CN102911965A - Method for transferring anti-borer (BT) and anti-herbicide (Bar) covalent exogenous genes into saccharum officinarum - Google Patents
Method for transferring anti-borer (BT) and anti-herbicide (Bar) covalent exogenous genes into saccharum officinarum Download PDFInfo
- Publication number
- CN102911965A CN102911965A CN 201210462236 CN201210462236A CN102911965A CN 102911965 A CN102911965 A CN 102911965A CN 201210462236 CN201210462236 CN 201210462236 CN 201210462236 A CN201210462236 A CN 201210462236A CN 102911965 A CN102911965 A CN 102911965A
- Authority
- CN
- China
- Prior art keywords
- bar
- sugarcane
- antiweed
- snout moth
- larva
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the research field of plant adverse resistance, and discloses a method for transferring anti-borer (BT) and anti-herbicide (Bar) covalent exogenous genes into saccharum officinarum. The method mainly includes S1, construction of a plant expression vector; S2, transfer of exogenous genes; and S3, obtaining of positive transgenic plant of the saccharum officinarum through resistance screening, polymerase chain reaction (PCR) identification and the non-toxic processing. According to the method, hereditary characters of the saccharum officinarum are improved, and anti-borer and anti-herbicide capacities of the saccharum officinarum are improved to a certain extent.
Description
Technical field
The present invention relates to stress resistance of plant gene studies field, be specifically related to the method that a kind of anti-snout moth's larva (BT) and antiweed (Bar) covalency foreign gene import sugarcane.
Background technology
Sugarcane (Saccharum officinarum L.) is main in the world sugar crop, and sucrose accounts for 70% of sugared ultimate production.And China is one of sugarcane source region, the world, also is in the world main sucrose producing country, and sucrose yield occupies the third place in the world at present.Sugarcane also is used for papermaking and fuel ethanol production except sugaring.Equally, Sugar Industry occupies critical role in national economy, is to solve one of most important non-grain biomass energy crop of world energy sources crisis.In recent years, snout moth's larva is the cane kind insect that the generation of China sugarcane district is the most general, harm is the most serious, characteristics with full growing season, the harm of brill moth property etc., seedling stage, withered heart rate generally reached 10%～20%, serious in 60% (HuangY K, LiW F.SugarCropsChina, 2006 (4): 34-35), middle and later periods harm causes Sucrose significantly to reduce, and causes that the phenomenon such as windbreak produces.At present, the harm of control sugarcane moth borer has become a very important job on the current sugarcane production.And administer snout moth's larva with comprehensive and environmental protection viewpoint, be the new task that faces of current sugarcane field plant protection work person (Reagan TE.LouisianaAgric, 2001,44:16-18).Sugarcane genetically modified safe class is the I level, is again asexually propagated crop and insutrial crop, and therefore, transgene improvement has obvious advantage.External prevention practice proves, planting pest-resistant sugar cane breed is the safest, the cost-effective method of control sugarcane moth borer, have persistence and cumulative economic benefit, thereby be the first-selected measure (Smith C M.Wiley, New York.1989) of integrated control.And this measure do not conflict mutually with other prophylactico-therapeutic measures (Kogan M.Wiley, New York.1994.73-128).If establishing in large scale can also reduce whole zone the snout moth's larva Population (Reay-JonesFPF, ShowlerAT, ReaganTE, et al.EnvironmentEntomology, 2005,34:1558-1565).From (the Arencibia A such as Arencibia in 1999, Carmona E, Cornide M T, et a.l TransgenRes, 1999,8 (5): 349-360) report has turned since the anti-snout moth's larva proterties of cry1A improvement of genes sugarcane the earliest, has reported again that successively 3 examples turn the resistance research of cry1Ab, gna and the anti-stem snout moth's larva of pinII improvement of genes or Mexico rice borer.
