CN100451113C - Gene transfer method for promoting insect resistance of soybean - Google Patents
Gene transfer method for promoting insect resistance of soybean Download PDFInfo
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Abstract
The present invention relates to a transgenic method for improving the insect resisting capacity of soy beans, which belongs to a transgenic breeding method for the soy beans. The method comprises: transferring a gene g2 ps1 of pyranone synthase to the soy beans in a pollen-tube pathway mode; executing the operation of amkin identification to transgenic plants in a seedling stage; screening out plants which can resist amkin; further executing the operation of PCR identification and RT-PCR identification; breeding transgenic insect resisting soy bean materials through combining the identification of the insect resisting capacity of indoor feeding tobacco cutworms. The consequence of breeding the tobacco cutworms shows that significant differences (significant level P>P0.05)exist in the weight of larva and pupae fed by two transgenic materials and the weight of larva and pupae fed for comparison. The comparison of the consequence of breeding the tobacco cutworms shows that the transgenic materials can be used for obviously inhibiting the growth and the development of the tobacco cutworms.
Description
One, technical field
A kind of transgenic method that improves the soybean insect-resistance of the present invention's----belongs to the soybean transgene breeding method.
Two, technical background
Soybean rich in proteins and grease are people's important protein matter and edible oil sources, also are important industrial raw material, and long plantation history is arranged, and people are also being perplexed in the harm of various disease and pests simultaneously always.Wherein soybean leaf-feeding insect is the primary pest of soybean in China, and all there is soybean leaf-feeding pest damage in each soybean producing region of China, especially south and the Yellow River and Huai He River soybean district.Nineteen ninety, greenish brown hawk moth broke out (Liu Zhen, 1991) in Xinxiang, Henan; The 1992-1993 bollworm is big generation in North China; The big bridging worm in Pei County broke out in 1966; Along the river the big bridging worm repeating transmission in area in 1989; 1992 and bean pyralid in 1993 break out; The prodenia litura generating capacity was bigger in 1994 and 1998.Surplus only three counties of Heihe City soybean injured area in 2001,2,002 two reaches 80 ten thousand mu, wherein three one-tenth of the underproduction are above just reaches 400,000 mu (Zhang Daoming etc., 2002).Area, clear and definite so far Nanjing soybean leaf-feeding insect has 49 kinds from 21 sections, prodenia litura wherein, and soybean bridging worm, bean pyralid is most important worm kind.Soybean leaf-feeding insect usually becomes one of limiting factor of high yield of soybean stable yields.Therefore, the kind of the high pest-resistant evil of seed selection seems particularly important.
Cultivate pest-resistant soybean varieties following two kinds of approach arranged:
The one, use conventional art.Promptly extensively screening from existing soybean resource (comprising the new resources that Applied Physics or chemomorphosis technology obtain) obtains pest-resistant material, and carries out transformation with technology such as hybridizing, backcross, thereby cultivates new variety.Traditional breeding technology can not satisfy the requirement of the disease-resistant worm kind of agriculture production seed selection owing to reasons such as anti-source lack, breeding cycle is long.Therefore, have in the production use agricultural chemicals in a large number, cause the vicious cycle of environmental pollution, eubiosis destruction, disease and pest aggravation.
The 2nd, utilize genetic engineering technique.Import anti insect gene in the crop plant cells and make its stably express and heredity with genetic engineering means, thereby cultivate the direction that pest-resistant new crop varieties has become modern agriculture and crop breeding development.
The research of transfer destination gene on soybean, people use agriculture bacillus mediated or particle bombardment, but because the tissue culture of soybean is a problem that is difficult to break through always, so on soybean, utilize agriculture bacillus mediated or the particle bombardment transgenosis, be difficult to obtain regeneration plant, utilize pollen tube passage method transgene then directly the zygote of soybean transformation or zygote obtain transgenic seed, obtain regeneration plant by seed easily, overcome this difficulty of tissue culture.But utilize pollen tube passage method to change goal gene over to soybean research, do not see a lot of reports in the document at home and abroad.
Three, summary of the invention
Technical problem
The objective of the invention is a kind of transgenic method that improves the soybean insect-resistance; introduce foreign gene by genetic engineering technique; the pest-resistant soybean new lines of quickly breeding (kind) is is effectively prevented and treated soybean insect pest, protection environment, is improved China's soybean quality and the output and the market competitiveness thereof.
