CN101831459A - Method for acquiring transgene cotton - Google Patents
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Abstract
The invention discloses a method for acquiring transgene cottons. The method comprises the following steps of: (1) cutting cotyledons, leaf stalks and a part of hypocotyl of cotton cotyledon seedlings and keeping r 1-3 cm of the hypocoty to obtain tissues to be infected at 25-30 DEG C and with the relative humidity 40-60%, and culturing for 2-3 days under dark environment; (2) scratching double surfaces from the top of a stem tip to the base part of the hypocotyl with the cut of 0.8-1.2 mm in depth; (3) dip-dyeing for 15-45 min in solution A which contains a recombination agrobacterium carrying exogenous genes; (4) co-culturing for 2-4 days and then carrying out resistance screening to obtain the resistant cotton seedling; and (5) grafting the resistant cotton seedling on a cotton rootstock to obtain the transgene cotton. The invention improves the conversion efficiency through adjusting the growth states of the stem tip, determining the appropriate depth of the agrobacterium-converted stem tip infection, adding acetosyringone and adjusting the proper pH value. Transgene plants can be quickly obtained by using the method, and the higher transgene efficiency is higher.
Description
Technical field
The invention belongs to the plant transgenic technology field, relate to a kind of method that obtains transgene cotton, be specifically related to a kind of with the cotton stem apex be acceptor, novel method by agriculture bacillus mediated acquisition transgenic cotton plant.
Background technology
Cotton is a kind of important cash crop, occupies critical role in national economy.The cotton transgenic technology makes substantial progress aspect genetic improvement, becomes the example of biotechnology successful Application in crop genetic improvement.At present, the genetic transforming method of transgenic plant is most widely used with the agrobacterium mediation converted method.By agriculture bacillus mediated gene transformation technology have be not subject to seasonal restrictions, goal gene mostly is single copy or low copy, insert the border and mostly be border, the T-DNA left and right sides, the rearrangement rate is low, easy gets rid of redundant plasmid dna sequence, obtain the advantage such as transfer-gen plant of marker-free easily and gain great popularity.Since reported first such as Umbeck in 1987 by agrobacterium-mediated transformation NPT II gene is imported cotton, the virtuous grade of Chen Zhi also utilized this method to obtain to import 2 in 1994, the upland cotton plant of 4-D resistant gene, Bt gene, CPTI gene also import cotton in succession thereupon, are thisly developed rapidly by agriculture bacillus mediated gene transformation technology.
According to the type of cotton genetic transformation acceptor, cotton has with agriculture bacillus mediated nucleus genetic transformation major technique system: be the transformation system of acceptor with the embryo callus, be the transformation system of acceptor with the hypocotyl, be the transformation system of acceptor with the shoot apical meristem.With embryo callus and hypocotyl is that two kinds of transformation systems of acceptor all must take place and plant regeneration through somatic embryo, because will be in the evoked callus process through differentiation, atomization again, can produce a lot of heritable variations and somatic variation, the plant regeneration difficulty, transformation period is long, reports that at present the used time of the shortest plant regeneration is more than five months; Genotype is given birth to by the cotton somatic embryos fetal hair and plant regeneration has decisive role, the somatic embryo generation is different with the ability of plant regeneration between the different cotton seeds of same Gossypium, between the same cotton seed different varieties, have only the material of minority kind isolated culture can induce embryo callus, limited the variety range of genetic transformation.
The Agrobacterium-mediated Transformation method that with the shoot apical meristem is acceptor is without dedifferentiation, atomization again, and heritable variation and somatic variation degree are low; Be subjected to the influence of cotton gene type restriction less, manyly fail to realize that somatic embryo takes place and the cotton variety of plant regeneration also can successfully carry out genetic transformation.Since reports such as Renfroe utilized the cotton shoot apical meristem to obtain regeneration plant, lot of domestic and international scholar utilized stem apex or shoot apical meristem to obtain transgenic regenerated plant.But usually transformation efficiency is low, and because the acquisition of the limitations affect regrowth of factor such as indefinite bud originates from many cells, and mosaic is many.
Summary of the invention
The purpose of this invention is to provide a kind of method that obtains transgene cotton.
The method of acquisition transgene cotton provided by the invention comprises the steps:
(1) cotyledon, petiole and the part hypocotyl of excision cotton cotyledon seedling, the length that hypocotyl keeps 0.8-3cm (can be 0.8-2cm, as 0.8-1.5cm, 1.5-2cm), obtains tissue to be infected; 25-30 ℃ then (as 25-28 ℃, 28-30 ℃), relative humidity 40%-60% (as 40%-50%, 50%-60%), secretly cultivate 2-3 days (as 2-2.5 days, 2.5-3 days); Described seed plantlet is that cotyledon half launches, hypocotyl elongation to 4-6cm (as 4-5cm, aseptic seedling 5-6cm);
(2) after will secretly cultivating wait to infect organize two-sided scuffing, scuffing scope be from the stem apex top to the hypocotyl base portion, the dark 0.8-1.2mm of wound is (as 0.8-1mm, 1-1.2mm);
(3) will scratch wait to infect is organized in contaminates 15-45 minute (as 15-30 minute, 30-35 minute) in the solution A; Contain the reorganization Agrobacterium of carrying foreign gene in the described solution A;
(4) infect and cultivate 2-4 days (as 2-3 days, 3-4 days) altogether after finishing, carry out resistance screening then, obtain the resistant cotton seedling;
(5) with the grafting of resistant cotton seedling on the cotton stock, obtain transgene cotton.
