CN101831459B - Method for acquiring transgene cotton - Google Patents
Method for acquiring transgene cotton Download PDFInfo
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- CN101831459B CN101831459B CN2010101685578A CN201010168557A CN101831459B CN 101831459 B CN101831459 B CN 101831459B CN 2010101685578 A CN2010101685578 A CN 2010101685578A CN 201010168557 A CN201010168557 A CN 201010168557A CN 101831459 B CN101831459 B CN 101831459B
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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 through agriculture bacillus mediated acquisition transgenic cotton plant.
Background technology
Cotton is a kind of important cash crop, in national economy, occupies critical role.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 used the most extensive with the agrobacterium mediation converted method.Have the season limit of not receiving, goal gene by agriculture bacillus mediated gene transformation technology and be mostly single copy or low copy, insert the border and be mostly 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 through 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 pass through somatic embryo and take place and plant regeneration; Owing in the evoked callus process, will pass through differentiation, atomization again; Can produce a lot of heritable variations and somatic variation; The plant regeneration difficulty, the 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; Receive 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, like 0.8-1.5cm, 1.5-2cm), obtains tissue to be infected; 25-30 ℃ then (as 25-28 ℃, 28-30 ℃), relative humidity 40%-60% (like 40%-50%, 50%-60%), secretly cultivate 2-3 days (as 2-2.5 days, 2.5-3 days); Said seed plantlet is that cotyledon half launches, hypocotyl elongation to 4-6cm (like 4-5cm, aseptic seedling 5-6cm);
(2) wait to infect that to organize two-sided scuffing, scuffing scope be from stem apex top to hypocotyl base portion after will secretly cultivating, the dark 0.8-1.2mm of wound is (like 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 said solution A;
(4) cultivate 2-4 days (as 2-3 days, 3-4 days) altogether after infecting completion, 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 said solution A is that 5.3-5.8 (like 5.3-5.6,5.6-5.8), is made up of solute and water; The solute and the concentration thereof of said solution A are following: Syringylethanone 40-120 μ lL
-1(like 40-80 μ lL
-1, 80-120 μ lL
-1), 2-N-morpholino ethane sulfonic acid (MES) 65-85mM is (like 65-75mM, 75-85mM), glucose 2-3% (w/w, quality percentage composition) is (like 2-2.5%, 2.5-3%), kinetin (KT) 0.08-0.12mgL
-1(like 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), said resistance screening can carry out on screening culture medium.Said screening culture medium specifically can be made up of MS basic culture solution, agar, glycocoll, 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 glycocoll is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1, the final concentration of kantlex is 50-100mgL
-1(like 50-75mgL
-1, 75-100mgL
-1), the final concentration of cephamycin is 250-500mgL
-1(like 250-375mgL
-1, 375-500mgL
-1).
In the step (4), said cultivation altogether can carried out on the substratum altogether.Said substratum altogether specifically can be made up of MS basic culture solution, agar, glycocoll and kinetin; In the substratum, the final concentration of agar is 7.5gL altogether
-1, the final concentration of glycocoll is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
The solvent of said 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) |
| 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) |
| Glycocoll | 2.0 |
| Vitamin | 0.1 |
| Pyridoxine hydrochloride | 0.5 |
| Nicotinic acid VB5 | 0.5 |
| Inositol | 100 |
Said foreign gene specifically can be anti insect gene Cry2A
*, AGP promotor or GFP gene; Said anti insect gene Cry2A
*Nucleotide sequence shown in the sequence 1 of sequence table; The nucleotide sequence of said AGP promotor is shown in the sequence 2 of sequence table; The nucleotide sequence of said GFP gene is shown in the sequence 3 of sequence table.
Said reorganization Agrobacterium can be the reorganization Agrobacterium that obtains with the recombinant plasmid transformed Agrobacterium of carrying said foreign gene.Said reorganization Agrobacterium specifically can be as follows (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; Said 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; Said 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.
Said cotton specifically can be cotton variety Ji rich 197 or cotton variety Hubei Province anti-cotton 12.
More than arbitrary said method can be applicable to cotton breeding.
The present invention is directed in the present cotton genetic transformation that plant regeneration difficulty, genetic mutation rate are high, genotype is restricted strong and the transformation period is long, proposing a kind of is the novel method of acceptor through agriculture bacillus mediated cotton genetic transformation with the cotton stem apex.This method is through adjusting the stem apex growth conditions, confirming that the stem apex of Agrobacterium-mediated Transformation infects appropriate depth, adds Syringylethanone and the suitable pH value of adjustment, raising 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 like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, 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 germinated seeds.