The weeds of harm sugarcane have kind more than 100 at present, subtropical zone high temperature, high humidity weather and sugarcane ground is the exposed weed growth that is particularly suitable for for a long time, need manually to carry out in a large number weeding, greatly increased the input of sugarcane production, simultaneously also bring heavy losses (Li Song, Liao Jiangxiong, Xie Tinglin to sugarcane production, Deng. sugar material sugarcane production weed control technology [J]. China's sugar material, 2010 (3): 49-51).Plant genetically modified anti-weeding crop and can greatly reduce workload, realize mechanized, reduce cost, the soybean of antiweed is widely used on producing at present.After through the development in 15 years, genetically modified crops accumulative total cultivated area had exceeded 1,000,000,000 hm in 2010
2, antiweed genetically modified crops area also increases the genetic resources that sugarcane does not have antiweed year by year, can only could strengthen the antiweed ability of sugarcane by importing foreign gene.Sugarcane genetically modified research mainly concentrates on the fields such as Resistant, drought resisting and antiweed.In sugarcane antiweed transgenic breeding; can make the free amino group acetylize of the effective ingredient PPT of careless fourth phosphine from the Bar gene of soil streptomyces hygroscopicus and the phosphinothricin acetyl transferase of can encoding; the activity that can not suppress GS; thereby make plant obtain the ability (Duan Faping of antiweed; the joist support Ye, an ancient place in Henan Province; Li Yuan celebrating .Bar gene and the progress [J] that turns the Bar gene crops. GUIHAIA, 2001,21 (2): 166-172.).At present the Bar gene is imported sugarcane and have successfully and reports, obtained to have anti-careless fourth phosphine the sugarcane plant (Manickavasagam M, Ganapathi A, Anbazhagan V R, et al.Plant Cell Rep, 2004,23:134-143).
Therefore, for problem set forth above, be necessary to design the method for a kind of anti-snout moth's larva (BT) and antiweed (Bar) covalency foreign gene importing sugarcane.
Summary of the invention
The object of the present invention is to provide a kind of anti-snout moth's larva (BT) and antiweed (Bar) covalency foreign gene to import the method for sugarcane.
In order to solve problem set forth above, the technical solution used in the present invention is:
A kind of anti-snout moth's larva (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, may further comprise the steps:
The construction step of S1, plant expression vector, be specially, reference supports pCambia1300 multiple clone site, design respectively the combination of anti-snout moth's larva (BT) and antiweed (Bar) gene and promoter primer, carry out respectively again the plasmid of the corresponding BT gene of pcr amplification, Bar gene and promotor with combination of primers; The PCR product is behind agarose gel electrophoresis, reclaim respectively, again behind T-clone, positive-selecting, check order, characteristics according to pCambia1300 multiple clone site (MCS) and primer two ends restriction enzyme site, anti-snout moth's larva (BT) and antiweed (Bar) gene and Ubi-1 promoter fragment insertion vector pCambia1300 that order-checking is correct, thus sugarcane genetically modified plant expression vector pCambia1300-BT-bar obtained;
S2, importing foreign gene step are specially, and choose sugarcane plant acceptor, make embryo callus; Plasmid DNA and the purifying of plant expression vector pCambia1300-BT-bar among the extraction step S1 carry out the particle gun bombardment processing to embryo callus.
S3, behind bombardment processing, identify and take off the step that obtains positive sugarcane genetically modified plant after bacterium is processed by resistance screening, PCR.
Preferably, among the described step S1, the PCR product is through 1% agarose gel electrophoresis.
Preferably, among the described step S2, the concrete steps of making embryo callus are:
Choose robust growth from the field and without No. 22 sugarcane plant of new platform sugar of disease and pest, remove the taper blade, wipes of alcohol wash disinfection with 75% under aseptic condition, strips the lobus cardiacus in the above 10cm of vegetative point, it is disk about 2mm that lobus cardiacus is cut into thickness, be inoculated on the inducing culture CM1, under 26 ℃ of-28 ℃ of dark, cultivate, induce the generation callus, then subculture 1-2 time in subculture medium CM2 makes it produce embryo callus.
Preferably, among the described step S2, behind the plasmid DNA of plant expression vector pCambia1300-BT-bar and the purifying quantitatively to 1 μ g μ L
-1
Preferably, among the described step S2, the concrete steps of embryo callus being carried out the particle gun bombardment processing are:
The eugonic embryo callus of 8h picking before bombardment (faint yellow, dry and comfortable, diameter 0.3-0.5cm) is inoculated in high infiltration substratum (MS+2mg
L-12,4-D+0.2mol/L sorbyl alcohol+0.2mol/L N.F,USP MANNITOL+30g L
-1Sucrose+6gL
-1Agar powder, pH5.8) in, carry out pre-treatment;
30min concentrates on the embryo callus of osmotic treated the position, center (2.5cm) of culture dish before bombardment;
Then with particle gun culture dish is carried out bombardment processing.
Preferably, the model of described particle gun is the PDS-1000/He type.
Preferably, the bombardment parameters of described particle gun is: target distance 6cm, air pressure 1100psi, tungsten powder/DNA consumption are 600 μ g μ
L-1, the precipitation agent CaCl of 2.5mol/L
2Spermidine with 0.1mol/L.
