CN103667342B - A kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton - Google Patents
A kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton Download PDFInfo
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Abstract
The invention discloses a kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton.One disclosed by the invention infects reagent, and this infects reagent and is made up of solvent and solute, and solvent is 1/2MS substratum, does is solute SILWET? L-77, sucrose, Syringylethanone and KT.Tool of the present invention has the following advantages: 1. utilize the particular stage of the cotyledon period of cotton seedling directly to infect, and avoid cotton tissue to cultivate regeneration by genotype restricted problem, all cotton varieties all can transform.2. the transformation period is short, obtains transfer-gen plant and only needs about about a 4 months cotton growth cycle.3. transformation efficiency is high, can reach 30%.
Description
Technical field
The present invention relates to a kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton.
Background technology
At present, in the transformation technology of the transgenic research of cotton, Agrobacterium tumefaciens mediated tissue culture plants regeneration of transgenic method, particle bombardment, pollen tube passage method is established.
Agriculture bacillus mediated transgenic method is current most study, and mechanism is the clearest, the transgenic method that technology is the most ripe, is the natural genetic transformation system of nature.After agriculture bacillus mediated foreign gene enters vegetable cell, the form mainly with single copy is integrated on karyomit(e), and transgenic progeny genetic stability is good, and less generation is reticent, and offspring's majority meets mendelian inheritance, is convenient to follow-up research.The T-DNA of Ti-plasmids can hold the insertion of the large fragment DNA molecule reaching 5Okb simultaneously.In the transgenic plant of current acquisition, more than 80% is realized by agriculture bacillus mediated mode.
The shortcoming of Agrobacterium tumefaciens mediated tissue culture Cotton Transformation is the tissue culture technique relying on cotton, tissue culture mutation rate is high, by genotypic restriction, the kind (or material) that can regenerate now is the kind of having eliminated on producing mostly, and main breed is often not easy to obtain regeneration, need to obtain transgenic regenerated plant by proceeding to type material, then by the method for hybridizing or backcross goal gene transformation in excellent kind.And the tissue culture regenerates cycle of cotton is very long, approximately takes 12-18 month, add the time of hybridization transformation, at least 2 years.
Agriculture bacillus mediated transgenic method technical scheme is as follows:
1, goal gene is obtained.
2, the structure of expression vector.Goal gene and carrier (great majority select plasmid) are coupled together with DNA ligase.
3, goal gene is imported recipient cell.Recombinant plasmid containing goal gene is imported Agrobacterium (Agrobacterium is recipient cell).
4, Agrobacterium infects type material callus, tissue culture subculture general 6 ~ 8 generations, per about about 45 days generation, Regenerated Plants From Somatic Cells.
5, the qualification of the marker gene of regeneration plant, the Detection and Identification of goal gene.
6, pattern transgenic line and target material hybridize transformation, filial generation seed selection, obtain transgenic line.
Summary of the invention
The object of this invention is to provide a kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton.
The invention provides one and infect reagent, this infects reagent and is made up of solvent and solute, and solvent is 1/2MS substratum, and solute is SILWET L-77, sucrose, Syringylethanone and KT;
Described SILWET L-77 is 0.02-0.04% at the described volumn concentration infected in reagent;
Described sucrose is 0.4g/100ml in the described concentration infected in reagent;
Described Syringylethanone is 50mg/L in the described concentration infected in reagent;
Described KT is 0.1mg/L in the described concentration infected in reagent.
One infects liquid and also belongs to protection scope of the present invention, and this infects liquid and is infected reagent by described and formed containing the recombinational agrobacterium of goal gene.
Above-mentionedly infect in liquid, described in infect liquid OD600 be 0.4-0.6.