Technical scheme
A kind of transgenic method that improves the soybean insect-resistance of the present invention's----comprises:
1) to include the Agrobacterium phtt376 of pyrone synthase gene g2ps1, utilizes pollen tube passage method soybean transformation wave height, Nan Nong 73-935.
2) from T1 for, transfer-gen plant is carried out kalamycin resistance seedling stage identifies, will resist that plant of card further to do the PCR evaluation, the pcr amplification system contains 0.25mM dNTP, MgCl2,1U Taq enzyme, 0.5 μ M Primer, 100-200ng DNA, cumulative volume 25 μ l, the pcr amplification reaction condition: pre-95 ℃ of 5min of sex change, after 95 ℃ of 15s, 52 ℃ of 59s, 72 ℃ of 2min, 30 circulations, 72 ℃ are extended 10min, select the positive material of PCR qualification result;
3) T2 will block that resistant plant and do the PCR evaluation for still transfer-gen plant being carried out the kalamycin resistance evaluation seedling stage, and the pcr amplification system is the same, selects the positive material of PCR qualification result.Further be RT-PCR with this material again and identify, the total RNA of soybean adopts ' sky is that Time Inc.'s plant RNA extracts test kit ' to extract, and the pcr amplification system is the same, selects the positive material of RT-PCR qualification result;
4) T2 is carried out the resistance identification experiment of indoor feeding prodenia litura for transgenic line, insect-resistance is identified and is adopted indoor feeding method.In soybean bloom, pluck on the plant and launch leaf fully, supporting the worm container is the disposable plastic cup that covers of large size, inoculate 6 of 1 age in days Spodoptera litura larvae for every glass, triplicate is gathered the blade (transfer-gen plant and adjoining tree) of each material and is fed 28 ℃, illumination 14h/d changes twice in fresh blade every day.Nursing to the claims every glass of larva heavy in the time of ten days.Observe the prodenia litura upgrowth situation simultaneously, the prodenia litura that adjoining tree is fed in the time of the tenth day grew to for 5 ages, and the prodenia litura that transfer-gen plant is fed grew to for 4 ages, and the larvae pupation situation is write down every day in the back of weighing, and treated that every glass of larva back of pupating fully claimed on the 4th day that pupa was heavy.Larva is heavily reached pupa to be weighed two groups of data and carries out variance analysis.Change the resistance index of g2ps1 gene soybean as evaluation with this.The larva that two transgenic line wave height, Nan Nong 73-935 feed heavily reach pupa heavy with contrast feed between all have significant difference, conspicuous level P＞0.05, this material is the transgenic line that passes through the resistance evaluation of acquisition.
Beneficial effect
Compare with traditional technology, a kind of transgenic method that improves the soybean insect-resistance provided by the present invention has following advantage:
Method seed selection provided by the invention the insect-resistant transgenic soybean material, the larva that transgenic line is fed heavily reach pupa heavy with the contrast nursing between all have significant difference (conspicuous level P＞P0.05), illustrate that transgenic line can obviously suppress growing of prodenia litura.Utilize present method can significantly improve the breeding efficiency of insect-resistance soybean.
Four, description of drawings
Fig. 1: the g2ps1 gene PCR amplification figure in genetically engineered soybean T1 generation
M: standard molecular weight; P: positive control; C: negative control; H: blank 1-6: transfer-gen plant
Fig. 2: the g2ps1 gene PCR amplification figure in genetically engineered soybean T2 generation
M: standard molecular weight; P: positive control; C: negative control; 1-13: transfer-gen plant
Arrow is depicted as the purpose band
Fig. 3: genetically engineered soybean T2 is for g2ps1 Gene RT-PCR amplification figure
CK: contrast; M: standard molecular weight; C: negative control T: transfer-gen plant
Arrow is depicted as the purpose band
Fig. 4: the kind wave height is fed the tenth day Spodoptera litura larvae upgrowth situation comparison diagram
CK: contrast wave height kind is fed TG: transgenosis wave height plant is fed
Fig. 5: the Spodoptera litura larvae that the kind wave height the is fed comparison diagram of pupating
CK: contrast wave height kind is fed TG: transgenosis wave height plant is fed
Five, embodiment
1) Agrobacterium phtt376 (the Stefan Eckermann et al to include pyrone synthase gene g2ps1,1998, Nature 396:p387～390), utilize pollen tube passage method soybean transformation kind wave height and Nan Nong 73-935 (soybean varieties wave height and Nan Nong 73-935 are public kind, and national modified soybeans center (Nanjing) is for sale).