Relative humidity, represent airborne absolute humidity and synthermal under the ratio of saturated humidity ratio, total is a per-cent (just refers within a certain period of time, in the air of somewhere under contained vapour quantity and this temperature per-cent of saturation vapour amount).
In the step (3), the pH of described solution A is that 5.3-5.8 (as 5.3-5.6,5.6-5.8), is made up of solute and water; The solute and the concentration thereof of described solution A are as follows: Syringylethanone 40-120 μ lL
-1(as 40-80 μ lL
-1, 80-120 μ lL
-1), 2-N-morpholino ethane sulfonic acid (MES) 65-85mM is (as 65-75mM, 75-85mM), glucose 2-3% (w/w, quality percentage composition) is (as 2-2.5%, 2.5-3%), kinetin (KT) 0.08-0.12mgL
-1(as 0.08-0.1mgL
-1, 0.1-0.12mgL
-1), contain the reorganization Agrobacterium OD of foreign gene
600=0.6-0.8.
In the step (4), described resistance screening can carry out on screening culture medium.Described screening culture medium specifically can be made up of MS basic culture solution, agar, glycine, kinetin (KT), kantlex (Kan) and cephamycin (Cef); In the screening culture medium, the final concentration of agar is 7.5gL
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1, the final concentration of kantlex is 50-100mgL
-1(as 50-75mgL
-1, 75-100mgL
-1), the final concentration of cephamycin is 250-500mgL
-1(as 250-375mgL
-1, 375-500mgL
-1).
In the step (4), described cultivation altogether can carried out on the substratum altogether.Described substratum altogether specifically can be made up of MS basic culture solution, agar, glycine and kinetin; In the substratum, the final concentration of agar is 7.5gL altogether
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
The solvent of described MS basic culture solution is a water, concrete solute can be as shown in table 1.
The solute of table 1 MS basic culture solution
| Macroelement | Concentration (gL in the substratum -1) |
| ??NH 4NO 3 | ??1.65 |
| ??KNO 3 | ??1.9 |
| ??KH 2PO 4 | ??0.17 |
| ??MgSO 4·7H 2O | ??0.37 |
| ??CaCl 2 | ??0.44 |
| Trace element | Concentration (mgL in the substratum -1) |
| Macroelement | Concentration (gL in the substratum -1) |
| ??FeSO 4·7H 2O | ??27.8 |
| ??Na 2EDTA | ??37.3 |
| ??MnSO 4·4H 2O | ??22.3 |
| ??ZnSO 4·4H 2O | ??8.6 |
| ??H 3BO 3 | ??6.2 |
| ??KI | ??0.83 |
| ??Na 2MoO 4·2H 2O | ??0.25 |
| ??CuSO 4·5H 2O | ??0.025 |
| ??CoCl 2·6H 2O | ??0.025 |
| Organic composition | Concentration (mgL in the substratum -1) |
| Glycine | ??2.0 |
| Vitamin | ??0.1 |
| Pyridoxine hydrochloride | ??0.5 |
| Nicotinic acid VB5 | ??0.5 |
| Inositol | ??100 |
Described foreign gene specifically can be anti insect gene Cry2A
*, AGP promotor or GFP gene; Described anti insect gene Cry2A
*Nucleotide sequence shown in the sequence 1 of sequence table; The nucleotide sequence of described AGP promotor is shown in the sequence 2 of sequence table; The nucleotide sequence of described GFP gene is shown in the sequence 3 of sequence table.
Described reorganization Agrobacterium can be the reorganization Agrobacterium that obtains with the recombinant plasmid transformed Agrobacterium of carrying described foreign gene.Described reorganization Agrobacterium specifically can be following (a) or (b) or (c):
(a) transform Agrobacterium EHA105 with recombinant plasmid pTiBO542 and obtain the Agrobacterium of recombinating;
(b) transform Agrobacterium EHA105 with recombinant plasmid pBI α GUS and obtain the Agrobacterium of recombinating; Described recombinant plasmid pBI α GUS is substituted by the recombinant plasmid that the DNA shown in the sequence 2 obtains with the small segment that HindIII and the Xba I enzyme of pBI121 are cut between the recognition site;
(c) transform Agrobacterium LBA4404 with recombinant plasmid pBI α GFP and obtain the Agrobacterium of recombinating; Described recombinant plasmid pBI α GFP is substituted by the recombinant plasmid that the DNA shown in the sequence 3 obtains with the small segment that Xba I and the Sac I enzyme of pBI α GUS are cut between the recognition site.
Described cotton specifically can be cotton variety Ji rich 197 or cotton variety Hubei Province anti-cotton 12.
More than arbitrary described method can be applicable to cotton breeding.