The method preparing transgenic plant also belongs to a protection scope of the present invention, comprises the steps:
(1) launch with two panels cotyledon and the plant seedlings that stretches out of rough leaf for infecting object;
(2) destroy the stem apex terminal bud of seedling, make stem apex can not differential growth further, the division of promotor leaf auxiliary primodia;
(3) cotyledon axillalry bud place scratch mouth, and wound drip containing recombinational agrobacterium infect liquid;
Described recombinational agrobacterium is imported in Agrobacterium by the recombinant expression vector containing external source goal gene to obtain;
(4) cultivating 1 day by dripping the seedling moisturizing after infecting liquid, then continuing to cultivate;
(5) stem apex infecting the cotyledon of rear seedling stops growing, cotyledon auxiliary primodia merisis, forms sprouting, continues to cultivate the plant grown up to and is transfer-gen plant.
In aforesaid method, the described cotyledon axillalry bud in described step (3) is one-sided or bilateral cotyledon axillalry bud.
In above-mentioned arbitrary described method, the degree of depth of the described wound in described step (3) is 2-5mm.
In above-mentioned arbitrary described method, to infect liquid described in described step (3) be that 50ul is above-mentioned infects liquid.
In above-mentioned arbitrary described method, described in described step (4) and step (5), the condition of cultivation is 25 DEG C of illumination cultivation;
The condition of moisturizing described in described step (4) is the seedling after liquid is infected in described dropping be placed in seedling pan, is placed in the plastics casing sealed with overlay by seedling pan.
In above-mentioned arbitrary described method, described plant is cotton.
Above-mentioned infect reagent preparation transgenic plant in application also belong to protection scope of the present invention;
And/or,
The above-mentioned arbitrary described application of liquid in preparation transgenic plant of infecting also belongs to protection scope of the present invention;
And/or,
The application of above-mentioned arbitrary described method in preparation transgenic plant also belongs to protection scope of the present invention.
Tool of the present invention has the following advantages:
1, utilize the particular stage of the cotyledon period of cotton seedling directly to infect, avoid cotton tissue to cultivate regeneration by genotype restricted problem, all cotton varieties all can transform.
2, the transformation period is short, obtains transfer-gen plant and only needs about about a 4 months cotton growth cycle.
3, transformation efficiency is high, can reach about 30%.
Accompanying drawing explanation
Fig. 1 is preinfective seedling.
Fig. 2 is the regeneration of infecting rear cotyledon axillalry bud.
Fig. 3 is the PCR Molecular Detection of transfer-gen plant.
Fig. 4 is that the southern blotting of transfer-gen plant detects.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In YEP liquid nutrient medium solute and concentration in the medium as follows:
YEP solid medium: add 1.5% agar powder (% represents quality volumn concentration, g/100ml) in YEP liquid nutrient medium, pH=7.2,121 DEG C, autoclaving 20min.
10mM CaCl
2the aqueous solution is prepared as follows: get 0.15g CaCl
22H
2o adds about 80ml distilled water and dissolves, and glycerol adding 15ml, is settled to 100ml with distilled water, adjusts pH=5.8,121 DEG C, autoclaving 20min.
Agrobacterium LBA4404 is purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd, and catalog number is MCC026.
PCAMBIA1300(is containing hygromycin gene) purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd, catalog number is MCV033.
Baimian 1 document " Wang Qinglian, Zhang Jinbao, Lu Yuzhen. state examines new cotton variety Baimian 1 [J]. China seed industry, 2010, (2): 75-76 " in be disclosed, the public can obtain from Henan Science and Technology College.
CCRI 41 document " Liu Jinhai, Zhou Guanyin, Diao Yupeng. bivalent transgenic cotton against pests new variety CCRI 41 [J]. China seed industry, 2003, (1): 51 " in be disclosed, the public can obtain from Henan Science and Technology College.
Jade-like stone word 312 document " Jia Peipei, Hu Genhai, Zhang Jinbao; etc. different plant growth regulator treatment is on the impact [J] of upland cotton cotyledon node regeneration. Chinese agronomy circular; 2012,28 (15): 31-35 " in be disclosed, the public can obtain from Henan Science and Technology College.
SILWET L-77 is purchased from Canadian GE Healthcare Bio-Sciences AB company, and catalog number is SL770080596.
KT(6-chaff aminopurine) purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd, catalog number is MB-K5762.