2) from T1 for, transfer-gen plant is carried out kalamycin resistance seedling stage identifies, antagonism kantlex plant further carries out PCR and identifies, the pcr amplification system contains 0.25mM dNTP, MgCl2,1U Taq enzyme, 0.5 μ M Primer, 100-200ng DNA, cumulative volume 25 μ l, the pcr amplification reaction condition: pre-95 ℃ of 5min of sex change, after 95 ℃ of 15s, 52 ℃ of 59s, 72 ℃ of 2min, 30 circulations, 72 ℃ are extended 10min, select the positive material (Fig. 1) of PCR qualification result.
3) in T2 generation, still carry out kalamycin resistance to transfer-gen plant identify seedling stage, to block that resistant plant and do the PCR evaluation, the pcr amplification system is the same, select the positive material (Fig. 2) of PCR qualification result, further being RT-PCR with this material again identifies, the total RNA of soybean extracts and adopts sky, Beijing is ' RNApure nontoxicity RNA extracts test kit ' of Time Technology company limited, and the pcr amplification system is the same, selects the positive material (Fig. 3) of RT-PCR qualification result.
4) T2 adopts the indoor feeding method to identify the resistance of transgenic line to prodenia litura (purchasing in Institute of Plant Protection, academy of agricultural sciences, Jiangsu Province) for transgenic line, claims every glass of larva heavy (Fig. 4) when feeding to the tenth day.Observe the prodenia litura upgrowth situation simultaneously, the prodenia litura that adjoining tree is fed in the time of the tenth day grew to for 5 ages, the prodenia litura that transfer-gen plant is fed grew to for 4 ages, and the larvae pupation situation is write down every day in the back of weighing, and treated that every glass of larva back of pupating fully claimed pupa heavy (Fig. 5) on the 4th day.Larva is heavily reached pupa to be weighed two groups of data and carries out variance analysis, the larva that two transgenic line wave height, Nan Nong 73-935 feed heavily reach pupa heavy with the contrast nursing between all have significant difference, conspicuous level P＞P0.05, this material are the transgenic line that resistance is identified that passes through of acquisition.
Concrete grammar is as follows:
1. pollen tube passage method soybean transformation
At 7-8 month soybean full-bloom stage, (this moment, the corolla color was vivider to select the unopened bud in plant middle and upper part about 6 o'clock mornings, contour or the corolla of corolla and calyx is higher than calyx 1mm), the playing cards of finishing writing are suspended to bennet portion, and with unnecessary having spent of same position.Afternoon, 3:00 carried out the pollen tube channel importing later on.Left hand is clamped the bottom (do not deflorate lobe) of anthocaulus by flower, and scratches keel lightly with the scissors in the right hand, with an amount of the cutting of the column cap of big Tofu pudding, has both guaranteed that pollen tube existed, and guarantees that again drop subsequently can enter ovary along pollen tube.Aim at the column cap cut off with microsyringe, injection plasmid DNA solution (ice chest is deposited) treats that the damping fluid volatilization of dripping for the first time does back (about 10 minutes), drips for the second time, about per injection 10 μ l again.
2. seedbed screening
Seedling stage, preliminary screening was identified with kalamycin resistance.Method is that absorbent cotton is torn into little kantlex solution that picks 400ppm, adheres on two true leaves of soybean, adopts the identical blank of isoacceptor material, smears.Tangible yellow patch all appears in all soybean leaves that do not contain foreign gene that speckle with kantlex solution after 3 days, and the blade that changes foreign gene over to does not have any symptom still for normal green.After the preliminary screening of seedbed, obtain the big bean seedlings of that resistance of card, the primary part observation record of listing, the management of conventional land for growing field crops ordinary production.The performing PCR Markers for Detection of going forward side by side.