It is restricted strong and the transformation period is long to the present invention is directed to plant regeneration difficulty in the present cotton genetic transformation, genetic mutation rate height, genotype, and proposing a kind of is the novel method of acceptor by agriculture bacillus mediated cotton genetic transformation with the cotton stem apex.This method is by adjusting the stem apex growth conditions, determining that the stem apex of Agrobacterium-mediated Transformation infects appropriate depth, adds Syringylethanone and adjusts suitable pH value, improves transformation efficiency.Utilize method of the present invention to obtain transfer-gen plant fast and have higher transgene efficiency.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
Embodiment 1, application method of the present invention obtain transgene cotton
One, the acquisition of reorganization Agrobacterium
Recombinant plasmid pTiBO542: China Agricultural University guarantees to provide to the public; Reference: the detailed building process of recombinant plasmid is seen (2005) paper " Transgenic indica rice plants harboring a syntheticcry2A such as Chen H
*Gene of Bacillus thuringiensis exhibit enhanced resistance against lepidopteran ricepests. " in the 1131st to 1132 page; Carry the anti insect gene Cry2A shown in the sequence 1 of sequence table in this recombinant plasmid
*
Agrobacterium EHA105: China Agricultural University guarantees to provide to the public; Reference: Torregrosa L., et.al.Influence of Agrobacterium Strain, Culture Medium, and Cultivar on the TransformationEfficiency of Vitis vinifera L.Am.J.Enol.Vitic, 53,3:183-190.
Transform Agrobacterium EHA105 with recombinant plasmid pTiBO542, obtain the Agrobacterium of recombinating.
Two, the acquisition of transgene cotton
Hubei Province anti-cotton 12: Hubei Prov. Seed Group Co. is Hubei Province's crop varietal approval committee kind, and variety certification is numbered Hubei Province and examines cotton 2005010.
(1) acquisition of seed plantlet
1, cotton seeds lint
50g cotton (Hubei Province anti-cotton 12) seed adds the 15ml left and right sides vitriol oil, stirs 2-3min fast, and liming neutralization is then constantly washed by rubbing with the hands, and flowing water flushing 10-12min obtains clean lint seed.
2, with the lint seed through 20% (volumn concentration) hydrogen peroxide solution sterilization and on shaking table, shook 5 hours with 130 rev/mins, clean seed 5-6 time with aqua sterilisa then, add the 150ml aqua sterilisa at last, be placed under 28 ℃ of environment and cultivated 24 hours, obtain germinating seed.
3, germinating seed will be planted shell under gnotobasis and peel, the cotton embryo that removes kind of shell will be planted on the germination substratum, dark culturing, 28 ℃ of temperature; Obtain that cotyledon half launches, hypocotyl elongation is to the seed plantlet of 4cm.
(2) acquisition of transgene cotton
1, the prescription of related solution and substratum
Solution A: form pH5.3 by solute and water; Solute and concentration thereof are as follows: Syringylethanone 40 μ l/L, 2-N-morpholino ethane sulfonic acid 65mM, glucose 2% (w/w, quality percentage composition), kinetin 0.08mgL
-1, step 1 reorganization Agrobacterium OD
600=0.6-0.8.
Substratum is made up of MS basic culture solution, agar, glycine and kinetin altogether; The solvent of MS basic culture solution is that water, solute are as shown in table 1; In the substratum, the final concentration of agar is 7.5gL altogether
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
Screening culture medium is made up of MS basic culture solution, agar, glycine, kinetin (KT), kantlex (Kan) and cephamycin (Cef); The solvent of MS basic culture solution is that water, solute are as shown in table 1; In the screening culture medium, the final concentration of agar is 7.5gL
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1, the final concentration of kantlex is 50mgL
-1, the final concentration of cephamycin is 250mgL
-1
2, the acquisition of transgene cotton
(1) cultivates seed plantlet, remove cotyledon, petiole and part hypocotyl, retain about plumular axis 0.8cm; 25 ℃, relative humidity 40%, secretly cultivate 2 days (the stem apex elongation becomes straight).
(2) with the dissecting needle of sharp-pointed band 1mm crotch with the two-sided scuffing of stem apex, the scuffing scope to the hypocotyl base portion, marks the wound about dark 0.8mm from the top of stem apex, obtains to scratch stem apex.
(3) stem apex that scratches was contaminated in solution A 15 minutes; Blot stem apex surface bacterium liquid with sterilization filter paper, again stem apex is placed on the common substratum and cultivated 2 days, obtain the stem apex of cultivating altogether that infects.
(4) will infect stem apex and transfer on the screening culture medium, screen 3 times, change once fresh screening culture medium around every pact, obtain the resistant cotton seedling.
(5) with the grafting of resistance seedling on the cotton stock, obtain regeneration plant (number is N).
(3) calculate transformation efficiency
Whole steps two about 3 months, the regeneration plant (number is N) that (5) of step 2 are obtained carries out the PCR checking, obtains transfer-gen plant (number is n).Transformation efficiency=n/N * 100%.