3, germinated seeds 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 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 following: 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, glycocoll 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 glycocoll is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
Screening culture medium is made up of MS basic culture solution, agar, glycocoll, 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 glycocoll 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, stem apex is placed on the common substratum cultivated 2 days again, 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 following:
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 big fragment that 1. step is reclaimed is cut product with the enzyme that 2. step reclaims 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 ltd, 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 germinated seeds.
3, germinated seeds 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 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 following: 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, glycocoll 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 glycocoll is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
Screening culture medium is made up of MS basic culture solution, agar, glycocoll, 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 glycocoll 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, stem apex is placed on the common substratum cultivated 3 days again, 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 following: 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. the pBI α GUS that obtains with 2 of the step 2 of restriction enzyme Xba I and Sac I double digestion embodiment 2 reclaims 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 big fragment that 1. step is reclaimed is cut product with the enzyme that 2. step reclaims and is connected, and obtains recombinant plasmid pBI α GFP (having replaced the 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 ltd, 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 germinated seeds.
3, germinated seeds 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 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 following: 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, glycocoll 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 glycocoll is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
Screening culture medium is made up of MS basic culture solution, agar, glycocoll, 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 glycocoll 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, stem apex is placed on the common substratum cultivated 4 days again, 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 following: 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 (8)
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; Said seed plantlet is that cotyledon half launches, hypocotyl elongation to the aseptic seedling of 4-6cm;
(2) wait to infect that to organize two-sided scuffing, scuffing scope be from stem apex top to hypocotyl base portion, the dark 0.8-1.2mm of wound after will secretly cultivating;
(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 said solution A;
(4) cultivated altogether 2-4 days after infecting completion, 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;
In the step (3), the pH of said solution A is 5.3-5.8, is made up of solute and water; The solute and the concentration thereof of said solution A are following: 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.
2. the method for claim 1, it is characterized in that: in the step (4), said resistance screening carries out on screening culture medium; Said screening culture medium is made up of MS basic culture solution, agar, glycocoll, kinetin, kantlex and cephamycin; In the screening culture medium, the final concentration of agar is 7.5gL
-1, the final concentration of glycocoll 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
3. the method for claim 1 is characterized in that: in the step (4), the said cultivation altogether carried out on the substratum altogether; Said substratum altogether is made up of MS basic culture solution, agar, glycocoll and kinetin; In the substratum, the final concentration of agar is 7.5gL altogether
-1, the final concentration of glycocoll is 2mgL
-1, the final concentration of kinetin is 0.1mgL
-1
4. the method for claim 1, it is characterized in that: said foreign gene is anti insect gene Cry2A*, AGP promotor or GFP gene; The nucleotide sequence of said anti insect gene Cry2A* is shown in the sequence 1 of sequence table; The nucleotide sequence of said AGP promotor is shown in the sequence 2 of sequence table; The nucleotide sequence of said GFP gene is shown in the sequence 3 of sequence table.
5. method as claimed in claim 4 is characterized in that: the reorganization Agrobacterium of said reorganization Agrobacterium for obtaining with the recombinant plasmid transformed Agrobacterium of carrying said foreign gene.
6. method as claimed in claim 5 is characterized in that: said reorganization Agrobacterium for (a) as follows 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; Said 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; Said 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.
7. like arbitrary described method in the claim 1 to 6, it is characterized in that: said cotton is cotton variety Ji rich 197 or cotton variety Hubei Province anti-cotton 12.
8. the application of arbitrary said method in cotton breeding in the claim 1 to 7.
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| 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 |
| 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 |
| CN103820489B (en) * | 2014-03-24 | 2016-04-06 | 山东大学 | Improve the method for cotton etiolated seedling shoot apical meristem gene transformation rate |
| CN108486150A (en) * | 2018-04-20 | 2018-09-04 | 刘寒冬 | A kind of Cotton Transformation method |
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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 |
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Non-Patent Citations (2)
| Title |
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| 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β-（1，3）-glucanase基因的棉花再生植株.《中国优秀博硕士学位论文全文数据库（硕士） 农业科技辑》.2003,(第4期),第1-39页. * |
| 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.《西南师范大学学报》.2002,第27卷(第5期),751-754. * |
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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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