3. the extraction of soy bean DNA and purifying
The CTAB method is extracted soy bean DNA:
1) 1g is frozen leaf (or bright leaf) and put into the mortar of precooling, add liquid nitrogen grinding.The blade that mill is good is poured in the 50ml centrifuge tube, adds extracting solution [composition of damping fluid: CTAB 2% (W/V), the Tris-HCl 0.1M of CTAB, EDTA (pH8.0) 0.05M, NaCl 1.0M], every 10min stirs once, 8000rpm/min is centrifugal, and supernatant is transferred to new pipe.
2) add isopyknic chloroform extracting, low temperature shook on shaking table one hour, and supernatant is transferred to new pipe, with isopyknic isopropanol precipitating DNA, at room temperature placed 10-20min.
3) DNA is chosen with spillikin, washing is 1 hour in 70% alcohol, and is centrifugal, abandons alcohol, and dries up on clean Bechtop.With 10ml HS-TE solution dissolution precipitation, add an amount of 10mg/ml RNase and spend the night.
4) with PH 7.0 saturated phenol extractings once, chloroform extracting twice, supernatant with 2V straight alcohol precipitation, is shaken up the back and places 30min for-20 ℃, DNA is chosen with spillikin, washing is 1 hour in 70% alcohol, and is centrifugal, abandons alcohol, and dries up on clean Bechtop.With an amount of TE buffered soln dissolution precipitation, the DNA after the dissolving meets DNA cloning and requires the back packing to preserve through ultraviolet spectroscopy A260/230 and A260/280.
4. a large amount of extractions and the purifying of plasmid DNA
A large amount of extractions of plasmid DNA and purifying are with reference to Sa nurse Brooker, and the D.W. Russell is outstanding, and 2002, molecular cloning experiment guide (third edition). Beijing: Science Press.Specific as follows:
1) preparation 1000mlLB substratum (Nacl 10g transfers to pH7.0 with 10M NaOH for Tryptone 10g, Yeast extract 5g, adding distil water to 1000ml), every 200mlLB average mark is loaded in the triangular flask of 500ml, and high pressure autosterilization pot is sterilized.
2) etc. the substratum of sterilization cooling back ammonification benzyl to final concentration on Bechtop is 100ppm.In every bottle, add then the pre-culture of intestinal bacteria that 1ml contains goal gene (first picking on the LB solid medium, cultivate contain the single bacterium colony of goal gene intestinal bacteria, in liquid LB 37 ℃, concussion is cultured to logarithmic phase), shaking culture (200 times/min) 12h.
3) culture is changed in the centrifugal bottle of 500ml, 4000rpm/min removes supernatant, and the bacterium colony branch that is deposited in the bottom is installed in the 50ml centrifuge tube.
4) add 5ml solution I [10mMEDTA (pH8.0), autoclaving, 4 ℃ store for future use for 50mM Glucose, 25mM Tris-HCL (pH8.0)] in the centrifuge tube, resuspended precipitation, fully vibration is with dissolution precipitation.
5) add the new solution II of preparing [0.2M NaOH, 1%SDS] of 10ml, the tight pipe lid of lid is slowly put upside down the centrifuge tube several, with abundant mixing Dissolve things inside, ice bath 10-20min.
6) add the ice-cold solution III of 7.5ml [containing the 3M potassium acetate among the 100ml, the 11.5ml glacial acetic acid], the tight pipe lid of lid is slowly put upside down centrifuge tube for several times, and with abundant mixing content, at this moment ice bath 10-20min should have white precipitate.
7) 8000rpm/min, 4 ℃ of centrifugal 20min, with the gauzes of the 4 layers of bacterium of going out supernatant liquid filtering in a 50ml centrifuge tube, add the Virahol of 0.6V, abundant mixing, room temperature is placed 10-20min, 4500rpm/min reclaims plasmid DNA in the centrifugal 20min of room temperature.
8) carefully outwell supernatant, open wide the mouth of pipe inversion centrifuge tube supernatant is flow to end, with 70% washing with alcohol precipitation and tube wall.Pour out 70% ethanol, and centrifuge tube is upside down on the clean paper handkerchief on the Bechtop, so that last remaining trace ethanol volatilization totally.With 4mlTE damping fluid [10mM Tris-HCl (pH8.0), 1mM Na
2-EDTA (pH8.0)] dissolution precipitation, and add 10mg/ml RNase to final concentration 20ug/ml, spend the night.