PCR verifies that used primer is as follows:
5′-CTCGCTCGTGTCAATGCT-3′；5′-CTCTGGGTTTGCTGTGGG-3′。
Transformation efficiency reaches 6.2%.
Embodiment 2, application method of the present invention obtain transgene cotton
One, the acquisition of reorganization Agrobacterium
1, the seed specific expression promotor AGP shown in the sequence 2 of composition sequence table.
2, the acquisition of recombinant plasmid (pBI α GUS)
1. use restriction enzyme HindIII and Xba I double digestion pBI121 (Beijing Baeyer enlightening Bioisystech Co., Ltd, product article No.: MP-091), reclaim big fragment;
2. use the seed specific expression promotor AGP of restriction enzyme HindIII and Xba I double digestion step 1, reclaim enzyme and cut product;
3. the enzyme that 2. big fragment and the step that 1. step is reclaimed reclaim is cut product and is connected, and obtains recombinant plasmid pBI α GUS (having replaced the composition type expression promoter CaMV35S among the pBI121 with the seed specific expression promotor AGP shown in the sequence 2).Among the recombinant plasmid pBI α GUS, gus gene is a seed specific expression.
3, the acquisition of reorganization Agrobacterium
Transform Agrobacterium EHA105 with recombinant plasmid pBI α GUS, obtain the Agrobacterium of recombinating.
Two, the acquisition of transgene cotton
Ji rich 197: available from Hebei Ji Feng kind industry company limited, be the Hebei province crop varietal approval committee kind, variety certification is numbered the Ji and examines cotton No. 2004007.
(1) acquisition of seed plantlet
1, cotton seeds lint
50g cotton (Ji Feng 197) seed adds the 15ml left and right sides vitriol oil, stirs 2-3min fast, and liming neutralization is then constantly washed by rubbing with the hands, and flowing water flushing 10-12min obtains clean lint seed.
2, with the lint seed through 20% (volumn concentration) hydrogen peroxide solution sterilization and on shaking table, shook 5 hours with 130 rev/mins, clean seed 5-6 time with aqua sterilisa then, add the 150ml aqua sterilisa at last, be placed under 28 ℃ of environment and cultivated 24 hours, obtain germinating seed.
3, germinating seed will be planted shell under gnotobasis and peel, the cotton embryo that removes kind of shell will be planted on the germination substratum, dark culturing, 28 ℃ of temperature; Obtain that cotyledon half launches, hypocotyl elongation is to the seed plantlet of 5cm.
(2) acquisition of transgene cotton
1, the prescription of related solution and substratum
Solution A: form pH5.6 by solute and water; Solute and concentration thereof are as follows: Syringylethanone 80 μ lL
-1, 2-N-morpholino ethane sulfonic acid 75mM, glucose 2.5% (w/w, quality percentage composition), kinetin 0.1mgL
-1, step 1 reorganization Agrobacterium OD
600=0.6-0.8.
Substratum is made up of MS basic culture solution, agar, glycine and kinetin altogether; The solvent of MS basic culture solution is that water, solute are as shown in table 1; In the substratum, the final concentration of agar is 7.5gL altogether
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
Screening culture medium is made up of MS basic culture solution, agar, glycine, kinetin, kantlex and cephamycin; The solvent of MS basic culture solution is that water, solute are as shown in table 1; In the screening culture medium, the final concentration of agar is 7.5gL
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1, the final concentration of kantlex is 75mgL
-1, the final concentration of cephamycin is 375mgL
-1
2, the acquisition of transgene cotton
(1) cultivates seed plantlet, remove cotyledon, petiole and part hypocotyl, retain about plumular axis 1.5cm; 28 ℃, relative humidity 50%, secretly cultivate 2.5 days (the stem apex elongation becomes straight).
(2) with the dissecting needle of sharp-pointed band 1mm crotch with the two-sided scuffing of stem apex, the scuffing scope to the hypocotyl base portion, marks the wound about dark 1mm from the top of stem apex, obtains to scratch stem apex.
(3) stem apex that scratches was contaminated in solution A 30 minutes; Blot stem apex surface bacterium liquid with sterilization filter paper, again stem apex is placed on the common substratum and cultivated 3 days, obtain the stem apex of cultivating altogether that infects.
(4) will infect stem apex and transfer on the screening culture medium, screen 3 times, change once fresh screening culture medium around every pact, obtain the resistant cotton seedling.
(5) with the grafting of resistance seedling on the cotton stock, obtain regeneration plant (number is N).
(3) calculate transformation efficiency
Whole steps two about 3 months, the regeneration plant that (5) of step 2 are obtained carries out the PCR checking, obtains transfer-gen plant (number is n).Transformation efficiency=n/N * 100%.PCR verifies that used primer is as follows: 5 '-TCGGCTATGACTGGGCACAACAGA-3 '; 5 '-AAGAAGGCGATAGAAGGCGATGCG-3 '.
Transfer-gen plant is carried out GUS dyeing, all show positive.
Transformation efficiency reaches 5.9%.
Embodiment 3, application method of the present invention obtain transgene cotton
One, the acquisition of reorganization Agrobacterium
1, the GFP gene shown in the composition sequence 3.
