CN102703499B - Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers - Google Patents
Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers Download PDFInfo
- Publication number
- CN102703499B CN102703499B CN201210223328.0A CN201210223328A CN102703499B CN 102703499 B CN102703499 B CN 102703499B CN 201210223328 A CN201210223328 A CN 201210223328A CN 102703499 B CN102703499 B CN 102703499B
- Authority
- CN
- China
- Prior art keywords
- rice
- gene
- carrier
- sequence
- disease
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers. The method comprises the following steps: first, cloning green fluorescent protein (GFP) and hygromycin phosphotransferase (HPT) on a marker gene carrier, placing a target gene in other T-DNA carrier, mixing strains of agrobacterium tumefaciens carrying the marker gene carrier and strains of agrobacterium tumefaciens carrying the target gene carrier and converting callus of the rice, performing PCR (polymerase chain reaction) detection on the disease-resistance genes of the target gene carrier by a specific primer, and screening to obtain co-transformation plants (T0); then, screening the marker gene plants with positive GFP rapidly and massively by means of a desk lamp fluorescence detector in segregative generations (T1 or T2) of the co-transformation plant for removing, and performing PCR detection for disease-resistance genes to the plants with negative GFP so as to obtain the individuals without selective markers of the transgenic disease-resistance genes. The method can be applied to transgenic breeding without selective markers of rice blast-resistant genes or other functional genes of the rice, and enhances the disease resistance of the rice or improves other agronomic traits.
Description
Technical field
The present invention relates to the transgenic technology of a kind of paddy rice, especially a kind of rice transformation disease-resistant gene also obtains non selecting sign transgene offspring's method.
Background technology
Cultivating disease-resistant variety is one of major objective of crop breeding.Transgenic technology, as the important supplement of traditional breeding way, can be avoided the complicated processes such as hybridization and many generation selections, by, from Plant Genome clone's disease-resistant gene conversion susceptible variety, utilizes the disease resistance of shorter time improvement acceptor kind.Aspect the Breeding Application of paddy disease-resistant gene, perfect along with Rice Genomics and map-based cloning, a series of paddy disease-resistant genes are by separated and research successively, for transgenic paddy rice research provides abundant genetic resources.
By transgenic technology, cultivate disease-resistant variety and also depend on efficient marker-free transformation system.Two bacterial strains (two carrier) cotransformation, as a kind of conventional marker-free method for transformation, has carrier simply and is easy to the advantages such as structure.Compare with " double T-DNA carrier " co-transformation method of particle, although the latter only uses single conversion carrier, single carrier contains 2 T-DNA districts simultaneously, makes vector construction difficulty relatively.For example, if the goal gene transforming is the paddy rice NBS-LRR class disease-resistant gene (rice blast resistant gene Pi9) that sequence is longer, the difficulty that builds double T-DNA carrier can be larger.
On the other hand, the existing research about two bacterial strain cotransformations shows, the change of frequency of marker gene carrier and goal gene carrier generation cotransformation is larger, affected by vegetable material physiological status and transforming condition of agricillin etc.At transgenosis segregating generation (T1 or T2), need from a large amount of rice plants, to extract DNA and carry out the PCR detection of marker gene and goal gene respectively.In addition, in the genome of about 50% cotransformation plant, the T-DNA of marker gene and goal gene occurs chain, is difficult to obtain separated non selecting sign transgene from its offspring's mass screening individual.So the analytical work of two bacterial strain cotransformation progeny populations is very heavy, need the screening efficiency that improves its non selecting sign transgene individuality badly, so that on efficient, safe transgenic technology basis, cultivate the farm crop new germ plasm that conforms with breeding objective.
2004, Kolesnik etc. carried out transposon tagging research in paddy rice, and green fluorescent protein (GFP), as negative selectable marker, the paddy rice that screens out band T-DNA (simultaneously containing GFP, HPT marker gene and Ac/Ds transposable element) is separated individual.2005, Chen etc. utilized a double T-DNA carrier transformation of tobacco, and wherein the T-DNA of marker gene is with GFP and kalamycin resistance selective marker (NPTII), and the T-DNA of goal gene is only containing gus reporter gene.By GFP fluoroscopic examination, screen out the T1 plant of the GFP positive, then from the negative plant screening of GFP GUS positive individuals, obtained not and to have turned gus gene tobacco containing NPTII mark.