9) with above-mentioned nucleic acid solution once, twice of chloroform extracting with the saturated phenol extracting of isopyknic alkalescence.4 ℃, the centrifugal 10min of 8000rpm/min, extremely no white protein matter in the middle layering.Supernatant is drawn onto the new centrifuge tube of another,, shakes up the back and placed 2 hours for-20 ℃ with 2V dehydrated alcohol and 0.1V NaCl (2M) precipitation.Carefully outwell supernatant, open wide the mouth of pipe inversion centrifuge tube supernatant is flow to end, with 70% washing with alcohol precipitation and tube wall.Pour out ethanol, and centrifuge tube is upside down on the clean paper handkerchief on the Bechtop, so that last remaining trace ethanol volatilization totally.With 2mlTE damping fluid dissolution precipitation.The quality of the plasmid DNA of 1.0% agarose electrophoresis Detection and Extraction and estimated concentration, the DNA after the dissolving meet as packing after the quality of foreign DNA importing requirement and preserve through ultraviolet spectroscopy A260/230 and A260/280.
5.PCR detect
The pcr amplification system contains 0.25mM dNTP, MgCl
2, 1U Taq enzyme, 0.5 μ M Primer, 100-200ng DNA, cumulative volume 25 μ l.The pcr amplification reaction condition: pre-95 ℃ of 5min of sex change, after 95 ℃ of 15s, 52 ℃ of 59s, 72 ℃ of 2min, 30 circulations, 72 ℃ are extended 10min.Amplification back 1%EB agarose gel electrophoresis or 8%SDS-PAGE polyacrylamide gel detect, and the result can amplify specific band in genetically engineered soybean (molecular weight is: g2ps1 1209bp), does not then have in the contrast.As Fig. 1, Fig. 2.
6.RT-PCR detect
The total RNA of soybean extracts and adopts ' sky is that the total RNA of epoch plant extracts ' test kit, and method is as follows:
1) gets 50-100mg flesh tissue or 30mg dry weight tissue, add 900 μ l lysate RA, in mortar, fully grind.
2) take out ground homogenate, change in the centrifuge tube, add 300 μ l solution RB, put upside down centrifuge tube for several times, mix, be positioned over 5 minutes on ice.
3) 12,000 * g room temperature is centrifugal 3 minutes, changes supernatant liquor over to new centrifuge tube, adds 600 μ l dehydrated alcohol mixings.
Get adsorption column CR and add in the centrifuge tube, change solution and the precipitation that obtains over to adsorption column CR for twice.Centrifugal 30 seconds of 12,000 * g discards the waste liquid in the collection tube.
4) add 0.7ml rinsing liquid RW, centrifugal 30 seconds of 12,000 * g abandons waste liquid.
5) add 0.5ml rinsing liquid RW, centrifugal 30 seconds of 12,000 * g abandons waste liquid.
6) adsorption column is put back in the collection tube, centrifugal 2 minutes of 12,000 * g removes rinsing liquid as far as possible.
7) adsorption column is changed in the new centrifuge tube, add 50-200 μ l RNase-free water, room temperature was placed 2 minutes, centrifugal 2 minutes of 12,000 * g.
8) if RNA expection yield greater than 20 μ g, can repeat to drill and do once, merge obtain for twice solution.
The pcr amplification system contains 0.25mM dNTP, MgCl
2, 1U Taq enzyme, 0.5 μ M Primer, 100-200ng DNA, cumulative volume 25 μ l.The pcr amplification reaction condition: pre-95 ℃ of 5min of sex change, after 95 ℃ of 15s, 52 ℃ of 59s, 72 ℃ of 2min, 30 circulations, 72 ℃ are extended 10min.Amplification back 8%SDS-PAGE polyacrylamide gel detects, and the result can amplify specific band in genetically engineered soybean (molecular weight is: g2ps1 1209bp), does not then have in the contrast.As Fig. 3.