Beneficial effect of the present invention is: anti-snout moth's larva disclosed by the invention (BT) and antiweed (Bar) covalency foreign gene import the protein behind the sugarcane, the overexpression of this covalency gene, anti-snout moth's larva and two kinds of resistances of antiweed of sugarcane have been improved to a certain extent, can increase production and improve the agricultural operation, reduce to produce and drop into, have great commercial production and be worth.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited to this.
The invention provides the method that a kind of anti-snout moth's larva (BT) and antiweed (Bar) covalency foreign gene import sugarcane, the method for its specific implementation is:
(1) selects acceptor material
The acceptor material that is used for sugarcane (Saccharum officinarum L.) genetic transformation is No. 22, new platform sugar and osmanthus sugar 03-1403 sugarcane plant, is provided by academy of agricultural sciences, Guangxi sugarcane institute in the present embodiment.
Bacterial strain and plasmid: the cry1Ac gene can directly obtain, and plant expression carrier plasmid is introduced by Australian Cambia company, and intestinal bacteria (E.coli) DH5 α bacterial strain is provided by Guangxi Sugarcane genetic improvement emphasis open laboratory in the present embodiment.
Substratum: inducing culture CM
1Be MS+3mg L
-12,4-D+30g
L-1 sucrose+6gL
-1Agar powder, pH5.8; Subculture medium CM
2Be MS+2mg L
-12,4-D+30g L
-1Sucrose+6gL
-1Agar powder, pH5.8; Division culture medium CM
3Be MS+2.0mg L
-1BA+0.5mg L
-1KT+0.2mgL
-1NAA+30gL
-1Sucrose+6g L
-1Agar powder, H5.8; Root media CM
4: 1/2MS+0.2mgL
-16-BA+3mg L
-1NAA+60gL
-1Sucrose+6gL
-1Agar powder, pH5.8.
(2) structure of plant expression vector
Reference supports pCambia1300 multiple clone site, design respectively the combination of anti-snout moth's larva (BT) and antiweed (Bar) gene and promoter primer, primer is synthetic by Nanjing Genscript Biotechnology Co., Ltd., order-checking is all finished by giving birth to worker's biotechnology (Shanghai) limited-liability company, and primer sequence is as follows:
Carry out the corresponding BT gene of pcr amplification with combination of primers respectively, the plasmid of Bar gene and promotor, the PCR product is behind 1% agarose gel electrophoresis, reclaim respectively, the T-clone, positive-selecting, send the order-checking of living worker's biotechnology (Shanghai) limited-liability company, characteristics according to pCambia1300 multiple clone site (MCS) and primer two ends restriction enzyme site, anti-snout moth's larva (BT) and antiweed (Bar) gene and Ubi-1 promoter fragment insertion vector pCambia1300 that order-checking is correct obtain sugarcane genetically modified plant expression vector pCambia1300-BT-bar.
(3) import foreign gene:
Choose robust growth from the field and without the sugared No. 22 sugarcane plant of the new platform of disease and pest, remove the taper blade, the wipes of alcohol wash disinfection with 75%, under aseptic condition, strip the lobus cardiacus in the above 10cm of vegetative point, it is disk about 2mm that lobus cardiacus is cut into thickness, is inoculated in inducing culture CM
1On, under 26 ℃ of-28 ℃ of dark, cultivate, induce the generation callus, then at subculture medium CM
2Middle subculture 1-2 time makes it produce embryo callus.
Extract plasmid DNA and the purifying of plant expression vector pCambia1300-BT-bar, quantitatively to 1 μ g μ L
-1The eugonic embryo callus of 8h picking before bombardment (faint yellow, dry and comfortable, diameter 0.3-0.5cm) is inoculated in high infiltration substratum (MS+2mgL
-12,4-D+0.2mol/L sorbyl alcohol+0.2mol/L N.F,USP MANNITOL+30g L
-1Sucrose+6gL
-1Agar powder, pH5.8) in, carry out pre-treatment.30min concentrates on the embryo callus of osmotic treated the position, center (2.5cm) of culture dish before the bombardment.Particle gun is the PDS-1000/He of Bio-Rad company type, every ware bombardment 1 time.Adopt the bombardment parameters of optimizing: target distance 6cm, air pressure 1100psi.Tungsten powder/DNA consumption is 600 μ g μ L
-1, the precipitation agent CaCl of 2.5mol/L
2With the spermidine (now with the current) of 0.1mol/L, its concrete steps are carried out according to PDS-1000/He type particle gun process specifications.