The preparation of embodiment 1, recombinational agrobacterium
One, the competent preparation of LBA4404
(1) a little Agrobacterium LBA4404 of transfering loop picking, is inoculated in 5ml YEP liquid nutrient medium (containing 50mg/L Rifampin), 28 DEG C, 200r/mim overnight incubation.
(2) get 2ml culture to continue to cultivate, until OD600 is about 0.5 in YEP liquid nutrient medium (containing 50mg/L Rifampin).
(3) culture of step (two) is put 30min in ice bath, 4 DEG C, 5000r/min, centrifugal 5min, abandoning supernatant.
(4) with the 0.1mol/L NaCl solution suspension bacteria liquid that 10ml is cold.
(5) suspension bacteria liquid 4 DEG C, the 5000r/min that step (four) are obtained, centrifugal 5min, abandoning supernatant.
(6) with the 10mM CaCl2 solution suspension bacterium liquid that 1ml is cold, be distributed into 50 μ l/ and manage, to-80 DEG C of preservations after freezing in liquid nitrogen.
Two, pCAMBIA1300 transforms LBA4404
(1) 1ug pCAMBIA1300 is added in 200ul LBA4404 competent cell, place 30 minutes on ice.
(2) by the cell of step () in liquid nitrogen freezing 1 minute.
Cell is dissolved in (three) 37 DEG C of water-baths.
(4) in cell, add 1ml YEP liquid nutrient medium, 2-4hr(low speed cultivated by 28 DEG C of shaking tables).
(5) centrifugal 1 minute, suspension cell was in 100ul LB substratum.
(6) coated plate (YEP solid plate substratum, containing 50mg/L Rifampin, 30mg/L Totomycin), cultivate 2-3 days for 28 DEG C, the clone grown is the LBA4404 Agrobacterium with pCAMBIA1300, is denoted as recombinational agrobacterium.
Embodiment 2, infect the preparation of liquid
One, bacterium liquid expands numerous
The recombinational agrobacterium list bacterium colony that picking embodiment 1 obtains contains in 50mL in the LB substratum of Totomycin (30mg/L) and Rifampin (25mg/L), 28 DEG C, and 300r/min concussion is cultured to OD
600value is 2.0-2.5,4 DEG C, and the centrifugal 5min of 8000r/min, collects thalline.
Two, the preparation of liquid is infected
Infect reagent: this reagent is made up of solvent and solute; Solvent is 1/2MS substratum; Solute is SILWET L-77, sucrose, Syringylethanone and KT; SILWET L-77 is 0.02%-0.04% infecting the volumn concentration in reagent, sucrose is that 0.4% (% represents quality volumn concentration infecting the concentration in reagent, g/100ml), Syringylethanone is 0.1mg/L to infect the concentration in reagent be 50mg/L, KT infecting the concentration in reagent.
Infect after preparation of reagents completes, with infecting the centrifugal recombinational agrobacterium thalline obtained of reagent resuspending step one, make OD600 be about 0.4-0.6, what be recombinational agrobacterium infects liquid, be placed on 4 DEG C for subsequent use.
The preparation of embodiment 3, transgene cotton
One, the preparation of cotton seedling: the seed choosing full Baimian 1, in seedling pan program request, room temperature 25 DEG C sprouting, emerges for about 3 days.
Two, under 25 DEG C of conditions, Baimian 1 is deployed into when rough leaf grows at two panels cotyledon and is about 7-9 days, stretch out for the optimal conversion phase to rough leaf after two panels cotyledon launches, the cotyledon axillalry bud of cotton is in dormant state in this stage, generally can not grow, only have and destroy the suppression that stem apex terminal bud just can remove antithetical phrase axil bud.
A slice cotyledon is drawn in a have gentle hands, makes gap between two cotyledons bigger, stirs the middle tender stem apex of children of two panels cotyledon, to destroy stem apex terminal bud, and ensure the complete of cotyledon, for promotor leaf auxiliary primodia merisis creates conditions with scalper is first gently horizontal.
Three, be about the wound of 2-5mm in cotyledon axillalry bud place cutting depth, can both sides be cut, also can, the region of interest of preinfective seedling is as shown in Figure 1.