7. the insect-resistance of transfer-gen plant is identified
Insect-resistance is identified and is adopted indoor feeding method:
The soybean chamber planting flowering period, is plucked on the plant and is launched leaf fully, and makes every effort to same position to guarantee the consistence of the fresh and tender degree of blade.Behind the picking blade, wilt, should install with sealed bag immediately, put into ice chest for avoiding influenced by strong sunshine or pyritous.Supporting the worm container is the disposable plastic cup that covers of large size, and bowl cover gets out a plurality of apertures with pin, breathes freely guaranteeing.Inoculate 6 of 1 age in days Spodoptera litura larvae for every glass, triplicate is gathered the blade (transfer-gen plant and adjoining tree) of each material and is fed, and 28 ℃, illumination 14h/d changes twice in fresh blade every day.
Indoor feeding prodenia litura to the claims every glass of larva heavy in the time of ten days, observe the prodenia litura upgrowth situation simultaneously, and the prodenia litura that adjoining tree is fed in the time of the tenth day grew to for 5 ages, and the prodenia litura that transfer-gen plant is fed only grew to for 4 ages, saw Fig. 4.Weigh and write down the larvae pupation situation back every day, treat that every glass of larva back of pupating fully claimed pupa heavy on the 4th day, write down all larvae pupation fates, the larvae pupation time that transfer-gen plant is fed obviously contrast lag behind, and the individual sizes of both pupas also have notable difference, see Fig. 5.The variance analysis larva heavily reaches pupa and weighs two groups of data, and the result shows, the larva that two transgenic line wave height, Nan Nong 73-935 feed heavily reach pupa heavy with the contrast nursing between all have significant difference (conspicuous level P＞P0.05).Illustrate that compared with the control transgenic line can obviously suppress growing of prodenia litura.
Claims (1)
1, a kind of transgenic method that improves the soybean insect-resistance comprises:
1) uses the Agrobacterium phtt376 that includes pyrone synthase gene g2ps1, utilize pollen tube passage method soybean transformation wave height and Nan Nong 73-935;
2) from T1 for, transfer-gen plant is carried out kalamycin resistance seedling stage identifies, resistant plant further is PCR identifies that the pcr amplification system contains 0.25mM dNTP, MgCl
2, the 1UTaq enzyme, 0.5 μ M primer, 100-200ngDNA, cumulative volume 25 μ l, the pcr amplification reaction condition: pre-95 ℃ of 5min of sex change, after 95 ℃ of 15s, 52 ℃ of 59s, 72 ℃ of 2min, 30 circulations, 72 ℃ are extended 10min, select the positive material of PCR qualification result;
3) T2 will block that resistant plant and do the PCR evaluation for still transfer-gen plant being carried out the kalamycin resistance evaluation seedling stage, and the pcr amplification system is the same, selects the positive material of PCR qualification result; Further be RT-PCR with this material again and identify, the total RNA of soybean adopts ' sky is Time Inc.'s plant total RNA extraction reagent box ' to extract, and the pcr amplification system is the same, selects the positive material of RT-PCR qualification result;
4) T2 is identified for the resistance worm that transgenic line carries out the indoor feeding prodenia litura: in soybean bloom, pluck on the plant and launch leaf fully, supporting the worm container is the disposable plastic cup that covers of large size, inoculate 6 of 1 age in days Spodoptera litura larvae for every glass, triplicate is gathered the blade of transfer-gen plant and each material of adjoining tree and is fed 28 ℃, illumination 14h/d changes twice in fresh blade every day; Nursing to the claims every glass of larva heavy in the time of ten days; Observe the prodenia litura upgrowth situation simultaneously, the prodenia litura that adjoining tree is fed in the time of the tenth day grew to for 5 ages, and the prodenia litura that transfer-gen plant is fed grew to for 4 ages, and the larvae pupation situation is write down every day in the back of weighing, and treated that every glass of larva back of pupating fully claimed on the 4th day that pupa was heavy; Larva is heavily reached pupa to be weighed two groups of data and carries out variance analysis, the larva that two transgenic line wave height, Nan Nong 73-935 feed heavily reach pupa heavy with the contrast nursing between all have significant difference, conspicuous level P＞0.05, this material are the transgenic line that resistance is identified that passes through of acquisition.
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| CN102219838B (en) * | 2011-04-22 | 2012-11-28 | 南京农业大学 | Soybean MYC (Myelocytomatosis) transcription factor as well as coding gene and application thereof |
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