(4) transgenic regenerated plant obtains
Identify and take off bacterium and process and obtain positive sugarcane genetically modified plant by resistance screening, PCR.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. an anti-snout moth's larva (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, it is characterized in that, may further comprise the steps:
The construction step of S1, plant expression vector, be specially: the multiple clone site of reference supports pCambia1300, insert respectively suitable restriction enzyme site according to anti-snout moth's larva (BT), antiweed (Bar) gene and promoter sequence, to resist snout moth's larva (BT), antiweed (Bar) gene and promotor insertion vector pCambia1300 by gene recombination technology, obtain sugarcane genetically modified plant expression vector pCambia1300-BT-bar.
S2, importing foreign gene step are specially: utilize particle gun will wrap up little bullet bombardment Sugarcane Embryogenic Callus of plant expression vector pCambia1300-BT-bar.
Behind S3, the bombardment processing, identify and take off the sugarcane genetically modified plant of the rear acquisition positive of bacterium processing by resistance screening, PCR.
2. anti-snout moth's larva according to claim 1 (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, and it is characterized in that: among the described step S1, the PCR product is through 1% agarose gel electrophoresis.
3. anti-snout moth's larva according to claim 1 (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, it is characterized in that: among the described step S2, the concrete steps of making embryo callus are:
Choose robust growth from the field and without No. 22 sugarcane plant of new platform sugar of disease and pest, remove the taper blade, wipes of alcohol wash disinfection with 75% under aseptic condition, strips the lobus cardiacus in the above 10cm of vegetative point, it is disk about 2mm that lobus cardiacus is cut into thickness, be inoculated on the inducing culture CM1, under 26 ℃ of-28 ℃ of dark, cultivate, induce the generation callus, then subculture 1-2 time in subculture medium CM2 makes it produce embryo callus.
4. anti-snout moth's larva according to claim 1 (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, it is characterized in that: among the described step S2, behind the plasmid DNA of plant expression vector pCambia1300-BT-bar and the purifying quantitatively to 1 μ g μ L
-1
5. anti-snout moth's larva according to claim 1 (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, and it is characterized in that: among the described step S2, the concrete steps of embryo callus being carried out the particle gun bombardment processing are:
The eugonic embryo callus of 8h picking before bombardment (faint yellow, dry and comfortable, diameter 0.3-0.5cm) is inoculated in high infiltration substratum (MS+2mgL
-12,4-D+0.2mol/L sorbyl alcohol+0.2mol/L N.F,USP MANNITOL+30g L
-1Sucrose+6gL
-1Agar powder, pH5.8) in, carry out pre-treatment;
30min concentrates on the embryo callus of osmotic treated the position, center (2.5cm) of culture dish before bombardment;
Then with particle gun culture dish is carried out bombardment processing.
6. anti-snout moth's larva according to claim 5 (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, and it is characterized in that: the model of described particle gun is the PDS-1000/He type.
7. anti-snout moth's larva according to claim 5 (BT) and antiweed (Bar) covalency foreign gene import the method for sugarcane, it is characterized in that: the bombardment parameters of described particle gun is: target distance 6cm, air pressure 1100psi, tungsten powder/DNA consumption are 600 μ g μ L
-1, the precipitation agent CaCl of 2.5mol/L
2Spermidine with 0.1mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210462236 CN102911965A (en) | 2012-11-16 | 2012-11-16 | Method for transferring anti-borer (BT) and anti-herbicide (Bar) covalent exogenous genes into saccharum officinarum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210462236 CN102911965A (en) | 2012-11-16 | 2012-11-16 | Method for transferring anti-borer (BT) and anti-herbicide (Bar) covalent exogenous genes into saccharum officinarum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102911965A true CN102911965A (en) | 2013-02-06 |
Family
ID=47610545
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210462236 Pending CN102911965A (en) | 2012-11-16 | 2012-11-16 | Method for transferring anti-borer (BT) and anti-herbicide (Bar) covalent exogenous genes into saccharum officinarum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102911965A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070616A (en) * | 2016-11-07 | 2018-05-25 | 沈阳农业大学 | A kind of In Planta transformation method of low pressure particle gun mediation |
| CN109355311A (en) * | 2018-11-30 | 2019-02-19 | 广西大学 | A method of passing through via Particle Bombardment Transformation fast culture sugarcane resistant plant |