(two panels cotyledon axil respectively has 1 sub-axil bud, but cuts 2 limits simultaneously and have certain difficulty, can only process.）
Four, infect, in the wound of scalper cutting, what instill the recombinational agrobacterium of embodiment 2 preparation with pipettor infects liquid one (about 50 microlitre), after contaminating, seedling pan (is placed in the plastics casing of 17cm × 45cm × 70cm in 1 day by moisturizing, box mouth overlay seals, 25 DEG C of illumination cultivation), then the seedling after infecting is moved out of plastics casing, continue to cultivate under 25 DEG C of illumination.
Five, due to the destroyed of stem apex terminal bud, through about 15 emperor's axil bud fissions regeneration, develop into sprouting by auxiliary primodia position, as shown in Figure 2, after growing plant, be turn pCAMBIA1300 T0 for plant.
Six, the qualification of transfer-gen plant
(1) resistance screening
Preparing Totomycin solution to concentration with distilled water is 250mg/L, after the plant (namely turning the T0 of pCAMBIA1300 for plant) formed after cotyledon axillary bud development grows true leaf, with pipettor or dropper, rule on true leaf blade face, cultivate about 7 days observation experiment results, what spot was burnt in generation is negative plant, and what do not burn spot is positive plant.
(2) PCR Molecular Detection
Carry out nest-type PRC Molecular Detection to positive plant DNA, concrete steps are as follows:
It is pcr template that extraction step () is accredited as the positive T0 turning pCAMBIA1300 for the DNA of the blade of plant and wild-type Baimian 1 plant, and arrange simultaneously water belongs with yin contrast and pCAMBIA1300 plasmid be positive control, design two pairs of primers according to the hygromycin gene on pCAMBIA1300 plasmid and carry out nested PCR amplification.Two-wheeled amplimer nested designs, second takes turns amplimer on the binding site of gene nucleic acid sequence (GenBank:EU849664.1) compared with first round amplimer, and upstream primer moves inward about 50bp, and downstream primer moves inward about 200bp.
First round PCR:
Pcr amplification primer:
Upstream primer hpt1:5'-CGCGGATCCATGAAAAAGCCTGAA-3'; (SEQ ID No.1)
Downstream primer hpt2:5'-CCCAAGCTTTCTATTTCTTTGCCCTC-3'.（SEQ ID No.2）
PCR amplification system: upstream and downstream primer (20mmol/L) each 1uL, 1U Taq archaeal dna polymerase, 10 × PCR reaction buffer 2ul, dNTP (2.5mmol/L) 1.6uL, concentration is the template DNA 2.0ul of 0.1-0.25ug/ul, and deionized water supplies system to 20ul.
Pcr amplification program: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 52 DEG C of annealing 40s, 72 DEG C extend 1min, totally 35 circulations; 72 DEG C extend 5min.
Second takes turns PCR:
Pcr amplification primer:
Upstream primer hpt3:5'-TACTTCTACACAGCCATCGGTCC-3'; (SEQ ID No.3)
Downstream primer hpt4:5'-AGTGCTTGACATTGGGGAGTTTAG-3'.（SEQ ID No.4）
PCR amplification system: upstream and downstream primer (20mmol/L) each 1uL, 1U Taq archaeal dna polymerase, 10 × PCR reaction buffer 2uL, dNTP (2.5mmol/L) 1.6uL, template is first round pcr amplification product 1.0ul, and deionized water supplies system to 20ul.
Pcr amplification program: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 54 DEG C of annealing 40s, 72 DEG C extend 50s, totally 35 circulations; 72 DEG C extend 5min.
Second pcr amplification product of taking turns all detects with 1% sepharose, and have the sample of single purpose band (about 750bp) to contain Totomycin reporter gene (namely having proceeded to pCAMBIA1300 plasmid), result as shown in Figure 3.
In Fig. 3,1 is DNA marker, and stripe size is followed successively by 100bp from the bottom to top, 250bp, 500bp, 750bp, 1000bp, 2000bp; 2 is negative control (water); 3 is positive control (pCAMBIA1300 plasmid); 4 is wild-type Baimian 1; 5-7 is that three strain steps () are accredited as the positive T0 turning pCAMBIA1300 for plant.