2, the acquisition of recombinant plasmid (pBI α GFP)
1. with the 2 pBI α GUS that obtain of the step 2 of restriction enzyme Xba I and Sac I double digestion embodiment 2, reclaim big fragment;
2. use the GFP gene of restriction enzyme Xba I and Sac I double digestion step 1, reclaim enzyme and cut product;
3. the enzyme that 2. big fragment and the step that 1. step is reclaimed reclaim is cut product and is connected, and obtains recombinant plasmid pBI α GFP (having replaced gus gene among the pBI α GUS with the GFP gene shown in the sequence 3).
3, the acquisition of reorganization Agrobacterium
Agrobacterium LBA4404: China Agricultural University guarantees to provide to the public; Reference: Torregrosa L., et.al.Influence of Agrobacterium Strain, Culture Medium, and Cultivar on the TransformationEfficiency of Vitis vinifera L.Am.J.Enol.Vitic, 53,3:183-190.
Transform Agrobacterium LBA4404 with recombinant plasmid pBI α GFP, obtain the Agrobacterium of recombinating.
Two, the acquisition of transgene cotton
Ji rich 197: available from Hebei Ji Feng kind industry company limited, be the Hebei province crop varietal approval committee kind, variety certification is numbered the Ji and examines cotton No. 2004007.
(1) acquisition of seed plantlet
1, cotton seeds lint
50g cotton (Ji Feng 197) seed adds the 15ml left and right sides vitriol oil, stirs 2-3min fast, and liming neutralization is then constantly washed by rubbing with the hands, and flowing water flushing 10-12min obtains clean lint seed.
2, with the lint seed through 20% (volumn concentration) hydrogen peroxide solution sterilization and on shaking table, shook 5 hours with 130 rev/mins, clean seed 5-6 time with aqua sterilisa then, add the 150ml aqua sterilisa at last, be placed under 28 ℃ of environment and cultivated 24 hours, obtain germinating seed.
3, germinating seed will be planted shell under gnotobasis and peel, the cotton embryo that removes kind of shell will be planted on the germination substratum, dark culturing, 28 ℃ of temperature; Obtain that cotyledon half launches, hypocotyl elongation is to the seed plantlet of 6cm.
(2) acquisition of transgene cotton
Solution A: form pH5.8 by solute and water; Solute and concentration thereof are as follows: Syringylethanone 120 μ l/L, 2-N-morpholino ethane sulfonic acid 85mM, glucose 3% (w/w, quality percentage composition), kinetin 0.12mgL
-1, step 1 reorganization Agrobacterium OD
600=0.6-0.8.
Substratum is made up of MS basic culture solution, agar, glycine and kinetin altogether; The solvent of MS basic culture solution is that water, solute are as shown in table 1; In the substratum, the final concentration of agar is 7.5gL altogether
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
Screening culture medium is made up of MS basic culture solution, agar, glycine, kinetin, kantlex and cephamycin; The solvent of MS basic culture solution is that water, solute are as shown in table 1; In the screening culture medium, the final concentration of agar is 7.5gL
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1, the final concentration of kantlex is 100mgL
-1, the final concentration of cephamycin is 500mgL
-1
(1) cultivates seed plantlet, remove cotyledon, petiole and part hypocotyl, retain about plumular axis 2cm; 30 ℃, relative humidity 60%, secretly cultivate 3 days (the stem apex elongation becomes straight).
(2) with the dissecting needle of sharp-pointed band 1mm crotch with the two-sided scuffing of stem apex, the scuffing scope to the hypocotyl base portion, marks the wound about dark 1.2mm from the top of stem apex, obtains to scratch stem apex.
(3) stem apex that scratches was contaminated in solution A 45 minutes; Blot stem apex surface bacterium liquid with sterilization filter paper, again stem apex is placed on the common substratum and cultivated 4 days, obtain the stem apex of cultivating altogether that infects.
(4) will infect stem apex and transfer on the screening culture medium, screen 3 times, change once fresh screening culture medium around every pact, obtain the resistant cotton seedling.
(5) with the grafting of resistance seedling on the cotton stock, obtain regeneration plant (number is N).
(3) calculate transformation efficiency
Whole steps two about 3 months, the regeneration plant that (5) of step 2 are obtained carries out the PCR checking, obtains transfer-gen plant (number is n).Transformation efficiency=n/N * 100%.PCR verifies that used primer is as follows: 5 '-TCGGCTATGACTGGGCACAACAGA-3 '; 5 '-AAGAAGGCGATAGAAGGCGATGCG-3 '.
Transfer-gen plant is carried out the GFP fluoroscopic examination, all show positive.
Transformation efficiency reaches 5.3%.
