterium in position, the hole of bead and infect conversion, significantly can increase foreign gene import and be integrated into the genomic probability of zygote, reduce the blindness of the corn gene method such as pollen tube passage method, Ovary injection in external source channel genes and integration;
4, simple operating steps is efficiently easy to grasp, and time saving and energy saving repeatability is high, without the need to expensive plant and instrument, is suitable for extensive, industrialization corn gene breeding.
Accompanying drawing explanation
Fig. 1 corn ovary (a) and ovule (b) sectional view arrow indication are that ovule wall is by admicropylar perforation position.
Fig. 2 Agrobacterium infects corn ovule 3(a), 9(b), 15(c), 21(d) growth of rataria and transgenic protocol behind sky.
The rataria regeneration that Fig. 3 Agrobacterium obtains after infecting corn ovule is divided into transfer-gen plant.
The PCR Molecular Identification result M:marker of Fig. 4 transfer-gen plant; +: pTF102 plasmid (positive control); –: unconverted plant (negative control); 1-16: transfer-gen plant (T
0-19,36,43,97,106,111,151,200,226,229,231,248,259,261,265,267), wherein 2,3,8,9,11,15 is transgene negative plant, and 1,4,5,6,7,10,12,13,14,16 is transgenic positive plant.
The Southern hybrid molecule qualification result WT of Fig. 5 transfer-gen plant: unconverted plant (negative control); 1-4: 4 transgenic lines of Stochastic choice.
Embodiment
The present invention is further described by following examples, and do not limit the present invention in any way, under the prerequisite not deviating from technical solution of the present invention, any change that those of ordinary skill in the art made for the present invention easily realize or change all will fall in right of the present invention.
Embodiment: to utilize with corn ovule be acceptor, and transgenic method transforms self-mating system
(1) preparation of substratum
The present invention used substratum row in Table 1.
The substratum that the transgenic method that table 1 is acceptor with corn ovule relates to
(2) agrobacterium strains and plasmid
Agrobacterium strains is EHA101, and plasmid is pTF102, and riddled basins is
barherbicide resistance gene (cauliflower mosaic virus 35 S promoter, soybean nutrient storage protein terminator), target gene is
gusreporter gene (cauliflower mosaic virus 35 S promoter, cauliflower mosaic virus 35S terminator).
(3) Agrobacterium infects the preparation of liquid
From-70 DEG C of frozen Agrobacterium EHA101(pTF102) take a morsel thalline, is inoculated in the YEP solid medium containing 0.025 g/l Rifampin, 0.05 g/l kantlex and 0.1 g/l spectinomycin, cultivates 2-3 days for 28 DEG C; Picking is containing the single colony inoculation of Agrobacterium in YEP liquid nutrient medium, and 28 DEG C, 180rpm shaking culture is spent the night, 4000rpm, and 4 DEG C of centrifugal 10min collect thalline, are resuspended in and infect substratum, regulates bacterial concentration to OD
600be stored in after=0.3-0.4 4 DEG C for subsequent use.
(4) separation of corn ovule acceptor
Bagging before self-mating system A188, Zheng 58, red 340 female fringes reel off raw silk from cocoons, the self-pollination when 5-10cm extracted out by filigree, after filigree pollination, 24h takes off female fringe, first 70% alcohol wipe bract is used, and the inner bract of careful strip off, then female fringe is soaked 15min in 1.5% clorox, sterilized water at least washes 3 times.Female fringe Fig. 1 (a) to be taken off middle part ovule Fig. 1 (b) with scalper by Bechtop in maltose solution (90 g/l), with diameter be the entry needle of 0.4mm at ovule wall by admicropylar site plan 1(b) arrow pointed location pierces through ovule wall.
(5) Agrobacterium is infected and transforms and cultivate
10 μ l Agrobacteriums are infected liquid and is placed in ovule surface, after infecting 5min, absorb excess surface bacterium liquid; Agrobacterium is infected the ovule after conversion and proceed to Dual culture substratum, 23 DEG C of light culture 3 days; After the Dual culture stage terminates, ovule is transferred to inducing culture, in 25 DEG C of light culture 2-3 weeks, be transferred to weekly new inducing culture last, until rataria forms (Fig. 2); By microscope, isolate the rataria formed in ovule, and be placed in regeneration culture medium, 25 DEG C of illumination cultivation 1-2 weeks, light/dark cycle is 16/8h, until stem appears in seedling; Seedling is transferred to root media, 25 DEG C of illumination cultivation 2-4 weeks, light/dark cycle is 16/8h, when seedling grows to 10cm (Fig. 3), transplants in the flowerpot of band soil.
(6) Molecular Identification of transfer-gen plant
From T
0genomic dna is extracted in transgenic corn plant blade, and with it for template, with 5 '-GACTCGTCCGTCCTGTAGAAACC-3 ' and 5 '-ATCTGCCCAGTCGAGCATCTC-3 ' for primer, in amplification transfer-gen plant
gusgene.PCR reaction conditions is: 95 DEG C of 5min; 94 DEG C of 30Sec, 60 DEG C of 30Sec, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min.Agarose gel electrophoresis pcr amplification product being carried out to 1% detects, and as shown in Figure 4, transgenic positive plant obtains the pcr amplification product of 189bp to PCR Molecular Identification result.