| WO2019061621A1 (en) * | 2017-09-26 | 2019-04-04 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for plasma high-throughput directed mutagenesis of sugarcane against glyphosate |
| CN114164230A (en) * | 2022-01-11 | 2022-03-11 | 广西壮族自治区农业科学院 | Expression vector suitable for sugarcane genetic transformation and construction method and application thereof |
-
2012
- 2012-11-16 CN CN 201210462236 patent/CN102911965A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108070616A (en) * | 2016-11-07 | 2018-05-25 | 沈阳农业大学 | A kind of In Planta transformation method of low pressure particle gun mediation |
| WO2019061621A1 (en) * | 2017-09-26 | 2019-04-04 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for plasma high-throughput directed mutagenesis of sugarcane against glyphosate |
| US11516979B2 (en) | 2017-09-26 | 2022-12-06 | Institute of Nanfan & Seed Industry, Guangdong Academy of Sciences | Method for conducting high-throughput and directed mutagenesis for sugarcane resistance to glyphosate by plasma |
| CN109355311A (en) * | 2018-11-30 | 2019-02-19 | 广西大学 | A method of passing through via Particle Bombardment Transformation fast culture sugarcane resistant plant |
| CN114164230A (en) * | 2022-01-11 | 2022-03-11 | 广西壮族自治区农业科学院 | Expression vector suitable for sugarcane genetic transformation and construction method and application thereof |
| CN114164230B (en) * | 2022-01-11 | 2022-10-14 | 广西壮族自治区农业科学院 | Expression vector suitable for sugarcane genetic transformation and construction method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102660560B (en) | Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof | |
| CN101358190B (en) | Artificially synthetic high gene sequence expressing high virulence protein for lepidoptera pest and use thereof | |
| CN103266132A (en) | Bacillus thuringiensis cry1Ah/cry1Ie bivalent gene expression vector and application thereof | |
| CN112724214A (en) | Xanthoceras sorbifolia drought induction transcription factor XsMYB308L and application thereof | |
| CN102816773A (en) | Application of OsWRKY28 transcription factor gene of rice in improvement of plant disease resistance | |
| CN102911965A (en) | Method for transferring anti-borer (BT) and anti-herbicide (Bar) covalent exogenous genes into saccharum officinarum | |
| CN103288943B (en) | Protein bHLH13 (Basic Helix Loop Helix 13) as well as coding gene and application thereof | |
| CN102154337B (en) | Gossypium hirsutum mitogen-activated protein kinas 6 (GhMAPK6) gene and application thereof | |
| CN101775409A (en) | Method for obtaining transgenic corns by infecting young ears with agrobacterium | |
| CN110607307A (en) | Sedum lineare salt-tolerant gene SlNAC and application thereof | |
| CN110982817A (en) | amiRNA for resisting wheat yellow mosaic virus and application thereof | |
| WO2019091390A1 (en) | Nucleotide sequence and application thereof in enhancing pest-resistant ability of plant | |
| CN1768575A (en) | Method for improving beet salt-resistance, draught-resistance and anti-herbicide chlorsulfuron characteristics by multi-gene transformation and application | |
| CN104726488A (en) | Method for culturing stress-resistance herbicide-resistance transgenic aerobic rice | |
| CN100372935C (en) | Cloning of gene against meloidogyne of capsicum and application thereof | |
| Lei et al. | Agrobacterium-mediated transformation of cotton shoot apex with SNC1 gene and resistance to cotton Fusarium wilt in T1 generation | |
| CN113637678A (en) | Application of gene GhSWEET42 in prevention and treatment of cotton verticillium wilt | |
| ZHANG et al. | Breeding and identification of insect-resistant rice by transferring two insecticidal genes, sbk and sck | |
| Xu et al. | Construction of expression vector of CryIA (c) gene and its transformation in sugarcane | |
| CN101463393A (en) | Method for screening transgenic plate with safety marker gene | |
| CN109837298A (en) | A kind of degeneration-resistant genetic conversion system of sweet cherry roots and its construction method | |
| CN102329817B (en) | Agrobacterium-mediated method for culturing transgenic populus wutunensis plants | |
| CN103667294B (en) | DNA molecule for expressing hairpin RNA for inhibiting macrosiphum avenae lipoprotein lipase and applications thereof | |
| CN114032247B (en) | Application of insecticidal gene cry2Ah-vp and cry9Ee combination in insect-resistant plants | |
| CN102220347B (en) | Wheat TaTOC1 genes, cloning method and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| DD01 | Delivery of document by public notice |
Addressee: Nanjing Oyang Hi-Tech Agriculture Co.,Ltd. Document name: Notification of Passing Preliminary Examination of the Application for Invention |
|
| DD01 | Delivery of document by public notice |
Addressee: Nanjing Oyang Hi-Tech Agriculture Co.,Ltd. Document name: Notification of Publication of the Application for Invention |
|
| DD01 | Delivery of document by public notice |
Addressee: Nanjing Oyang Hi-Tech Agriculture Co.,Ltd. Document name: Notification that Application Deemed to be Withdrawn |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130206 |