Sequence table
＜110〉China Agricultural University
＜120〉a kind of method that obtains transgene cotton
<130>CCGNARY102282
<160>3
<210>1
<211>2343
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
cggatccaga?ctcactctga?gcgtcgtcac?acgcagcttg?tgcgggatat?catttgcctg????60
taaccggttt?ccttaaagcg?aaaacccccc?cacccaaagg?taaggctatg?aacaacgtgc????120
tgaacagcgg?caggaccacc?atctgcgacg?cctacaatgt?cgtggcccac?gaccccttca????180
gcttcgagca?caagagcctg?gatacgatcc?agaaggaatg?gatggagtgg?aagcgcacgg????240
accacagcct?ctacgtcgcc?ccagtggtcg?gcactgtgtc?gagcttcctg?ctgaagaagg????300
tgggtagcct?catcggcaag?cgcatcctgt?ccgagctctg?gggcatcatc?ttccccagcg????360
gtagcaccaa?cctgatgcag?gatatcctgc?gcgagaccga?acagttcctg?aaccagcgcc????420
tgaacactga?caccctcgct?cgtgtcaatg?ctgacctgat?cggcctgcag?gccaacatca????480
gggagttcaa?tcaacaggtg?gacaacttcc?tcaaccccac?ccagaaccca?gtgccgagct????540
ccatcacgag?ctccgtgaac?accatgcagc?agctgttcct?gaatcgcctc?ccgcagttcc????600
agatccaagg?ctaccagctc?ttgctgctgc?ccctcttcgc?tcaggcggcc?aacatgcacc????660
tgagcttcat?ccgcgacgtg?atcctgaacg?ctgacgagtg?gggtatctcc?gccgccaccc????720
tcaggaccta?ccgcgattac?ctgcgcaact?acacccgtga?ctattccaac?tactgcatca???780
acacctacca?gaccgccttc?aggggcctca?acacccgcct?gcacgacatg?cttgagttcc???840
gcacatacat?gttcctgaac?gtgttcgaat?acgtctccat?ctggagcctc?ttcaagtacc???900
agagcctgat?ggtgagctcc?ggcgctaacc?tctacgccag?cggttccggc?ccacagcaaa???960
cccagagctt?caccgcccag?aactggccct?tcctctacag?cctgttccaa?gtgaatagca???1020
actacatcct?gtccggcatc?tccggtacca?ggctgtcgat?caccttcccc?aacatcggcg???1080
gtctgccagg?cagcacgacc?actcactccc?tgaacagcgc?cagggtgaac?tacagcggcg???1140
gtgtgagcag?cggtctcatc?ggcgccacca?atctcaacca?caacttcaac?tgcagcaccg???1200
tgctgccacc?cctgtccacc?cccttcgttc?gcagctggct?ggacagcggc?accgataggg???1260
agggcgtggc?taccagcacc?aactggcaga?ccgaatcctt?ccagaccact?ctgagcctca???1320
ggtgcggtgc?cttcagcgcc?cgcggcaata?gcaactactt?ccccgactac?ttcatccgca???1380
acattagcgg?cgtcccactc?gtgatccgca?acgaggacct?gaccaggccc?ctccactaca???1440
accaaatccg?caacatcgag?tcccccagcg?gcagcccagg?tggcgctagg?gcctacctgg???1500
tgagcgtgca?caaccgcaag?aacaatatct?acgccgctaa?cgagaacggc?accatgatcc???1560
atctggcccc?cgaagactac?accggcttca?ccatcagccc?aatccacgcc?acgcaggtga???1620
acaatcaaac?ccgcactttc?atcagcgaga?agttcggcaa?ccagggcgac?agcctgaggt???1680
tcgagcagag?caacaccaca?gcccgctaca?ccctgcgtgg?caatggtaac?tcctacaacc???1740
tctacctgag?ggtcagcagc?atcggcaaca?gcaccatccg?cgtgaccatt?aacggccgtg???1800
tgtacaccgt?gagcaacgtg?aacaccacta?cgaacaacga?cggtgtcaac?gataacggcg???1860
ctcgcttctc?cgacatcaac?atcggtaata?tcgtggccag?cgataacacc?aacgtcaccc???1920
tggacatcaa?cgtgaccctc?aactccggca?cccccttcga?cctgatgaac?atcatgttcg???1980
tgcccaccaa?cctgccgcca?ctctactaat?gacgaattcc?cgatctagta?acatagatga???2040
caccgcgcgc?gataatttat?cctagtttgc?gcgctatatt?ttgttttcta?tcgcgtatta???2100
aatgtataat?tgcgggactc?taatcataaa?aacccatctc?ataaataacg?tcatgcacct???2160
gaatagatct?tggacaagcg?ttaggcctat?ctgtgcatta?catgttaatt?attacatgct???2220
taacgtaatt?caacagaaat?tatatgataa?tcatcgcaag?accggcaaca?ggattcaatc???2280
ttaagaaact?ttattgccaa?atgtttgaac?gatcggggaa?attcgagctc?ggatccaagc???2340
ttc?????????????????????????????????????????????????????????????????2343
<210>2
<211>1163
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
cccaagcttc?tattttcatc?ctatttagaa?atccaagttg?acacctaaaa?tttagttgga????60
ctgccatgta?ggattatcgt?tagagagata?acggagctta?acggtagagt?gatcactttg????120
taacaaaata?ataacaaaag?tgactaaagt?gtaacatttc?aaacataaat?gattaaaata????180
taacctgagg?caaacaaaaa?tgactatttt?tatagattac?cctaaaatta?aagagtcatg????240
gccctagccc?ctcgcctact?tgtttgtttt?taataaacta?acatagtata?atatattgtt????300