The present invention has introduced GFP fluorescent mark to two bacterial strain cotransformation carrier systems.GFP and hygromix phosphotransferase (HPT) are cloned in to marker gene carrier, and goal gene (being rice blast resistant gene Pi9) is placed in another T-DNA carrier, by two bacterial strain methods, obtains cotransformation plant.At segregating generation (T1 or T2), screen out the marker gene plant of the GFP positive, then the negative plant of GFP is carried out the PCR detection of Pi9 gene, thereby obtain the marker-free individuality that turns Pi9.And by means of desk lamp type fluorimetric detector, screen out in a large number, rapidly GFP positive individuals, reduce the workload of follow-up evaluation Pi9 gene, to improve the screening efficiency of marker-free plant.
Summary of the invention
The present invention will solve the shortcoming of above-mentioned prior art, the rice transformation disease-resistant gene that a kind of screening efficiency is high is provided and obtains non selecting sign transgene offspring's method.
The present invention solves the technical scheme that its technical problem adopts: first, green fluorescent protein (GFP) and hygromix phosphotransferase (HPT) are cloned in to marker gene carrier, goal gene is placed in another T-DNA carrier, the agrobacterium strains that carries marker gene carrier mixes with the agrobacterium strains that carries goal gene carrier and rice transformation callus, utilize special primer to carry out PCR detection to the disease-resistant gene of goal gene carrier, screening obtains cotransformation plant (T0).Then, segregating generation (T1 or T2) cotransformation plant, by means of desk lamp type fluorimetric detector, screens out the marker gene plant of the GFP positive in a large number, rapidly, and the negative plant of GFP is carried out to the PCR detection of disease-resistant gene, thereby obtain the marker-free individuality that turns disease-resistant gene.Genome existence and the sequence that is converted disease-resistant gene homology due to acceptor paddy rice, so according to the disease-resistant gene sequence of goal gene carrier and both sides T-DNA sequence thereof, many cover PCR primers have been designed, between T-DNA border sequence and disease-resistant gene upstream or downstream sequence, increase respectively, goal gene is effectively detected.
Inventing useful effect is: the present invention has introduced GFP fluorescent mark to two bacterial strain cotransformation carrier systems GFP and hygromix phosphotransferase (HPT) are cloned in to marker gene carrier, and by means of desk lamp type fluorimetric detector, in a large number, screen out rapidly GFP positive individuals, reduce the workload of follow-up evaluation Pi9 gene, to improve the screening efficiency of marker-free plant, can be applied to the non selecting sign transgene breeding of rice blast resistant gene or other functional gene, improve paddy disease-resistant ability or improve other economical character.
Accompanying drawing explanation
Fig. 1: the screening strategy of the carrier system of two bacterial strain cotransformations and cotransformation rice plant
Pi9, paddy rice Pi9 blast resistant gene; GFP, green fluorescent protein; HPT, hygromix phosphotransferase; LB and RB, T-DNA left margin and right border sequence;
Fig. 2: the green fluorescence transformant group producing from the Rice Callus of common cultivation
A: through 3d Agrobacterium cultivate altogether with 10 ~ 15d Totomycin substratum on selection cultivate, under lamp formula fluorimetric detector, a plurality of callus produce GPT+ transformant group; B: the GFP+ transformant group producing from single callus;
Fig. 3: in cotransformation rice plant, the PCR of Pi9 blast resistant gene detects
The DNA sequence dna in A:pLJ42 carrier T-DNA district and the PCR primer of Pi9 gene.RB and LB sequence represent with bold-faced letter, and near RB and LB, the homologous sequence of the special primer (RB-1, RB-2, LB-1 and LB-2) of sequence represents with underscore, and the SalI restriction enzyme site of Pi9 both sides represents with tilted letter.Pi9-1, Pi9-2 and Pi9-3, the special primer of Pi9 gene.The amplification direction of primer represents with arrow.B and C: be respectively the PCR (Pi9/RB-PCR) of RB-1 and Pi9-1 primer and the PCR (Pi9/LB-PCR) of LB-1 and Pi9-2 primer.M, DNA molecular amount standard; 1, negative contrast (water); 2, positive control (pLJ42 plasmid); 3, negative contrast (unconverted paddy rice); 4-11,8 independent transformed plants (T0) that Japan is fine.D: sky educates 131, Pi9/RB-PCR and the Pi9/LB-PCR of the T0 plant in the safe B in 11B, Guangdong, Zhejiang and Zhejiang extensive 414 analyze.M, DNA molecular amount standard; 1-2 (sky educates 131); 3-6 (Zhejiang 11B); 7 (the safe B in Guangdong); 8-12 (Zhejiang extensive 414);