To T
1carry out the qualification of Southern hybrid molecule for transgenic corn plant, genomic dna is used
drai enzyme is cut, and transfers to (Amersham Hybond-N+, GE Healthcare Life Sciences) on nylon membrane after 0.8% sepharose 3V/cm electrophoresis spends the night, and with
gusfull length gene sequence is as hybridization probe, according to Random Primer DNA Labeling kit (TaKaRa, Japan) test kit carries out probe mark and crossover operation, the results are shown in Figure 5, the ovule of three different genotype all obtains the transgenic line of genetic stability after being infected conversion by Agrobacterium.
(7) cultivation of different genotype ovule acceptor is compared with transformation efficiency
After Agrobacterium infects corn ovule, the cultivation of statistics different genotype ovule acceptor and transformation efficiency, the results are shown in Table 2, there is not significant difference in cultivation and the transformation efficiency of ovule between different genotype, and the transgenic method that to illustrate with corn ovule be acceptor does not limit by genotype.
Table 2 Agrobacterium infects cultivation and the transformation efficiency of corn ovule
Claims (6)
1., with the transgenic method that corn ovule is acceptor, it is characterized in that concrete steps are as follows:
1) Agrobacterium infects the preparation of liquid: picking contains the single colony inoculation of Agrobacterium of target gene in YEP liquid nutrient medium, and at 28 DEG C, 180rpm shaking culture is spent the night, 4000rpm, collect thalline after 4 DEG C of centrifugal 10min, what be resuspended in pH 5.2 infects in substratum, regulates bacterial concentration to OD
600be stored in after=0.3-0.4 4 DEG C for subsequent use;
2) separation of corn ovule acceptor: bagging before maize ear reels off raw silk from cocoons, the self-pollination when 5-10cm extracted out by filigree, after filigree pollination, 24h takes off female fringe, first 70% alcohol wipe bract is used, and the inner bract of careful strip off, then female fringe is soaked 15min in 1.5% clorox, sterilized water at least washes 3 times; Female fringe is taken off middle part ovule with scalper by Bechtop in maltose solution 90 g/l, is that the entry needle of 0.4mm pierces through ovule wall at ovule wall by admicropylar position with diameter;
3) Agrobacterium is infected and transforms and cultivate: 10 μ l Agrobacteriums are infected liquid and is placed in ovule surface, after infecting 5min, absorbs excess surface bacterium liquid; Agrobacterium is infected the Dual culture substratum that the ovule after conversion proceeds to pH 5.8,25 ovules about inoculated by each culture dish, 23 DEG C of light culture 3 days; After the Dual culture stage terminates, transferred to by ovule in the inducing culture of pH 5.8,25 DEG C of illumination cultivation 1-2 weeks, light/dark cycle is 16/8h, until stem appears in seedling; Seedling is transferred to the root media of pH 5.8, through 25 DEG C of illumination cultivation 2-4 weeks, light/dark cycle is 16/8h, when seedling grows to 10cm, transplants in the flowerpot of band soil;
4) Molecular Identification of transfer-gen plant: adopt PCR or Southern hybridization check transfer-gen plant, positive plant is cultured to maturation in greenhouse.
2. the transgenic method that is acceptor with corn ovule according to claim 1, is characterized in that the described component infecting substratum is as follows: 1/2 N6 is a large amount of, 1/2 B5 trace, MS VITAMIN, 0.001 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 68.4 g/l sucrose, 36 g/l glucose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.01962 g/l Syringylethanone, 0.1% plant surface promoting agent Silwet-L77.
3. the transgenic method that is acceptor with corn ovule according to claim 1, it is characterized in that the component of described Dual culture substratum is as follows: 1/2 N6 is a large amount of, 1/2 B5 trace, MS VITAMIN, 0.001 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 150 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.03924 g/l Syringylethanone, 3g/l plant gel.
4. the transgenic method that is acceptor with corn ovule according to claim 1, is characterized in that the component of described inducing culture is as follows: N6 is a large amount of, B5 trace, MS VITAMIN, 1 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 150 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.25 g/l Pyocianil, 0.0015 g/l bialaphos, 3 g/l plant gels.
5. the transgenic method that is acceptor with corn ovule according to claim 1, it is characterized in that the component of described regeneration culture medium is as follows: N6 is a large amount of, B5 trace, MS VITAMIN, 0.25 g/l caseinhydrolysate, 0.7 g/l proline(Pro), 0.15 g/l inositol, 60 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.001 g/l zeatin, 0.003 g/l bialaphos, 3 g/l plant gels.
6. the transgenic method that is acceptor with corn ovule according to claim 1, it is characterized in that the raw material of described root media is composed as follows: N6 is a large amount of, B5 trace, MS VITAMIN, 0.25 g/l caseinhydrolysate, 30 g/l sucrose, 0.5 g/l 2-N-morpholine-ethylsulfonic acid, 0.003 g/l bialaphos, 3g/l plant gel.
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| US6998517B1 (en) * | 1998-06-25 | 2006-02-14 | The Regents Of The University Of California | Control of fruit dehiscence in Arabidopsis by indehiscent1 genes |
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