Fig. 3 shows, positive control (pCAMBIA1300 plasmid) and step () are accredited as the positive T0 turning pCAMBIA1300 all can amplify object band for plant, is accredited as the positive T0 turning pCAMBIA1300 is defined as positive transgenic plant further for plant through PCR Molecular Detection three strain step ().
(3) southern blotting detects
The positive T0 turning pCAMBIA1300 is accredited as to step (two) and carries out agarose gel electrophoresis for the nested PCR amplification product of the hygromycin gene of plant, and transferring film, be that label probe is southern blotting and detects with Totomycin, the DIG DNA Labeling and Detection Kit of Roche company is adopted to carry out, operation is see test kit specification sheets, and result as shown in Figure 4.
The sequence of this probe is as shown in SEQ ID No.5.
Fig. 4 shows, the nest-type PRC of step (two) is accredited as the positive T0 turning pCAMBIA1300 and is defined as positive transgenic plant for plant further through southern blotting detection.
The step one calculating the present embodiment is about 30% to the transformation efficiency of the method for step 5, and result is as shown in table 1.
Application aforesaid method transforms CCRI 41 and jade-like stone word 312 again and carries out the qualification of positive plant, and result is as shown in table 1.
Table 1 transformation efficiency
Table 1 shows, utilizes the step one of the present embodiment very high to the transgenic method transformation efficiency of step 5.
Claims (8)
1. one grow cotton and infect reagent, this infects reagent and is made up of solvent and solute, and solvent is 1/2MS substratum, and solute is SILWET L-77, sucrose, Syringylethanone and KT;
Described SILWET L-77 is 0.02-0.04% at the described volumn concentration infected in reagent;
Described sucrose is 0.4g/100ml in the described concentration infected in reagent;
Described Syringylethanone is 50mg/L in the described concentration infected in reagent;
Described KT is 0.1mg/L in the described concentration infected in reagent.
2. one grow cotton and infect liquid, this infects liquid and infects reagent and the recombinational agrobacterium containing goal gene forms by according to claim 1.
3. according to claim 2ly infect liquid, it is characterized in that: described in infect the OD of liquid
600for 0.4-0.6.
4. prepare a method for transgene cotton, comprise the steps:
(1) launch with two panels cotyledon and the cotton seedling that stretches out of rough leaf for infecting object;
(2) destroy the stem apex terminal bud of seedling, make stem apex can not differential growth further, the division of promotor leaf auxiliary primodia;
(3) cotyledon axillalry bud place scratch mouth, and wound drip containing recombinational agrobacterium infect liquid;
Described recombinational agrobacterium is imported in Agrobacterium by the recombinant expression vector containing external source goal gene to obtain;
(4) cultivate 1 day by dripping the seedling moisturizing after infecting liquid, and continue to cultivate;
(5) stem apex infecting the cotyledon of rear seedling stops growing, cotyledon auxiliary primodia merisis, forms sprouting, continues to cultivate the cotton plants grown up to and is transgenic cotton plant;
The degree of depth of the described wound in described step (3) is 2-5mm;
To infect liquid described in described step (3) be that 50ul is according to claim 3 infects liquid.
The condition of cultivating described in described step (4) and step (5) is 25 DEG C of illumination cultivation;
The condition of moisturizing described in described step (4) is the seedling after liquid is infected in described dropping be placed in seedling pan, is placed in the plastics casing sealed with overlay by seedling pan.
5. method according to claim 4, is characterized in that: the described cotyledon axillalry bud in described step (3) is one-sided or bilateral cotyledon axillalry bud.
6. the application in transgene cotton prepared by the reagent that infects according to claim 1;
7. the liquid that infects described in Claims 2 or 3 is preparing the application in transgene cotton;
8. the method described in claim 4 or 5 is preparing the application in transgene cotton.
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| CN115605082A (en) * | 2019-11-26 | 2023-01-13 | 先正达农作物保护股份公司（Ch） | Transformation method |
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