Fig. 4 Zhejiang round-grained rice 22T1 is for the pcr analysis of plant Pi9, HPT and GFP gene
Zhejiang round-grained rice 22: rice conversion acceptor; WX207 and WX208: the independent cotransformation rice strain transforming; Pi9+HPT-GFP-: turn the marker-free T1 of Pi9 for plant;
The cotransformation T1 of Fig. 5 Zhejiang extensive 414 and Zhejiang round-grained rice 22 is for plant rice blast fungus inoculated identification
A: the inoculation result of Philippines rice blast fungus strain PO6-6, the Pi9 gene plant performance of marker-free is disease-resistant, and transformation receptor Zhejiang extensive 414 and the T1 plant that do not contain Pi9 all show susceptible; B: 12, Zhejiang Province rice blast fungus strain (B1, B15, C13, D1-1, D1-2, D3-1, D3-2, D3-3, D7-1, D7-2, E1 and E3) mixes the inoculation result of spore, the marker-free plant performance that turns Pi9 gene is disease-resistant, and Zhejiang round-grained rice 22 and not susceptible containing the T1 plant performance of Pi9.Former rich morning, Co39 and the black paddy of Lijing: susceptible variety; Zhejiang extensive 414 and Zhejiang round-grained rice 22: rice conversion acceptor; 75-1-127: containing the rice strain of Pi9 blast resistant gene; WX409: 1 cotransformation strain in Zhejiang extensive 414; WX207: 1 cotransformation strain of Zhejiang round-grained rice 22.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described:
Embodiment: take rice transformation Broad-spectrum blast resistance gene Pi9 as example, by improved pair of bacterial strain (two carrier) cotransformation system, initiative turns the marker-free rice germplasm material of disease-resistant gene.
Vector construction: cotransformation carrier system comprises marker gene carrier (pRB01) and goal gene carrier (pLJ42), as Fig. 1.Green fluorescent protein (GFP) gene clone that corn ubiquitin promoter Ubi is controlled is to HindIII and the EcoRI restriction enzyme site of Agrobacterium-mediated Transformation carrier pCAMBIA1300 (Genbank AF234296), produce marker gene carrier pRB01, pRB01 also carries the hygromycin phosphotransferase gene (HPT) of being controlled by cauliflower mosaic virus CaMV35S promotor.HPT is as hygromycin resistance selective marker, and for Screening of Rice transformant, GFP gene is as visual genetic marker, for following the tracks of rice conversion cell, tissue and the plant of carrying pRB01T-DNA, to improve screening efficiency.Meanwhile, the Nos-polyA sequence in pCAMBIA0380 carrier (Genbank AF234290) T-DNA district is removed, obtain T-DNA district and do not contain the derivative vector pZJ06 of any gene, then the SalI site that the genomic fragment of broad-spectrum rice-blast resistant gene of paddy rice Pi9 (13.5kb) is cloned into pZJ06 carrier (its T-DNA district is not containing any gene), produces goal gene carrier pLJ42.By electrization, pBR01 and pLJ42 are transformed respectively to Agrobacterium EHA105 fungus strain.Each carrier is got 1 Agrobacterium-mediated Transformation at random, extract plasmid DNA and transform e.colistraindh5α, on conversion flat board, choose at random 8-10 and transform bacterium colony, enlarged culturing, extract plasmid and carry out enzyme and cut evaluation, enzyme is cut result and is cut consistent Agrobacterium-mediated Transformation of banding pattern for rice conversion with the enzyme of former pBR01 or pLJ42 carrier.
The inducing culture of Rice Callus: 6 of paddy rice acceptor kinds (being), wherein extensive 414, the Zhejiang round-grained rice 22 in Zhejiang, Zhejiang 11B, Japanese warm and fine sky educate 131 for japonica rice (Oryza sativa L.subsp.japonica), and the safe B in Guangdong is long-grained nonglutinous rice (Oryza sativa L.subsp.indica).Get Mature seed of rice and shell, 70% alcohol-pickled 2min, chlorine bleach liquor's (available chlorine is more than 5%) soaks 2 times, each 20min, aseptic water washing 5 times.Then in the upper cultivation of NB substratum (N6 macroelement, B5 trace element and VITAMIN, 2mg/L2,4-D) 4-5 week.Culturing room's light and temperature condition is 26 ℃, illumination 12h, dark 12h.The embryo callus of induction is transferred to fresh NB substratum, succeeding transfer culture 10d.