aggattatat?aaaattatta?ataaatagta?taattaattt?aaaatttatg?aaaaaataaa????360
ctaccatatt?tcttaatacg?tggcacctta?tgttagattg?gactgtataa?cttatatact????420
attatctata?ttgaatccaa?atccttgctt?ttaagcgttt?ttagtgaaac?attttatttt????480
ccattcttat?tatataaatt?tatataatga?tataatatgt?aatacttaga?taatattatt????540
gaaaaagaat?aaaaatacct?caaactttga?aaggactaat?ttgtatgagc?atcaaacgta????600
caggatacca?aaagtataca?tatctgaatt?tgttcatatc?tcctgcaact?catagatcat????660
caccatgcac?agcaacatgt?gtacacttga?cttgtcctcc?atcaactcaa?cccttaactc????720
agtgaatcgg?gacatctctg?tctcacttta?aaacccttcc?cagtttcaac?actctttgaa????780
ttcaactgag?ttcacataca?acacaacacg?gtccatcatc?tttctgctgt?taaagcatca????840
tcatttcgcc?ccttccagtt?acagatgcaa?ccatgaaccc?ccctgcaaca?aagtttgtcc????900
gaaccttgct?cgtaccatgt?gaagggatgt?ggcatctcga?tatctaccca?ccactataca????960
aaaaaaaaaa?aaaagagaca?atatttcgtc?ttctttaatt?tgcacactcg?tcatcttgca????1020
tgtcaatgtc?ttcaacacgt?tgatgaagat?ttgcatgcaa?aaatatcacc?ttccacagct????1080
ccaccttcta?taaatacatt?accactcttt?gctattacca?tcacacagta?acaaaataca????1140
gagcttatcg?taatctctag?agc????????????????????????????????????????????1163
<210>3
<211>732
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
gctctagaat?gagtaaagga?gaagaacttt?tcactggagt?tgtcccaatt?cttgttgaat????60
tagatggtga?tgttaatggg?cacaaatttt?ctgtcagtgg?agagggtgaa?ggtgatgcaa????120
catacggaaa?acttaccctt?aaatttattt?gcactactgg?aaaactacct?gttccatggc????180
caacacttgt?cactactttc?ggttatggtg?ttcaatgctt?tgcgagatac?ccagatcata????240
tgaaacagca?tgactttttc?aagagtgcca?tgcctgaagg?ttatgtacag?gaaagaacta????300
tatttttcaa?agatgacggg?aactacaaga?cacgtgctga?agtcaagttt?gaaggtgata????360
cccttgttaa?tagaatcgag?ttaaaaggta?ttgattttaa?agaagatgga?aacattcttg????420
gacacaaatt?ggaatacaac?tataactcac?acaatgtata?catcatggca?gacaaacaaa????480
agaatggaat?caaagttaac?ttcaaaatta?gacacaacat?tgaagatgga?agcgttcaac????540
tagcagacca?ttatcaacaa?aatactccaa?ttggcgatgg?ccctgtcctt?ttaccagaca????600
accattacct?gtccacacaa?tctgcccttt?cgaaagatcc?caacgaaaag?agagaccaca????660
tggtccttct?tgagtttgta?acagctgctg?ggattacaca?tggcatggat?gaactataca????720
aataagagct?cg????????????????????????????????????????????????????????732
Claims (10)
1. a method that obtains transgene cotton comprises the steps:
(1) cotyledon, petiole and the part hypocotyl of excision cotton cotyledon seedling, hypocotyl keeps the length of 0.8-3cm, obtains tissue to be infected; 25-30 ℃ then, relative humidity 40%-60%, secretly cultivated 2-3 days; Described seed plantlet is that cotyledon half launches, hypocotyl elongation is to the aseptic seedling of 4-6cm;
(2) after will secretly cultivating wait to infect organize two-sided scuffing, scuffing scope be from the stem apex top to the hypocotyl base portion, the dark 0.8-1.2mm of wound;
(3) will scratch wait to infect is organized in the solution A contaminated 15-45 minute; Contain the reorganization Agrobacterium of carrying foreign gene in the described solution A;
(4) infect and cultivated altogether 2-4 days after finishing, carry out resistance screening then, obtain the resistant cotton seedling;
(5) with the grafting of resistant cotton seedling on the cotton stock, obtain transgene cotton.
2. the method for claim 1, it is characterized in that: in the step (3), the pH of described solution A is 5.3-5.8, is made up of solute and water; The solute and the concentration thereof of described solution A are as follows: Syringylethanone 40-120 μ lL
-1, 2-N-morpholino ethane sulfonic acid 65-85mM, glucose 2-3% (quality percentage composition), kinetin 0.08-0.12mgL
-1, contain the reorganization Agrobacterium OD of foreign gene
600=0.6-0.8.