Agrobacterium is cultivated: get the single bacterium colony of Agrobacterium of conversion carrier pRB01 and pLJ42, single culture (28 ℃, 200r/min, 1-2d) in containing the YEP liquid nutrient medium of 50mg/L kantlex.In bacterium liquid OD600 value, reach at 1.4 ~ 1.8 o'clock, the bacterium liquid higher to OD value adds YEP, until be diluted bacterium liquid and another bacterium liquid OD value equate.Then the pRB01 bacterium liquid of above-mentioned same cell concentration and pLJ42 bacterium liquid are pressed to 1:4 volume mixture.Centrifugal collection thalline, and be suspended in the AAM nutrient solution that contains 200mM Syringylethanone (acetosyringone, Sigma).With AAM, the OD600 value of mixed bacteria liquid is transferred to 0.7 left and right, for infecting Rice Callus.
Rice conversion: as mentioned above, from the seed maturity embryonal induction callus of each rice varieties (being).The Agrobacterium of carrying goal gene carrier pLJ42 mixes in 4:1 ratio with the Agrobacterium of carrying marker gene carrier pRB01, and each rice varieties (being) is got 100 ~ 300 callus, in 10ml Agrobacterium mixed solution, soaks 10min.Suck bacterium liquid, callus is transferred on sterilizing filter paper, dry 15min on Bechtop.Again callus is transferred to the common culture medium that surface coverage has aseptic filter paper, under 20 ℃ of dark conditions, cultivated 3d.Through the callus of cultivating altogether, first with aseptic washing 4-5 time, then with the aseptic aqueous solution containing 300mg/L Pyocianil, wash 1-2 time 30min.With aseptic filter paper, blot callus surface moisture content, callus is transferred to and selected substratum (NB, 50mg/L Totomycin, 300mg/L cephamycin).Selection for the first time through 15 ~ 20d is cultivated, and under lamp formula fluorimetric detector, the surface of callus grows green fluorescence transformant group (Fig. 2).Then the transformed calli of GFP green fluorescence is expressed in screening, and transfers to fresh selection substratum, selects for the second time to cultivate (continuing 15-20d).The positive callus of GFP is at the upper 25 ~ 30d that cultivates of division culture medium (NB, 3mg/L 6-BA, 0.5mg/LNAA), wait breaking up, transfers to root media (1/2NB, without hormone) root induction when seedling grows to 4cm left and right.
The GFP fluoroscopic examination of rice transformation callus and plant (T0): for the larger transformed calli of volume, the green fluorescence that transformed calli is expressed in the lower directly observation of desk lamp type fluorimetric detector (modular fluorescence desk lamp detector, Hungary BLS company) culture dish.GFP excitation light source is FHS/LS-1B (460-495nm), and spectral filter is FHS/EF-2G2 (505-515nm).For the transformant group of small volume, at the lower GFP fluorescence of observing of come card fluor stereomicroscope (LeicaM165FC).Transformed plant GFP fluorescence detects by desk lamp type detector.
The PCR detection of goal gene and the screening of cotransformation rice plant: transformed plant (T0) is expressed HPT enzyme and GFP fluorescin, performance hygromycin resistance, and by green fluorescence, detect to obtain and confirm.From the T0 plant of the GFP positive, get leaf texture again, extract oryza sativa genomic dna, utilize the special primer of goal gene (rice blast resistant gene Pi9) to carry out pcr analysis, screening is containing the cotransformation rice plant (Pi9+HPT+GFP+) of Pi9, as Fig. 1.Due to the genome existence of acceptor paddy rice and the sequence of Pi9 blast resistant gene homology, so according to the T-DNA sequence of goal gene carrier disease-resistant gene sequence and both sides thereof, designed many cover PCR primer (Fig. 3 A; Table 1), between T-DNA border sequence and disease-resistant gene upstream or downstream sequence, increase respectively (Fig. 3 B, 3C and 3D), thereby goal gene is effectively detected: the amplification of Pi9 upstream sequence, adopt near the homologous primer RB-1 (5'-CGCTCTTTTCTTAGGTTTACC-3') of carrier sequence of T-DNA right margin (RB) and the homologous primer Pi9-1 (5'-AACCGTAAGTAGTATAGCATAGCT-3') of Pi9 gene 5' sequence, or adopt RB-2 (5'-ATATCCTGTCAAACACTGATAGT-3') and Pi9-1.The amplification of Pi9 downstream sequence, adopt the homologous primer Pi9-2 (5'-TCTTTGGACATATGCATGGACC-3') of Pi9 gene 3' sequence and near the homologous primer LB-1 (5'-GTAAACAAATTGACGCTTAGACAAC-3') of the carrier sequence of T-DNA left margin (LB), or adopt Pi9-3 (5'-TGTATATTCTGGCCTTGTTTAGTT-3 ') and LB-2 (5'-ATTGCGGACGTTTTTAATGTACTGA-3 ').Detect the PCR reaction conditions of Pi9: 94 ℃, 3min; 94 ℃, 30s; 62 ℃, 30s; 72 ℃, 1min; 32 circulations; 72 ℃, 5min.For the Pi9 downstream sequence of high GC content, use 2 * GC PCR damping fluid (TAKARA company).RTaq archaeal dna polymerase (TAKARA company) is used in PCR reaction.
Turn the screening of Pi9 disease-resistant gene marker-free offspring plant: from cotransformation plant (T0), gather in the crops T1 for seed, after germination, under desk lamp type fluorimetric detector, detect green fluorescence, distinguish GFP positive (GFP+) and negative (GFP-) plant, then the marker gene plant of the reject GFP positive.Again according to above about the PCR detection method of goal gene (Pi9), from the marker-free plant (table 2) of GFP-plant mass screening band goal gene.Utilize the special primer of HPT and GFP simultaneously, marker-free offspring and other T1 plant are detected, further confirm marker-free plant (Pi9+HPT-GFP-) (Fig. 4); T2 is similar to T1 generation for the screening method of non selecting sign transgene plant: from T1 for Pi9+GFP+ plant results T2 seed, T2 plant is carried out to green fluorescence detection, and then GFP-plant is carried out to the PCR detection of Pi9 gene, screen the Pi9 gene plant (table 3) of marker-free.
T1 is for the blast resistance identification of rice plant: adopt spore suspension spray method to turning Pi9 gene marker-free rice plant inoculation rice blast fungus strain PO6-6 (Fig. 5 A).For transformation receptor, be the WX207 strain (T1) of Zhejiang round-grained rice 22, utilize the mixing spore inoculating (Fig. 5 B) of 12 rice blast fungus strains (B1, B15, C13, D1-1, D1-2, D3-1, D3-2, D3-3, D7-1, D7-2, E1 and E3).Rice blast inoculation method: rice paddy seed shells, surface sterilization, germinates and growth 3-5d on without hormone culture-medium at 1/2MS, is transplanted to the rice soil of sterilizing.The rice seedling 12-14d that grows in growth cabinet.Growth conditions is: illumination cultivation 12h (28 ℃ of temperature, relative humidity 80%), dark culturing 12h (20 ℃ of temperature, relative humidity 60%).On the same day of seed germination, start to cultivate rice blast fungus simultaneously, the filter paper with rice blast fungus spore of-20 ℃ of preservations is inoculated into oat medium (24g/L rolled oats, 12g/L agar, 100mg/L Pyocianil), 27 ℃ of dark 5d that cultivate, then under uniform temp, 5-7d is cultivated in continuous illumination.Rice blast fungus culture rinses with the aqua sterilisa containing 0.02%Tween 20, collects spore suspension, and through Miracloth filtering with microporous membrane.Before inoculation, spore concentration is adjusted to 5 * 105spores/ml.Adopt spray method to rice seedling inoculation rice blast fungus spore, Inoculated Rice seedling, at the 25-26 ℃ of dark 24h that cultivates, is then transferred in growth cabinet and cultivates 6-7d, investigates rice blast incidence.