3. method as claimed in claim 1 or 2 is characterized in that: in the step (4), described resistance screening carries out on screening culture medium; Described screening culture medium is made up of MS basic culture solution, agar, glycine, kinetin, kantlex and cephamycin; In the screening culture medium, the final concentration of agar is 7.5gL
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1, the final concentration of kantlex is 50-100mgL
-1, the final concentration of cephamycin is 250-500mgL
-1
4. as claim 1 or 2 or 3 described methods, it is characterized in that: in the step (4), the described cultivation altogether carried out on the substratum altogether; Described substratum altogether is made up of MS basic culture solution, agar, glycine and kinetin; In the substratum, the final concentration of agar is 7.5gL altogether
-1, the final concentration of glycine is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
5. as claim 3 or 4 described methods, it is characterized in that: the solvent of described MS basic culture solution is that water, solute are as shown in table 1.
6. as arbitrary described method in the claim 1 to 5, it is characterized in that: described foreign gene is anti insect gene Cry2A
*, AGP promotor or GFP gene; Described anti insect gene Cry2A
*Nucleotide sequence shown in the sequence 1 of sequence table; The nucleotide sequence of described AGP promotor is shown in the sequence 2 of sequence table; The nucleotide sequence of described GFP gene is shown in the sequence 3 of sequence table.
7. method as claimed in claim 6 is characterized in that: the reorganization Agrobacterium of described reorganization Agrobacterium for obtaining with the recombinant plasmid transformed Agrobacterium of carrying described foreign gene.
8. method as claimed in claim 7 is characterized in that: described reorganization Agrobacterium is following (a) or (b) or (c):
(a) transform Agrobacterium EHA105 with recombinant plasmid pTiBO542 and obtain the Agrobacterium of recombinating;
(b) transform Agrobacterium EHA105 with recombinant plasmid pBI α GUS and obtain the Agrobacterium of recombinating; Described recombinant plasmid pBI α GUS is substituted by the recombinant plasmid that the DNA shown in the sequence 2 obtains with the small segment that HindIII and the Xba I enzyme of pBI121 are cut between the recognition site;
(c) transform Agrobacterium LBA4404 with recombinant plasmid pBI α GFP and obtain the Agrobacterium of recombinating; Described recombinant plasmid pBI α GFP is substituted by the recombinant plasmid that the DNA shown in the sequence 3 obtains with the small segment that Xba I and the Sac I enzyme of pBI α GUS are cut between the recognition site.
9. as arbitrary described method in the claim 1 to 8, it is characterized in that: described cotton is cotton variety Ji rich 197 or cotton variety Hubei Province anti-cotton 12.
10. the application of arbitrary described method in cotton breeding in the claim 1 to 9.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719475A (en) * | 2011-03-30 | 2012-10-10 | 中国农业科学院生物技术研究所 | Genetic transformation method by utilization of cotton meristematic tissue |
| CN103667342A (en) * | 2013-11-29 | 2014-03-26 | 河南科技学院 | Method for preparing genetically modified cotton from axillary bud of cotton cotyledon |
| CN103820489A (en) * | 2014-03-24 | 2014-05-28 | 山东大学 | Method for improving conversion rate of foreign gene of stem tip meristem of yellowing seedling of cotton |
| CN108486150A (en) * | 2018-04-20 | 2018-09-04 | 刘寒冬 | A kind of Cotton Transformation method |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104099367A (en) * | 2013-04-15 | 2014-10-15 | 华中农业大学 | Method for culturing transgenic insect-resistant paddy rice |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101445808A (en) * | 2008-11-24 | 2009-06-03 | 广东省农业科学院作物研究所 | Agrobacterium-mediated genetic transformation method with peanut seed domant bud hypocotyl as explant |
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| CN101445808A (en) * | 2008-11-24 | 2009-06-03 | 广东省农业科学院作物研究所 | Agrobacterium-mediated genetic transformation method with peanut seed domant bud hypocotyl as explant |
Non-Patent Citations (2)
| Title |
|---|
| 《中国优秀博硕士学位论文全文数据库（硕士） 农业科技辑》 20031215 李忠 根癌农杆菌介导转化鄂抗棉9号愈伤组织获得含Chitinase和beta-（1，3）-glucanase基因的棉花再生植株 第1-39页 1-10 , 第4期 2 * |
| 《西南师范大学学报》 20021031 杨秀荣等 乙酰丁香酮对根癌农杆菌介导的甘薯遗传转化的影响 751-754 1-10 第27卷, 第5期 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719475A (en) * | 2011-03-30 | 2012-10-10 | 中国农业科学院生物技术研究所 | Genetic transformation method by utilization of cotton meristematic tissue |
| CN103667342A (en) * | 2013-11-29 | 2014-03-26 | 河南科技学院 | Method for preparing genetically modified cotton from axillary bud of cotton cotyledon |
| CN103667342B (en) * | 2013-11-29 | 2015-10-28 | 河南科技学院 | A kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton |
| CN103820489A (en) * | 2014-03-24 | 2014-05-28 | 山东大学 | Method for improving conversion rate of foreign gene of stem tip meristem of yellowing seedling of cotton |
| CN108486150A (en) * | 2018-04-20 | 2018-09-04 | 刘寒冬 | A kind of Cotton Transformation method |
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