Table 1 utilizes Pi9 and T-DNA special primer to detect cotransformation T
0rice plant
*pi9/RB-PCR: utilize Pi9-1, RB-1 and RB-2 primer
Pi9/RB-PCR：primers?Pi9-1,RB-1and?RB-2were?used
*pi9/LB-PCR: utilize Pi9-2, Pi9-3, LB-1 and LB-2 primer
Pi9/LB-PCR：primers?Pi9-2,Pi9-3,LB-1and?LB-2were?used
Table 2 turns the marker-free T of Pi9 blast resistant gene
1screening for plant
Table 3 turns the marker-free T of Pi9 blast resistant gene
2screening for plant
*derive from 1 Pi9 of WX409 transgenic lines
+gFP
+hPT
+t
1plant
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (7)
1. a rice transformation disease-resistant gene obtain non selecting sign transgene offspring's method, comprise following steps: 1) green fluorescent protein GFP and hygromix phosphotransferase HPT are cloned in to marker gene carrier, goal gene rice blast resistant gene Pi9 is placed in another T-DNA carrier, 2) agrobacterium strains that carries marker gene carrier mixes with the agrobacterium strains that carries goal gene carrier and rice transformation callus, 3) by green fluorescence, detect confirmation, from the T0 plant of the GFP positive, get leaf texture, extract oryza sativa genomic dna, utilize the special primer of goal gene rice blast resistant gene Pi9 to carry out pcr analysis, screening is containing the cotransformation rice plant Pi9+HPT+GFP+ of Pi9, wherein pcr analysis method is specially: according to the T-DNA sequence of goal gene carrier disease-resistant gene sequence and both sides thereof, many cover PCR primers have been designed, between T-DNA border sequence and disease-resistant gene upstream or downstream sequence, increase respectively, thereby goal gene is effectively detected, the wherein amplification of Pi9 upstream sequence, adopt near the homologous primer RB-1 of the carrier sequence of T-DNA right margin RB, its sequence is 5'-CGCTCTTTTCTTAGGTTTACC-3', homologous primer Pi9-1 with Pi9 gene 5' sequence, its sequence is 5'-AACCGTAAGTAGTATAGCATAGCT-3', or employing RB-2, its sequence is 5'-ATATCCTGTCAAACACTGATAGT-3', and Pi9-1, the amplification of Pi9 downstream sequence, adopt the homologous primer Pi9-2 of Pi9 gene 3' sequence, its sequence is 5'-TCTTTGGACATATGCATGGACC-3', near and the homologous primer LB-1 of carrier sequence T-DNA left margin LB, its sequence is 5'-GTAAACAAATTGACGCTTAGACAAC-3', or adopts Pi9-3, and its sequence is 5'-TGTATATTCTGGCCTTGTTTAGTT-3', and LB-2, its sequence is 5'-ATTGCGGACGTTTTTAATGTACTGA-3', 4) at the segregating generation T1 of cotransformation plant or the chitting piece of T2, by means of desk lamp type fluorimetric detector, screen out the marker gene plant of the GFP positive, and the negative plant of GFP is carried out to the PCR detection of disease-resistant gene, thereby obtain the marker-free individuality that turns disease-resistant gene.
2. rice transformation disease-resistant gene according to claim 1 obtain non selecting sign transgene offspring's method, it is characterized in that: described step 1) specifically, the green fluorescent protein GFP gene clone that corn ubiquitin promoter Ubi is controlled is to HindIII and the EcoRI restriction enzyme site of Agrobacterium-mediated Transformation carrier pCAMBIA1300, produce marker gene carrier pRB01, pRB01 also carries the hygromycin phosphotransferase gene HPT being controlled by cauliflower mosaic virus CaMV35S promotor; The Nos-polyA sequence in pCAMBIA0380 carrier T-DNA district is removed, obtain T-DNA district and do not contain the derivative vector pZJ06 of any gene, then the genomic fragment 13.5kb of broad-spectrum rice-blast resistant gene of paddy rice Pi9 is cloned into the SalI site of pZJ06 carrier, produces goal gene carrier pLJ42.
3. rice transformation disease-resistant gene according to claim 2 obtain non selecting sign transgene offspring's method, it is characterized in that: described step 2) specifically, the Agrobacterium of carrying goal gene carrier pLJ42 mixes with the Agrobacterium bacterium liquid that carries marker gene carrier pRB01, each rice variety system is got 100～300 callus, in 10ml Agrobacterium mixed solution, soaks 10min; Suck bacterium liquid, callus is transferred on sterilizing filter paper, dry 15min on Bechtop; Again callus is transferred to the common culture medium that surface coverage has aseptic filter paper, under 20 ℃ of dark conditions, cultivated 3d; Through the callus of cultivating altogether, first with aseptic washing 4-5 time, then with the aseptic aqueous solution containing 300mg/L Pyocianil, wash 1-2 time 30min; With aseptic filter paper, blot callus surface moisture content, callus is transferred to formula for NB, 50mg/L Totomycin, the selection substratum of 300mg/L cephamycin, selection for the first time through 15～20d is cultivated, and the transformed calli of GFP green fluorescence is expressed in screening, and transfers to fresh selection substratum, select for the second time to cultivate, continue 15-20d; The positive callus of GFP is NB at formula, and 3mg/L6-BA, cultivates 25～30d on the division culture medium of 0.5mg/L NAA, wait breaking up when seedling grows to 4cm left and right, transfers to formula for 1/2NB, without the root media root induction of hormone.
4. rice transformation disease-resistant gene according to claim 3 obtain non selecting sign transgene offspring's method, is characterized in that: described rice variety system comprises that Zhejiang is extensive 414, Zhejiang round-grained rice 22, Zhejiang 11B, Japan are fine, sky educates 131 and the safe B in Guangdong.
5. rice transformation disease-resistant gene according to claim 3 obtain non selecting sign transgene offspring's method, it is characterized in that: the cultivation of described Agrobacterium mixed solution specifically, get the single bacterium colony of Agrobacterium of conversion carrier pRB01 and pLJ42, single culture in containing the YEP liquid nutrient medium of 50mg/L kantlex, culture condition is 28 ℃, 200r/min, 1-2d, in bacterium liquid OD600 value, reach at 1.4～1.8 o'clock, the bacterium liquid higher to OD value adds YEP, until be diluted bacterium liquid and another bacterium liquid OD value equate, then the pRB01 bacterium liquid of above-mentioned same cell concentration and pLJ42 bacterium liquid are pressed to 1:4 volume mixture, centrifugal collection thalline, and be suspended in the AAM nutrient solution that contains 200mM Syringylethanone, with AAM, the OD600 value of mixed bacteria liquid is transferred to 0.7 left and right.
6. rice transformation disease-resistant gene according to claim 3 obtain non selecting sign transgene offspring's method, it is characterized in that: the inducing culture of described Rice Callus specifically, getting Mature seed of rice shells, 70% alcohol-pickled 2min, more than 5% chlorine bleach liquor of available chlorine soaks 2 times, each 20min, aseptic water washing 5 times, then at formula, be N6 macroelement, B5 trace element and VITAMIN, 2mg/L2, on the NB substratum of 4-D, cultivate 4-5 week, culturing room's light and temperature condition is 26 ℃, illumination 12h, dark 12h, the embryo callus of induction is transferred to fresh NB substratum, succeeding transfer culture 10d.
7. rice transformation disease-resistant gene according to claim 1 obtain non selecting sign transgene offspring's method, is characterized in that: the PCR reaction conditions that detects Pi9: 94 ℃, and 3min; 94 ℃, 30s; 62 ℃, 30s; 72 ℃, 1min; 32 circulations; 72 ℃, 5min, for the Pi9 downstream sequence of high GC content, is used 2 * GC PCR damping fluid, and rTaq archaeal dna polymerase is used in PCR reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210223328.0A CN102703499B (en) | 2012-06-27 | 2012-06-27 | Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210223328.0A CN102703499B (en) | 2012-06-27 | 2012-06-27 | Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102703499A CN102703499A (en) | 2012-10-03 |
| CN102703499B true CN102703499B (en) | 2014-02-12 |
Family
ID=46896546
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210223328.0A Expired - Fee Related CN102703499B (en) | 2012-06-27 | 2012-06-27 | Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102703499B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525861B (en) * | 2013-05-07 | 2016-07-20 | 江苏省农业科学院 | Possess bidirectional promoter trapping and the binary Ti plasmid of plasmid rescue function and construction method thereof |
| CN103352049A (en) * | 2013-07-09 | 2013-10-16 | 浙江省农业科学院 | Transposable element vector and screening method of transgenic offspring of transposable element vector |
| CN103952426B (en) * | 2014-04-28 | 2016-06-08 | 四川农业大学 | A kind of double T-DNA carrier containing double; two reporter genes and construction method thereof and application |
| CN104429928A (en) * | 2014-11-27 | 2015-03-25 | 重庆师范大学 | Method for improving identification accuracy of rice blast resistance |
| CN105950743B (en) * | 2016-05-31 | 2019-07-12 | 南京林业大学 | A kind of rice blast resistance gene Pi9 specific Function molecular labeling and its application |
| CN106010986A (en) * | 2016-07-25 | 2016-10-12 | 西南大学 | Separation and identification method of citrus gummosis |
| CN106967784A (en) * | 2017-04-10 | 2017-07-21 | 浙江省农业科学院 | It is a kind of that the method for carrying out blast resistance identification is inoculated with using paddy rice microchamber |
| CN113201555A (en) * | 2021-04-01 | 2021-08-03 | 云南师范大学 | Construction method of binary vector containing eGFP marker and hygromycin resistance |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597969A (en) * | 2004-09-20 | 2005-03-23 | 扬州大学 | Double T-DNA carrier and its application in cultivating of non selecting sign transgene rice |
-
2012
- 2012-06-27 CN CN201210223328.0A patent/CN102703499B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597969A (en) * | 2004-09-20 | 2005-03-23 | 扬州大学 | Double T-DNA carrier and its application in cultivating of non selecting sign transgene rice |
Non-Patent Citations (4)
| Title |
|---|
| 何海燕等.提出转基因植物中选择标记的研究进展.《浙江农业学报》.2012,第24卷(第2期),摘要、第355页右栏第2段、第356页1共转化法. |
| 刘峰等.双农杆菌共转化获得无标记转pepc基因的水稻植株.《应用于环境生物学报》.2005,第11卷(第4期),摘要、第393页跨栏段、第394页1.1-1.4、第395-396页2.1-2.3、图1. |
| 双农杆菌共转化获得无标记转pepc基因的水稻植株;刘峰等;《应用于环境生物学报》;20050825;第11卷(第4期);摘要、第393页跨栏段、第394页1.1-1.4、第395-396页2.1-2.3、图1 * |
| 提出转基因植物中选择标记的研究进展;何海燕等;《浙江农业学报》;20120325;第24卷(第2期);摘要、第355页右栏第2段、第356页1共转化法 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102703499A (en) | 2012-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102703499B (en) | Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers | |
| CN101875689B (en) | New gene for rice zinc-finger protein transcription factor and application thereof in drought resistance and salt tolerance | |
| WO2014036946A1 (en) | Rice brown planthopper resistance gene bph9 and molecular markers, and uses thereof | |
| CN102766618B (en) | Rice OsICL protein and coding gene thereof, and application of the two | |
| CN106399323A (en) | Paddy rice leaf color regulation and control gene YL1 and use thereof | |
| Hensel et al. | Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack) | |
| CN106244607A (en) | The application in resisting verticillium of the cotton cells cytochrome p 450 CYP94C1 gene | |
| CN101693891B (en) | Promoter of shepherd spurse CBF path key gene CbCBF and applications thereof | |
| CN106148390A (en) | CHY zinc finger protein transcriptional activation cofactor and application thereof | |
| CN108642065A (en) | A kind of paddy endosperm silty related gene OsSecY2 and its coding protein and application | |
| CN104593381B (en) | A kind of corn resistant gene of salt and its application | |
| CN104593380B (en) | For the gene ZmHKT1 for the coding corn HKT transport proteins for improving plant salt endurance;1a and its application | |
| CN101942455B (en) | TT16 gene families of brassica napus as well as application thereof | |
| CN101698854A (en) | Application of transcription thellungiella halophila CBF1 gene in improving drought resistance and salt tolerance of corn and wheat | |
| CN101643745A (en) | Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof | |
| CN103172715A (en) | Plant epidermal hair controlling gene and application thereof | |
| CN103951740A (en) | Bermuda grass CCAAT transcription factor CdtNF-YC1 as well as coding gene and application thereof | |
| CN102010864B (en) | Maize pollen tissue specific promoter and expression vector thereof | |
| CN101979551A (en) | Cloning and application of promoter sequence of corn malate dehydrogenase gene | |
| CN103667290A (en) | Corn nutritive organ-specific promoter and application thereof | |
| CN102911941A (en) | Root-specific promoter and application thereof | |
| CN102304526A (en) | Application of Malus xiaojinensis Cheng et Jiang Fe-regulation transporter gene in improvement of Fe content in plant | |
| CN105505984A (en) | Vector of rice respiratory burst oxidase gene OsRboh(LOC_Os01g25820) and application thereof | |
| CN112342218B (en) | Application of BOC1 protein in regulation and control of rice callus browning | |
| CN114989275B (en) | Application of OsERF940 protein in improving rice blast resistance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140212 Termination date: 20180627 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |